bluuebunny/crossref_metadata_2025 huggingface dataset:
The redox state of the nicotinamide–adenine dinucleotides in rat liver homogenates
Authors: Krebs H. A. , Gascoyne T.
Published: August 2015

1. The redox state of the NAD couple of rat liver mitochondria, as measured by the [β-hydroxybutyrate]/[acetoacetate] ratio, rapidly changed in the direction of oxidation during the preparation of homogenates in a saline medium. The value of the [β-hydroxybutyrate]/[acetoacetate] ratio fell from 2·3 to 0·15 in 10min. EDTA diminished the fall and succinate prevented it. 2. The redox state of the rat liver cytoplasm, as measured by the [lactate]/[pyruvate] ratio, changed slightly in the direction of reduction during the preparation of homogenate. This was prevented by succinate. 3. In unsupplemented homogenates the differences in the redox states of mitochondria and cytoplasm decreased. Succinate and EDTA together maintained the differences within the physiological range. A measure of the ability of the mitochondria to maintain different redox states in mitochondria and cytoplasm is the value of the expression [lactate][acetoacetate]/[pyruvate][β-hydroxybutyrate]. If there are no differences in the redox states of the NAD in the two cell compartments the value of the expression is 444 at 37°. The value in the intact rat liver is between 4·7 and 21. 4. α-Oxoglutarate or glutamate were still more effective than succinate in maintaining high [β-hydroxybutyrate]/[acetoacetate] ratios in the homogenates because these substrates supply a reducing agent of NAD+ and, through succinate, an inhibitor of the oxidation of NADH. 5. When supplemented with α-oxoglutarate and EDTA, homogenates readily adjust the redox state of the β-hydroxybutyrate dehydrogenase system after it has been upset by the addition of either acetoacetate or β-hydroxybutyrate. 6. Amytal and rotenone raised the value of the [β-hydroxybutyrate]/[acetoacetate] ratio. This is taken to indicate that the reduction of acetoacetate in the homogenates was not an energy-linked process. 7. 2,4-Dinitrophenol shifted the [β-hydroxybutyrate]/[acetoacetate] ratio in the presence of succinate in favour of oxidation because it inhibited the oxidation of succinate and accelerated the oxidation of NADH. 8. Rotenone increased the rate of ketone-body formation of liver homogenates, though it decreased the rate of oxygen uptake.
The stereospecificity of α-chymotrypsin
Authors: Ingles D. W. , Knowles J. R.
Published: August 2015

1. The rates of deacylation of acyl-α-chymotrypsins in which the hydrogen-bonding capacity of the acylamino group of the substrate has been systematically removed were measured. 2. The ratio of deacylation rates of l- and d-acyl-enzymes is found to depend largely on the existence in the substrate of an amido –NH– group. 3. The data presented agree with the postulate that the stereospecificity of α-chymotrypsin is exercised in catalytic rather than binding steps, and that the active site of the enzyme presents three loci to the substrate: the site containing the catalytic functionalities (including serine-195), the hydrophobic area for amino acid side-chain binding, and a hydrogen-bond acceptor site for acylamino group binding. 4. It is noted that, though the hydrogen-bonding site is crucial for the stereospecificity, the free energy of binding of substrates and inhibitors is dominated by the hydrophobic interaction. 5. It is tentatively proposed that α-chymotrypsin selects a high-energy conformation of the substrate when the latter binds at the enzyme's active site.
Two forms of aspartate aminotransferase in rat liver and kidney mitochondria
Authors: Bhargava M. M. , Sreenivasan A.
Published: August 2015

1. Butan-1-ol solubilizes that portion of rat liver mitochondrial aspartate aminotransferase (EC 2.6.1.1) that cannot be solubilized by ultrasonics and other treatments. 2. A difference in electrophoretic mobilities, chromatographic behaviour and solubility characteristics between the enzymes solubilized by ultrasonic treatment and by butan-1-ol was observed, suggesting the occurrence of two forms of this enzyme in rat liver mitochondria. 3. Half the aspartate aminotransferase activity of rat kidney homogenate was present in a high-speed supernatant fraction, the remainder being in the mitochondria. 4. A considerable increase in aspartate aminotransferase activity was observed when kidney mitochondrial suspensions were treated with ultrasonics or detergents. 5. All the activity after maximum activation was recoverable in the supernatant after centrifugation at 105000g for 1hr. 6. The electrophoretic mobility of the kidney mitochondrial enzyme was cathodic and that of the supernatant enzyme anodic. 7. Cortisone administration increased the activities of both mitochondrial and supernatant aspartate aminotransferases of liver, but only that of the supernatant enzyme of kidney.
Studies on the role of polyamines associated with the ribosomes from Bacillus stearothermophilus
Authors: Stevens L. , Morrison Marcelle R.
Published: August 2015

1. Spermine and spermidine were the main polyamines detectable in Bacillus stearothermophilus. 2. When grown at 65° B. stearothermophilus contained lower concentrations of polyamines per mg. of RNA than when grown at 45° or at 55°. 3. Ribosomes isolated from B. stearothermophilus in 0·01m-tris–hydrochloric acid buffer (pH7·4)–0·01m-magnesium chloride contained sufficient polyamines to neutralize between 4% and 9% of their RNA phosphorus. 4. Removal of polyamines from the ribosomes by dialysis against m-potassium chloride did not appreciably alter the hypochromicity or thermal denaturation profiles of the ribosomes when measured in 0·01m-tris–hydrochloric acid buffer (pH7·4)–0·01m-magnesium chloride, though it did cause a loss of ribosome particles sedimenting at greater than 78s. 5. When ribosomes were dialysed against acridine orange solutions acridine orange bound to the ribosomes and did not displace spermine, but when a mixture of ribosomal RNA and spermine was dialysed against acridine orange the acridine orange displaced the spermine. It is concluded that polyamines in the ribosomes are less accessible for displacement by acridine orange than when polyamines are bound to ribosomal RNA.
Citrate-cleavage enzyme, ‘malic’ enzyme and certain dehydrogenases in embryonic and growing chicks
Authors: Goodridge Alan G.
Published: August 2015

