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The H+/Ca++ ratio during the aerobic uptake of Ca++ by rat liver mitochondria, oxidizing endogenous substrates or succinate, increased with the increase of the Ca++/protein ratio. At high protein concentrations an apparent ‘reuptake’ of the H+ ejected was observed. Mitochondrial aging also resulted in a decrease of the H+/Ca++ ratio. Succinate caused a decrease of the H+ ejection. The effect of succinate was dependent on the pH and on the concentration of Ca++. A binding of succinate to the mitochondria was observed parallel to the decrease of the H+/Ca++ ratio from 2 to 1. Addition of Ca++ caused a release of K+ which was slow in fresh mitochondria and rapid in valinomycin treated mitochondria. The K+/Ca++ ratio was 2 in both cases. A H+/Ca++ ratio of about 2 was usually observed when the aerobic uptake of Ca++ was started either by the addition of mitochondria to a Ca++ containing medium or by the addition of succinate to Ca++- and rotenone-treated mitochondria. [ABSTRACT FROM AUTHOR]

An endonuclease showing no apparent base specificity, nor any preference for the sugar moiety has been isolated from sheep kidney. The products of the digestion are 5′-P-ended oligonucleotides, and not mononucleotides. Preparation of short oligo A and oligo U (di-, tri-. tetra-) with a good yield is described. The enzyme requires Mg ions, is inhibited at high ionic strength and destroyed by temperatures of 60° and above. The enzyme displays an extremely rigorous specificity with regard to the secondary structure of its substrate. This structural specificity is apparent from the fact that native DNA, complexes between poly A and poly U, and polyinosinic acid in high salt are completely resistant to the enzyme. Even poly A and poly C, at neutral pH, denatured DNA, ribosomal DNA, and especially tRNA, are only slowly attacked. Preliminary experiments on the effect of addition of poly U on the hydrolysis of a copolymer A, C suggest that the enzyme will hydrolyze the unstructured loop of polyribonucleotide complexes. The analysis of the hydrolysis of tRNAs by sheep kidney nuclease shows that the enzyme attacks all chains and produces mainly large fragments (average chain length, 40 nucleotide units), containing pGp terminal and TpΨpCpG sequences; and small oligonucleotides (average chain length 4.5 nucleotide units), containing a large amount of fragments with a CpCpA terminal. These results arc discussed in relation to the conformation of tRNA. [ABSTRACT FROM AUTHOR]

Template activity of RNA from rat liver and the distribution of this activity along a sucrose gradient have been studied. The experiments were performed with RNA extracted from polysomes, monosomes and ribosomal subunits. There is no correlation between template activity of RNA extracted from polysomes and early-labelled RNA, since template activity is distributed all along a sucrose gradient while the radioactivity after a 20 min pulse with [14C]orotic acid is almost exclusively localized in the 18-4 S region. Stimulatory activity in the heavy regions of the gradient is due either to unique large molecules of messenger RNA or to aggregation of ribosomal RNA with messenger RNA. Our experimental results, however, seem to exclude aggregation due to the presence of bivalent ions as well as aggregates held by hydrogen bonds. Ribosomal RNA is itself responsible for part of the template activity found in different fractions of the gradient. Ribosomal RNA extracted from monosomes prepared from polysomes by treatment with RNase, and from subunits, showed a stimulatory activity which, although inferior to that due to messenger RNA, was consistent. Experiments were performed which excluded the possibility that residual messenger still attached to ribosomal RNA was responsible for this activity. Treatment of ribosomal RNA with chemical agents such as EDTA and urea increased the template activity in those cases where a permanent increase in hyperchromicity could be demonstrated. Although neomycin also increased stimulatory activity, it is thought unlikely, on the basis of our experiments, that this is due to a separation of the strands of RNA. [ABSTRACT FROM AUTHOR]

Crude extracts of Candida utilis contain two types of 6-phosphogluconate dehydrogenase. One can be obtained in the crystalline form and has already been described and studied. In the present paper the purification of the second one, together with the differences in the properties between the two purified proteins, is reported. The two types of enzyme have identical pH optimum, same Km for the substrates and same specificity. They differ in amino acid composition, molecular weight, electrophoretic mobility, stability to pH and heat treatment, sensitivity to chlorodinitrobenzene and proteolytic treatment. All evidence indicates a greater instability of the crystalline enzyme in respect to the non-crystalline one and seems to exclude that the two forms of enzyme are an artefact due to the extraction or the purification procedures. [ABSTRACT FROM AUTHOR]

The modification of one cysteine residue of transamidinase by reaction with 5,5′-dithiobis-2-nitrobenzoic acid alters the catalytic properties of the enzyme. The Michaelis constant of transamidinase for arginine is increased and the capacity to utilize glycine and hydroxylamine as amidine acceptors is lost. Arginine, the amidine donor substrate, protects the cysteine residue and prevents the changes in catalytic activity induced by the treatment with 5,5′-dithiobis-2-nitrobenzoic acid. [ABSTRACT FROM AUTHOR]

From kinetic studies on the reaction sequence of substrate level phosphorylation in rat liver mitochondria, using anaerobic ketoglutarate dismutation in the presence of oligomycin and [32P]phosphate, phosphohistidine appears to be the first intermediate to be labelled, followed by GTP. [32P]ADP rather than [32P]ATP is shown to be the main product of the reaction. The phosphorylatin of AMP requires ketoglutarate and is stimulated by 2,4-dinitrophenol. GTP-AMP-P transferase is localized in the mitochondria. This conclusion is based on enzymatic assays of fractionally ectracted rat liver and of isolated mitochondria and microsomes. Mean values for the activities of GTP-AMP-P transferase, nucleoside diphosphate kinase and succinic thiokinase in rat liver mitochondria are given and arc compared with the rate of ketoglutarate oxidation. A possible function of GTP-AMP-P transferase for the phosphorylation of endogenous AMP is discussed with regard to the compartmentation of nucleotides in the mitochondria. A new chromatographic assay for GTP-AMP-P transferase is reported, an assay which is not affected by nucleoside diphosphate kinase and adenylate kinase occurring in liver homogenates. An optical enzymatic assay for nucleoside diphosphate kinase is also described. [ABSTRACT FROM AUTHOR]

