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A ribonucleotide reductase has been purified to homogeneity from Euglena gracilis. The enzyme will reduce the four common ribonucleoside triphosphates when a dithiol and 5′-deoxyadenosylcobalamin, required for activity, are added. Magnesium ions can stimulate the reaction. CTP reduction can be stimulated by dATP or ATP, and can be inhibited, as can ATP and GTP reduction, by dTTP. Reduction of UTP is unaffected by deoxyribonucleotides. The molecular weight of the enzyme from Euglena, as determined from gel filtration, is 440,000. Sodium dodecyl sulfate gels indicate subunits of approximately 100,000.

Acetyl-CoA: S-(β-aminoethyl)-l-cysteine ω-N-acetyltransferase, an enzyme catalyzing the transfer of acetyl group from acetyl-CoA to the terminal amino group of S-(β-aminoethyl)-l-cysteine, a metabolic antagonist of l-lysine, has been purified about 450-fold from the cell-free extracts of Aerobacter aerogenes. The purified enzyme is homogeneous by the criterion of disc gel electrophoresis and has an approximate molecular weight of 100,000. In addition to S-(β-aminoethyl)-l-cysteine, its d-enantiomer, sulfoxide, sulfone, and higher homolog, and an oxygen analog of lysine, O-(β-aminoethyl)-dl-serine act as acetyl acceptors. l- or d-Lysine and l- or d-ornithine also can accept an acetyl group to a certain extent, but α- or ω-N-acetylated derivatives of both S-(β-aminoethyl)-l-cysteine and l-lysine are not active. Acetyl-CoA is the exclusive acyl donor in the transfer reaction. The following apparent Michaelis constants were determined: S-(β-aminoethyl)-l-cysteine, 2.1 x 10-3 m; S-(β-aminoethyl)-d-cysteine, 1.6 x 10-3 m; O-(β-aminoethyl)-dl-serine, 1.7 x 10-3 m; S-(β-aminoethyl)-l-homocysteine, 7.1 x 10-4; and acetyl-CoA, 4.5 x 10-3 m. The enzyme activity is inhibited competitively in propionyl-CoA, butyryl-CoA, and benzoyl-CoA against acetyl-CoA. S-(β-Aminoethyl)-α-N-acetyl-l-cysteine and S-(β-aminoethyl)-mercaptopropionate are strong competitive inhibitors of S-(β-aminoethyl)-l-cysteine acetylation.

Highly purified, stable preparations of adenosine monophosphate nucleosidase (EC 3.2.2.4) have been studied by initial velocity kinetics in order to determine the kinetic parameters of the enzyme and the relationships between the substrate and modifier sites. The results indicate that AMP nucleosidase contains interdependent modifier sites which can be occupied by MgATP2-, ATP4-, or MgPPi to give catalytically competent enzyme-activator complexes. MgATP2- is the most effective activator with ATP4- and MgPPi being less effective. The presence of saturating concentrations of activator increased the initial velocity from 100- to 400-fold over that in the absence of activator. The Hill coefficients for all three activator species are between 2.5 and 3.5 and are not affected by the concentration of AMP, the substrate of the reaction. The substrate sites exhibit interdependent kinetic behavior only at low activator concentrations and give linear double reciprocal plots of initial velocity as a function of AMP concentration at higher concentrations of activator. At high concentrations of the substrate, AMP, apparent substrate inhibition occurs. Kinetic results are consistent with this inhibition occurring by AMP combination at the modifier sites, thereby displacing the activator. Such inhibition occurs with each of the activators tested. Kinetic studies of the inhibition by AMP showed that the enzyme complex with AMP saturating at both the substrate and modifier sites retains a basal level of activity. This finding was substantiated by the demonstration of equivalent basal activity in the absence of activators. Kinetic studies of the activator-free AMP hydrolysis were consistent with the substrate sites maintaining a low rate of hydrolytic activity when the modifier sites are unoccupied. Saturation of the modifier sites with AMP leads to an increase in the rate of hydrolysis at the substrate sites. The enzyme is strongly inhibited by low concentrations of inorganic phosphate and by higher concentrations of sulfate. This inhibition occurs by competition with respect to the modifier site. From the kinetic parameters obtained, it is suggested that AMP nucleosidase is regulated in vivo by the intracellular ratios of MgATP2- and inorganic phosphate.

In crude homogenates of the submaxillary gland of the male mouse, epidermal growth factor activity is found almost entirely in a high molecular weight complex. The complex has a molecular weight of about 74,000 and appears to be composed of 2 molecules of epidermal growth factor (6,045 molecular weight) and 2 molecules of a binding protein (29,300 molecular weight). The complex is stable only in the range of pH 5.0 to 8.0, and its behavior during isoelectric focusing, gel filtration, and ultracentrifugation suggests that it may be in a slow equilibrium with its subunits. In addition, the complex appears to undergo a concentration-dependent aggregation. The biological activity of the high molecular weight complex is proportional to its fractional content of the low molecular weight factor. No evidence was found which indicated that the binding protein might affect the biological activity of the low molecular weight epidermal growth factor. It is postulated that the high molecular weight form of epidermal growth factor is an enzyme-product complex. The substrate is presumed to be the epidermal growth factor precursor which is recognized by a specific arginine esterase, the binding protein.

Rat kidney contains two distinct glutaminase isoenzymes; one of which is phosphate-dependent, the other is phosphate-independent but is strongly activated by maleate. The phosphate-dependent glutaminase is contained within mitochondria. Its activity cofractionates with mitochondrial marker activities during differential centrifugation and on sucrose gradients using both isopycnic and sedimentation velocity techniques. Incubation of mitochondria with borate buffer results in increased phosphate-dependent glutaminase, cytochrome oxidase, and malate dehydrogenase activities, indicating that this glutaminase activity may be latent. Submitochondrial fractionation by digitonin-Lubrol treatment in the presence of borate indicated that this glutaminase isoenzyme is contained in the inner mitochondrial membrane. This conclusion was confirmed by a swell-shrink, sonication procedure carried out in the absence of borate. Only 25 to 35 % of the phosphate-independent glutaminase was associated with the mitochondrial fraction obtained by differential centrifugation; the remaining activity was pelleted by subsequent centrifugation at 40,000 x g for 30 min (heavy microsomes). The phosphate-independent glutaminase in both fractions banded during isopycnic centrifugation with a mean density (1.166) similar to that of light mitochondria. However, the results of sedimentation velocity centrifugation studies showed that this isoenzyme was contained in an organelle distinct from mitochondria. The phosphate-independent glutaminase did not fractionate with marker activities for lysosomes, microsomes, or plasma membranes during differential centrifugation or isopycnic gradient analysis and may be contained in some as yet unidentified organelle.

The methylglucose-containing lipopolysaccharide (MGLP) of Mycobacterium phlei is an acidic molecule with 18 hexose units. Four forms (MGLP-I to MGLP-IV) exist with from 0 to 3 moles of esterified succinate. Each form is also acylated with acetate, propionate, isobutyrate, and octanoate, totaling 6 moles. Eight positions of acylation have been ascertained by methyl replacement (Gray, G. R., and Ballou, C. E. (1972) J. Biol. Chem. 247, 8129–8135). Six of these positions are now shown to be acylated specifically with monobasic acids and two others specifically with acidic succinyl groups. In addition, another site of succinylation has been found, which accounts for the nine positions in MGLP-IV. Characterization of newly methylated fragments produced by methyl replacement and Smith degradation of MGLP-I, which does not contain succinate, was used to determine placement of the neutral acyl groups. To complement these studies, we characterized the neutral acylated fragments isolated from a mixture of Smith-degraded MGLP-I and II, which were isotopically labeled in the various monobasic acyl groups. The positions at which succinate was esterified were determined by characterization of acidic acylated products resulting from Smith degradation of MGLP-II which contained labeled succinate. Our results indicate that the monobasic acids were esterified at positions 4 and 6 of the 3-O-methylglucose unit, and at position 6 of the 3 adjacent glucose residues at the 3-O-methylglucose end of the chain. An additional neutral acyl group (probably the octanoate) was esterified to position 3 of the glyceric acid. Propionate was found in two of the positions of acylation nearest the 3-O-methylglucose end of MGLP, indicating that there was some heterogeneity in the placement of monobasic acids among these sites since only 1 mole of propionate is normally present. Succinate was esterified at position 6 of the remaining glucose residues near the glyceric acid end of the molecule. MGLP-II, which contains only 1 mole of succinate per mole of polysaccharide, was succinylated to some extent at each of these three positions. The nonrandom distribution of monobasic versus dibasic acids provides support for the concept that a defined placement of esters is in some way related to the biological function of the lipopolysaccharide.

