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Under physiological conditions, manganese(II) exhibits catalase-like activity. However, at elevated concentrations, it induces apoptosis via a non-mitochondria-mediated mechanism (Oubrahim, H., Stadtman, E. R., and Chock, P. B. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 9505-9510). In this study, we show that the Mn(II)-induced apoptosis, as monitored by caspase-3-like activity, in NIH3T3 cells was inhibited by calpain inhibitors I and II or the p38 MAP kinase inhibitor, SB202190. The control experiments showed that each of these inhibitors in the concentration ranges used exerted no effect on activated caspase-3-like activity. Furthermore, caspase-12 was cleaved in Mn(II)-treated cells, suggesting that the Mn(II)-induced apoptosis is mediated by caspase-12. This notion is confirmed by the observations that pretreatment of NIH3T3 cells with either caspase-12 antisense RNA or dsRNA corresponding to the full-length caspase-12 led to a dramatic decrease in caspase-3-like activity induced by Mn(II). The precise mechanism by which Mn(II) induced the apoptosis is not clear. Nevertheless, Mn(II), in part, exerts its effect via its ability to replace Ca(II) in the activation of m-calpain, which in turn activates caspase-12 and degrades Bcl-xL. In addition, the dsRNA(i) method serves as an effective technique for knocking out caspase-12 in NIH3T3 cells without causing apoptosis.

The thiol-specific antioxidant protein (TSA) protects glutamine synthetase from inactivation by a metal-catalyzed oxidation (MCO) system comprised of dithiothreitol (DTT)/Fe3+/O2 but not by the ascorbate/Fe3+/O2 MCO system. The removal of sulfur-centered radicals or H2O2 has been proposed as the protective mechanism of TSA. Like catalase, TSA prevents the initiation of the rapid O2 uptake phase during MCO of DTT but causes only partial inhibition when added after the reaction is well into the propagation phase. Stoichiometric studies showed that the antioxidant property of TSA is, at least in part, due to its ability to catalyze the destruction of H2O2 by the overall reaction 2 RSH + H2O2→ RSSR + H2O. Results of kinetic studies demonstrate that the removal of H2O2 by TSA correlates with its ability to protect glutamine synthetase from inactivation. In the presence of thioredoxin, TSA is more active, whereas C170S (an active mutant of TSA in which cysteine 170 was replaced by a serine) and open reading frame 6 (a human antioxidant protein homologous to TSA with only one conserved cysteine residue) are only slightly affected. The thiol specificity of the protective activity of TSA derives from the fact that the oxidized form of TSA can be converted back to its sulfhydryl form by treatment with thiols but not by ascorbate.

During exposure to ozone, the methionine and aromatic amino acid residues of Escherichia coli glutamine synthetase (GS) and bovine serum albumin (BSA) are oxidized rapidly in the order Met > Trp > Tyr approximately His > Phe. The loss of His is matched by a nearly equivalent formation of aspartate or of a derivative that is converted to aspartic acid upon acid hydrolysis. Conversion of His to aspartate was confirmed by showing that the oxidation of E. coli protein in which all His residues were uniformly labeled with 14C gave rise to 14C-labeled aspartic acid in 80% yield and also by the demonstration that His residues in the tripeptides Ala-His-Ala or Ala-Ala-His gave rise to nearly stoichiometric amounts of aspartic acid whereas oxidation of His-Ala-Ala yielded only 36% aspartate. The oxidation of BSA and GS led to formation, respectively, of 11 and 3.3 eq of carbonyl groups and 0.5 and 0.3 eq of quinoprotein per subunit. Although BSA and GS contain nearly identical amounts of each kind of aromatic amino acid residues, oxidation of these residues in BSA was about 1.5-2.0 times faster than in GS indicating that the susceptibility to oxidation is dependent on the primary, secondary, tertiary, and quaternary structure of the protein.

Incubation of glucose-6-phosphate dehydrogenase (Glu-6-PDH) from Leuconostoc mesenteroides with the lipid peroxidation product 4-hydroxy-2-nonenal leads to the formation of cross-linked protein. This is accompanied by the appearance of protein-associated fluorescence with excitation and emission maxima of 340 and 415 nm, respectively, and with the disappearance of histidine and lysine residues. Cross-linked protein is less susceptible than native Glu-6-PDH to proteolysis by the multicatalytic protease, a multienzymic proteolytic complex involved in the intracellular degradation of damaged proteins. In addition, 4-hydroxy-2-nonenal-modified Glu-6-PDH inhibits the multicatalytic protease and can therefore prevent the efficient degradation of oxidized protein. These findings may have important implications for the accumulation of altered protein and fluorescent material in vivo, processes that are believed to be involved in age- and disease-related impairment of cellular function.