The activities of several enzymes possibly implicated in lipogenesis were measured in the soluble fraction of homogenates of liver and adipose tissue of embryonic and growing chicks. The activities of adipose-tissue enzymes showed little or no change. The activities of hepatic hexose monophosphate-shunt dehydrogenases, malate dehydrogenase, 3-phosphoglyceraldehyde dehydrogenase and NAD-linked α-glycerophosphate dehydrogenase also showed little or no change. Isocitrate dehydrogenase activity in liver rose to a peak on the day of hatching and fell to half the peak value during the next 12 days, where it remained to 26 days after hatching. The activities of ‘malic’ enzyme and citrate-cleavage enzyme showed very low stable values in embryonic liver and remarkable rises during the early part of the post-hatching period. An 85-fold increase in the activity of ‘malic’ enzyme activity was completed in 7 days and a 15-fold increase in that of citrate-cleavage enzyme in 5 days. The activities then attained were maintained up to 26 days after hatching. 2. The increases in the activities of hepatic citrate-cleavage enzyme and ‘malic’ enzyme occurred simultaneously with a marked increase in lipogenesis, suggesting a relationship of these enzymes to lipogenesis in chick liver. By contrast, activity of the hexose monophosphate-shunt dehydrogenases does not appear to be thus associated.
Lifetime of messenger ribonucleic acid for penicillinase synthesis in several strains of bacilli
Authors: Yudkin M D
Published: August 2015

1. Previous studies of penicillinase synthesis in Bacillus licheniformis showed that enzyme synthesis after the addition of actinomycin continues for far longer in the constitutive mutant 749/C than in the parental inducible strain (Yudkin, 1966). This result was interpreted as indicating a difference in the lifetime of specific messenger RNA in the two strains. Other bacilli have now been examined in an attempt to see whether this difference is general. 2. There was no difference in the lifetime of messenger RNA for penicillinase synthesis between an inducible and a constitutive strain of Bacillus cereus. 3. Three freshly isolated constitutive mutants of B. licheniformis also had short-lived messenger RNA, like their inducible parent. 4. A reinvestigation of mutant 749/C confirmed the original finding that, on treatment with actinomycin, it continued to synthesize penicillinase far longer than did its parent. 5. An inducible revertant of mutant 749/C was indistinguishable from the original inducible strain, and appeared to have lost both constitutivity and long-lived messenger RNA in the back mutation.
Properties of renin substrate in rabbit plasma with a note on its assay
Authors: Ryan J W , McKenzie J. K.
Published: August 2015

1. Rabbit plasma enzymes that degrade angiotensin I are inhibited completely by the combination of 2,3-dimercaptopropan-1-ol (10mm), EDTA (10mm) and chlorhexidine gluconate (0·005%, w/v). These compounds do not modify the reaction of renin with renin substrate and are termed the selective inhibitors. 2. The renin substrate concentration of plasma can be measured as angiotensin I content by incubating plasma plus the selective inhibitors with renin for a time sufficient to allow complete utilization of renin substrate. 3. This reaction obeys first-order kinetics to substrate concentrations of at least 1000ng. of angiotensin I content/ml. In general, the renin substrate concentrations of normal rabbit plasmas are less than 1000ng. of angiotensin I content/ml. Thus the time required for the complete release of angiotensin I from normal plasma is inversely related to renin activity and is independent of renin substrate concentration. 4. A method for the assay of renin substrate, taking these reaction kinetics into account, is presented.
Nucleic acid and protein synthesis associated with the induction of nitrate reductase activity in radish cotyledons
Authors: Ingle J.
Published: August 2015

1. RNA and protein synthesis was studied during the incubation of excised radish cotyledons in nitrate, conditions that induced nitrate reductase activity in the tissue. 2. Synthesis of total RNA and protein, as measured by the incorporation of radioactive precursor, was significantly stimulated in the presence of nitrate (compared with chloride control), but was decreased in the presence of ammonium nitrate, which induced higher enzyme activity. 3. Synthesis of RNA and protein was required for induction of enzyme activity, as determined by using the inhibitors actinomycin D, puromycin and cycloheximide. 4. On the basis of 5-fluorouracil inhibition, the synthesis of only DNA-like RNA was required for induction, but no differences, either quantitative or qualitative, were observed in DNA-like RNA synthesis in the presence or absence of induction. 5. A 100-fold purification of the nitrate reductase activity showed no increase in nitrate reductase protein, nor any increased incorporation of radioactive precursor into nitrate reductase protein in the induced versus the control system. Such results suggested that the protein synthesis required for induction may be for a protein other than nitrate reductase.
Purification of the alliin lyase of garlic, Allium sativum L.
Authors: Mazelis Mendel , Crews Laurie
Published: August 2015