Longitudinal and transverse relaxation rates of water protons were measured as a function of temperature (1–40°) and frequency (6 to 48 MHz) for Mn(III)- and Fe(III)-containing superoxide dismutases. For the Mn(III) superoxide dismutase, the bulk of the molar relaxivity can be accounted for by 1 water molecule in the first hydration shell. In contrast, a portion of the water relaxivity for the Fe(III) superoxide dismutase comes from 1 proton in the first coordination shell and up to 50% from a water molecule in the second shell. For both enzymes a mechanism involving diffusion of O2- into the second coordination shell but probably not into the primary coordination shell is compatible with kcat = 3 x 109 m-1 s-1. These data are supported by the observation that KCN does not diminish the water relaxation rates for either the Mn(III) or Fe(III) enzymes.

Glutamine synthetase purified to apparent homogeneity from Bacillus subtilis is subject to feedback inhibition by multiple end products of glutamine metabolism, as well as by the reaction product l-glutamine. AMP, glutamine, and histidine are potent inhibitors when any of the substrates glutamate, MnATP, or ammonia are present in limiting concentration during assay, but the strong inhibition by glycine and alanine is influenced far less by substrate concentration. In the Mg2+-dependent biosynthetic assay, AMP and glutamine are potent inhibitors of glutamine synthetase, whereas alanine and glycine do not inhibit this activity at all. Tryptophan, which is not an inhibitor in the Mn2+ biosynthetic assay, exhibits some inhibition in the Mg2+ assay. Synergistic inhibition of the Mn2+-dependent glutamine synthetase activity is present when AMP and glutamine are studied together; glutamine alone is not inhibitory when present at 5 mm concentration in the saturating assay, but produces 85% inhibition when studied in the presence of 2.5 mm AMP. Histidine and AMP are also synergistic, but glutamine acts independently of histidine. Alanine and glycine reciprocally reduce the effectiveness of one another as inhibitors. Glutamine appears to play a major role in the over-all regulation of its own synthesis. In Escherichia coli, glutamine modulates the glutamine synthetase adenylylation-deadenylylation system, thereby controlling over-all enzyme activity as well as responsiveness to feedback inhibitors. B. subtilis appears to lack the mechanisms to adenylylate and deadenylylate preformed glutamine synthetase. The organism does, however, appear to achieve similar regulatory effects by the direct inhibition of glutamine synthetase by glutamine, and by the striking potentiation of AMP inhibition that occurs with low concentrations of glutamine. Therefore, in B. subtilis as well as E. coli, intracellular glutamine levels play an important role in the fine control of the over-all glutamine synthetase activity, and in the control of cellular anabolic nitrogen metabolism.

The autoxidation of dithiothreitol, 2-mercaptoethanol, reduced glutathione, and ethyl mercaptan has been seen to involve the univalent reduction of oxygen. The superoxide radicals, so generated, were detected by their ability to reduce p-nitroblue tetrazolium to a formazan and by the inhibition of this reaction by superoxide dismutase. The rate of autoxidation of these thiols and the sensitivity of the reduction of nitroblue tetrazolium to inhibition by superoxide dismutase were both augmented, as the pH was raised from 7.8 to 10.2. These results, lead to the proposal that thiols reduce nitroblue tetrazolium by at least two different pathways, one of which involves O2- and is therefore inhibitable by superoxide dismutase.

The mechanism for regulation of α-ketoglutarate metabolism by the phosphorylation state of the intramitochondrial nucleotides has been investigated using intact rat and guinea pig liver mitochondria and isolated α-ketoglutarate dehydrogenase. Depletion of ATP under conditions of either high or low intramitochondrial NADH levels was achieved by addition of oligomycin plus ADP and glucose-hexokinase as an extramitochondrial ATP trapping system to State 4 or uncoupled mitochondria, or by a stimulation of the rate of GTP utilization for P-enolpyruvate synthesis by malate addition. A decrease of the ATP:ADP ratio in rat liver mitochondria resulted in a decrease of the succinyl-CoA to CoA-SH ratio. The GTP:GDP ratio, calculated by assuming equilibrium at succinate thiokinase, also decreased over a 4-fold range but the measured GTP content (approximately 0.2 nmoles per mg of protein) remained constant within analytical error. Nucleoside diphosphate kinase was calculated to be displaced from equilibrium by about two orders of magnitude. Flux through α-ketoglutarate dehydrogenase, as measured by α-ketoglutarate uptake, varied inversely with the succinyl-CoA to CoA-SH ratio at both high and low levels of intramitochondrial NADH. The rate of α-ketoglutarate uptake by mitochondria having approximately equal intramitochondrial phosphate potentials and succinyl-CoA to CoA-SH ratios was greater at low than at high ratios of NADH:NAD. Addition of malate to guinea pig liver mitochondria incubated with α-ketoglutarate in the presence of uncoupler and oligomycin caused a 2-fold stimulation of α-ketoglutarate uptake, a 4.4-fold increase of P-enolpyruvate production and a 4-fold decrease of the succinyl-CoA to CoA-SH ratio. The ATP content decreased from 3.5 to 1.1 nmoles per mg of protein while the GTP content changed insignificantly from 0.21 to 0.18 nmoles per mg of protein. With guinea pig liver mitochondria at low NADH levels, a linear relationship was obtained between the reciprocal of the α-ketoglutarate uptake and the succinyl-CoA to CoA-SH ratio, which provided a value of 1.6 for the ratio of Michaelis constant for CoA-SH to the inhibition constant for succinylCoA. Initial velocity kinetics with purified pig heart α-ketoglutarate dehydrogenase showed that succinyl-CoA and NADH were strong inhibitors, but no inhibitory effects were obtained with GTP or ATP. The inhibition by succinyl-CoA was independent of the NADH:NAD ratio and was competitive with CoA-SH. At high α-ketoglutarate and NAD concentrations, the Km for CoA-SH was 2.7 µm and the Ki for succinyl-CoA was 6.9 µm. Inhibition by NADH was noncompetitive with respect to both NAD and α-ketoglutarate. At high α-ketoglutarate and CoA-SH concentrations, the Km for NAD was 21 µm and the Ki for NADH was 4.5 µm. It is concluded that regulation of flux through α-ketoglutarate dehydrogenase by the state of phosphorylation of the nucleotide systems in rat and guinea pig liver mitochondria is achieved primarily by variations of the succinyl-CoA to CoA-SH ratio and to a lesser extent by variations of the NAD oxidation-reduction state. There are no grounds for supposing a direct effect via GTP or ATP.

Specific binding of 1α,25-dihydroxycholecalciferol in the cytosol and purified chromatin fractions of chick small intestinal mucosa was studied to investigate the mechanism by which this hormone arrives at its chromatin binding site. The cytosol fraction of the mucosa contains a macromolecule with a high affinity (Kd = 2.2 x 10-9 m) for 1α,25-dihydroxycholecalciferol and has a limited number of binding sites which are saturated at low hormone concentrations (≃ 10-8 m). The association of the sterol with the cytoplasmic component is thermolabile, and is destroyed by treatment with pronase, but not by nucleases, and the receptor component is therefore presumed to be a protein. The protein-sterol complex was isolated and characterized by sucrose gradient centrifugation and sediments at 3 to 3.5 S in the presence of 0.15 m KCl. The interaction of 1α,25-dihydroxy[3H] cholecalciferol with the cytosol protein is reversed by unlabeled 1α,25-dihydroxycholecalciferol g 25-hydroxycholecalciferol g 1α,25-hydroxycholecalciferol g cholecalciferol ≃ estradiol-17β. The subcellular localization of 1α,25-dihydroxycholecalciferol is temperature-dependent. The hormone remains bound to the cytosol fraction at 0° in whole intestines in vitro, and a dramatic movement of 1α,25-dihydroxycholecalciferol to the nuclear chromatin occurs at 37°. The temperature dependence of the hormone localization is also evident in intestinal mucosa homogenates and reconstituted cytosol-chromatin systems. This observation suggests that the cell membrane and nuclear membrane are not involved in the temperature-dependent process. It is postulated that the specific cytosol receptor for 1α,25-dihydroxycholecalciferol functions to transport the hormone to its ultimate site of action in the nuclear chromatin.

4-Aminobutanal dehydrogenase has been purified from a Pseudomonas species. The enzyme has high activity toward aminoaldehydes (4-aminobutanal or 3-aminopropanal) and low activity toward succinic semialdehyde as substrate. The kinetic constants, effect of p-hydroxymercuribenzoate, and pH profile with different substrates are documented. The enzyme has a molecular weight of 228,000 as determined by gel filtration and polyacrylamide gel electrophoresis with reference proteins. Electrophoresis in sodium dodecyl sulfate gave a subunit molecular weight of 75,000 indicating that this is a three-subunit enzyme.

Sterols derived from cholesterol by hydroxylation of the side chain in the 20α, 22α, 22β, or 25 position inhibited sterol synthesis from acetate and depressed the level of 3-hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34) activity in primary cultures of mouse fetal liver cells and in L cell cultures. Rates of acetate metabolism to fatty acids and CO2, and rates of RNA and protein synthesis were not affected. Following the addition of the most potent inhibitors of the group, 25-hydroxycholesterol and 20α-hydroxycholesterol, to l cell cultures the enzyme activity diminished to one-half of the original amount within a period of 1 to 1.3 hours. Inhibitory potency was influenced by the location of the hydroxyl function on the side chain, by the completeness of the side chain, and by the introduction of a third functional group into the molecule.