Cytotoxic action of membrane lipid peroxidation product 4-hydroxynonenal (HNE) is due mainly to its facile reactivity with proteins (Esterbauer, H., Schaur, R. J., and Zollner, H. (1991) Free Radical Biol. Med. 11, 77-80). In the present study, the detailed mechanism of HNE modification of a key enzyme in intermediary metabolism, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), is studied mainly focusing on the formation of HNE-amino acid adducts in the enzyme. When GAPDH (1 mg/ml) was treated with 0-2 mM HNE in sodium phosphate buffer (pH 7.2) for 2 h at 37 degrees C, the enzyme was inactivated by HNE in a concentration-dependent manner. The loss of enzyme activity was associated with the loss of free sulfhydryl groups. Following its reduction with NaBH4, amino acid analysis of the HNE-modified enzyme demonstrated that histidine and lysine residues were also modified. At concentrations lower than 0.5 mM, HNE reacts preferentially with cysteine and lysine residues. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the HNE-modified enzyme suggested the formation of intra- and intermolecular cross-links of the enzyme subunit. The HNE-dependent loss of amino acid residues was accompanied by the generation of protein-linked carbonyl derivatives as assessed by reduction with NaB[3H]H4 and reaction with 2,4-dinitrophenylhydrazine. Thus, the conjugation of all the amino acids appears to involve Michael addition type reactions in which the carbonyl function of HNE would be preserved. The modified histidine residues were quantitatively recovered as the HNE-histidine adduct. However, only 28% of the missing lysine could be accounted for as the HNE-lysine derivative, and only 15.6% of the modified cysteine could be accounted for as the HNE-cysteine thioether derivative. It is proposed that the carbonyl groups of the HNE-derived Michael addition products may undergo secondary reactions with the amino acid groups of lysine residues to yield inter- and intrasubunit cross-links.

The peroxidative activity of Cu,Zn-containing superoxide dismutase (Cu, Zn-SOD) was studied by using a chromogen, 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) which reacts with .OH radicals to form ABTS+, and the spin traps, N-tert-butyl-alpha-phenylnitrone (PBN) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The formation of ABTS+. in this study required both active Cu,Zn-SOD and H2O2 and followed first order kinetics with respect to SOD and H2O2. However, it showed a binding isotherm with ABTS that yielded a dissociation constant of ABTS-enzyme as Kd = 7.1 +/- 0.5 microM. The Kd values for DMPO and PBN were obtained as 0.63 and 11 mM, respectively, by competition reactions. A radical scavenger, formate anion, inhibits the formation of ABTS+., whereas ethanol does not. The results together indicate that DMPO and anionic scavengers have easy access inside the positively charged active channel of Cu,Zn-SOD and are thus in a position to intercept the newly formed .OH radicals. PBN and ethanol, however, stay outside of the channel and are not able to compete with ABTS for .OH radicals. We trapped free radicals, which were produced in the presence of free radical scavengers, with PBN. The formation curve of PBN-hydroxyethyl radical adduct observed in the presence of ethanol indicated that the enzyme became inactivated in a relatively short period. In contrast, in the presence of anionic scavenger formate, formyl radicals were produced catalytically, and the enzyme activity was protected by the formate against H2O2 inactivation. Thus Cu,Zn-SOD behaves as an enzyme that catalyzes the formation of free radicals using anionic scavengers and H2O2 as substrates.

Incubation of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides with 4-hydroxy-2-nonenal (HNE) results in a pseudo first-order loss of enzyme activity. The pH dependence of the inactivation rate exhibits an inflection around pH 10, and the enzyme is protected from inactivation by glucose 6-phosphate. Loss of enzyme activity corresponds with the formation of one carbonyl function per enzyme subunit and the appearance of a lysine-HNE adduct. The data presented in this paper are consistent with the view that the epsilon-amino group of a lysine residue in the glucose 6-phosphate-binding site reacts with the double bond (C3) of HNE, resulting in the formation of a stable secondary amine derivative and loss of enzyme activity. We have described a mechanism by which HNE may, in part, mediate free radical damage. In addition, a method for the detection of the lysine-HNE adduct is introduced.