1. Alliin lyase (EC 4.4.1.4) was purified up to sevenfold from garlic-bulb homogenates. The enzyme was unstable to storage at −10°, particularly in dilute concentrations, but the addition of glycerol (final concentration 10%, v/v) stabilized the activity completely for at least 30 days. 2. The purified enzyme had an optimum pH for activity at 6·5. The addition of pyridoxal phosphate stimulated the reaction rate and the stimulation became more marked as the purification proceeded. 3. Hydroxylamine (10μm) and cysteine (0·5mm) inhibited the enzyme activity by more than 80%. Spectral studies indicated that cysteine reacted with pyridoxal phosphate bound to the protein. 4. The Km values for S-methyl-, S-ethyl-, S-propyl-, S-butyl- and S-allyl-l-cysteine sulphoxides were determined. With S-allyl-l-cysteine sulphoxide the Km was 6mm and the Vmax. was greater than those with the other substrates tested. 5. The thioether analogues of the substrates were competitive inhibitors for the lyase reaction. The Ki decreased with increasing chain length of the alkyl substituent. With S-ethyl-l-cysteine sulphoxide as substrate the Ki was 33, 8 and 5mm respectively for S-methyl-, S-ethyl- and S-propyl-l-cysteine. 6. The addition of EDTA or Mg2+, Mn2+, Co2+ or Fe2+ stimulated the reaction rate. Other bivalent cations either had no effect or gave a strong inhibition. In the presence of EDTA no further increase of activity was observed with added Mg2+.
Incorporation of 5-methylorcylaldehyde and methionine into the acetogenin (polyketide) gliorosein in Gliocladium roseum I.M.I. 93065
Authors: Steward M. W. , Packter N. M.
Published: August 2015

1. methyl−14C-labelled 1,3-dihydroxy-4,5-dimethylbenzene, 5-methylorcylaldehyde and 5-methylorsellinic acid were synthesized from orcinol and sodium [14C]cyanide and tested for activity as precursors of gliorosein. ring−14C-labelled orcylaldehyde was also prepared. 5[14C]-Methylorcylaldehyde was incorporated into gliorosein (36% conversion); all the radioactivity was located in the C-methyl groups. 5-Methylorsellinic acid was decarboxylated by Gliocladium roseum and the resulting phenol was secreted into the medium. 2. The formation of an enzyme-bound derivative of 5-methylorsellinic acid as the first aromatic compound in the biosynthesis of gliorosein is suggested to explain these results. 3. ring−14C-labelled 3,4-dihydroxy-6-methyltoluquinone was also effectively incorporated into gliorosein and related products (20% conversion). 4. Sodium [14C]formate and [Me−14C]-methionine were incorporated into gliorosein and related products (15·4 and 22·2% conversion respectively). Isolation and estimation of the radioactivity in the O-methyl and C-methyl groups in the 14C-labelled gliorosein thus formed showed an appreciable difference in the specific activities of the two types of methyl group (14 and 15% respectively). The results in the doubly-labelled methionine experiment indicate that the C-methyl group arises in the same manner as that in ergosterol; one of the original hydrogen atoms of the methyl group is lost. This confirms that C-methylation occurs at an ethylenic group at the aliphatic level. 5. The sequence of reactions at the aromatic level leading to the formation of gliorosein is proposed as 5-methylorsellinyl-enzyme→3-hydroxy-5-methylorsellinyl-enzyme→3,4-dihydroxy-6-methyltoluquinol→3,4-dimethoxy-6-methyltoluquinol→gliorosein.
Amino acid sequences around the cystine residues in horse growth hormone
Authors: Oliver Louise , Hartree Anne Stockell
Published: August 2015

The cystine-containing peptides of horse growth hormone were isolated and their amino acid sequences determined. Four unique half-cystine residues occur in two peptides, one containing 11 and the other, at the C-terminus of the protein, 15 amino acids. These sequences are compared with published data on growth hormones from other species.
Glucose metabolism in the mucosa of the small intestine. A study of hexokinase activity
Authors: Srivastava L M , Shakespeare P. , Hübscher G.
Published: August 2015

1. The intracellular distribution of hexokinase activity was studied in the mucosa of rat and guinea-pig small intestine. In the rat 60% and in the guinea pig 45% of the hexokinase activity of homogenates were recovered in a total particulate fraction that contained only 5–17% of the homogenate activity of hexose phosphate isomerase, pyruvate kinase, lactate dehydrogenase and overall glycolysis (formation of lactate from glucose). 2. Fractionation of homogenates from guineapig small intestine showed that the particulate hexokinase activity was chiefly in the mitochondrial fraction with a small proportion in the nuclei plus brush-border fraction. 3. After chromatography of the particle-free supernatants on DEAE-cellulose, hexokinase types I and II were determined quantitatively. No evidence was obtained for the presence of hexokinase type III or glucokinase. In the preparations from guinea pigs, hexokinase types I and II amounted to 69% and 31% respectively of the eluted activity; the corresponding values for preparations from rats were 5·8% and 94·2%. 4. Total and specific hexokinase activities decreased significantly in homogenates and particle-free supernatants prepared from the intestinal mucosa of rats starved for 36hr. and increased again after re-feeding. The decrease in hexokinase activity in the particle-free supernatant from starved rats was chiefly due to a decrease in the type II enzyme.
The presence of glycopeptide material in cultured mouse mast-cell tumours
Authors: Thomas D. B.
Published: August 2015