In the previous paper [1] we proposed a mechanism for the reaction of pancreatic ribonuclease with nucleotide diesters. in which the kinetic constants k+1 and k-1 + k+2 are assigned to a base catalysed step for the formation, and a proton catalysed step by the conjugate acid for the breakdown, of an enzyme stabilized intermediate. In the hydrolysis reaction of the 2′,3′- cyclic diesters the rates depend on the structure of the catalysing base, and in particular on its polarisability. in addition to this effect, we found that, in the dinucleoside phosphate series, the rates increased up to three orders of magnitude, depending on the nature of the base of the second nucleoside [4]. According to our suggested mechanism I-VI the accelerated rates could be due to (a) the second base influencing the polarisability of the catalysing system by π-electron interactions between the stacked bases. This effect should be reflected in the enthalpy term and should parallel the hypochromicity of the dinucleoside-phosphates exactly: (b) pre-ordering of the reacting atoms (C-2-oxygen, 2′-OH-group and phosphorus) by the base stacking, which would be reflected in a lowering of the activation energy in the entropy term and should only roughly parallel the hypochromicity. To differentiate between these two possibilities we prepared the diesters IX -XXXIV, determined the hypochromic effect and also the values of km and k+2 at pH 7. There is only a partial correlation between k+3 values and the hypochromic effect. The k+2 values vary with the structure of the second nucleoside and show a particular dependence upon the nature of the link between base and phosphate group. Highest values are found with adenosine and deoxyadenosine. Elongating the ribose with mercaptoethanol (XIX) or substituting the relatively rigid ribofuranose ring with flexible n-hexyl-, pentyl- or butyl-chains (XX-XXIII appreciably reduces the hypochromicities and also the k+2, values, in the case of a propyl link (XXIII, XXIV( or in the analogously structured 3′-3′-dinucleosidephosphate XXV no hypochromic effect is found and the k+2 values are as low as that of 3′-cytidylic acid benzyl ester. A second nucleoside attached to the 5′ end of a substrate (XXXIII. XXXIV) does not result in an increase of the rate of diester hydrolysis at the 3′ end though there is a normal hypochromicity. Therefore a pre-ordering of the reacting atoms (demonstrable on models) appears to be the accelerating factor. A specific interaction between the second nucleoside and the enzyme being responsible for the pre-ordering effect can be excluded by either blocking or varying all the potential interaction sites. The concept of a pre-ordering in the substrate due to bass stacking agrees with the results from measurements of the temperature dependence of k+2 and calculations of the enthalpy (ΔH≠) and entropy (Δ,S≠) term in the activation energy of this step. When diesters with an exactly analogous structure of the intermediate II such as CpA (IX) and Cp-butyladenine (XXII) are compared, the differences are found in the entropy term only. Between the first and second step substrates such as CpA and 2′3′-Cp the differences are also found in the entropy term with a small factor in the enthalpy term. From a comparison of Up- and Cp-diesters it can be seen that due to the differences in the enthalpy term the Up-diesters should react 5–10 times faster than Cp-diesters, but that due to the differences in the entropy term Cp-diesters react 20 times faster than Up-diesters.… [ABSTRACT FROM AUTHOR]

We have studied the repression of the first enzymes of the main branch and the pathways after the branchpoint in the aromatic biosynthesis of Saccharomyces cerevisiae. Anthranilate synthetase is repressed by L-tryptophan and prephenate dehydratase by L-phenylalanine. 3-Deoxy-D-arabinoheptulosonic acid 7-phosphate (DAHP) synthetases are not repressed by L-phenylalanine, L-tyrosine and L-tryptophan, chorismate mutases are repressed neither by L-phenylalanine nor by L-tyrosine, and prephenate dehydrogenase shows no repression by L-tyrosine. L-Tryptophan exhibits an induction of the chorismate mutase. Besides chorismate mutase is activated by L-tryptophan. L-Phenylalanine exhibits an induction and also an activation of the prephenate dehydrogenase. By simultaneous addition of L-tryptophan, L-phenylalanine, and L-tyrosine p-aminobenzoic acid was accumulated and the production of p-aminobenzoate synthetase was induced. The tyrosine-sensitive and phenylalanine-sensitive isoenzymes of DAHP synthetase belong to the V-system of the allosteric enzymes. Chorismate mutase and prephenate dehydrogenase are members of the K-system. In an earlier publication we have discussed the feedback inhibition of the described enzymes. In this paper we discuss the whole events of the regulation phenomena in Saccharomyces cerevisiae. We also considered the degradation of L-tryptophan, L-phenylalanine, and L-tyrosine to tryptophol, phenylethanol, and tyrosol respectively in connection with the regulatory events. [ABSTRACT FROM AUTHOR]

Exposure of liver microsomes to high concentration of neutral salts resulted in the conversion of the microsomal hemoprotein, P-450, to a modified from called P-420. The efficiency of various salts in causing the conversion abeyed the order known as Hofmeister's lyotropic series of ions, i.e. SCN -> I- > Br- > CI- > SO4--, CH3COO- for anions, and Li+ > K+, Na+ for cations. The conversion induced by salts proceeded more rapidly in dithionite-treated microsomes, in which the hemoprotein was kept reduced, than in aerobic microsomes. Even in the basence of added salts the conversion took place to some extent when pH was moved far from neutrality. The addition of salts narrowed the pH region where the hemoprotein was resistant to the conversion. Guanidine hydrochloride, lysolecithin, and high conentrations of aniline also caused the conversion of P-450. Although iodine was an effective concersion agent, it caused considerable destruction of the hemoprotein. Based on these and other findings, the structure around the heme group of P-450 as well as the mechanism of its conversion to P-420 are discussed. [ABSTRACT FROM AUTHOR]

The inhibition of mitochondrial respiration by azide under various steady states was analyzed and found to be controlled by an active accumulation of azide. The azide inhibition is found to be uncompetitive with the respiratory rate. Thus the 50% inhibitiong concentration of azide decreases inversely to the respiratoory rate, independent of the substrate. The respiration activated by ion uptake is about 10 times more sensitive to azide than the uncoupled respiration. The respiration of particles is about 20-fold less sensitive to azide when compared to mitochondria under identical conditions (ion pumping state) and at equal respiratory rates. Aside can be demonstrated to be a permeant anion under conditions of passive swelling. An active accumulation of azide, as deduced from these results, is demonstrated by respiratory assays in sedimented mitochondria. Azide does not inhibit electron transfer to ferricyanide but rather stimulates it by activation of ion uptake. It is concluded that azide on mitochondria acts also by means of its accumulation and that its interference with energy transfer is a secondary result of its accumulation as a permeant anion. [ABSTRACT FROM AUTHOR]

1.e amino acid sequence of a dodecapeptide containing the essential SH-group of pig heat l-lactate dehydrogenase, isoenzyme I, was determined as Val-Ile-Gly-Ser-Gly-Cys-Asn-Leu-Asp-Ser-Arg. This sequence shows three differences with that of an SH-peptide from chicken LDH-I. 2. It is shown that the possible condons for the amino acids occupying equivalent positions in the heptapeptides around the essential SH-groups of glyceraldehyde 3-phosphate-, yeast alcohol-, liver alcohol- and lactate dehydrogenases can be related to one another by a chain of point mutations. [ABSTRACT FROM AUTHOR]

A non-oxidative pentose phosphate pathway starting from 3,4--labeled hexoses leads in growing organisms to the formation of ribose with a number of marked positions (n) varying between 1.0 and 1.67. n depends on the percentage (100 × b) of erythrose-4-phosphate that is branched off for the synthesis of aromatic amino acids; it can be calculated using the equation n +(5--4b)/ (3--2b). This method is demonstrated with growing cultures of Clostridium kluyveri. Data are presented showing that under normal regulatory conditions about 70% of the erythrose-4-phosphate is utilized for aromatic amino acid synthesis while in the presence of tyrosine or phenylalanine or all three aromatic amino acids respectively about 50%, 40% or almost none of the tetrose is branched off iinto the synthesis of aromatic compounds. It is shown by analysis of the cell constituents that in cells growing in media supplemented with aromatic amino acids the decreasing percentage of branched-off tetrose is quantitatively correlated to the decreasing requirement for aromatic amino acids. [ABSTRACT FROM AUTHOR]