As a variety of eukaryotic cells age, the specific activity of glucose-6-phosphate dehydrogenase (Glu-6-PDH) declines as much as 50%. Because of the central role of this enzyme in metabolism, it is important to define factors responsible for this loss in enzyme activity. We report that Glu-6-PDH from Leuconostoc mesenteroides is rapidly inactivated by micromolar concentrations of Fe2+ and H2O2. Inactivation correlated with the formation of one carbonyl functionality/enzyme subunit, indicating that inactivation is the result of site-specific oxidative modification. Our results suggest that Fe2+ binds to the glucose 6-phosphate binding site and that interaction of the enzyme-bound Fe2+ with H2O2 leads to the oxidative modification of amino acids essential for enzyme activity. Partially inactivated enzyme remained predominantly in the dimeric form, and no change in the apparent affinity of the remaining active subunits for substrate was observed. Partial inactivation did, however, lead to a decrease in the thermal stability of the remaining activity. This decrease in thermal stability could be largely overcome by the addition of glucose 6-phosphate. Thus, although exposure to H2O2 and Fe2+ results in the irreversible inactivation of Glu-6-PDH, the resulting modification is selective, leads to the formation of heterodimers of both active and inactive subunits, and does not appear to cause large scale structural changes. Our results demonstrate the inherent susceptibility of Glu-6-PDH from L. mesenteroides to modification by an oxidation system known to exist in vivo. An assessment of the physiological significance of Fe(2+)-catalyzed oxidation of Glu-6-PDH awaits extension of these studies to mammalian sources known to accumulate less active or inactive forms of the enzyme as a function of age.

Metal catalyzed oxidation of specific amino acid residues has been proposed to be an important physiological mechanism of marking proteins for proteolytic degradation. After initial oxidative inactivation of dodecameric Escherichia coli glutamine synthetase (GS), the integrity of the GS active site and protein structure was assessed by monitoring ATP binding, observing a susceptibility of GS to tryptic cleavage, and comparative thermodynamic analysis. The tryptic cleavage rates of an active site linked central loop were significantly accelerated for the oxidized conformer. This tryptic cleavage was essentially prevented in the presence of glutamate for native GS but not for the oxidized conformer. The integrity of the ATP binding site in the oxidized GS was substantially altered as indicated by the reduction in fluorescence enhancement associated with ATP binding. Decreases in the free energies of quaternary protein structure and subunit interactions due to oxidative modification were determined by temperature and urea induced unfolding equilibrium measurements. Comparative thermal stability measurements of a partial unfolding transition indicated that the loss in stabilization free energy for the oxidized GS conformer was 1.3 kcal/mol dodecamer. Under alkaline conditions, the urea-induced disruption of quaternary and tertiary structures of oxidized and native GS were examined. This comparative analysis revealed that the free energies of the subunit interactions and unfolding of the dissociated monomers for oxidized GS were decreased by 1.5 and 1.7 kcal/mol, respectively. Our results suggest that small free energy decreases in GS protein structural stability of only 1-2 kcal/mol may be responsible for the selective proteolytic turnover of the oxidized GS.

The oxidation of amino acids by Fenton reagent (H2O2 + Fe(II] leads mainly to the formation of NH+4, alpha-ketoacids, CO2, oximes, and aldehydes or carboxylic acids containing one less carbon atom. Oxidation is almost completely dependent on the presence of bicarbonate ion and is stimulated by iron chelators at levels which are substoichiometric with respect to the iron concentration but is inhibited at higher concentrations. The stimulatory effect of chelators is not due merely to solubilization of catalytically inactive polymeric forms of Fe(OH)3 nor to the conversion of Fe(II) to complexes incapable of scavenging hydroxyl radicals. The results suggest that an iron chelate and another as yet unidentified form of iron are both required for maximal rates of amino acid oxidation. The metal ion-catalyzed oxidation of amino acids is likely a "caged" process, since the oxidation is not inhibited by hydroxyl radical scavengers, and the relative rates of oxidation of various amino acids by the Fenton system as well as the distribution of products formed (especially products of aromatic amino acids) are significantly different from those reported for amino acid oxidation by ionizing radiation. Several iron-binding proteins, peptides, and hemoglobin degradation products can replace Fe(II) or Fe(III) in the bicarbonate-dependent oxidation of amino acids. In view of their ability to sequester metal ions and their susceptibility to oxidation by H2O2 in the presence of physiological concentrations of bicarbonate, amino acids may serve an important role in antioxidant defense against tissue damage.

Point mutations of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) have been linked to familial amyotrophic lateral sclerosis (FALS). We reported that Cu,Zn-SOD can catalyze free radical generation and a FALS mutant, G93A, exhibits an enhanced free radical-generating activity, while its dismutation activity is identical to that of the wild-type enzyme (Yim, M. B., Kang, J.-H., Yim, H.-S., Kwak, H.-S., Chock, P. B., and Stadtman, E. R. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5709-5714). The A4V mutation is both the most commonly detected of FALS-associated SOD1 mutations and among the most clinically severe (Rosen, D. R., Bowling, A. C., Patterson, D., Usdin, T. B., Sapp, P., Mezey, E., McKenna-Yasek, D., O'Regan, J. P., Rahmani, Z., Ferrante, R. J., Brownstein, M. J., Kowall, N. W., Beal, M. F., Horvitz, H. R., and Brown, R. H., Jr. (1994) Hum. Mol. Genet. 3, 981-987). We cloned the cDNA for the FALS A4V mutant, overexpressed the protein in Sf9 insect cells, purified the protein, and studied its enzymic activities. Our results show that the mutant and wild-type enzymes contain one copper ion per subunit and have identical dismutation activities. However, the free radical-generating activity of the mutant, as measured by the spin trapping method at low H2O2 concentration, is enhanced relative to that of the wild-type and G93A enzyme (wild-type G93A > A4V, while the kcat is identical for these enzymes. Thus, the FALS symptoms are not associated with the reduction in the dismutation activity of the mutant enzyme. The fact that the A4V mutant has the lowest Km for H2O2 is correlated to the clinical severity observed with the A4V patients, if FALS is associated with a differential gain of the free radical-generating function of the Cu,Zn-SOD mutant.