1. Mouse mast-cell tumours P815 Y and HC were shown to contain glycoprotein material composed of glucosamine, galactosamine, sialic acid, galactose and mannose. 2. The major amino acids released after acid hydrolysis of Pronase-treated digests of the glycoprotein are aspartic acid, glutamic acid, serine, threonine, proline, glycine and alanine. The Pronase-digested material is not degraded in alkaline solution. 3. On incubation of mast cells with [35S]sulphate, heparin is the major radioactive product. However, [1−14C]glucosamine and d-[14C]glucose are incorporated largely into the glycoprotein. 4. The fate of [35S]sulphate-labelled and [1−14C]glucosamine-labelled material was studied. In each case high-molecular-weight radioactive material is released from the cells into the culture medium. The t½ of [35S]sulphate-labelled material in cells is 70hr. and that of [1−14C]-glucosamine-labelled material in cells is 40hr. 5. About 60% of the [35S]sulphate-labelled material is present in the mitochondrial and granular fraction. [1−14C]-Glucosamine-labelled material is present in both microsomal and mitochondrial and granular fractions, [14C]sialic acid being concentrated in the microsomal fraction.
The preparation and properties of pyruvate kinase attached to porous sheets, and the operation of a two-enzyme continuous-feed reactor
Authors: Wilson R. J. H. , Kay G. , Lilly M. D.
Published: August 2015

1. The covalent attachment of pyruvate kinase to filter-paper disks and some kinetic properties of the resultant enzymically active porous sheets are described. 2. A two-enzyme reactor was constructed by using water-insoluble sheets of lactate dehydrogenease in conjunction with the above-mentioned sheets possessing pyruvate kinase activity.
The lactose synthetase particles of lactating bovine mammary gland. Preparation of particles with intact lactose synthetase
Authors: Coffey R. G. , Reithel F. J.
Published: August 2015

1. The particulate form of lactating bovine mammary lactose synthetase activity is shown to be more highly organized than previously reported. 2. A novel method of shattering frozen mammary tissue with effective cell disruption is described. 3. The apparent subcellular distribution of lactose synthetase was shown to reflect the method of homogenization. 4. After mild homogenization particles associated with a high content of intact lactose synthetase activity sedimented in the lysosome size range between 5×104 and 3×105g-min. 5. Lactose synthetase was dissociated and solubilized by VirTis homogenization and ultrasonic treatment. The activities and behaviour of UDP-galactose hydrolase, succinate dehydrogenase, β-glucuronidase and phosphodiesterase I were compared. 6. Inhibition of UDP-galactose hydrolase by UTP and α-lactalbumin was observed.
16s and 23s components in the ribosomal ribonucleic acid of Rhodopseudomonas spheroides
Authors: Szilágyi J. F.
Published: August 2015

1. Ribosomal RNA was extracted from lysates of Rhodopseudomonas spheroides without prior isolation of ribosomes. 2. The composition of this RNA was investigated by using gradient centrifugation, showing that the proportion present as 23s component depended on the method of extraction. 3. The highest proportion of 23s component was found when cells were disrupted by ultrasonic treatment in the presence of ribonuclease inhibitors. 4. The results indicated that a ribonuclease is active in the cell lysate; this could account for the previous report (Lessie, 1965) that ribosomes of Rhodopseudomonas spheroides do not contain a 23s component.
The hybridization capacity of ribonucleic acid produced during hormone action
Authors: Wyatt G. R. , Tata J. R.
Published: August 2015

1. Measurements of hybridization with homologous DNA were used to assess the nature of the RNA synthesized during hormone action in several systems. 2. When increasing amounts of pulse-labelled rat liver nuclear RNA were annealed with constant amounts of DNA, saturation was not achieved even with RNA/DNA ratios of up to 180:1, which is taken to indicate great diversity in the species of labelled RNA molecules. In the converse experiment, when the DNA/RNA ratio was varied up to 20:1, a plateau of hybridization was observed, and the non-hybridizing RNA is believed to represent chiefly ribosomal and ribosomal precursor species. 3. In the livers of hypophysectomized and thyroidectomized rats treated with growth hormone and tri-iodothyronine, and in whole Xenopus larvae during induced metamorphosis, the synthesis of non-hybridizing RNA was consistently stimulated more than that of hybridizing RNA. This is interpreted as reflecting preferential synthesis of ribosomal RNA in response to these hormones.
Studies on medium-chain fatty acyl-coenzyme A synthetase. Purification and properties
Authors: Bar-Tana J. , Rose G. , Shapiro B.
Published: August 2015

1. Medium-chain fatty acyl-CoA synthetase (EC 6.2.1.2) was isolated by the method of Mahler, Wakil & Bock (1953) and the enzyme activity determined by the disappearance of CoA in the presence either of butyrate and ATP or of butyryl-AMP, as well as by ATP formation from butyryl-AMP and PPi. 2. Preincubation of the enzyme with CoA and ATP alone or together, followed by the removal of these substrates by gel filtration, caused a marked inhibition of ATP formation, contrary to results previously obtained with palmitoyl-CoA synthetase. 3. The effect of ATP on butyryl-AMP-dependent CoA disappearance was inconsistent. Low concentrations of ATP (0·1–0·5mm) always caused inhibition, whereas higher concentrations (5–10mm) activated in some enzyme preparations and inhibited in others. 4. This inconsistency was shown to be due to the presence of two enzyme fractions. Both fractions had similar activities when assayed by the butyryl-AMP- or butyrate-plus-ATP-dependent CoA disappearance. However, fraction I was activated by ATP as measured by butyryl-AMP-dependent CoA disappearance whereas fraction II was inhibited by it. Fraction I also catalysed ATP formation from butyryl-AMP and PPi whereas fraction II was lacking in such activity. 5. The relationship of these observations with respect to other known mechanisms of fatty acid-activating systems is discussed.
Preparation, properties and metabolism of retro-3-dehydroretinyl acetate
Authors: Mallia A. Krishna , Lakshmanan M. R. , John K. V. , Cama H. R.
Published: August 2015