Within the last few years, the quinoline alkaloids viridicatine (I), viridicatol (II) and 3-O-me thyl viridicatine (III) have been isolated from the mycelium and culture medium of Penicillium cyclopium Westling, Penicillium puberulum Bainier and Penicillium viridicatum Westling. Two further alkaloids found in the culture medium of P. cyclopium und P. viridicatum, cyclopenine (IV) and cyctopenol (V) are benzodiazepine derivatives. We have shown, using labelled precursors with P. viridicatum strain V41 and P. cyctopium strain SM72 that cyclopenine, cyclopenol, viridicatine and viridicatol are synthesised from anthranilic acid and phenylalanine. These results, together with the easy conversion of cyclopenine and cyclopenol into viridicatine and viridicatol respectively by means of acid or alkali, suggested that an in vivo relationship could exist between these two groups of alkaloids. in this paper we show that cyclopenine and cyclopenol are intermediates in the formation of viridicatine and viridicatol and that the transformation of the former to the latter is effected by an enzyme which we have named cyctopenase. This transformation occurs with fresh homogenised mycelium with dried lyophilised mycelium and with mycelium free extract, that with the dead mycelium being much faster than with the other preparations. Using the mycelium free extract it was shown that the "agent of activity" depends upon pH, is not dialysable and can be destroyed by heating at 60° for 3 min, thus indicating the enzymatic nature of the transformation. Additional products of the transformation are carbon dioxide and methylamine. Cyclopenase activity is shown to be strongest in the surface cultured mycelium of P. viridicatum, strain V41. In the surface cultured P. cyclopium, strain SM72 mycelium the activity was much tess and in the submerged culture of both strains practically no activity was detected. This agrees with the observation that, under normal conditions only P. viridicatum strain V41 produces an appreciable amount of viridicatine and viridicatol. These experiments, together with previous work, indicate that anthranilic acid and a phenylpropane (e.g. phenylalanine) combine to give the benzodiazepines, cyclopenine and cyclopenol and these are transformed by the enzyme cyclopenase to the quinoline derivatives viridicatine and viridicatol. The detailed mechanism of this transformation is being investigated. [ABSTRACT FROM AUTHOR]

1. A new diagonal etectrophoretic technique for determining the order of the lysine-containing tryptic peptides of a protein is described. The protein is converted into its trifluoracetyl derivative, digested enzymatically (or chemically), and the resulting peptides separated by paper electrophoresis. The paper is then treated with ammonia vapour, which re-exposes the ε-amino groups of the lysine residues, and submitted to a second electrophoresis at right angles to the first direction. Peptides containing lysine residues, together with the N-terminal peptide of the protein, are found to lie off a diagonal formed by all other peptides, whence they may be readily purified. A study of these peptides enables the order of the lysine-containing tryptic peptides in the protein to be deduced. 2. The technique has been successfully tested with insulin. 3. When the method was applied to porcine pepsin, the four tryptic peptides isolated were easily ordered and the carboxyl-terminal sequence of the protein shown to be Arg-Gln-Tyr-TyrThr-Val-Phe-Asp-Arg-Ala-Asn-Asn-Lys-Val-Gly-Leu-Ala-Pro-Val-Ala. The thee basic ammo acid residues in the molecule are thus found clustering towards the C-terminus of the polypeptide chain. 4. A common ancestral gene for porcine pepsin and bovine (calf) rennin is suggested by the close homology between the C-terminal sequents of the two proteins. [ABSTRACT FROM AUTHOR]

In a preceding publication Le Minor and Staub [8] predicted from phagic and immunological studies, that the repeating unit, of the group D2 polysaccharide should be: [This equation cannot be presented in ASCII format] In this paper the specific polysaccharide was prepared according to Freeman's technique and hydrolysed. High yields of 3 oligosaccharides have been obtained whose analysis gave: [This equation cannot be presented in ASCII format] Periodate oxydation of the polysaccharide intact or degraded by Slight hydrolysis (N CH3COOH, 4 hours at 100°), confirmed these data and showed that tyvelose is linked to carbon 3 of mannose. No O-acetyl was found. Immunological data showed that tyvelose was the immunodominant sugar for both factors (9) and 46. No other analysis of these factors was possible since we did not possess oligosaccharides containing tyvelose. The position d the different factors of Salmonella strasbourg ((3), (9), (10), 46) on the polysaccharide is schematised on Fig. 6. [ABSTRACT FROM AUTHOR]

It is known from previous work that the regulation of Glutaminesynthetase in coli is effected by two mechanisms. First, the synthesis of GS is repressed by NH4+; and secondly, the activity of GS is regulated by inactivation and reactivation of the enzyme molecule. In the present paper we demonstrate and characterize the reactivation of inactivated GS in intact cells of E. coli. GS is inactivated after addition of NH4+ to the culture medium. Experiments with a cell flee extract from E. coli have shown, that the inactivation is mediated by a specific "inactivating enzyme", ATP, Mg2+, and Glutamine which is probably formed from the added NH4+, The reactivation is observed, when the inactivated ceils are centrifuged from the NH4+ containing medium and then resuspended in an NH4+ free medium. In about 5–7 minutes the activity increases 5–30-fold. Inhibitors of protein synthesis such as chloramphenicol and p-fluoro-phenylalanine do not affect the reactivation process. Only a slight retardation of the reactivation is observed. A carbon source is required for reactivation. When inactivated cells are incubated in a medium containing only salts and buffer no reactivation is observed. Probably the carbon sources is metabolized to form intermediates and energy, which are needed for glutamine catabolizing reactions or for prevention of glutamine synthesis. Reactivation does not take place m the presence of NH4+ i.e. under inactivating conditions. However if inactivation is inhabited by fluoride, reactivation can be observed in the presence of NH4+. When inactivated cells are incubated with fluoride only, without a carbon source in the reactivation medium, a reactivation is found. Inactivation as well as reactivation arc inhibited by thiol-group reagents. From these results we conclude that: (a) Reactivation is independent of protein synthesis; (b) Reactivation does not require energy; (c) Probably thiol-groups are involuted in the reactivation process. [ABSTRACT FROM AUTHOR]

This paper describes mass spectrometric studies of the peracetylated derivatives of synthetic and natural 6,6′-diesters of trehalose containing acyl radicals varying from C22 to C90. The peracetylated synthetic 6,6′-dieicosanoyl trehalose (I) gave a molecular ion at m/e 1238. The synthetic 2,3,4,2′,3′,4′-hexaacetyl-6,6′-di(α-eicosanyl-β-acetoxy tetracosanoyl)-trehalose (IIb) showed no molecular ion but a peak (M-60) at m/e 1910 (Fig. l). The cleavage of the glycosidic bond leads to typical oxonium ions which are at m/e 611 (A) for (I) and at m/e 977 (B2) for (IIb). The three natural trehalose 6,6′-diesters examined are the ‘cord factors’ of a human virulent strain of Mycobacterium tuberculosis (strain Prévois), of a bovine attenuated strain (BCG) and of Mycobacterium butyricum. The mycolic acids isolated after saponification of these compounds have also been studied by mass spectrometry. Formulae III, IV, V and VI are proposed for their methyl esters. The molecular weights of the paracetylated ‘cord factors’ are above 3000; no molecular ion could be detected. The oxonium ions, however, give significant peaks which are useful for structural determination. Oxonium ions for the glycolipid of the human virulent strain Prévois are found at m/e 1360–1528 (Fig.2). The peracetylated glycolipid of BCG contains two different series of homologous mycolic acids (acyl ions at m/e 1144–1284 and 1072–1184) and gives oxonium ions at m/e 1432–1572 for one series and at m/e 1360–1472 for the second one. The ‘cord factor’ of M. butyricum gives homologous oxonium ions from m/e 1361 to 1445 corresponding to acyl ions from m/e 1073 to 1157. [ABSTRACT FROM AUTHOR]