Glutaminase B from Escherichia coli is modulated by nucleotides and divalent cations. ATP and ADP inhibit glutaminase B whereas AMP and divalent cations activate it. Inhibition and activation required preincubation of the nucleotides with glutaminase B at 4 degrees. Mg2+, Mn2+, and Ca2+ activated the enzyme and prevented the inhibition by ATP. Dialysis in the presence of an activator ligand reversed the ATP inhibition of glutaminase B. The modulation of glutaminase B by energy charge is similar to that observed with other catabolic enzymes. We suggest that a pattern of reciprocal regulation of glutaminase B and glutamine synthetase by adenine nucleotides prevents the formation of a "futile cycle" of amide synthesis and degradation.

Escherichia coli contains two glutaminases, A and B, with pH optima below pH 5 and above pH 7, respectively. Neither glutaminase A nor B is released from E. coli by osmotic shock. Glutaminase B has been purified 6,000-fold and the purified preparation is estimated to contain about 40% glutaminase B. The enzyme has a molecular weight of 90,000 and an isoelectric point of 5.4. Glutaminase B exhibits a broad pH optimum between 7.1 and 9.0. Only L-glutamine is deamidated by glutaminase B, L-asparagine and D-glutamine are not deamidated. The substrate saturation curve for glutaminase B shows an intermediary plateau region. Like many regulatory enzymes, glutaminase B is cold-labile. The enzyme is inactivated by cooling and activated by warming; both processes are first order with respect to time. The activation energy for activation by warming was calculated to be 5900 cal/mol. Activation by warming increased the Vmax and decreased the S0.5 for L-glutamine, but did not alter the molecular weight of the catalytically active enzyme. Borate and glutamate protected glutaminase B from inactivation by cold.

Glutaminase B is both activated and inhibited by L-glutamate, the product of the reaction. The activation process is time- and temperature-dependent. Activation by L-glutamate alters the Vmax, So.5, and shape of the substrate saturation curve. The activation decays as a first order process with time after separation of L-glutamate from glutaminase B. L-Glutamate inhibits both glutaminase B and the glutamate-activated enzyme. Like L-glutamate, borate activates and inhibits glutaminase B. Inhibition of the enzyme by glutamine and glutamate analogs is also examined and similarities between the glutamate activation and activation by warming at 23 degrees are noted.

A number of metal-catalyzed oxidation (MCO) systems mediate the oxidative inactivation of enzymes. This oxidation is accompanied by conversion of the side chains of some amino acid residues to carbonyl derivatives (for review, see Stadtman, E. R. (1986) Trends Biochem. Sci. 11, 11-12). To identify the amino acid residues which are sensitive to MCO oxidation, several enzymes/proteins and amino acid homopolymers were exposed to various MCO systems. The carbonyl groups which were formed were converted to their corresponding 3H-labeled hydroxy derivatives. After acid hydrolysis, the labeled free amino acids were separated by ion exchange chromatography. Each protein or polymer gave rise to several different labeled amino acids. The elution profiles of the labeled amino acids obtained from preparations of Escherichia coli glutamine synthetase which had been oxidized by MCO systems comprised of either Fe(II)/O2 or ascorbate/Fe(II)/O2 both in the presence and absence of EDTA were qualitatively the same. From a comparison of the elution profiles of labeled amino acids from various proteins with those obtained from homopolymers, it is evident that the side chains of histidine, arginine, lysine, and proline are particularly sensitive to oxidation by the MCO systems. This conclusion is supported also by direct amino acid analysis of acid hydrolysates which shows that the oxidation of glutamine synthetase, enolase, and phosphoglycerate kinase is associated with the loss of at least 1 histidine residue per subunit. From the results of studies with homopolymers, it is apparent that glutamic semialdehyde is a major product of both proline and arginine residues. In addition, hydroxyproline and unlabeled glutamic acid were identified among the hydrolysis products of oxidized poly-L-proline, and unlabeled aspartic acid was identified as a product of poly-L-histidine oxidation.