1. Treatment of 3-dehydroretinyl acetate with aqueous hydrobromic acid resulted in the formation of retro-3-dehydroretinyl acetate, which, on alkaline hydrolysis, gave the corresponding alcohol. 2. retro-3-Dehydroretinyl acetate was isomerized to 3-dehydrovitamin A when fed to vitamin A-deficient rats. 3. When retro-3-dehydroretinyl acetate was administered orally, it was hydrolysed to retro-3-dehydroretinol in the rat intestine, isomerized to 3-dehydroretinol and esterified before being transported to the liver for storage. 4. When administered intraperitoneally, both 3-dehydrovitamin A and retro-3-dehydrovitamin A were accumulated in liver and other tissues, whereas after enterectomy 3-dehydrovitamin A was not detected anywhere in the body. 5. The small intestine was shown to be the major site of conversion of retro-3-dehydrovitamin A into 3-dehydrovitamin A. 6. The extent of conversion of retro-3-dehydroretinyl acetate into 3-dehydrovitamin A was much smaller than that of the conversion of retro-retinyl acetate into vitamin A. 7. The biological potency of retro-3-dehydroretinyl acetate, determined by the rat-growth assay, was 2·6% of that all-trans-retinyl acetate, when given orally.
Exo-β-glucanases in yeast
Authors: Abd-El-Al Ahmed T. H. , Phaff H. J.
Published: August 2015

1. A number of yeast species were examined for the presence of β-glucanases. Extracts obtained by cell disruption of Saccharomyces cerevisiae, Fabospora fragilis and Hansenula anomala hydrolysed laminarin and pustulan with the production of glucose. Enzymic activities were also detected in the culture fluids of F. fragilis and H. anomala grown aerobically in buffered mineral medium with glucose as the carbon source. 2. F. fragilis and H. anomala possessed approximately sevenfold higher β-(1→3)-glucanase activity than S. cerevisiae. 3. Intracellular exo-β-glucanase from baker's yeast was purified 344-fold from the dialysed cell extract. 4. Exo-β-glucanase from F. fragilis was purified 114-fold from the dialysed culture fluid and 423-fold from the dialysed intracellular extract. The purified extracellular and intracellular enzymes had similar properties and essentially the same specific activity, 79 enzyme units/mg. of protein. 5. Extracellular exo-β-glucanase of H. anomala was purified 600-fold. 6. The optimum pH of the enzymes from F. fragilis, S. cerevisiae and H. anomala was 5·5 in each case. Chromatographic evidence indicated that the three enzymes remove glucosyl units sequentially from laminarin as well as pustulan. 7. The ratio of activities towards laminarin and pustulan remained constant during purification of the exo-β-glucanase obtained from the three species, suggesting a single enzyme. Additional evidence for its unienzymic nature are: (i) the two activities were destroyed at exactly the same rate on heating of the purified enzyme from F. fragilis at three different temperatures; (ii) the competitive inhibitor glucono-δ-lactone gave the same value of Ki when tested with either substrate; (iii) quantitative application of the ‘mixed-substrate’ method with the purified enzyme of S. cerevisiae gave data that were in excellent agreement with those calculated on the assumption of a single enzyme. 8. The purified exo-β-glucanases of the different species of yeast had different kinetic constants. The ratios of maximal velocities and Km values with laminarin and pustulan differed markedly. Comparison of Vmax. and Km values suggests that the rapid release of spores from asci in F. fragilis might be explained in terms of an enzyme with higher maximal velocity and higher affinity to the ascus wall than that present in baker's yeast. 9. The estimated molecular weights for exo-β-glucanases from F. fragilis, S. cerevisiae and H. anomala were 22000, 40000 and 30000 respectively.
Effect of pH on the inhibition of the nicotinamide–adenine dinucleotide-specific isocitrate dehydrogenase from baker's yeast by anions
Authors: Cennamo C. , Montecuccoli G. , Bonaretti G. , Razzoli L.
Published: August 2015

1. The sensitivity of the NAD+-specific isocitrate dehydrogenase from baker's yeast towards inhibition by anions decreases with decrease in pH. The patterns of the pH-dependence of the enzymic activity can be explained by this effect. 2. In the presence of a high isocitrate concentration, citrate, unlike AMP, has no antagonizing effect on the inhibition of the enzyme by anions. In the presence of AMP, citrate inhibits the enzyme at high isocitrate concentration and activates at low isocitrate concentration. 3. The effects on the enzymic activity of the previous incubation of the enzyme were studied in relation to the substrate concentration, the chloride concentration and the presence of citrate and AMP.
Effect of alloxan-diabetes and treatment with anti-insulin serum on pathways of glucose metabolism in lactating rat mammary gland
Authors: Walters Eileen , McLean Patricia
Published: August 2015