When undergoing nucleophilic reactions with proteins, α-iodopropionic acid and its amide show greater specificity for SH groups than do iodoacetic acid and its amide. The kinetics and stereochemistry of the reactions of the D(+) and L(-) antipodes of α-iodopropionic acid and its amide with both cysteine and papain were investigated. The antipodes of the SH reagents reacted with these asymmetric SH compounds at different rates. In the reaction with papain, the L(-) antipode of the acid reacted faster than the D(+) antipode; the stereochemical preference was inverted when the amide was used, the D(+)antipode reacting faster than the L(-)antipode. Arguing from the differing reactivities of the SH reagents and their antipodes and the pH dependence of the reaction rates, it has been suggested, that L(-)alpha;-iodopropionic acid is specifically oriented onto the reaction center through the concerted attractive and repulsive actions of a cationic and an anionic group in the protein. These have been tentatively identified as a protonated imidazole residue and a carboxylate group respectively. Parallels between these stereospecific alkylations and the catalytic action of the enzyme are discussed. [ABSTRACT FROM AUTHOR]

Glucagon is a polypeptide of 29 residues, which can be crystallised. Optical rotatory dispersion in dilute neutral, acid and alkaline solution suggests that the poypeptide chain is largely random, but contains about one turn of α-hehx, which is elfininated in 6 M guanidine hydrochloride. Using thermal difference spectra, nuclear magnetic resonance, and the temperature-dependence of the optical rotatory dispersion as criteria, it appears that glucagon possesses no defined tertiary structure in solution, but may be regarded as being in a state of αhelix⇄ random coil equilibrium, near the high-temperature end of the transition range. That there is no steric objection to the formation of a highly α-helical conformation is demonstrated by optical rotatory dispersion measurements in 2-chloroethanol solution, in which the protein becomes largely helical. A rough calculation indicates that in aqueous solution at pH 2 the transition mid-point is at about —40°. From changes in the proton magnetic resonance spectrum when glucagon is transferred from an aqueous environment into 6 M guanidine hydrochloride, it is deduced that the short helical segment in the chain in the aqueous state contains most of the long aliphatic side chains, and since the sequence is known it has been possible to identify such a segment uniquely near the C-terminal end. On standing in acid solution the viscosity increases and a birefringent gel is formed. Sedimentation studies indicate the formation of large aggregates. On further standing, or warming, a precipitate appears which has the appearance of long fibrils in the electron microscope. Infrared spectra of the gel, of solid films and of the precipitated material show that in all these states the glucagon is in the form of antiparallel β-chains. [ABSTRACT FROM AUTHOR]

The RNA populations of rat liver and three rat hepatomas were compared by DNA-RNA hybridization studies in the presence and absence of competing RNA. The hepatoma nuclei were found to lack hybridizable RNA species present in normal liver nuclei, while the latter appeared to contain all the hepatoma RNA species. In normal liver, the cytoplasmic RNA could compete with nuclear RNA only to the extent of 20–30%. This indicates that a large number of nuclear RNA species are not transferred to the cytoplasm. In the case of the hepatomas nearly all the nuclear RNA species were detected in the cytoplasmic RNA preparations. A more extensive homology between nuclear and cytoplasmic RNA was also observed in regenerating liver. It is suggested that a selective mechanism governs the transfer of RNA species from the nucleus to the cytoplasm and that this mechanism is altered in the hepatomas. [ABSTRACT FROM AUTHOR]

Mutants of Moraxella calcoacetica in which the activity of protocatechuate oxygenase is lost or severely impaired synthesize the remaining four enzymes of the protocatechuate coordinate block—carboxymuconate lactonizing enzyme, carboxymuconolactone decarboxylase, β-ketoadipate enol-lactone hydrolase I and βl-ketoadipate succinyl-CoA transferaseI—at high differential rates in the absence of an exogenous inducer. Such mutants can be isolated directly from the wild type, by selection for the loss of protocatechuate oxygenase function. Similar mutants can also be isolated indirectly from the wild type by two-step selection. The first step involves selection for ability to grow with cis, cis-muconate, as a result of a mutation that confers increased permeability both to this compound and to βl-ketoadipate; the second step involves selection for ability to grow with β-ketoadipate as sole carbon source, as a result of the constitutive synthesis of a βl-ketoadipate succinyl-CoA transferase. Most mutants selected in this way for ability to grow with β-ketoadipate synthesize constitutively transferase I and other enzymes of the protocatechuate coordinate block, but have invariably lost protocatechuate oxygenase function. The properties of these constitutive mutants indicate that the greatly elevated rate of synthesis of enzymes of the protocatechuate coordinate block is not caused by mutation of a regulatory gene. Revertants selected for restored protocatechuate oxygenase function revert simultaneously to the inducible state; the magnitude of constitutive enzyme synthesis is directly related to the degree of impairment of protocatechuate oxygenase function; and most constitutive mutants become inducible when grown at a low temperature (20°), even though protocatechuate oxygenase function remains impaired. These traits suggest that the high rate of synthesis of the enzymes of the protocatechuate block is caused by elevated levels within the cell of the inducer, protoeatechuate, synthesized from endogenous sources and accumulated as a result of the absence or impairment of protocatechuate oxygenase. The endogenous source of protocatechuate appears to be the hydroaromatic intermediates in the pathway of aromatic biosynthesis. The wild type of Moraxella calcoacetica uses shikimate as a source of carbon and energy. Shikimate is dissimilated via protocatechuate by means of inducible enzymes. The initial step is mediated by shikimate dehydrogenase, an inducible enzyme which can be readily distinguished by its catalytic properties from the biosynthetic enzyme responsible for shikimate synthesis, 5-dehydroshikimate reductase. Shikimate dehydrogenase is product-induced by protocatechuate. In mutants lacking protocatechuate oxygenase, it is synthesized, like the enzymes of the protocatechuate block, at high rates in the absence of exogenous inducer, and it is restored to the inducible state when these mutants are cultivated at low temperature, or when they regain protocatechuate oxygenase function by reversion. Accordingly, protocatechuate plays a very important role as an inducer in M. calcoacetica, since it controls the synthesis of all the enzymes responsible for the conversion of shikimate to β-ketoadipyl-CoA. [ABSTRACT FROM AUTHOR]

Further evidence for coordinacy of enzymes operative in the protocatechuate branch of the β-ketoadipate pathway in Moraxella calcoacetica has been obtained through studies with an analog of protocatechuate, 3-hydroxy-4-methylbenzoate. This compound, which is not metabolizable by M. calcoacetica, inhibits the induced synthesis of shikimate dehydrogenase, protocatechuate oxygenase, carboxymuconate lactonizing enzyme, carboxymuconolactone decarboxylase, β-ketoadipate enol-lactone hydrolase I and β-ketoadipate succinyl-CoA transferase I, by the wild type. It also effectively abolishes constitutive synthesis of these enzymes by mutants with impaired protocatechuate oxygenase function. Mutants in which the regulatory control of these enzymes is modified have been isolated. Such mutants are still inducible, but are very poorly induced by protocatechuate, the metabolite which is the normal inducer of these enzymes in the wild type. However, 3-hydroxy-4-methylbenzoate, which interferes with induction in the wild type, has become an effective inducer for the mutants. [ABSTRACT FROM AUTHOR]