Following a freeze-thaw cycle, treatment of Escherichia coli with the nonionic detergent, Lubrol WX, renders the cells permeable to small molecules but not to cytosolic proteins. After such treatment, the permeabilized cell suspensions can be assayed directly by standard procedures both for intracellular levels of glutamine synthetase and the state of adenylylation (i.e. the average number, n, of adenylylated subunits/dodecameric molecule). Permeabilization of cells from cultures containing an adequate supply of glutamine as the sole nitrogen source led to complete retention of all protein components of the bicyclic cascade that regulates the interconversion of glutamine synthetase between adenylylated and unadenylylated forms; similar treatment of glutamine-starved cells leads to selective inactivation, only, of the uridylyltransferase. When suspended in buffers containing ATP and glutamine, the value of n in permeabilized cells increased to high values (n = 11), whereas in the presence of alpha-ketoglutarate, Pi, and ATP, the value of n decreased to approximately 2.0. Time-dependent changes in n that occur during incubations of permeabilized cells in buffers containing these effectors can be arrested either by sonication at 0-4 degrees C or by the addition of cetyltrimethylammonium bromide (to inactivate adenylyltransferase). It is thus evident that Lubrol-treated cells may be used to investigate the regulation of glutamine synthetase adenylylation in situ.

Mixed-function oxidation systems comprised of Fe3+, O2, and electron donors such as thiol compounds, ascorbate, NAD(P)H/NAD(P)H oxidase, and xanthine oxidase/hypoxanthine, catalyze the inactivation of many enzymes. This report describes the isolation and purification of a soluble protein from Saccharomyces cerevisiae, which specifically inhibits the inactivation of various enzymes by a nonenzymatic Fe3+/O2/thiol mixed-function oxidase system. When thiol is replaced with another electron donor (e.g. ascorbate), this specific protein no longer protects against iron (or copper)/O2-dependent radical-induced enzyme inactivation. Purification steps included a polyethylene glycol precipitation followed sequentially by a chromatography on DE52 and high pressure liquid chromatography on phenyl, DEAE, and gel-filtrated columns. The final gel filtration step yielded two protein peaks exhibiting protector activity and possessing a Mr of 500,000 and 90,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these two fractions gave a single band at 27 kDa suggesting that these protein species simply represent different oligomeric structures. The protector protein did not possess catalase, glutathione peroxidase, superoxide dismutase, or iron chelation activities. Since the protection activity reported herein is specific for mixed-function oxidation systems containing thiols, we propose that the protector protein functions as a sulfur radical scavenger.

When cell-free extracts of Clostridium thermoaceticum are incubated with pyruvate and 14CO2, radioactive carbon is incorporated into the carboxyl groups of pyruvate, acetate, formate, and an unidentified fatty acid. The unknown acid was isolated and identified as α-ketoisovalerate. A metabolic pathway was proposed to explain the formation of α-ketoisovalerate and the ability of C. thermoaceticum to ferment glucose to nearly 3 eq of acetate. Data are presented showing that α-ketoisovalerate is probably not an intermediate in the total synthesis of acetate from CO2 which is catalyzed by C. thermoaceticum. However, the evidence suggests that the proposed pathway of metabolism might account in part for the ability of this organism to catalyze incorporation of CO2 into the carboxyl group of acetate.

Cell-free extracts of Pseudomonas M.A., a nonphotosynthetic bacterium which grows on methylamine as its sole source of carbon, nitrogen, and energy, have been shown to catalyze a reaction between methylamine and α-ketoglutaric acid. The product of this reaction has been identified as the cyclic amide, 5-hydroxy-N-methylpyroglutamic acid. This reaction is catalyzed by an enzyme which differs from the one catalyzing the formation of N-methylglutamic acid, and it appears to be specific for α-ketoglutarate. The possibility of a two-step reaction is discussed.

SUMMARY Cofactor requirements for the reduction of diphosphopyridine nucleotide with molecular hydrogen by enzyme preparations of Clostridium kluyveri were reinvestigated. The results confirmed previous conclusions that flavin adenine dinucleotide and a factor in boiled extracts (“Magnesol co- factor”), separable from FAD by chromatography on Magnesol, were involved. Whereas the FAD requirement is absolute at all concentrations of reactants tested, activation by the Magnesol cofactor is observed only at relatively low concentrat,ions of FAD. In the absence of Magnesol cofactor, hydrogen-linked DPN reduction is a bimolecular function of the FAD concentration. However, when Magnesol cofactor is present, DPN reduction follows normal Michaelis-Menten saturation kinetics with re- spect to FAD, and the affinity of enzyme for FAD is much higher than when Magnesol cofactor is absent. Similar activa- tion of the hydrogenase system is obtained when the Magnesol cofactor is replaced by high concentrations of flavin mononu- cleotide, adenosine triphosphate, diphosphate, or any of a large number of other nucleoside di- or triphosphates. The results suggest the presence two binding sites on hydro- genase, namely, a catalytic site capable of binding FAD and an allosteric site capable of binding factors present in the Magnesol cofactor preparation and also nucleotides including FAD, flavin mononucleotide, adenosine triphosphate, and adenosine diphos- phate when these are present at high concentrations. The Magnesol preparation was resolved by diethylaminoethyl cellulose chromatography into two active fractions, neither of which contained a sufficient concentration of any of the common nucleotides to account for its activity. Previous suggestions that the Magnesol cofactor activity is due to flavin mononucleotide or ferredoxin were shown be incorrect. REFERENCES