1. The role of cytochrome b5 in the oxidative metabolism of benzphetamine and 3,4-benzpyrene in the presence of NADPH was studied with the reconstituted hydroxylation system isolated from rat liver microsomes whose components were cytochrome P-450 or P-448, NADPH-cytochrome c reductase, and lipid. In the absence of cytochrome b5, the turnover number for benzphetamine and 3,4-benzpyrene (nanomoles per min per nmole of P-450 or P-448) in the reconstituted system was at least as good as that in liver microsomes, indicating that cytochrome b5 is not an obligatory component in the NADPH-dependent benzphetamine or 3,4-benzpyrene hydroxylation system. 2. The addition of cytochrome b5 at a ratio of cytochrome P-450 to b5 normally found in microsomes inhibited NADPH-dependent benzphetamine N-demethylation by the three components (cytochrome P-450, NADPH-cytochrome c reductase, and lipid). This inhibition was shown to be due to competition between cytochrome b5 and cytochrome P-450 for NADPH-cytochrome c reductase. The addition of NADH and cytochrome b5 reductase to the reconstituted system reversed the inhibition of NADPH-dependent benzphetamine N-demethylation by cytochrome b5. Thus, the NADH synergistic effect observed in liver microsomes with benzphetamine as the substrate and NADPH as electron donor could be due to NADH and cytochrome b5 reductase preventing the inhibition by cytochrome b5. 3. The addition of cytochrome b5 at a ratio of cytochrome P-448 to b5 normally found in microsomes did not inhibit the NADPH-dependent hydroxylation of 3,4-benzpyrene by the three components (cytochrome P-448, NADPH-cytochrome c reductase, and lipid). In the presence of NADH, NADPH, cytochrome b5, cytochrome b5 reductase, and the three components, the rate of 3,4-benzpyrene hydroxylation was significantly increased. This increased rate was due to a NADH-dependent hydroxylation pathway via cytochrome b5 and cytochrome b5 reductase. 4. It is concluded that cytochrome b5 is not an obligatory component in the NADPH-dependent benzphetamine or 3,4-benzpyrene hydroxylation systems. However, this cytochrome plays an important role in regulating NADPH-dependent hydroxylation of these substrates and is also an essential component of the NADH-dependent hydroxylation pathway.

Fatty acid synthetase from lactating bovine mammary gland has an s020, w of 13.5 and a molecular weight of 530,000 as determined by sedimentation equilibrium. This enzyme is not cold-labile and can be stored at 4° with little loss in enzyme activity for up to 2 weeks in 0.25 m potassium phosphate buffer, pH 6.8, containing 1.0 mm dithiothreitol in an atmosphere of N2. At room temperature the activity was lost in 3 days. In Tris (10mm)-glycine (35 mm) buffer, pH 8.45, the enzyme dissociated completely in about 27 hours into obviously nonidentical, but inseparable 9 S subunits with molecular weights of 220,000 with a loss of most of the fatty acid synthetase and crotonyl-CoA reductase activities. On filtration of these subunits through Sephadex G-50, equilibrated with 0.25 m potassium phosphate buffer, pH 6.8, containing 10-3 m dithiothreitol, they reassociated with partial recovery of both fatty acid synthetic and crotonyl-CoA reductase activities. Prolonged dialysis of the enzyme against Tris buffer resulted in the loss of both activities completely. The ressociation, as judged by the recovery of the two activities of the enzyme, then took 14 to 19 days to be accomplished. The various size-shape parameters are consistent with a model in which the two half-molecular rod-shaped subcomplexes are held together in the native enzyme complex head to head with the catalytic sites of all the constitutive enzymes of the complex juxtaposed near each other. The crotonyl-CoA reductase activity is also believed to occur at a nearby site as the structural integrity of the native enzyme complex is essential for this activity also.

The general pattern of biosynthesis of the bluensidine (1D-1-O-carbamoyl-3-guanidino-3-deoxy-scyllo-inositol) moiety of bluensomycin, a monoguanidinated analogue of dihydrostreptomycin, has been studied in extracts of Streptomyces hygroscopicus forma glebosus ATCC 14607 (S. glebosus). Our results are consistent with the following biosynthetic pathway: myo-inositol (C)/→ keto-scyllo-inositol (D)/→ aminodeoxy-scyllo-inositol (E)/→ 1-amino-1-deoxy-scyllo-inositol-4-P (U)/→ 1D-1-O-carbamoyl-3-amino-3-deoxy-scyllo-inositol-6-P (F)/→ 1D-1-O-carbamoyl-3-guanidino-3-deoxy-scyllo-inositol-6-P →→ bluensomycin. A major uncertainty concerns the step (U) at which the carbamoyl group is introduced. Carbamoylation might occur, for example, prior to phosphorylation or following transamidination. myo-Inositol:NAD oxidoreductase activity (C) is reported for the first time in extracts of Streptomyces; activity was assayed by two radiochemical methods, both involving coupling with aminotransferase (D). l-Glutamine:keto-scyllo-inositol aminotransferase (D) was also found to catalyze aminodeoxy-scyllo-inositol:keto-scyllo-inositol, aminodeoxy-scyllo-inositol:pyruvate, and 1,3-diamino-1,3-dideoxy-scyllo-inositol:keto-scyllo-inositol transaminations. l-Arginine:inosamine-P amidinotransferase (F) catalyzed transamidinations with the following compounds as amidino acceptors: NH2OH, 1-amino-1-deoxy-scyllo-inositol-4-P, 1D-1-guanidino-3-amino-1,3-dideoxy-scyllo-inositol-6-P, 1D-1,3-diamino-1,3-dideoxy-scyllo-inositol-6-P, 1D-1,3-diamino-1,2,3-trideoxy-scyllo-inositol-6-P, and a compound present in S. glebosus extracts believed to be 1D-1-O-carbamoyl-3-amino-3-deoxy-scyllo-inositol-6-P. The latter transamidination is markedly enhanced in crude extracts by carbamoyl-P. S. glebosus extracts also have 1-guanidino-1-deoxy-scyllo-inositol-4-P phosphohydrolase activity; neither this enzyme nor the corresponding enzyme from streptomycin producers can dephosphorylate the transamidination product presumed to be bluensidine-6-P. Acid hydrolysis of the latter compound gave a compound which, unlike the unhydrolyzed compound, was converted to 1D-1-guanidino-3-amino-1,3-dideoxy-scyllo-inositol by enzymes from a streptomycin producing strain. S. glebosus cannot carry out the above conversion since it apparently lacks at least two enzymes which occur in streptomycin producers: guanidinodeoxy-(scyllo)-inositol dehydrogenase and l-alanine:1D-1-guanidino-3-keto-1-deoxy-scyllo-inositol aminotransferase. It is suggested that streptomycin producing strains might be descendents of an ancestral strain which, like S. glebosus, produced the monoguanidinated inositol derivative, bluensomycin. It is further suggested that gene duplication and subsequent evolutionary divergence resulted in biosynthesis of the diguanidinated inositol derivative, streptomycin, which is 10 times more effective than bluensomycin as an antibiotic and inhibitor of protein biosynthesis.

A simple model for the kinetics of adenylate cyclase activated by hormones or fluoride ion was developed. It was then tested on data, published by Birnbaumer et al. ((1969) J. Biol. Chem. 244, 3468–3476) for rat fat cell ghosts and data, reported by Drummond et al. ((1971) J. Biol. Chem. 246, 4166–4173) for membrane preparations of guinea pig ventricles. The model yields a satisfactory first approximation of the data. It was concluded that hormones as well as fluoride ion decrease the sensitivity of the enzyme toward inhibition by ATP not complexed to magnesium, thereby favoring the formation of the productive Michaelis complex. Parallel to this the maximal velocity is increased. In contrast to earlier interpretations, no evidence was found for a regulatory binding site for magnesium. Adenylate cyclase activated by hormones was found to be described as a system in equilibrium or in a steady state. Thus, it could be regarded as an allosteric enzyme system with hormones as heterotropic activators.

Protamine sulfate fractionation of crude extracts of Pseudomonas sp. (Shaw strain MA), containing malate ATP lyase activity, results in the separation of this activity into two protein components. The first component, identified as a malate thiokinase, has been purified to near homogeneity. The enzyme contains succinate thiokinase activity equal to that of malate thiokinase activity; however, this activity is distinct from succinate thiokinase itself. The second component of the malate ATP lyase reaction is a malyl coenzyme A lyase. This reversible reaction is specific for S-malyl-CoA. The equilibrium constant of this latter reaction is ∼3 x 10-3 m. Mixing together malate thiokinase and malyl-CoA lyase results in the reconstitution of the originally observed malate ATP lyase reaction.