Mutants of Moraxella caleoacetica which synthesize constitutively β-ketoadipate succinyl-CoA transferase, but in which all other enzymes of the β-ketoadipate pathway are still normally inducible, have been isolated by selection for the ability to grow with β-ketoadipate. The constitutively synthesized transferase was identified as transferase III. This enzyme is induced in the wild type by adipate and its physiological function is probably adipate activation. The properties of these constitutive mutants confirm that the synthesis of transferase III is regulated independently of the synthesis of enzymes that mediate reactions of the β-ketoadipate pathway. Two phenotypically different classes of mutants which synthesize transferase II constitutively have also been isolated by the same selective procedure. Both classes of mutants synthesize constitutively the five enzymes that convert catechol to β-ketoadipyl-CoA. Strict coordinacy between four of these enzymes (muconate lactonizing enzyme, muconolactone isomerase, β-ketoadipate enol-lactone hydrolase II and β-ketoadipate succinyl-CoA transferase II) is maintained in one class of mutants. However in the other class this coordinacy is destroyed and muconate lactonizing enzyme is synthesized in succinate-grown cells at a relative level much lower than those of the other three enzymes. These findings are discussed and evaluated in terms of earlier evidence concerning the regulation of the synthesis of enzymes of the catechol branch by M. calcoacetica. [ABSTRACT FROM AUTHOR]

Cholesterol side chain cleavage reaction has been studied in ovaries of immature female rats pretreated with pregnant mare serum gonadotrophin and human chorionic gonadotrophin. The cleavage enzymes were found to be in the mitochondria and to have the characteristics of a mixed function oxidase requiring NADPH and oxygen. Using [4-14C]cholesterol as substrate the main product obtained was progesterone while pregnenolone and 20α-hydroxypregn-4-en-3-one, were formed in smaller amounts. This shows that ovarian mitochondria contains a Δ5-3β-hydi-oxysteroid-dehydrogenase, a Δ5-6/4-5 isomerase and a 20α-hydroxy steroid dehydrogenase. The proposed intermediates in cholesterol side chain cleavage (20α-hydroxy cholesterol and 20α, 22ε-dihydroxy cholesterol) were not detected. Mitochondrial production of progesterone in the presence of NADPH was further stimulated by succinate while fumarate was without effect. In the presence of NADPH, NADH was found to be inhibitory while NAD+ gave rise to an increase in pregnenolone, the overall reaction being unaffected. Excess of NADPH causes an accumulation of pregnenolone and slightly decreases the overall reaction. The action of reduced glutathione (GSH) and ascorbic acid-like reducing agents of physiological occurrence has been studied. It was found that GSH in small amounts stimulates and in larger amounts inhibits the side chain cleavage reaction. Ascorbic acid in physiological amounts produced an inhibitory effect. The action of adenosine 3′,5′-cyclic-monophosphoric acid (3′5′AMP) and other nucleotides (AMP, ADP, ATP) was tested. 3′5′AMP markedly stimulates the conversion of cholesterol to pregnenolone and decreases the synthesis of progesterone. The action of AMP shows some similarities with that of 3′5′AMP but the action of ADP and ATP was found to be different. [ABSTRACT FROM AUTHOR]

Clostridium kluyveri was grown (a) on standard media, in which alternately [14C]bicarbonate, [U-14C], [1-14C], and 2-14C]acetate plus ethanol were the labeled substrates, and (b) on standard media supplemented with either formate, glycine or serine, in which bicarbonate or formate were 14C-labeled. Adenine, guanine and methionine were isolated from the cells and degraded. (a) Carbon atoms 2 and 8 of the purines were found to be synthesized from CO2 (about 70 %) and from the methyl group (about 20%) and the carboxyl group (about 10%) of acetate. The S-methyl group of methionine was shown to be derived almost equally from CO2 (about 50%) and from the two-carbon substrates, the methyl group of acetate accounting for about 35% and the carboxyl group for about 15%. (b) The participation of CO2 in the synthesis of the S-methyl group of methionine remained unaffected (about 50%) in the presence of serine, while it was decreased to below 5% in the presence of formate and increased to over 80% in the presence of glycine. The participation of formate was in the order of 55%. These data demonstrate that (1) two major competitive pathways for the synthesis of one-carbon units exist in Clostridium kluyveri: C1-units are formed predominantly by a reduction of CO2 and to a minor degree by a cleavage of serine, the usual pathway (C3-serine = C2-acetate) and (2) the relative rates of the two pathways are controlled by the glycine requirement of the growing organism. [ABSTRACT FROM AUTHOR]

Chemically synthesized TDP-D-[U-14C,5-3H]glucose was converted to TDP-4-keto-6-deoxy-D-[U-14C,6-3H]glucose by an enzyme system obtained from Escherichia coli B. The product contained only a minor amount of the tritium originally present in the starting material. Elimination of tritium from the starting material paralleled the formation of the keto-product. This result is interpreted as indicating that a 5,6-unsaturated hexose nucleotide is an intermediate in the overall conversion. Part of the tritium eliminated from the 5-position of D-glucose was reincorporated into the 6-position of the end product. The mechanistic implications of these results are discussed. [ABSTRACT FROM AUTHOR]

The relationship between oxidative phosphorylation and the energy-linked nicotinamide-adenine dinucleotide transhydrogenase reaction catalyzed by phosphorylating submitochondrial particles from beef heart has been investigated. The efficiency of the particles in exhibiting oxidative phosphorylation (P/O ratio) and NADH-linked NADP+ reduction (NADP+/O ratio) was measured, using succinate as oxidizable substrate and rotenone as inhibitor of NADH oxidase, under conditions where the phosphorylating and transhydrogenating systems were operating one by one or in parallel. When succinate oxidation was limited by increasing concentrations of malonate, the P/O ratio remained unchanged in the absence of an active phosphorylating system, and decreased in its presence. The NADP+/O ratio increased with increasing concentrations of malonate, both in the absence and presence of an active phosphorylating system. The (P + NADP+)/O ratio in the combined system approached 2, i.e., the generally accepted maximal ∼/0 ratio for succinate oxidase. Active transhydrogenation caused an increase in the apparent Km for Pi in the phosphorylating system from 1.7 to 4.5 mM, and active phosphorylation caused an increase in the apparent Km for NADH in the transhydrogenating system from 21 to 40 μM. It. is concluded that oxidative phosphorylation and the energy-linked transhydrogenase reaction are competing for a common nonphosphorylated high-energy intermediate generated by the respiratory chain. The kinetic evidence presented further suggests that the two systems interact with two different moieties of this high-energy intermediate. [ABSTRACT FROM AUTHOR]

Earlier studies had shown that the enzyme tryptophan pyrrolase which catalyzes the first irreversible step in the conversion of tryptophan to functional nicotinyl derivatives in rat liver, was strongly inhibited by NADPH which may be considered the ultimate distal product in the conversion of tryptophan to derivatives of nicotinamide. As an extension of this work the present study has demonstrated that the administration of nicotinamide in vivo to adrenalectomized animals resulted in a strong suppression of the tryptophan-mediated elevation of tryptophan pyrrolase but had little or no effect on the hormonal induction of this enzyme by corticosteroids. These effects of nicotinamide were in sofar specific as analogs of this compound had little or no effect on the specific tryptophan-mediated elevation of tryptophan pyrrolase. Furthermore, the effect of nicotinamide was shown to be specific for the enzyme, tryptophan pyrrolase, having no effect on the tryptophan-mediated increase in serine dehydratase. By means of anti-tryptophan pyrrolase antibodies prepared in rabbits, the pulse labeling of tryptophan pyrrolasc in vivo by [14C]phenylalanine showed that, in animals pre-fed a 0% protein diet, tryptophan administration resulted in a doubling of the [14C]phenylalanine incorporation into tryptophan pyrrolase. Nico-tinamide administration completely abolished this effect of tryptophan but had little effect on the dramatic stimulation of tryptophan pyrrolase synthesis by hydrocortisone. It is postulated that nicotinamide may exert its inhibitory effect on tryptophan induced synthesis of tryptophan pyrrolase at the level of genetic translation. [ABSTRACT FROM AUTHOR]