All of the cysteic acid residues (4.6 per subunit) found by amino acid analysis of performic acid-oxidized Escherichia coli glutamine synthetase are present as cysteine in the native enzyme but are buried in the interior of the molecule. They become exposed and accessible to titration by sulfhydryl reagents only when the enzyme is disaggregated into subunits or when divalent cation is removed with ethylenediaminetetraacetate. From the total number of peptides and the number of arginine-containing and 14C-carboxymethylated peptides that are produced upon tryptic digestion of 14C-carboxymethylated glutamine synthetase, it is suggested that the enzyme is composed of identical subunits. However, until an explanation is found for the wide variation in the observed specific radioactivity of the 14C-carboxymethylated peptides thus obtained, this conclusion is still uncertain. Glutamine synthetase can be inactivated by organic mercurials, and the extent of inactivation depends upon the amount of divalent cation in association with the enzyme. Two states of the enzyme are thus defined: "relaxed enzyme," without metal, which is susceptible to inactivation and disaggregation by p-chloromercuriphenylsulfonate, and "taut enzyme," which has bound manganese and is resistant to such treatment. The metabolic effectors of glutamine synthetase may cause alterations in the rate and extent of inactivation of the enzyme by mercurials. Of the inhibitors that are partially competitive with respect to glutamate, tryptophan diminishes the inactivation by mercurials, cytidine triphosphate enhances this inactivation, and glycine has little effect. Of the compounds that are partially competitive inhibitors with respect to ammonia, histidine is protective against inactivation and glucosamine-6-P has no effect. Similarly, with the noncompetitive inhibitors, AMP decreases the rate of inactivation while carbamyl phosphate and alanine have no influence. These results provide additional support for the occurrence in glutamine synthetase of separate binding sites for each inhibitor, as would be expected from the independent nature of the cumulative feedback inhibition phenomenon.

A purified preparation of clostridial ethanolamine deaminase was shown to be homogeneous by sedimentation in the ultracentrifuge and by disc gel electrophoresis. The enzyme has an s20,w of 14.5 S and a weight average molecular weight of 520,000 g. Treatment with 5 m guanidine hydrochloride results in dissociation of subunits with a weight average molecular weight of 51,000 g. Amino acid analysis of native and performic acid-oxidized deaminase indicated the presence of 3 moles of half-cystine residues per minimum molecular weight of 28,700 g. Titration with 5,5'-dithiobis(2-nitrobenzoic acid) yielded only 1 mole of free sulfhydryl group per mole of native enzyme (520,000 g) but in the presence of sodium lauryl sulfate 15 sulfhydryl groups per molecule were titratable. The spectrum of the enzyme contains a prominent peak at 470 mµ, with other peaks at 357, 410, and 530 mµ. The chromophore was resolved from the enzyme by treatment with acid-ammonium sulfate. On resolution, the 470 mµ peak of the enzyme disappeared and the dissociated chromophore had a spectrum similar to that of hydroxocobalamin and related cobamides. The chromophore was identified as an α-(adenylyl)cobamide by paper chromatography and paper electrophoresis. Between 1.35 and 3.1 moles of cobamide were found per mole of native enzyme. On resolution, the specific activity of the deaminase, when tested in the presence of added cobamide coenzyme, increased an average of 38%. Hydroxocobalamin, cyanocobalamin, and methylcobalamin were shown to be irreversible inhibitors of ethanolamine deaminase. Incubation of resolved enzyme with varying amounts of hydroxocobalamin resulted in complete inhibition of the enzyme after the binding of 2 to 3 moles of cobamide per mole of enzyme. The enzyme was able to bind about 7 moles of hydroxocobalamin per mole. In both the presence and absence of ethanolamine, incubation of the enzyme resulted in the slow appearance of a spectral peak at 352 mµ. Oxidation and Schmidt degradation of acetaldehyde produced by the enzymatic deamination of 1-14C-ethanolamine hydrochloride showed that the aldehyde carbon of the acetaldehyde is derived from the carbinol carbon of ethanolamine.