The presence of α-ketoglutaramate was shown in rat liver, kidney, and brain by application of an isotope dilution procedure; concentrations of 7.6, 4.7, and 10.7 µmoles per kg were found, respectively. The finding of increased concentrations of α-ketoglutaramate in cerebrospinal fluid in patients with hepatic coma was verified. Studies on rat kidney preparations, together with other considerations, provide no evidence for a physical linkage between glutamine transaminases and ω-amidase. The data suggest that α-ketoglutaramate is a normal tissue constituent formed by transamination of glutamine. Its accumulation in the cerebrospinal fluid in hepatic coma seems to reflect increased utilization of ammonia for glutamine synthesis and increased transamination of glutamine, as well as the relatively low ω-amidase activity of brain.

The synthesis, uptake, and efflux of some putative neurotransmitters by cultured rat glial tumor cells were studied. Three glial cell clones were capable of pyridoxal-dependent synthesis of γ-aminobutyrate both in cell-free homogenates and in intact cells in monolayers. Substantial catabolism of γ-aminobutyrate was not found. Synthesis of taurine and isethionic acid, but not β-alanine, by one of the glioma lines was also shown. Glioma lines were found to take up glutamate and exhibited Na+-dependent uptake of γ-aminobutyrate. The uptake of γ-aminobutyrate consisted of a slow saturable component (Kt ∼ 13 to 30 µm) and a rapid nonsaturable component. Both of these were antagonized by some structural analogs of γ-aminobutyrate as well as by taurine, β-alanine, bicuculline, and low temperature. Similar kinetic parameters were found for three different glioma lines. Fibroblast-like cells obtained from rat brain cell cultures had only the nonsaturable component of γ-aminobutyrate uptake. Taurine uptake also consisted of two Na+-dependent and temperature-dependent components: a rapid saturable component (Kt ∼ 10 to 17 µm) and a nonsaturable component which varied in magnitude between cell lines. These uptakes were antagonized by β-alanine but not by γ-aminobutyrate. The glioma lines also excreted the concentrated γ-aminobutyrate into the extracellular milieu, but could maintain cell to medium concentration ratios of g80-fold. In taurine efflux experiments, cell to medium concentration ratios in excess of 1500-fold could be maintained by one of the gliomas. The data are consistent with a possible role of central nervous system glial cells in the modulation of neuronal excitability via control of the levels of neuroactive substances in the extracellular milieu of neurons.

N-(n-Hexyl)-O-glucosylsphingosine completely inhibited β-glucosidase in cultured rat astrocytoma (RGC-6) and mouse neuroblastoma cell strains (NB41A and NB2a), creating a model for the infantile Gaucher nervous system. The inhibitor, at a concentration of 5 µg per ml, caused a 5-fold elevation of glucosylceramide in RGC-6 and a 6-fold elevation in NB41A cells. Under these conditions the growth rates of cells were unaffected. Other lysosomal enzymes were only depressed to approximately 80% of normal with the exception of β-glucuronidase (37% of normal); such reductions would not be anticipated to cause abnormal pathology and none was observed. Studies with glucosyl[14C]ceramide (labeled in the stearoyl moiety) showed that the glycolipid was rapidly taken up from the medium. In the presence of the β-glucosidase inhibitor the uptake of glucosylceramide was reduced in NB2a, NB41A, and RGC-6 cells, and in the latter two cell strains no subsequent catabolism could be detected. Studies with more complex glycolipids, such as gangliosides, showed that they were readily taken up and metabolized and that catabolism was not affected by the inhibitor. This was interpreted as evidence for two β-glucosidases, one which utilizes free glucosylceramide and is deficient in Gaucher's disease and the other which acts as an integral part of a multienzyme complex for the degradation of complex glycolipids and has normal activity in Gaucher's disease. Results indicated that specific catabolic reactions may be inhibited by substrate analogs to simulate the pathology of certain genetic diseases in neurons and glia in tissue culture.

In hepatomas, the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-controlling enzyme in cholesterol biosynthesis, is not normally suppressed by cholesterol. To examine the biochemical mechanism of this loss of feedback control of cholesterol synthesis, a comparison was made of the properties of 3-hydroxy-3-methylglutaryl coenzyme A reductase after solubilization and partial purification from microsomes of normal rat liver and of a minimal deviation rat hepatoma. The solubilized enzyme from the two sources behaved identically with respect to substrate kinetics, heat inactivation, cold inactivation, and gel filtration. By the use of a new method that permits assay of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity after polyacrylamide gel electrophoresis, it was shown that the solubilized hepatoma enzyme and the normal liver enzyme had similar electrophoretic mobility. It is concluded that the failure of hepatomas to suppress 3-hydroxy-3-methyl-glutaryl coenzyme A reductase activity is not due to the production of an altered form of the enzyme.

Incubation of HeLa cells with dimethylsulfoxide results in a partial or complete inhibition of protein synthesis. At a concentration of 12% dimethylsulfoxide, a complete inhibition of protein synthesis occurs within 3 to 4 min. This inhibition is reversible; on removal of dimethylsulfoxide, cells resume their normal rate of protein synthesis. Inhibition of protein synthesis by dimethylsulfoxide is accompanied by a complete breakdown of polyribosomes and release of nascent polypeptides. The breakdown of polyribosomes can be prevented by preincubating cells either with cycloheximide or anisomycin. These studies suggest that dimethylsulfoxide may be a useful tool to synchronize chain initiation in cultured cells.

Modification of erythrocyte membrane sialoglycoprotein(s) in isolated form, or when in membrane, is described using sequential sodium periodate oxidation and tritiated sodium borohydride reduction, essentially as suggested by Van Lenten and Ashwell ((1971) J. Biol. Chem. 246, 1889). Conditions of modification were investigated; under the optimal but nondestructive conditions selected, the modification appears specific for sialyl residues and leads to incorporation of tritium into the modified product, 5-acetamido-3,5-dideoxy-l-arabino-2-heptulosonic acid, identified by its susceptibility to neuraminidase and mild acid hydrolysis, and by its identity with the authentic compound in chromatographic behavior, several colorimetric assays, and extent of tritium incorporation. The selectivity of the modification for the sialoglycoprotein(s) is further demonstrated by carrying out the modification on solubilized membrane proteins; only the sialoglycoprotein(s) incorporates the label. In the modification of the intact membranes some lipid components also become radioactive. The modification of intact erythrocytes, the membranes, or the isolated sialoglycoprotein(s) results in loss of the M blood group activity, but the A and B blood group activities and the phytohemagglutinin binding sites are not affected. The modification offers a handle for isolation of membrane sialoglycoprotein(s) and study of their disposition, and possibly, for evaluation of the physiological role of the terminal polyhydroxy side chains of their sialyl residues.

Initial rate studies were employed to investigate the kinetic mechanism of action of rat skeletal muscle type II hexokinase (EC 3.7.1.1). These investigations involved the use of substrate analogs which act as competitive inhibitors and product inhibition experiments. The findings indicate that substrates d-glucose and MgATP2- add randomly to hexokinase. Both products exhibit noncompetitive inhibition relative to either substrate. The kinetic results are compatible with a Random Bi Bi mechanism of either the rapid equilibrium or the steady state type. The kinetic data are qualitatively identical with those obtained for yeast hexokinase. Although muscle hexokinase does not respond as predicted to the model of adenylate energy charge, it is potently inhibited by levels of glucose-6-P and orthophosphate thought to exist in muscle tissue. The enzyme is inhibited 79% at the approximate intracellular concentrations of substrates, products, and phosphate. This inhibition is increased to 96% when the concentration of these compounds rises to those levels believed to exist during tetanic muscular contraction. The data suggest that the muscle type II hexokinase is not functional during periods of highly active glycogenolysis and glycolysis in rat muscle tissue.

The catalytically active form of bacterial glutamate decarboxylase has an absorption maximum at 420 nm due to the presence of the enzyme—pyridoxal 5′-phosphate Schiff base. Above pH 5.5 the absorption maximum is at 340 nm, and the enzyme is inactive. If the pH of a solution of the enzyme is rapidly changed from 6.5 to 4.0 in a stopped flow apparatus, the change in absorption spectrum occurs over a period of several seconds. The change is general acid catalyzed and occurs in two steps. The first is a rapid conformation change of the enzyme induced by the addition of 3 protons to the enzyme. The second is general acid catalyzed formation of the catalytically active form of the enzyme. If the pH of a solution of the enzyme is rapidly increased from 4.0 to 7.0, the rate of the change in absorption spectrum can also be measured. The change is not buffer catalyzed, but its rate increases rapidly with increasing pH. The mechanism of this change involves an initial rate-determining conformation change of the enzyme induced by dissociation of 3 protons, followed by rapid formation of the equilibrium high pH form of the enzyme. The rate of the conformation change can also be measured on enzyme which has been reduced by sodium cyanoborohydride. From the kinetics of the pH jump reactions and other factors we conclude that the high pH form and the low pH form of glutamate decarboxylase differ by a protein conformation change and by a change in the covalent structure of the bound coenzyme. At low pH the coenzyme is bound in the usual ketoenamine form. At high pH a sulfhydryl group of the enzyme adds to the aldehyde carbon forming an aldamine. Many anions affect the properties of glutamate decarboxylase. Chloride ion increases the rate of transformation of the high pH form of the enzyme into the low pH form and decreases the rate of transformation of the low pH form into the high pH form. These effects occur because anions are able to bind to the low pH conformation of the enzyme but not to the high pH conformation.