Mercury, copper and bismuth compounds react in vivo with insoluble collagen in a way similar to gold thiosulphate. The introduction of artificial cross-links increases the overall stability of the collagen and lowers its solubility. As a result the fibrils acquire a more finely detailed cross-striation pattern as seen under the electron microscope. [ABSTRACT FROM AUTHOR]

The λ-chains of normal pig γG-immunoglobulin were digested with pepsin in 1 M formic acid. Cystine-containing peptides were isolated by gel filtration on Sephadex G-25 and by chromatography on Dowex 50 and Dowex 1. The following structures were found:[this equation cannot be presented in ASCII text] In addition to these peptides obtained in a high yield similar peptides were found but their complete structures were not determined in view of their low yield. The structures found in the λ-chains display a high degree of similarity with corresponding structures known to exist in human Bence­Jones proteins of the L-type. [ABSTRACT FROM AUTHOR]

The complexes of poly A and poly U are interesting models for studying the structure of nucleic acids. In the first part of this paper, the thermal stability of these complexes has been studied as a function of both pH and ionic strength in alkaline solution. The thermal stability is measured by the half transition temperatures, or Tm's. There are four different kinds of transitions: Tm2-1 refers to the dissociation of the two-stranded complex, poly (A + U); Tm2-1 to the dissociation of the three-stranded complex, poly (A + 2U) [4]. Tm8-2 is the temperature of half dissociation of poly (A + 2U) into poly (A + U) [1], and Tm2-3 is the temperature of rearrangement of 2 poly (A + U) into poly (A + 2U) [3] (Fig. 1). It is found that Tm2-1, and Tm8-1 decrease as pH increases above neutrality (Fig. 2 and 3), but that Tm2-8 does not vary (Fig. 3). This effect is readily explained if it is assumed that the ionisation of uracil brings a change in the state of the free poly U, but that the complexes themselves are not modified. In the second part of the paper a simple thermodynamic formulation is given which represents satisfactorily the relationship between the four equilibria, as observed at neutral pH (Fig. 1). A more complex treatment is then presented. It includes quantitation of electrostatic interactions, according to the theory of Kotin [18] and of the free enthalpy term corresponding to the organisation (stacking) of poly A. This leads to an excellent representation of the experimental data (Fig. 5). In alkaline solution, one can calculate the free enthalpy change associated with the ionisation of poly U at the Tm. According to our observations, it is likely that this is the only factor responsible for the decrease of Tm. Therefore, we can write that the sum of the free enthalpy of complex formation in the same conditions, but at pH 7, and of the free enthalpy of ionisation is zero. This leads to a determination of ΔG both as a function of temperature and ionic strength: the variation of ΔG with these factors is shown to be in good agreement with our detailed model. However, the absolute values are different, probably because of uncertainties concerning the data for poly A stacking enthalpy and entropy. Apart from providing evaluation of the thermodynamic parameters for double and triple helix formation, our results lead us to emphasize the importance of the thermodynamic state of the unassociated polynucleotides. This may be particularly relevant when one considers the partially complementary secondary structures of RNA's. [ABSTRACT FROM AUTHOR]

In this paper the thermal stability of poly (A + U) and poly (A + 2U) in acid solution is studied. It is known that at low pH poly A associates with itself to form a double protonated hellcat structure, poly (A + A). The dissociation Tm's of poly (A + U) and poly (A + 2U) do not vary with pH as long as they are higher than TmA [the dissociation temperature of poly (A + A)]. TmA itself varies with the pH (it increases as the pit is lowered) and thus, there are pH values at which TmA equals the dissociation Tm'S of the complexes poly (A + U) and poly (A + 2U). Below such pH's, Tm2-3, Tm3-1, Tm2-1 decrease as the pit becomes more acid. In contrast, Tm3-2 does not exhibit such a variation. The transition corresponding to this last Tm [the partial dissociation of poly (A + 2U) into poly (A + U)] does not change the concentration of ‘free’ poly A. We conclude that the change in thermal stability of the complexes in this region of pH is due only to their competition with poly (A + A) and that poly (A + U) and poly (A + 2U) are not modified. At low ionic strength, the transitions observed change with the pH and, by identifying them by means of two independent methods (continuous variation method, and comparison of spectral variations) we have shown that the stability diagram (temperature versus pH) formally resembles the stability diagram (temperature versus ionic strength) at neutral pH (Fig.3), when pH is decreased and/or ionic strength is increased, poly (A + 2U) is relatively more stable than poly (A + U): The formation of poly (A + A) lowers the stability of both poly (A + U) and poly (A + 2U), but, since the proportion of adenylic residues is 1/3 in this last complex, against 1/2 in the former, poly (A + 2U) is less destabilized, and is more sensitive to the diminution of electrostatic repulsion brought about by the sodium ion concentration. These effects are summarized in a three-dimensional diagram temperature-pH-ionic strength (Fig. 9). Some of the transitions occuring in acidic solution appear to be irreversible, or very slowly reversible. Such is the case of the formation of poly (A + U). We have shown that this effect is due to the structure poly (A + A). When the dissociation temperature (TmA) of this structure is higher than that of poly (A + U), it is necessary to Porto two A — U pairs replacing one A — A pair in order to obtain the free energy decrease. Therefore, such a reaction cannot occur in two steps, and this suggests a third order mechanism. Another example is given by the rearrangement of poly (A + U) into poly (A + 2U) and poly (A + A). This observation leads us to a general rule concerning the possibility of reorganization in a system of polynucleotides where several associations are possible [for instance it answers the question as to why poly G cannot expel poly I from poly (I + C)]. [ABSTRACT FROM AUTHOR]

The enzymatic synthesis of the polyribonucleotide ApUp(ApUp)n by the use of RNA polymerase is described. Snake venom and spleen phosphodiesterase degrade this polymer at a very slow rate compared to poly r(A—U). The dinucleotide resulting from digestion with pancreatic ribonuclease is resistant to spleen phosphodiesterase. [ABSTRACT FROM AUTHOR]

The pH-dependence of the inhibition of cobalt carbonic anhydrase by three different sulfonamides has been measured employing p-nitrophenyl acetate as substrate. The binding of these inhibitors is linked to two ionizing groups. One of these seems to be identical to the group in the enzyme previously shown to control the catalytic activity. The pK of the second group is characteristic for the individual inhibitor and agrees with pK-values obtained by titration of the sulfonamides. Sulfanilamide and the anionic inhibitor, NO3-, appear to compete for one binding site on the enzyme. When the sulfanilamide inhibition of the CO2-hydration activity was studied by the stopped-flow method, some anomalous behavior was observed. From the initial part of the reactions, apparent noncompetitive inhibition was obtained. The rates increased over a short time period, however, to give essentially a competitive pattern. The results can be explained by assuming that the substrate and the inhibitor are competitive, but that equilibration is not immediately achieved due to a comparatively slow rate of dissociation of the inhibitor from the enzyme. The apparent second order rate constant for the formation of the enzyme-sulfanilamide complex has been estimated as 7 × 104 M-1 sec-1 at pH 7.9 and 25°. In the presence of CO2, the reaction is slower to an extent which is compatible with a competition for a common binding site. A simple, though not unique, mechanism for the sulfonamide inhibition of bovine cobalt carbonic anhydrase, accounting for both rate and equilibrium data, implies the formation of the enzyme-inhibitor complex through a direct reaction of the ionized sulfonamide with the inactive, acid form of the enzyme. [ABSTRACT FROM AUTHOR]