1. The overall metabolic changes in lactating mammary gland in alloxan-diabetic and anti-insulin-serum-treated rats were assessed by measurement of the incorporation of 14C from specifically labelled glucose, pyruvate and acetate into carbon dioxide and lipid, together with measurements of enzymes concerned with the pentose phosphate pathway and with citrate metabolism. 2. Alloxan-diabetes depressed the rate of formation of 14CO2 from [1−14C]glucose and [2−14C]glucose to approx. 10% of the control rate; this was partially reversed by addition of insulin in vitro. The quotient Oxidation of [1−14C]glucose/Oxidation of [6−14C]glucose fell from a value of 17·6 in the control group to 3·9 in the diabetic group and was restored to 14·3 in the presence of insulin in vitro. In keeping with these results it was shown that glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities were significantly decreased in alloxan-diabetic rats. 3. Alloxan-diabetes depressed the decarboxylation and the oxidation of labelled pyruvate, but not the oxidation of labelled acetate. 4. The synthesis of lipid from specifically labelled glucose was greatly decreased, that from [2−14C]pyruvate was almost unchanged and that from [1−14C]acetate alone was increased in alloxandiabetic rats. However, the stimulation of lipid synthesis from acetate by glucose was small in the alloxan-diabetic rats compared with the controls. Insulin in vitro partially reversed all these effects. Both citrate-cleavage enzyme and acetate thiokinase activities were decreased in alloxan-diabetic rats. 5. Treatment of rats with anti-insulin serum depressed the formation of 14CO2 from [1−14C]glucose and [2−14C]glucose, but increased that from [6−14C]glucose. This was completely restored by the presence of insulin in vitro. The quotient Oxidation of [1−14C]glucose/Oxidation of [6−14C]glucose fell from a value of 17·6 in the control group to 3·8 in the anti-insulin-serum-treated group. There were no changes in the activity of glucose 6-phosphate dehydrogenase or 6-phosphogluconate dehydrogenase, but the hexokinase distribution changed and the content of the soluble fraction increased significantly. 6. The synthesis of lipid from specifically labelled glucose was depressed in anti-insulin-serum-treated rats; this effect was completely reversed by addition of insulin in vitro to the tissue slices.
The structure of a glycopeptide isolated from the yeast cell wall
Authors: Sentandreu R. , Northcote D. H.
Published: August 2015

1. Glycopeptides containing mannose were extracted from isolated yeast cell walls by ethylenediamine and purified by treatment with Pronase and fractionation on a Sephadex column. 2. A glycopeptide that appeared homogeneous on electrophoresis and ultracentrifugation had a molecular weight of 76000, and contained a high-molecular-weight mannan and approx. 4% of amino acids. 3. The amino acid composition of the peptide was determined. It was rich in serine and threonine and also contained glucosamine. No cystine and methionine were detected. 4. The glycopeptide underwent a β-elimination reaction when treated with dilute alkali at low temperatures. The reaction resulted in the release of mannose, mannose disaccharides and possibly other low-molecular-weight mannose oligosaccharides. During the β-elimination reaction the dehydro derivatives of serine and threonine were formed. One of the linkages between carbohydrate and amino acids in the glycopeptide is an O-mannosyl bond from mannose and mannose oligosaccharides to serine and threonine. 5. After the β-elimination reaction the bulk of the mannose in the form of the large mannan component was still covalently linked to the peptide. This polysaccharide was therefore attached to the amino acids by a linkage different from the O-mannosyl bonds to serine and threonine that attach the low-molecular-weight sugars. 6. Mannan was prepared from the glycopeptide and from the yeast cell wall by treatment of the fractions with hot solutions of alkali. The mannan contained aspartic acid and glucosamine and some other amino acids. The aspartic acid and glucosamine were present in equimolar amounts; the aspartic acid was the only amino acid present in an amount equivalent to that of glucosamine. Thus there is the possibility of a linkage between the mannan and the peptide via glucosamine and aspartic acid. 7. Mannose 6-phosphate was shown to be part of the mannan structure. Information about the structure of the mannan and the linkage of the glucosamine was obtained by periodate oxidation studies. 8. The glucosamine present in the glycopeptide could not be released by treatment with an enzyme preparation obtained from the gut of Helix pomatia. This enzyme released glucosamine from the intact cell wall. Thus there are probably at least two polymers containing glucosamine in the cell wall. 9. The biosynthesis of the mannan polymer in the yeast cell wall is discussed with regard to the two types of carbohydrate–amino acid linkages found in the glycoprotein.
The purification and some properties of a β-lactamase (cephalosporinase) synthesized by Enterobacter cloacae
Authors: Hennessey T. D. , Richmond M H
Published: August 2015

1. A β-lactamase has been purified from a strain of Enterobacter cloacae. 2. This enzyme is about eighty times as active against cephaloridine as against benzylpenicillin or ampicillin. 3. The enzyme has a net positive charge at pH8·0 and a molecular weight of about 14000. 4. An approximate amino acid composition of the enzyme is reported.
Inhibition of protein synthesis in Krebs 2 ascites cells and cell-free systems by phenylalanine and its effect on leucine and lysine in the amino acid pool
Authors: Roscoe Joan P. , Eaton M. D. , Choy Gladys Chin
Published: August 2015

1. The inhibition of incorporation of 14C-labelled amino acids into protein of whole cells by phenylalanine has been reproduced in a cell-free system. In both cases only the l-isomer was inhibitory. 2. The effect of phenylalanine on incorporation of [14C]leucine and [14C]lysine into protein was different in both whole cells and cell-free systems. 3. In whole cells inhibition of incorporation of leucine at 2·5μg./ml. was very rapid, but when the concentration was increased to 100μg./ml. the inhibition was not apparent for about 1hr. The kinetics of inhibition of lysine was the same at both these concentrations and was similar to that found with leucine at 100μg./ml. 4. Neither a lower specific radioactivity of the two amino acids in the pool nor a decrease in their pool size could be consistently related with inhibition of protein synthesis. 5. In the cell-free system l-phenylalanine inhibited the incorporation of leucine but not of lysine. 6. Charging of transfer RNA by leucine was markedly decreased in the presence of phenylalanine, whereas charging of transfer RNA by lysine was not.
The determination of glucosamine and galactosamine in some glycoproteins by radioisotope dilution
Authors: Graham E. R. B. , Neuberger A.
Published: August 2015