The flavoprotein lactate oxidase (decarboxylating) from Mycobacterium smegmatis catalyzes the formation of two different products from a novel substrate, β-chlorolactate: (a) combined oxidation and decarboxylation to chloroacetate and carbon dioxide (the normal sequence), and (b) intramolecular elimination of the β-chloro group of the substrate to form the keto acid pyruvate. Product composition depends on oxygen. Pyruvate is formed catalytically under anaerobic conditions. Chloroacetate production begins at low levels of oxygen and comprises 70 to 80% of the products at 100% oxygen saturation levels. Oxygen uptake and pyruvate formation are reciprocally related, suggesting a common precursor for the two reaction pathways. The ability of lactate oxidase to catalyze the elimination reaction suggests that catalysis occurs by initial abstraction of the α-hydrogen as a proton, followed by a subsequent 2-electron transfer in oxidation of the substrates. These data are similar to those obtained for reaction of β-chloroamino acid substrates with d- and l-amino acid oxidases (Walsh, C. T., Schonbrunn, A., and Abeles, R. H. (1971) J. Biol. Chem. 246, 6855) and suggest that these three flavoprotein oxidases have similar reaction mechanisms. Evidence for shielding from the solvent of the substratederived α-proton on the enzyme during catalysis was obtained with β-chloro[α-3H]lactate. During the elimination reaction with this substrate in H2O, 30% of the tritium originally present at the carbon was found in the methyl group (β carbon) of the pyruvate formed. The ability of this flavoprotein oxidase to catalyze such direct hydrogen transfer with β-chlorolactate provides indirect evidence for the formation and then the enzyme-catalyzed ketonization of the enol form of pyruvate at the active site during the elimination reaction. When [α-2H]lactate is used as substrate for lactate oxidase a deuterium kinetic isotope effect of 1.7 is noted; when α-3H substrate is used, there is a selection of 3.2 against tritiated substrate molecules, suggesting some transfer of the α-hydrogen is a slow step during catalysis.

Bacitracin was tritiated by the Wilzbach technique and purified by carboxymethylcellulose chromatography. Binding studies between [3H]bacitracin and Micrococcus lysodeikticus cells and protoplasts indicated that the cells or protoplasts were saturated at 2 x 105 molecules per cell. This value correlates well with the number of C55-isoprenyl pyrophosphate molecules found in the membrane and adds further support to the hypothesis that the antibiotic activity of this peptide is due to complex formation with this functional lipid. The Km for binding of bacitracin to cells or protoplasts was 3.7 x 10-6 molar which is consistent with the in vitro binding constant of 1 x 106 m-1 observed for the interaction of bacitracin A with the purified C55-isoprenyl pyrophosphate. In vitro binding studies between bacitracin A and purified bacterial phospholipids indicated that the antibiotic does not interact strongly with these lipids. It is concluded from these studies that binding of bacitracin to the bacterial cells can be accounted for quantitatively by virtue of a specific interaction with the membrane C55-isoprenyl pyrophosphate. Furthermore, the protoplast permeability changes induced by bacitracin, under certain conditions, may arise from interaction between bacitracin and the C55-isoprenyl pyrophosphate molecules in the membrane.

SUMMARY Difference spectral assays of cytochromes P-450, P-420, and bs in Morris hepatoma microsomes are complicated by the presence of methemoglobin and hemoglobin. Alternate assays of cytochromes P-450 and P-420 have been developed. The specific contents of cytochrome P-450 present in micro- somes obtained from tumor lines 5123C, 7777, and R3B are: 0.31, 0.014, and 0.10 nmoles per mg of protein, respectively; the specific contents of cytochrome P-420 are: 0.038,0.14, and 0.042 nmoles per mg of protein, respectively. Cytochrome bs is present in all samples of tumor microsomes; the specific content is somewhat higher in microsomes from tumors that contain greater amounts of cytochrome P-450. Although rat hepatic cytochrome P-450 exists in at least three spectrally identifiable forms, only two forms are found in microsomes from the slower growing tumor line, 5123C, in which cytochrome P-450 is relatively abundant. In the rapidly growing tumor line, 7777, there is only one form of cytochrome P-450. Only one spectrally discernible form of the very abundant cytochrome P-420 is present in tumor line 7777, and this cytochrome P-420 is no longer converted to cytochrome P-450 simply by the addition of glycerol. The amounts of flavoproteins of the NADH- and NADPH- oxidative pathways of microsomes in the tumor microsomes are somewhat lower than those of liver. However, NADH- and NADPH-dependent reductions of cytochrome c and ferri- cyanide are relatively rapid, and the properties of flavoproteins of tumor are similar to hepatic microsomes. The rate of NADPH-dependent oxygen uptake by micro- somes from tumor line 7777 is too slow to measure; aerobic oxidation of NADPH is unmeasurable. Demethylation of aminopyrine and benzphetamine occurs at only very slow rates. Furthermore, phenobarbital and methylcholanthrene induce mixed function oxidase activity in liver of the host rats but not in microsomes prepared from tumor line 7777. There is a striking appearance of cytochrome P-448 (Pi-450) in * This work was supported in part by funds from Research Grant AM-10767 from the National Institute of Arthritis, Metab- olism, and Digestive Diseases, Grant CA-10729 from the National Cancer Institute, United States Public Health Service, and in part by funds made available through the State University of New York. $ On leave from the Department of Biochemistry, University of Osaka Medical School, Osaka, Japan. tumor microsomes obtained from methylcholanthrene-treated rats. However, cytochrome P-448-dependent arylhydro- carbon hydroxylase activity is undetectable, even in these induced preparations. Since the cytochromes and flavoproteins are present in tumor microsomes, inactivity of mixed function oxidases may be ascribed either to absence of a hitherto unknown compo- nent or to undetected alterations in one of the known com- ponents. Since the cytochromes apparently may not function as components of mixed function oxidases in this tissue, alternate functions of these microsomal hemoproteins must be considered. Liver microsomes contain mixed function oxidases that cata- lyze the enzymic attack of drugs, carcinogenic hydrocarbons, insecticides, and several other classes of xenobiotics that have the common property of being relatively hydrophobic (1, 2). The oxidases are relatively complex multienzymic systems, being composed of cytochrome and flavoprotein; in addition, they may require phospholipids, which are selectively attacked by phos- pholipases (3). The complex systems may be chemically induced in wivo (1,4, 5). Induction experiments have yielded considerable informa- tion about the nature of microsomal oxidases; however, the in- duction process is complex, and results may be beyond interpre- tation. Alternatively, hepatic microsomal mixed function oxi- dases have been studied by genetic selection (6), resolution and reconstitution of enzymic components (7), electron spin reso- nance (8) and visible spectral studies of components (9), studying oxidation-reduction potentials (lo), investigating changes upon combination with substrate (11, 12), and immunological methods (13). A different approach has been the investigation of several uninduced oxidases from sources ot.her than liver, e.g. adrenal gland mitochondria (14), kidney (15), bacteria (16), yeast (17), plants (18). Since the Morris hepatoma is derived from liver hepatocytes, comparisons to microsomal enzymes of liver are particularly attractive. The terminal oxidase in these multienzymic systems is cyto- chrome P-450. Several lines evidence suggest that cytochrome 1980

The interaction of estradiol-17β with mature bovine endometrial tissue, and with isolated nuclei has been studied. The hormone binds to an insoluble nuclear fraction. This fraction contains membrane and evidence is presented to show that estradiol is bound to the nuclear membrane. Incubation of isolated nuclei and microsome fractions with estradiol-17β shows that the hormone binds essentially instantaneously to microsomes and nuclei. Such binding is non-saturable up to estradiol-17β concentrations as great as 2.5 x 10-6 moles per mg of membrane protein and it is likely that this interaction is not biologically significant. A second form of binding is observed in nuclei which is of higher affinity and saturable, with 507 ± 47 sites per nucleus. This class of binding sites is found to be blocked in cattle that are maintained in artificially induced estrus by feeding with diethylstilbesterol. The high affinity binding site is present only in the mature endometrium and is absent in nuclei from the mature myometrium (the muscular tissue surrounding the endometrium in the uterus) and the immature uterus. Potent estrogenic agents compete effectively with estradiol-17β for binding to this site, whereas weak estrogenic steroids (such as progesterone and testosterone) are inefficient competitors. The sensitivity of the high affinity site to pH and hydrolytic enzymes has been studied and compared with the effect of such agents on the low affinity, nonspecific, membrane site. Such a comparison underscores the inherent differences between these two sites of interaction. The thermodynamic parameters of the high affinity binding site have been measured and compared to reported values for the specific cytoplasmic receptor binding site. The nuclear high affinity binding site possesses a favorable free energy of interaction as a result of entropic forces (ΔS0 = +36.4 cal deg-1 mole-1, ΔH0 = -0.66 Cal mole-1). The number of high affinity nuclear sites varies dramatically during the estrous cycle in a bicyclic fashion. Sites are available during estrus and diestrus, whereas they are blocked in later metestrus and proestrus.