When [1-14C]palmitic acid was incubated with young pea leaves or solutions of their acetone-dried powders, 2-hydroxy [1-14C]palmitic acid accumulated. This metabolite has been isolated as its methyl ester. When diluted with unlabelled synthetic methyl D-2-hydroxypalmitate to known specific activity, and recrystallised, the crystals and mother liquor residues retained the same specific activity. When diluted with unlabelled synthetic methyl L-2-hydroxypalmitate to known specific activity and recrystallised, the crystals were almost non-radioactive. The radioactive metabolite therefore has the D-configuration. Unlabelled 2-hydroxypalmitic acid isolated from pea leaves was shown to have the D-configuration by direct measurement. [ABSTRACT FROM AUTHOR]

The lysyl ribonucleic acid synthetase from Escherichia coli has been purified to apparent homogeneity as judged from polyacrylamide gel electrophoresis and sedimentation velocity centrifugation. A molecular weight of 100,000 has been calculated for the enzyme. [ABSTRACT FROM AUTHOR]

A study of threonine deaminase from Saccharomyces cerevisiae has been undertaken. The enzyme has been purified 50-fold, with a low resolution of pyridoxal phosphate coenzyme. The purified enzyme is very unstable under all conditions tried so far. Properties have been studied with crude extracts. Optimum pH has been found to be 8.2. Isoleucine has complex effects on the enzyme activity. At a concentration of 0.1 mM, it is able to stimulate the enzyme. At higher concentrations, isoleucine inhibits. The Ki values increase with increasing pH, but 100% inhibition can be reached at all ranges of pH studied. Affinity of the enzyme for its substrate is even more strongly modified by pH. a) Substrate cooperativity, nil at pH 7, intermediate at pH 8, seems to be maximum at pH 9. At any pH, the substrate cooperativity can be suppressed by addition of L-valine. In addition, rather low concentrations of isoleucine also act partly as valine does. b) Apparent Km values, measured in the presence of valine, vary with pH in the opposite direction to Ki values. Hill's coefficients for substrate and inhibitor reflect these findings. For threonine alone, they vary from 1.2 at pH 7 to 3 at pH 9, In the presence of valine, they remain close to 1, at all pH. Isoleucine, 0.1 mM, produces an incomplete effect compared to valine. For isoleucine inhibition, the cooperativity coefficients remain close to 3 at all pH. The molecular weight of the enzyme has been determined to be roughly 1.2 × 105, at, pH 6.8 and at pH 8.7. Thermal inactivation is unimodal and fails to show desensitization of the enzyme in any of the conditions tried. The implications of these results on the structure of the enzyme have been discussed. [ABSTRACT FROM AUTHOR]

Acetohydroxyacid synthetase activity has been characterized, independently of the acetoin-forming system in Saccharomyces cerevisiae by a differential colorimetric assay. Maximum pH and Km values have been established for both enzymes with pyruvate as the substrate. Acetolactate formation by acetohydroxyacid synthetase has been shown directly by the separation of dinitrophenylosazones by thin-layer chromatography. Evidence for the biosynthetic role of the activity measured resides in its presence in wild-type strains, in its absence in the is2 class of mutants (isoleucine-valine auxotrophs) and in its reappearance in prototrophic revertants of these mutants. [ABSTRACT FROM AUTHOR]

Regulation of activity of acetohydroxyacid synthetase has been studied. L-Valine, one of the end-products of the combined pathway, is the only amino acid to inhibit strongly the enzyme (Ki = 5 × 10-5 M). Sensitivity to the inhibitor is rapidly lost by heating at 35°. The desensitized enzyme is stable at this temperature. In crude extracts, the enzyme is always partly desensitized, but 100% inhibition by valine is reached when the enzyme is assayed in situ, in benzene permeabilized cells. In addition, valine inhibition is more strictly dependent upon pH than activity itself. Kinetic studies of affinity for the substrate or inhibitor show no cooperative effects and valine inhibition seems truly competitive with pyruvate. Specific feedback inhibition by valine constitutes an additional proof that the acetohydroxyacid synthetase activity characterized hi studies reported in the preceding paper, is operative for valine biosynthesis. [ABSTRACT FROM AUTHOR]

The distribution of the functional group of the pH indicator bromthymol blue across the osmotic barrier component (or M phase) of the cristae membrane separating the inner from the outer aqueous phase of rat liver mitochondria has been measured by three partially independent methods. In mitochondria in State 5, under the usual conditions, only about one third of the total bromthymol blue reacts in the inner phase. In State 3, in presence of a high concentration of valinomycin in a KCl medium, approximately the same proportion of the bromthymol blue reacts in the inner phase as in State 5; but in State 4, practically all the bromthymol blue is expelled across the M phase and registers the pH of the outer phase. The bromthymol blue technique is incompetent to measure the pH of the inner phase of mitochondria in State 4; but a method is described that will permit the approximate quantitative measurement of the intramitochondria] pH from the light extinction of bromthymol blue in State 5 (or in State 3 in presence of valinomycin) provided that the distribution of the indicator and the internal and external buffering powers of the mitochondrial suspension are known. The expulsion of the bromthymol blue from the mitochondria in State 4 is attributable to the development of an electric potential across the M phase. [ABSTRACT FROM AUTHOR]

The uptake of succinate by mitochondria and the rate of respiration in the presence of succinate plus rotenone have been analysed. In conditions in which Vmax is the same, the apparent Km for succinate is lower when respiration is activated by ion uptake than it is in the active state. The highest value is obtained in the presence of 2,4-dinitrophenol. Succinate uptake is maximum during ion uptake, is lower in the active state and lowest in the uncoupled state. With increasing concentrations of uncoupler, a decrease in the intramitochondrial concentration of succinate is observed parallel to the decrease in respiration. The inhibition of respiration caused by gramicidin under certain conditions is accompanied by a decrease in the intramitochondrial concentration of succinate. Citrate and other substrate anions are shown to be competitive with succinate with respect to both the oxidation and the uptake of succinate. The inhibition of respiration by citrate is less pronounced in particles than in mitochondria. It is concluded that the rate of respiration with succinate plus rotenone as substrate is controlled by succinate uptake. [ABSTRACT FROM AUTHOR]