1. The principle of radioisotope dilution was applied on a semi-micro scale to the determination of glucosamine and galactosamine in some glycoproteins, such as immunoglobulins, a urinary glycoprotein and blood-group-specific substances. 2. The glycoprotein was hydrolysed in the presence of [1−14C]glucosamine or [1−14C]galactosamine or both. The amino sugars were made to react with naphthyl isothiocyanate and the products formed were isolated by the method of Scott (1962). The specific radioactivities determined from liquid-scintillation counting and the extinction at 240mμ or 222mμ were used to calculate the content of amino sugars in the protein analysed. 3. Where the values could be compared with those found by other workers, differences were in general not very great. The advantages of the method are that high concentrations of acid can be employed and undesirable side reactions, which may occur with the free sugars, do not affect the results. A potential source of error of the method is discussed.
The stereochemistry of desaturations of long-chain fatty acids in Chlorella vulgaris
Authors: Morris L. J. , Harris R. V. , Kelly W. , James A T
Published: August 2015

1. A study was made of the stereospecificity of hydrogen removal in the sequential desaturations performed by intact cells of Chlorella vulgaris in the biosynthesis of oleic acid, linoleic acid and α-linolenic acid. 2. By use of erythro- and threo-9,10−2H2-, −12,13−2H2- and −15,16−2H2-labelled precursors, it was demonstrated that the pair of hydrogen atoms removed from each of these positions had the cis relative configuration. 3. That the hydrogen atoms removed in oleic acid and linoleic acid formation were of the d absolute configuration was proved by use of d- and l-9−3H-and −12−3H-labelled precursors. 4. The presence of a substantial kinetic isotope effect of deuterium at both positions of the putative double bond was indicated, suggesting that the mechanism of desaturation involves simultaneous concerted removal of the pair of hydrogen atoms.
Studies on halotolerance in a moderately halophilic bacterium. Effect of betaine on salt resistance of the respiratory system
Authors: Rafaeli-Eshkol Dvora , Avi-Dor Y.
Published: August 2015

The role of betaine as a factor influencing the salt resistance of the respiratory system in resting cells of the moderately halophilic halotolerant bacterium Ba1 was studied. Betaine accelerated succinate oxidation in cells obtained from low-salt medium, and stimulation of the respiratory rate was stronger the higher the sodium chloride concentration in the assay medium. The stimulatory effect also depended on the ratio of betaine concentration to the amount of bacteria present. Accumulation of labelled betaine by the bacterial cells was demonstrated; like the respiratory stimulation, it was favourably influenced by an increase in the sodium chloride concentration of the medium. In cells harvested from a high-salt medium and washed with 2·0m-sodium chloride, betaine caused no increase in the respiratory rate, nor was the already high salt resistance of the respiratory system further improved by the addition of betaine. When, however, these cells lost their salt resistance as a result of washing in the absence of sodium chloride, betaine was able to restore it to its original level. In contrast with respiration in low-salt-grown bacteria, that in high-salt-grown cells was not affected by betaine, even after they were washed in the absence of sodium chloride, when the sodium chloride concentration was optimum.
The isolation and characterization of 3-phosphoglycerate dehydrogenase from peas
Authors: Slaughter J. C. , Davies D. D.
Published: August 2015

1. 3-Phosphoglycerate dehydrogenase was purified 400-fold from crude extracts of etiolated pea epicotyls. 2. Michaelis constants were determined for all four substrates. 3. Loss of sensitivity to inhibition by l-serine occurs on purification. 4. The purified enzyme is inhibited by thiol-group reagents and, with N-ethyl-maleimide, protection is afforded by 3-phosphoglycerate though not by NAD+.
Inhibition of 3-phosphoglycerate dehydrogenase by l-serine
Authors: Slaughter J. C. , Davies D. D.
Published: August 2015

1. l-Serine was shown to be a highly specific inhibitor of 3-phosphoglycerate dehydrogenase. 2. 3-Phosphoglycerate dehydrogenase is cold-labile with respect to its catalytic activity and to sensitivity to serine. 3. l-Serine protects the catalytic site as well as the inhibitor site. 4. Glycerol protects the catalytic site as well as the inhibitor site. 5. Serine acts as a ‘classical’ non-competitive inhibitor of fresh preparations of 3-phosphoglycerate dehydrogenase. 6. ‘Aged’ preparations when assayed at pH6·5 show sigmoid inhibition curves at saturating substrate concentrations. 7. A generalized model is advanced to account for the variation of the catalytic activity and the inhibitory effect of l-serine with time and conditions. 8. The possibility that the sigmoid kinetics of inhibition observed are an artifact of isolation is discussed.
Properties of some reductase enzymes in the nitrifying bacteria and their relationship to the oxidase systems
Authors: Wallace W. , Nicholas D. J. D.
Published: August 2015

The reductase enzymes in Nitrosomonas and Nitrobacter were studied under anaerobic conditions when the oxidase enzymes were inactive. The most effective electron-donor systems for nitrate reductase in Nitrobacter were reduced benzyl viologen alone, phenazine methosulphate with either NADH or NADPH, and FMN or FAD with NADH. Nitrite and hydroxylamine reductases were found in both nitrifying bacteria, and optimum activity for each enzyme was obtained with NADH or NADPH with either FMN or FAD. The product of both these enzymes was identified as ammonia. In extracts of Nitrosomonas the ammonia was further utilized by an NADPH-specific glutamate dehydrogenase. 15N-labelled nitrite, hydroxylamine and ammonia were rapidly incorporated into cell protein by Nitrosomonas, and Nitrobacter in addition incorporated [15N]nitrate. Relatively gentle methods of cell disruption were compared with ultrasonic treatment, to enable a more exact study to be undertaken of the intracellular distribution of the oxidase and reductase enzymes. The functional relationship of these opposing enzyme systems in the nitrifying bacteria is considered.
Stimulation of liver cholesterol synthesis by actinomycin D
Authors: De Matteis F.
Published: August 2015