The primary structure of the high potential iron-sulfur protein of the purple sulfur photosynthetic bacterium, Thiocapsa pfennigii, has been determined, providing the second example of a complete amino acid sequence in this group of proteins. A degree of homology is apparent in comparison with the amino acid sequence of the prototype protein of the purple sulfur photosynthetic bacterium, Chromatium vinosum, although the percentage of identical residues is only about 38%. A postulated 4-residue deletion in the T. pfennigii protein which occurs near the 4-iron 4-sulfur chromophore could be readily accommodated in the three-dimensional structure by minor adjustments in the peptide backbone.

N-(phosphonacetyl)-l-aspartate (PALA) is an analog of the activated complex for the reaction catalyzed by aspartate transcarbamylase and has previously been shown to be a potent and specific inhibitor of the enzymes from Escherichia coli and mouse spleen. PALA is now shown to be a potent inhibitor of de novo pyrimidine nucleotide biosynthesis in five lines of mammalian cells in culture. When the concentration of PALA in the medium is 10-4 m, simian virus 40-transformed hamster cells do not divide and, within about 2 days, have detached from the culture dishes and appear dead. The toxicity of 10-4 m PALA is prevented completely if 10-4 m uridine is present in the medium, showing that PALA inhibits uridylic acid biosynthesis specifically. The same effect can be observed with the other cell lines. When the external concentration of radioactive PALA is in the range 10-4 to 10-5 m, its concentration in the cytosol is approximately equal to the external concentration. Most of the PALA recovered from cells remains undegraded. In crude cell extracts, the Ki for PALA is about 1 x 10-9 m at 37° and pH 7.5. Addition of 1 x 10-7 m PALA in vitro to an extract of transformed hamster cells inhibits by 60% the incorporation of radioactivity from CO2 into UMP and CMP; approximately 10-6 m PALA is required to give comparable inhibition of incorporation from carbamyl-P. Higher concentrations give virtually complete inhibition in both cases. These results suggest that PALA may find clinical use in suppressing cellular proliferation. A small number of the virus-transformed hamster cells become resistant to 10-4 m PALA in culture and retain resistance after they are grown for many generations in the absence of the drug. Resistance is accompanied by a large increase in aspartate transcarbamylase activity.

Actin, an apparently universal component of eukaryotic cells, has been purified from Dictyostelium amoebae to electrophoretic homogeneity. Purification was facilitated by newly discovered solubility properties of actomyosin in 30% sucrose. A quantitative assay for the Dictyostelium actin is described which is based on its ability to activate muscle heavy meromyosin ATPase activity. The specific activity of purified amoeba actin for activation of heavy meromyosin is nearly the same as that of purified muscle actin. The molecular weight of the Dictyostelium actin is identical with that of muscle actin, as judged by co-electrophoresis on sodium dodecyl sulfate acrylamide gels. The amoeba actin, like muscle actin, forms Mg2+-paracrystals with a helical repeat of about 360 A. Some of the Dictyostelium actin is associated with membrane in a MgATP-stable linkage. Myosin is found in membrane preparations in a MgATP-labile linkage, presumably due to association with the membranelinked actin.

Regulation of protein synthesis in BHK21/13 cells has been examined as a function of cell growth rate and density, both in vivo by pulse-labeling of cells with [3H]valine and in an in vitro cell-free protein-synthesizing system. The specific activity of [3H]valyl-tRNA in the pulse-labeled cells was determined and the true in vivo rate of [3H]valine incorporation into protein was measured. Unlike the reported behavior of many other cell lines, BHK21/13 cells did not shut off their protein synthesis at all while their growth rate declined 10-fold or more during density-dependent inhibition of growth or during arrest of growth by serum deprivation. In vitro assays of the cells' protein synthetic activity with both synthetic and natural messenger RNA confirmed that all of the components needed for protein synthesis were equally active in rapidly growing and in density-inhibited or serum-deprived cells. Hence, in these cells the protein synthesis rate and the cell division rate are not coupled, and the amount of protein in the cell is not regulated at the level of synthesis, but rather at the level of protein degradation or export.

Nucleolar phosphoproteins are extensively labeled 2 hours after injection of 50 mCi per rat of [32P]orthophosphate into rats bearing Novikoff hepatoma ascites tumor cells. The nucleolar proteins soluble in 0.4 n H2SO4 were analyzed by two-dimensional electrophoresis and autoradiography. Of the 97 proteins visualized by staining, phosphorylation was detected in approximately 40 proteins by autoradiography and by scintillation counting of spots cut from the gels. Several high molecular weight protein spots designated C23, C24, C26, and C27 incorporated as much as 500 times more 32P per spot as some low molecular weight spots. Analysis for phosphoamino acids in 30 spots cut out of the gel slab indicated that radioactive phosphoserine was present in all the spots analyzed.

A particulate enzyme preparation was obtained from Mycobacterium phlei cells which had the activity of a polysaccharide:acyl coenzyme A acyltransferse. We conclude that the enzyme system is involved in the biosynthesis of the methylglucose-containing lipopolysaccharide (MGLP), since it catalyzed the transfer of acetyl, propionyl, isobutyryl, octanoyl, and succinyl groups, all of which are known to be present in the lipopolysaccharide. Moreover, the enzyme preparation used α-(1→4)-d-glucooligosaccharides as acceptors, a result consistent with the fact that a major part of the polysaccharide component of the lipopolysaccharide has the same amylose-like structure. Both MGLP and its deacylated product, MGP, acted as acceptors of acyl groups, but MGP which had been partially acetylated by acetic anhydride was still more active. The random acetylation probably increased slightly the hydrophobic character of MGP and promoted its binding to the enzyme. In agreement with this idea was the fact that artificial d-glucooligosaccharide acceptors were completely inactive unless partially acetylated. Previous studies have established that five of the eight positions of acylation in MGLP-III are located on the four sugar units at the nonreducing end of the polysaccharide. From analysis of the product obtained in the enzymic reaction between [14C]acetyl-CoA and MGP, we now have evidence that at least two of the sites for acetylation occur in this tetrasaccharide unit. Removal of the four sugar units from MGP by the combined action of α-amylase and glucoamylase I destroyed the capacity of the polysaccharide to accept acetyl groups from acetyl-CoA. However, this partially degraded MGP retained acceptor activity for succinyl groups, which suggests that the sites for succinylation are located elsewhere in the polysaccharide, a result which is in agreement with direct chemical analysis of the lipopolysaccharide (Smith, W. L., and Ballou, C. E. (1973) J. Biol. Chem. 248, 7118–7125).

The effects of the bactericidal protein colicin K have been compared on a normal strain of Escherichia coli and a derivative, uncA, that is defective in Ca2+, Mg2+-activated ATPase activity. The syntheses of DNA, RNA, and proteins, which are completely arrested by colicin K in the normal E. coli cells, are only reduced by 30 to 50% in the ATPase-deficient cells. Intracellular ATP levels, which are reduced in the normal cells, are substantially increased by colicin K in the uncA mutant. The active transport of glutamine, which is coupled to phosphate bond energy, is drastically inhibited in both strains as is the transport of proline, a transport system that is coupled to an energized membrane state. These results provide the basis for the proposal that the primary action of colicin K is to de-energize the cytoplasmic membrane systems that are essential for a variety of active transport processes in E. coli. In cells having ATPase activity, ATP levels are diminished by the action of ATPase attempting to re-energize the membrane. This reduction in ATP levels in turn leads to an arrest of macromolecular biosyntheses. These secondary effects of colicin K on ATP levels and on synthetic processes are not present in uncA cells because they lack ATPase activity.