The glutamine synthetase of Bacillus subtilis has an absolute requirement for divalent cation. Either Mg2+ or Mn2+ will serve as an activator, but each affects various catalytic parameters differently. The concentration of either divalent cation required to produce half-maximal activity (the S0.5) is a function of the ATP concentration. Optimal activity with Mn2+ occurs when the ratio of ATP to Mn2+ is 1.0, and decreases sharply with any deviation from this ratio. With Mg2+, optimal activity is achieved when the concentration of Mg2+ is 4- to 5-fold in excess of the ATP concentration, and it is not substantially affected by large excess of this cation. The saturation functions for the substrates are generally complex and vary with the kind of divalent cation used to activate the enzyme. With Mn2+, the activity increases with increasing concentration of glutamate or ammonia, but reaches a maximum at relatively low levels of substrate and then decreases with higher substrate concentrations. In contrast, with Mg2+, the enzyme is not saturated even at high concentration (100 mm) of either substrate. In the Mg2+ system, double-reciprocal plots of reaction velocity versus substrate concentration are concave downward, suggesting that negative cooperativity might be involved in substrate binding. The enzyme is inhibited by end products of glutamine metabolism, AMP, CTP, histidine, tryptophan, alanine, and glycine. Both the kinetics and sensitivity to feedback inhibition by each of these products are dependent upon whether Mg2+ or Mn2+ is used as the activating cation. The capacity of B. subtilis glutamine synthetase to be activated by Mn2+ and by Mg2+ is apparently an intrinsic property of the enzyme; it is independent of growth conditions and is not determined by adenylylation of the enzyme as it is for the enzyme from Escherichia coli. Efforts to demonstrate adenylylated forms of the B. subtilis enzymes were unsuccessful. In contrast to the E. coli enzyme, the glutamine synthetase from B. subtilis is inhibited by glutamine and this inhibition varies with the divalent cation used to activate the enzyme; with Mn2+ activation, inhibition by glutamine is greatly potentiated by AMP. From the standpoint of cellular regulation, it seems that in both E. coli and B. subtilis, the intracellular level of glutamine is the most important single factor in determining glutamine synthetase activity; however, in B. subtilis, glutamine is a direct inhibitor of glutamine synthetase activity, whereas, in E. coli, it exerts its affect indirectly through activation and inhibition of the adenylylating and deadenylylating enzymes, respectively.

An enzyme that catalyzes the reversible hydroxylation of nicotinic acid to 6-hydroxynicotinic acid has been purified from extracts of a nicotinic acid-fermenting clostridium. The enzyme appears to be a flavin adenine dinucleotide-containing non-heme iron protein and utilizes triphosphopyridine nucleotide as the ultimate electron acceptor. The purified enzyme also exhibits reduced triphosphopyridine nucleotide oxidase and diaphorase activity.

A procedure is described for the purification of glutamine synthetase from Bacillus subtilis, grown under conditions of nitrogen limitation, to a nearly homogeneous state. Although the purified enzyme differs from the glutamine synthetase from Escherichia coli in catalytic properties and stability, the physical characteristics of these two enzymes are quite similar. The B. subtilis enzyme has a molecular weight of ∼600,000, a sedimentation coefficient at infinite dilution of 19.3 S, and is seen in electronmicrographs as a molecule composed of 12 subunits arranged in 2 superimposed hexagonal rings. Studies with the dissociated enzyme suggest that the subunit molecular weight is ∼50,000, in agreement with a dodecameric aggregate structure of the native enzyme which contains 12 subunits of similar size. Both Mn2+ and Mg2+ activate the enzyme in the biosynthesis of l-glutamine, but, unlike the E. coli system, the Mg2+-dependent activity is intrinsically less stable than the Mn2+-dependent activity. Differences between the B. subtilis and E. coli enzymes are apparent also in the amino acid compositions, in the susceptibility to digestion by carboxypeptidase A, in the C-terminal amino acid of the subunit polypeptide chains, in immunochemical properties, and in that the E. coli adenylylating enzyme system does not incorporate 5'-14C-adenylyl groups into the B. subtilis glutamine synthetase.

6-Hydroxynicotinic acid was demonstrated to be the first intermediate in the degradation of nicotinic acid by a clostridium. In the presence of pyruvate, other intermediates accumulated; two of these were identified, one as 1,4,5,6-tetrahydro-6-oxonicotinic acid, and the other as α-methyleneglutaric acid. The pathways for nicotinic acid degradation may be considered as involving successive oxidative and reductive steps to 1,4,5,6-tetrahydro-6-oxonicotinic acid, then through some unidentified steps to α-methyleneglutaric acid, which is subsequently converted to propionic and acetic acids and carbon dioxide. Pyruvate and lactate were shown not to be obligatory intermediates in the production of propionate. This microorganism has a high content of B12 coenzyme, but its function in nicotinic acid fermentation is not yet understood.