1. An eightfold increase in the incorporation of [2−14C]acetate into liver cholesterol in vivo was observed 24hr. after starved rats had been given actinomycin D (0·5mg./kg. of body wt.). Liver cholesterol radioactivity declined faster in the treated animals, suggesting a greater rate of cholesterol turnover. 2. Liver slices from treated animals showed a tenfold increase in the incorporation of [2−14C]acetate into cholesterol; conversion into CO2 and into fatty acids was less markedly increased, and conversion into ketone bodies was not significantly affected. 3. The patterns of conversion into liver cholesterol in vivo of the lactone and the sodium salt of mevalonic acid differed markedly. The former was converted at a faster rate and to a greater extent than the latter. Treatment with actinomycin D increased the conversion of both forms of mevalonic acid into liver cholesterol, but only to a small extent. 4. Stimulation of the incorporation of acetate into cholesterol occurred at 4hr. after the administration of actinomycin D but not at 2hr. The response was abolished by the simultaneous administration of dl-ethionine or puromycin. 5. Pre-feeding with a cholesterol-rich diet greatly diminished the stimulation of conversion of acetate into cholesterol caused by actinomycin D, though it did not completely suppress it. Adrenalectomized animals responded to the drug, but much less markedly. 6. It is concluded that actinomycin D stimulates the synthesis of cholesterol in the liver at a stage in the pathway before mevalonic acid, by a mechanism that probably requires protein synthesis. A likely site would be the β-hydroxy-β-methylglutaryl-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. Some possible mechanisms by which the drug may lead to increased activity of this enzyme are considered.
Sporulation in Bacillus subtilis. Antigenic changes during spore formation
Authors: Waites W. M.
Published: August 2015

1. Antisera, prepared against extracts of cells and spores of Bacillus subtilis, were used in immunoelectrophoretic studies of the changes occurring in cell extracts during the course of spore formation. 2. At least 15 antigens could be detected in vegetative-cell extracts by the antiserum prepared against cell extracts and at least seven could be demonstrated in spore extracts by the homologous antiserum. 3. Cross-absorption studies showed that two of these antigens were probably completely specific for vegetative-cell extracts and that one was probably completely specific for spore extracts. The remainder were probably present in very small quantities in the heterologous extract. 4. In extracts of cells sporulating in an ‘exhaustion medium’ those antigens characteristic of the spore began to appear about 1hr. after the end of exponential growth. 5. In cells sporulating in a resuspension medium, spore antigens were detected at 4hr., and by 7hr. a decrease in vegetative-cell antigens was observed. 6. In an asporogenous mutant blocked early in sporulation there was neither an increase in spore antigens nor a decrease in vegetative-cell antigens. 7. In an asporogenous mutant blocked later in sporulation, there was an increase in spore antigens similar to that which occurred in the sporogenous strain.
Sporulation in Bacillis subtilis. Biochemical changes
Authors: Warren S. C.
Published: August 2015

1. During the course of growth and sporulation of Bacillus subtilis in chemically defined media, measurements were made of 16 different parameters, including the specific activities of nine intracellular enzymes. 2. Towards the end of exponential growth, proteolytic activity increased and reached a maximum soon after growth ceased. 3. In the presence of an excess of phosphate the specific activity of alkaline phosphatase increased fivefold at the end of exponential growth. 4. The specific activity of malate dehydrogenase remained at a high constant level throughout sporulation, but the specific activity of fumarase showed a two- to three-fold increase 5–9hr. after the end of exponential growth. 5. Aconitase activity was barely detectable during exponential growth in a glucose–glutamate medium, but increased rapidly when glutamate was replaced by citrate or when the glucose in the medium was exhausted. 6. The specific activity of alanine dehydrogenase increased threefold 1–5hr. after the end of exponential growth. 7. The specific activity of soluble NADH oxidase doubled 4–6hr. after the end of exponential growth. 8. Glucose dehydrogenase was undetectable until 4hr. after the end of exponential growth, but its specific activity increased 20-fold over the next 3–4hr. 9. The onset of refractility, the synthesis of 2,6-dipicolinic acid and the appearance of heat-resistance occurred in this order some 6–12hr. after the end of exponential growth. 10. The significance of these changes is discussed in relation to the morphological development of the spore.
The binding of sodium dodecyl sulphate to various proteins
Authors: Pitt-Rivers Rosalind , Impiombato F. S. Ambesi
Published: August 2015

1. The binding of sodium dodecyl sulphate to proteins by equilibrium dialysis was investigated. 2. Most of the proteins studied bound 90–100% of their weight of sodium dodecyl sulphate. 3. The glycoproteins studied bound 70–100% of their weight of sodium dodecyl sulphate, calculated in terms of the polypeptide moiety of the molecule. 4. Proteins not containing S·S groups bound about 140% of their weight of sodium dodecyl sulphate. 5. Reduction of four proteins containing S·S groups caused a rise in sodium dodecyl sulphate binding to 140% of the weight of protein. 6. The apparent micellar molecular weights of the protein–sodium dodecyl sulphate complexes were measured by the dye-solubilization method; they were all found to have approximately the same micellar molecular weight (34000–41000) irrespective of the molecular weight of the protein to which they were attached.