The complete primary structure of reduced and S-carboxy-methylated bovine erythrocyte superoxide dismutase has been derived through analysis of peptides from peptic, plasmin, and hydroxylamine digests of the intact polypeptide chain, and from chymotryptic, subtilisin, and dilute acid digests of derived peptides. From these data, the following unique amino acid sequence of 151 residues was deduced, corresponding to a molecular weight of 15,600, in each of the 2 apparently identical subunits of the protein molecule. (Cmc denotes S-carboxymethylcysteine.) [see PDF for equation]

An in vitro system for studying the order and kinetics of interchain disulfide bond formation (covalent assembly) in immunoglobulins has been developed using subclasses 1 and 4 of human immunoglobulin G. Noncovalent association of reduced, but not alkylated, heavy and light chains was achieved at pH 4.8 and air oxidation initiated by rapid transfer to aerated buffer at pH 8.2. Disulfide-bonded species in samples taken at various times were detected, following alkylation, by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The principal intermediates in the covalent assembly of immunoglobulin G1, with either kappa or lambda light chains, were half-molecules (HL) and H2L. The half-time for the appearance of the completed molecule, H2L2, ranged from 70 to 95 min at 25°. Half-molecules were the predominant intermediate in the assembly of immunoglobulin G4. The disulfide bonds between the heavy chains formed slowly; H2L2 appeared with a half-time of 16 hours. The effects of temperature, pH, cupric ion concentration, and reducing agent on the air oxidation of reduced immunoglobulin were evaluated. Although all these parameters influenced the rate of H2L2 production, the order of disulfide bond formation was unaffected except in the presence of high concentrations of cupric ion. Covalent assembly utilizing a thiol-disulfide interchange system (oxidized and reduced glutathione) was also examined. Optimum rates of H2L2 production were observed with 10-2 m glutathione + 10-3 m oxidized glutathione. For immunoglobulin G1 assembly the half-time was reduced to 25 min at 25° and 10 min at 37° and to 30 min at 37° for subclass 4. These rates are comparable to those observed for covalent assembly in vivo.

A method which permits quantitation of steroid-binding proteins and determination of binding constants at steady state binding conditions is described. This method, which is called "steady state polyacrylamide gel electrophoresis" has been used to demonstrate and quantitate binding of radioactive steroid hormones to serum testosterone-binding globulin, corticosteroid-binding globulin, albumin, and testicular androgen-binding protein. Steady state gel electrophoresis may be used to study binding of any noncharged molecule. Radioactive steroid is dissolved in the acrylamide solution before polymerization. When the sample migrates into the gel during electrophoresis, steroid is bound by the individual proteins. Gels are sliced and the radioactivity is extracted from each slice and measured. The law of mass action can be applied when the rate of association equals the rate of dissociation. This permits determination of total number of binding sites, and dissociation constant (Kd) for the binding between the protein and the steroid. The presence of several binding components in the same sample can be demonstrated and quantitated in one steady state electrophoresis, under identical conditions. The precision is satisfactory in the range of 0.01 to 10 pmoles per sample (coefficient of variation l10 %), while the sensitivity is dependent on the concentration of steroid in the gel. If the rate of dissociation of radioactive steroid from the protein is slow, as for the binding of [3H]dihydrotestosterone to the testosterone-binding globulin of human and monkey serum (t1/2 at 0° g 60 min), accurate quantitation can also be performed by saturation of the protein by radioactive ligand before electrophoresis. Using steady state gel electrophoresis of nonlabeled samples and samples saturated with labeled steroid, concentrations of testicular androgen-binding protein in rat and rabbit, as well as concentration of rabbit, monkey, and human serum testosterone-binding globulin have been measured.

The control of total δ-aminolevulinic acid (ALA) synthetase activity in mammalian liver has been studied in isolated rat liver cell suspensions. Induction of ALA synthetase by the drug, allylisopropyl-acetamide (AIA) at a rate comparable to that of drug-stimulated induction in fasted rats could be achieved in the isolated cells; induction was largely dependent on the presence of adenosine 3' : 5'-monophosphate (cyclic AMP) or its dibutyryl derivative. At optimal concentrations either AIA (1.2 mm) or dibutyryl cyclic AMP (50 µm) alone caused little induction, but in combination caused a 4- to 5-fold increase of ALA synthetase activity in isolated cells over a 6-hour period. It is suggested that the cyclic AMP dependence of induction by drugs, also demonstrated for the drug 3,5-dicarbethoxydihydrocollidine, explains glucose repression of ALA synthetase observed in vivo. Glucose had no effect on induction by AIA ± dibutyryl cyclic AMP in isolated cells. Despite reports that glucocorticoids are required for induction in vivo, addition of hydrocortisone succinate to concentrations maximally effective in inducing hepatic tyrosine aminotransferase had little effect on the increase in total ALA synthetase activity stimulated by AIA ± dibutyryl cyclic AMP in isolated cells. Induction was prevented by addition of cycloheximide or actinomycin D to cell suspensions.

Extracts of the body walls of the adult Ascaris lumbricoides var. suis inhibit pepsin between pH 1 and 6. Four inhibitors of pepsin were isolated and purified as follows. The crude extract of Ascaris was incubated at 37° and pH 2.0 for 75 min. The pepsin-inhibiting activity was obtained in a fraction precipitated at 0.65 saturation with ammonium sulfate at pH 5.35, and sequentially chromatographed on BioGel P-30 at pH 2.1 and Cellex SE at pH 4.7 and resolved into four inhibitors on DEAE-Sephadex at pH 8.8. The degree of purity of each inhibitor was demonstrated by disc gel electrophoresis. The over-all yield of pepsin inhibitors was 44% of the initial extract. Inhibitor I was 7%; Inhibitor II, 10%; Inhibitor III, 20%; and Inhibitor IV, 7%. The over-all purification of Inhibitors I → III was 6,800-fold and that of Inhibitor IV, 3,300-fold. The molecular weights of these inhibitors were calculated from their amino acid composition and were in agreement with the values obtained from both 5% and 10% polyacrylamide gels in sodium dodecyl sulfate. The values obtained from the amino acid composition were: 17,515 (= 160 amino acid residues), 15,584 (= 142 amino acid residues), 16,124 (= 147 amino acid residues), and 31,719 (= 290 amino acid residues) for Inhibitors I, II, III, and IV, respectively. The NH2-terminal amino acid residue of each of these proteins was histidine. The ability of each of these inhibitors to inactivate pepsin was lost by its treatment with either trypsin or chymotrypsin. Each inhibitor inhibited porcine, bovine, and human pepsins, and porcine gastricsin, but not human gastricsin.

Glutamate dehydrogenase decreases the distribution coefficient of glutamate oxalacetate transaminase in Sephadex G-200. This is consistent with previous results which suggested that a complex is formed between these two enzymes. These gel filtration as well as kinetic experiments suggest that transaminase can react with monomeric but not polymer forms of glutamate dehydrogenase. When the levels of both mitochondrial enzymes are too low to form a complex, there is little TPNH and NH4+ generated by the combined aspartate aminotransferase and glutamate dehydrogenase reactions. This is because oxalacetate is a potent product inhibitor of transaminase, the reaction with glutamate and glutamate dehydrogenase is slow and α-ketoglutarate, oxalacetate, and aspartate all inhibit this latter reactions. When the levels of both enzymes are sufficiently high to form a complex, asparate can be dehydrogenated quite rapidly even in the absence of α-ketoglutarate. Furthermore, the aspartate dehydrogenase reaction catalyzed by the enzyme-enzyme complex is not markedly inhibited by oxalacetate, or activated by α-ketoglutarate, and can take place even in the presence of rather high levels of glutamate. Kinetic and gel filtration experiments suggest that the dissociation constant of the enzyme-enzyme complex is considerably lower than that of these substrates for the free enzymes. These results suggest that an important physiological function of the enzyme-enzyme complex is to catalyze the asparatate dehydrogenase reaction in organs as brain, liver, and kidney, where the mitochondrial levels of these enzymes are sufficiently high to form a complex. An advantage of catalysis by the complex over transamination with aspartate followed by dehydrogenation of glutamate is that the complex is not markedly inhibited by low levels of oxalacetate. When tyrosine is the substrate and the levels of these two enzymes are too low to form a significant amount of complex, the tyrosine aminotransferase and glutamate dehydrogenase reactions are coupled. This can occur with tyrosine but not aspartate because hydroxyphenylpyruvate is not a potent inhibitor. However, when the levels of both enzymes are sufficiently high to form a significant amount of the enzymeenzyme complex, the tyrosine dehydrogenase reaction can be catalyzed by this complex and again α-ketoglutarate has no effect on the reaction. Similar relationships occur when phenylalanine is the substrate. Thus, the enzyme-enzyme complex can facilitate dehydrogenation of these amino acids which do not react with glutamate dehydrogenase.