Isolated liver cells were treated with snake venom phospholipase A to obtain information on the relative importance of different phospholipids in the plasma membrane to its structural stability. The hydrolysis of phosphatidylethanolamine and lecithin by phospholipase A from Naia naia produced a change in the permeability of the liver cell plasma membrane which was arbitrarily assessed by measuring the amount of the intracellular enzyme ghitamic oxaloacetic transaminase released into the incubation medium. There was only a small leakage of glutamic oxaloacetic transaminase from the cell until 25–35% of the phosphatidylethanolamine had been hydrolysed. Further hydrolysis caused a rapid increase in the release of the enzyme. Very little lecithin was hydrolysed by the same enzyme concentrations. After treatment with phospholipase A the cells were stained with either acridine orange, eosin or nigrosin and examined by light, phase contrast and fluorescent microscopy. Their gross morphology appeared to be unaltered by the hydrolysis of up to 50–60% of the cell phosphatidylethanolamine. The results suggest that phosphatidylethanolamine is relatively more important than lecithin in maintaining, in terms of permeability to a protein molecule, the structural integrity of the liver cell plasma membrane. [ABSTRACT FROM AUTHOR]

Some properties of the phosphoinositide inositolphosphohydrolase of guinea-pig intestinal mucosa supernatant fraction are described. The enzyme has an absolute requirement for metal ions, the most efficient activator being Ca2+. The pH optimum is 5.3 in acetate buffer and 5.9 in maleate; activity is higher in the latter. The effects of detergents and various reagents are described. Corn phosphatidyl inositol is hydrolysed faster than brain phosphatidyl inositol; the fatty acid compositions of these lipids have been compared. The enzyme is remarkably specific for the phosphoinositides. The composition of the enzyme preparation has been studied. The enzyme appears to be widely distributed in animal tissues. [ABSTRACT FROM AUTHOR]

The paper describes a simple kinetic model of an open monosubstrate enzyme reaction with substrate inhibition and product activation. A comparison between the model and the phosphofructokinase reaction shows a close resemblance between their dynamical properties. This makes it possible to explain qualitatively most experimental data on single-frequency oscillations in glycolysis. A mathematical analysis of the model has shown the following. In the model, at a definite relationship between the parameters, self-oscillations arise. The condition of self-excitation is satisfied more readily with a lower source rate, larger product sink rate constants, lower product-enzyme affinity and higher enzyme activity. Self-oscillations exist only in a certain range of values of the parameter determining the degree of substrate inhibition. This range increases with decreasing source rate. Too strong or, conversely, too weak substrate inhibition leads to damped oscillations. The period of self-oscillations depends on the degree of substrate inhibition, the source rate, the sink rate constant, the enzyme activity, the affinity of the substrate and the product for the enzyme; it decreases with an increase in these values. With an increase in the relative sink rate constant the steady state amplitude of self-oscillations initially increases until a definite maximum is reached and then drops to zero. A self-oscillatory state in the phosphofructokinase reaction exists only when the maximum rate of this reaction is essentially higher than the source rate, and lower than the maximum rate of the reactions controlling the sink of the products. An experimental investigation of self-oscillations in the phosphofructokinase reaction may be considerably simplified by using a reconstituted system consisting of a small number of reactions with an irreversible sink of the products and artificial substrate supply. In this case the above relationship (section 6) should hold. [ABSTRACT FROM AUTHOR]

Rats were maintained for several days on a protein-free carbohydrate containing diet which was changed to a protein-rich diet approximately 14–17 h before decapitation. The ability to incorporate amino acids into protein was enhanced after the nutritional alteration, and this enhancement was unaffected by pretreatment in vivo with the antibiotics actinomycin D and chloramphenicol. Molecular sieving of microsomes and cell sap through Sephadex G-25 removed components or factors from the fractions. Under these conditions and in the presence of optimal amounts of cell sap, GTP and an amino acid mixture stimulated the incorporating activity of the preparations in a manner such that the originally observed differences between the two types of rats became less. These results were interpreted to suggest that the in vivo predetermined capacity of protein synthesis could be altered. These alterations implied an increase rather than a decrease of the originally in vitro observed amino acid incorporating activities. [ABSTRACT FROM AUTHOR]

L-6-Hydroxynicotine oxidase, an inducible enzyme of nicotine catabolism in Arthrobacter oxidans, was obtained as a homogeneous protein, as judged by electrophoresis and ultracentrifuge studies; it forms hexagonal crystals in ammonium sulfate solution. Its molecular weight was determined to be 93,000. It contains two moles of FAD per mole of enzyme. In the presence of guanidine hydrochloride, enzymatically inactive components of molecular weight 47,000 were formed which appear to represent the subunits of the protein. No evidence for a participation of metal ions in the electron transport of the enzyme was obtained. Although Hg2+ and p-chloromercuriphenylsulfonate strongly affect activity, N-ethylmaleimide and 5,5′-dithiobis-(2-nitrobenzoate) do not inhibit the enzyme. The latter compound reacts with one thiol group only after exposure of the enzyme to 7.5 M urea. [ABSTRACT FROM AUTHOR]

A simple procedure for the extraction and purification of monoamine oxidase from pig brain mitochondria is described. The enzyme purified in this way appears to be homogeneous by cellulose acetate electrophoresis and the molecular weight was estimated to be approximately 102,000 by gel-filtration. The purified enzyme is inhibited by iproniazid, chelating agents and a sulphydryl reagent. The Km value for tyramine has been determined as has its Ki value for high substrate inhibition. [ABSTRACT FROM AUTHOR]

Poly U-directed stimulation of preincubated calf lens ribosomes revealed that these particles from a mammalian source have a phenylalanine incorporation ability comparable to the Escherichia coli system. A search was made for optimal conditions. After variation of the magnesium concentration during preincubation two levels of maximal incorporation were observed. The region of highest relative stimulation did not coincide with these maxima. Preincubation under conditions of optimal amino acid incorporation yields 80S monomers and to a lesser extent 120S particles. No smaller units or breakdown products were observed in the analytical ultracentrifuge. Such particles could be demonstrated after prolonged dialysis of either polysomes or 80S monomers against 0.1 mM MgC12. Their sedimentation coefficients were 50S and 30S respectively but not 60 and 40 S. [ABSTRACT FROM AUTHOR]

Nitration of pancreatic trypsin inhibitor with tetranitromethane affords a mono- and a disubstituted derivative which was shown by sequential analysis to contain nitrotyrosine in position 10 and in positions 10 and 21, respectively. Both retain full trypsin inhibitor activity. Measurements of optical rotatory dispersion in the 230 nm region indicate that the secondary structure of the protein is unaffected by nitration. The nitrated derivatives exhibit a conformation-dependent side-chain Cotton effect in the region of nitroaromatic absorption. No such Cotton effect is shown by a nitrotyrosine-containing pentadecapeptide isolated from the tryptic digest from the nitrated and oxidized inhibitor. [ABSTRACT FROM AUTHOR]

From Salmonella R lipopolysaccharides of the chemotypes Rc, Rd1, and Rd2 oligosaccharides were prepared and their structure studied by the following methods: quantitative sugar analyses; periodate oxidation followed by borohydride reduction in order to transform the L-glycero-D-mannose residues into D-mannose residues; methylation of original and transformed oligosaccharides, methanolysis and gas chromatographic identification of the methylated heptoses or mannoses; the action of glycosidases; serological tests. The results of the study are in agreement with the formulae given below for the different oligosaccharides. The anomeric form of the glucose unit linked to heptose, as well as that part of the oligosaccharides which contains the 2-keto-3-deoxy-octonate residues [KDO] remain uncertain. Chemotypes Rd2: IIeptose pα 1 → [KDO]x; Rd1: Heptose pα 1 → 3 Heptose pα 1 → [KDO]x; Rc: Glucose 1 → 3 Heptose pα 1 → 3 Heptose pα 1 → [KDO]x. [ABSTRACT FROM AUTHOR]