Cell-free extracts of Clostridium thermoaceticum have been shown to synthesize acetate, labeled in both carbon atoms, from 14CO2. The extracts can also catalyze the conversion of the methyl group of Co-14CH3-cobalamin into the methyl group of acetate. This conversion is dependent upon coenzyme A and pyruvate, and is sensitive to sulfhydryl poisons, especially arsenite and cadmium. An exchange between CO2 and the carboxyl group of pyruvate is catalyzed by the extracts and is sensitive to iodoacetamide. Passage of the extracts over diethylaminoethyl cellulose resolves the system into two fractions which can be reactivated on recombination only by the further addition of ferredoxin.

Initial rates of adenine uptake by Escherichia coli resting cells were found to correlate closely with adenine phosphoribosyltransferase activity of cell homogenates when measured under optimal conditions. Cultural conditions, inhibitors, and subjecting the cells to osmotic shock altered both uptake and phosphoribosyltransferase activities pari passu. At appropriate concentrations virtually all of the adenine in the medium was taken up into the cells even under conditions in which no growth occurred and macromolecular synthesis was negligible. In energy-depleted cells glucose, ribose, or P-ribose-PP markedly stimulated uptake of adenine while several other sugar phosphates gave much less or no stimulation. P-ribose-PP also stimulated uptake of several additional purines and pyrimidines. For P-ribose-PP-stimulated adenine uptake the kinetics with respect to both P-ribose-PP and adenine concentrations was similar to those observed for adenine phosphoribosyltransferase. Iodo-acetamide (0.4 mm) caused virtually complete inhibition of the glucose- or ribose-stimulated uptake of adenine but did not affect the P-ribose-PP-stimulated uptake. Labeled adenine taken up was found intracellularly predominantly as AMP, IMP, and GMP while free adenine accounted for 0 to 2% of the total radioactivity taken up. Nucleotide inhibitors of adenine phosphoribosyltransferase activity were also found to be inhibitors of adenine uptake; free purines (which do not inhibit adenine phosphoribosyltransferase) also inhibited uptake, most probably acting after conversion to nucleotides. Several phosphoribosyltransferases are apparently located in the pericytoplasmic space since variable amounts (up to 70%) are released into the "shock fluid" when cells are subjected to osmotic shock by the method of Nossal and Heppel (J. Biol. Chem., 241, 3055 (1966)). This release is accompanied by a parallel loss in purine uptake capacity. Although all purine and pyrimidine uptake systems can be stimulated to a greater or lesser extent by P-ribose-PP, they are distinct and may be governed by completely different mechanisms.

In this paper, glycine uptake by isolated membrane preparations of Escherichia coli W (W+) and the d-serine-resistant mutant (WS) is examined in detail. Glycine uptake by W+ membrane preparations is dependent on pH and on the presence of Mg++ and an energy source. In W+ membranes, glycine is first taken up into an exchangeable pool, from which it is subsequently removed as the incubation proceeds. The exchangeable pool is built up within 30 sec and remains constant with time, although glycine uptake by the membranes continues. Glycine uptake into the exchangeable pool of W+ membranes is independent of temperature. Uptake and exchange in W+ membranes are specific for glycine, dl-alanine, dl-serine, and dl-threonine. Membrane preparations from the WS mutant are unable to catalyze either the uptake of glycine or the exchange of glycine between the incubation medium and the pool. Glycine uptake by W+ membranes is inhibited 50 to 70% by anaerobiosis, incubation at 0°, or incubation in the presence of 2,4-dinitrophenol, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, NaN3, KAsO4, KCN, iodoacetamide, and p-chloromercuriphenylsulfonate. No inhibition is oberved with NaF, sodium Amytal, hydroxyquinoline N-oxide, antimycin A, Chloromycetin, or ouabain. Although glycine uptake at 30 min is inhibited by the conditions listed above, there is no diminution of glycine uptake when measured at 30 sec. Under none of the conditions studied is the uptake (at either 30 sec or 30 min) decreased to the values found with WS membranes at 37° or 0°. W+ membranes incubated at 37° convert about 94% of the glycine carbon taken up into compounds that have the properties of phosphatidylethanolamine and phosphatidylserine. Under all of the conditions studied, the concentration of glycine in the intramembranal pool approximates that of the incubation medium. Incubation at 0° or in the presence of dinitrophenol or iodoacetamide inhibits the conversion of glycine to phospholipids, and the amount of glycine in the intramembranal pool is increased above that found for uninhibited (control) preparations. WS membranes convert significant amounts of glycine to phospholipids, but the concentration of glycine in the intramembranal pool is only 10 to 20% of that in the external pool. When the external glycine concentration is raised so that the rate of glycine uptake by WS membranes is comparable to the rate exhibited by W+ membranes, the WS membranes convert as much glycine to phospholipids as do W+ membranes, but the concentration of glycine in the intramembranal pool remains only 10 to 20% of that in the external pool. Hydroxylamine almost quantitatively inhibits the conversion of glycine to phospholipids, and in the presence of this inhibitor the WS membranes manifest the same defect.