Upon binding of pyridine nucleotides to Escherichia coli MB 1428 dihydrofolate reductase, absorption bands of both enzyme and pyridine nucleotide are changed. Titration of enzyme with TPNH confirms the presence of two TPNH binding sites. Similar titrations show two binding sites for TPNH on a 1:1 complex of enzyme with methotrexate, two sites for TPN+ on both enzyme and a 1:1 enzyme-methotrexate complex, and at least one DPNH binding site on the enzyme-methotrexate complex. Equilibrium ultrafiltration measurements confirm the presence of two TPN+ binding sites on both enzyme and 1:1 enzyme-methotrexate and demonstrate the presence of two DPNH binding sites on 1:1 enzyme-methotrexate. Titrations and comparison of 2:1 with 1:1 pyridine nucleotide-enzyme difference spectra reveal that most of the absorbance changes associated with binding occur upon binding at the higher affinity site. Dissociation constants for TPN+ and DPNH were calculated from these titrations: E·(TPN+)2, 80 ± 40 µm and 5 ± 3 µm; E·MTX·(TPN+)2, 1.7 ± 0.9 µm and 0.06 ± 0.03 µm; E·MTX·(DPNH)2, 100 ± 50 µm and 9 ± 5 µm. Absorbance changes at 280 and 290 nm suggest the involvement of at least 1 tryptophan residue in the binding of TPNH, and possibly TPN+, at the higher affinity site. Absorbance changes upon binding of DPNH, TPNH, and TPN+ to the enzyme-methotrexate complex indicate that all three cofactors are bound identically. The binding of pyridine nucleotides to the enzyme-methotrexate complex changes the absorption bands of methotrexate toward an even more acidic spectrum.

The steady state kinetic properties of l-lysine monooxygenase from Pseudomonas fluorescens have been investigated. The enzyme has been shown to exhibit both oxygenase and oxidase activity with different substrates, the oxidase activity being much less efficient. Lysine is an oxygenase-type substrate while ornithine is an oxidase-type substrate (Nakazawa, T., Hori, K. and Hayaishi, O. (1972) J. Biol. Chem. 247, 3439–3444). During steady state analysis, both substrates exhibit sigmoidal saturation curves. Hill plots for lysine and ornithine are biphasic, with the Hill coefficient n = 2.9 at low substrate concentrations and n = 1 at higher concentrations. With lysine as substrate, the reaction does not go to completion, and the per cent completion of the reaction is a function of the initial lysine concentration. e-Aminocaproic acid has been found to act as a nonsubstrate effector by permitting the reaction to reach completion. Furthermore, e-aminocaproate reduces the cooperative effect of lysine, converting the biphasic Hill plot to a monophasic one, with a Hill coefficient of 1 obtaining over most of the concentration range of lysine studied. e-Aminocaproate is also a competitive inhibitor toward lysine with a Ki of 2.6 x 10-4 m. e-N,N-Dimethyl-l-lysine is a poor substrate except in the presence of lysine, in which case the reaction proceeds with both substrates, but stops short of completion, as seen with lysine alone as substrate. These results indicate that lysine itself is an effector, and that at least two sites per flavin are important to catalysis, i.e. one catalytic site and one or more effector sites.

Reports in the literature indicate that during the exponential phase of growth of Candida utilis NADH oxidation is insensitive to rotenone, that rotenone sensitivity is acquired during the transition to the late stationary phase and is again lost on catabolite repression. The acquisition and loss of rotenone sensitivity appears to be accompanied by similar changes in Site 1 phosphorylation but does not appear to be reflected in the rate of oxidation of NADH (by mitochondria) or of NAD-linked substrates (by mitochondria or whole cells). In the present paper evidence is presented that these fluctuations in sensitivity to inhibitors of NADH oxidation reflect the presence of different types of inner membrane-bound NADH dehydrogenases in different phases of growth. Thus inner membrane preparations from exponential phase cells contain an NADH dehydrogenase which reacts equally well with ferricyanide and juglone as electron acceptor, appears to be very labile, and lacks EPR signals corresponding to iron-sulfur Centers 1 and 2, whereas a new species, probably an iron-sulfur protein, with resonances at g|| = 2.01, and g⊥ = 1.92 in the reduced state, is present. This species is not significantly reduced by NADH. In corresponding preparations from late stationary phase cells NADH-ferricyanide activity is high, juglone reductase activity is low, and the enzyme is stable and exhibits the EPR signals of iron-sulfur Centers 1 and 2, whereas the EPR signals of iron-sulfur Centers 3 + 4 change very little on transition from exponential to stationary phase cells. There is also a decrease in cytochrome concentration. Most prominent among these is a b-type cytochrome (g = 2.54; 2.23; 1.87) which decreases 2- to 3-fold. The EPR detectable species with g|| = 2.01 and g⊥ = 1.92 in the reduced state is no longer detected. On catabolite repression of late stationary phase cells there is an 80 to 90% decline in NADH-ferricyanide activity, of iron-sulfur Centers 1 and 2, a 50 to 60% decrease of Centers 3 + 4, and an increase in a b cytochrome, but the specific activity in NADH-juglone reductase and NADH oxidase assays increases, the enzyme becomes once again labile, and the EPR detectable species with g|| = 2.01 and g⊥ = 1.92 appears on reduction with dithionite. All these changes are prevented by cycloheximide. The data suggest that sensitivity to piericidin A and coupling to energy conservation Site 1 are properties of the type of NADH dehydrogenase present in late stationary phase cells but not in exponential phase or of catabolite-repressed cells.

Cinnamic acid 4-hydroxylase in etiolated sorghum seedlings was concentrated in the microsomal or light membrane fraction upon extraction. The three substrates required in the catalysis, cinnamic acid, TPNH, and O2 have Km values in the micromolar range. The same microsomal fraction contained cytochromes P-450 and b5 at 0.10 and 0.55 nmoles · mg of protein-1, respectively. Preparation of a photodissociation action spectrum on carbon monoxide inhibition of the cinnamic acid hydroxylase indicated a requirement for cytochrome P-450 in the catalysis. When challenged with a range of potential inhibitors, the hydroxylase behaved in a manner that is fairly typical of the more extensively studied P-450 monooxygenases of nonplant tissues.

The protein mixture discharged by guinea pig pancreatic lobules incubated in vitro has been analyzed by two complementary techniques: isoelectric focusing (which separates proteins according to charge), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (which separates proteins according to size). Isoelectric focusing columns spanning a set of pH ranges were used to fractionate secretory proteins biosynthetically labeled with 3H- or 14C-amino acids. Effluent fractions were used to determine radioactivity distribution and to locate (by specific enzyme assays) ribonuclease, amylase, chymotrypsinogen, trypsinogen, procarboxypeptidase A and procarboxypeptidase B either directly or after trypsin activation. There are single species of the first four proteins all of which are isoelectric above pH 9.2, and two forms of each procarboxypeptidase isoelectric near pH 5 (procarboxypeptidase A) and pH 7 (procarboxypeptidase B). The average isoelectric point of secretion mass-labeled with a mixture of 15 14C-amino acids is at pH 9.2. The most acidic species isoelectric near pH 3.4 can be labeled with both 35SO42- and amino acids. Its charge complementarity with the bulk of the secretory proteins is discussed with reference to the concentration and packaging of secretory proteins in the condensing vacuoles of the acinar cells. Sodium dodecyl sulfate-polyacrylamide gels resolve eight major proteins from the secretion. Six of these have been identified by cross-referencing aliquots from the isoelectric focusing columns onto the gels. No enzyme activities or potential activities have been assigned to two of the major proteins: the first has a molecular weight of 68,000 and the second appears to be slightly smaller than chymotrypsinogen. The results indicate that the guinea pig pancreas produces essentially the same complement of enzymes and zymogens as found in other species; they also demonstrate that the zymogen granules of the acinar cells contain the same proteins as the secretion discharged by pancreatic lobules upon carbamylcholine stimulation. The procedures described can be used to analyze on a protein-specific basis a variety of events in the secretory process.

Tay-Sachs disease is an inherited disorder in which β-d-N-acetylhexosaminidase activity is absent. Sandhoff's disease is a clinically identical disorder in which both hexosaminidase A and hexosaminidase B activity is missing. Antibodies to purified hexosaminidase A and hexosaminidase B have been produced in rabbits. Each antiserum was reactive against both isozymes on immunodiffusion. No hexosaminidase A antigen was present in the livers of the two Tay-Sachs disease patients and only a possible trace of antigen was found in the third case. The liver from one patient with Sandoff's disease contained both hexosaminidase A and hexosaminidase B antigens, even though no enzymatic activity was present, whereas the liver from the other patient with Sandhoff's disease contained only hexosaminidase A antigen. We suggest that hexosaminidase A and hexosaminidase B share a common subunit and the other subunit in the two hexosaminidases may be different.

Calf brain microtubule protein (tubulin) was characterized chemically. Amino acid analysis and hydrogen ion titration in 5 m guanidine hydrochloride yielded a chemical composition similar to that of tubulin from other sources. The NH2-terminal residue was identified as methionine. When the protein was denatured and reduced in 8 m urea, two distinct protein bands were observed in gel electrophoresis in the presence of urea, suggesting the existence of two nonidentical subunits. The molecular weight of the tubulin subunits was determined by a variety of techniques, including sedimentation equilibrium and light scattering in 6 m guanidine hydrochloride, sodium dodecyl sulfate gel electrophoresis, chromatrography on an agarose column in the presence of 6 m guanidine hydrochloride, and viscometry of S-carboxymethylated, reduced protein in 6 m guanidine hydrochloride. All methods yielded an average molecular weight value of (54,000 ± 1,000), whether the measurements were done with or without reducing agents, indicating that all disulfide bonds present are intrachain.