bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies on the thiol group of lactose synthetase A protein from human milk and on the binding of uridine diphosphate galactose to the enzyme
Authors: Kitchen B. J. , Andrews P.
Published: August 2015

The lactose synthetase activity of A protein from human milk was much decreased but not abolished by reaction with thiol-group reagents. Protection experiments indicated that a free thiol group on the enzyme is situated near the UDP-galactose binding site and inactivation of the enzyme with p-hydroxymercuribenzoate was probably due to prevention of UDP-galactose binding. Affinity chromatography showed that the mercuribenzoate substituent also decreased the affinity of A protein for N-acetylglucosamine but complex-formation between A protein–N-acetylglucosamine and α-lactalbumin was relatively unaffected. UDP-galactose appears to be bound to the enzyme mainly through its pyrophosphate group with Mn2+ ion and through the cis hydroxyls of ribose, whereas its hexose moiety has little if any affinity for the enzyme. Lactose synthetase activity remaining after the reaction with thiol-group reagents indicates that a free thiol group is not an essential part of the A protein active site.
Distribution of reducing equivalents on xanthine oxidase molecules and the rates of the intramolecular electron-transfer reactions
Authors: Bray Robert C. , Lowe David J. , Barber Michael J.
Published: August 2015

When xanthine oxidase turns over 1-methylxanthine aerobically at pH8.2, the time-sequence in development of its electron-paramagnetic-resonance signals is not primarily due to slow intramolecular reactions among its centres. It derives instead from gross differences in electron distribution within enzyme molecules reduced by the substrate in comparison with those that have subsequently been partly reoxidized by one-electron reaction with oxygen.
Specificity of anti-nucleoside antibodies
Authors: Humayun Mir Z. , Jacob T. Mathai
Published: August 2015

The method of conjugation of a nucleoside or related compound to a carrier protein may have a significant effect on the specificity of the antibodies elicited. It is demonstrated, by means of the membrane-filtration assay, that anti-isopentenyladenosine antibodies produced by the ‘periodate procedure’ are much more reactive with the periodate-oxidized form of the nucleoside than with the parent compound. In addition, the simplicity and specificity of the assay used suggests its use as a sensitive radioimmunoassay for this multifunctional nucleoside.
Affinity chromatography of bovine trypsin. A rapid separation of bovine α- and β-trypsin
Authors: Jameson G. W. , Elmore D. T.
Published: August 2015

Affinity adsorbents for bovine trypsin were prepared by covalently coupling p-(p′-amino-phenoxypropoxy)benzamidine to cellulose and to agarose. Trypsin binds to both adsorbents at pH6–8 and is released at low pH values or in the presence of n-butylamine hydrochloride. Pure β-trypsin may be eluted from crude trypsin bound at pH8.0 to the cellulose adsorbent by stepwise elution with an acetate buffer, pH5.0. Both α- and β-trypsin may be isolated by chromatography of crude trypsin on the agarose derivative in an acetate buffer, pH4.0. These two methods for purifying the trypsin are specific to the particular adsorbents. They are rapid and convenient in use. Both methods leave a mixture of the two enzymes bound to the adsorbent and release occurs only at low pH values. The effects of pH, composition and ionic strength of buffer and other variables on both purification methods are described. Affinity adsorbents of soya-bean trypsin inhibitor and of N-α-(N′-methyl-N′-sulphanilyl) sulphanilylagmatine bound to agarose were prepared, but were found to be of limited usefulness in the purification of trypsin.
NN′-Diacetylchitobiase activity in Tay–Sachs disease and Sandhoff's disease
Authors: Stirling John L.
Published: August 2015

In Tay–Sachs disease and Sandhoff's disease respectively, one of the N-acetyl-β-hexosaminidases (form A) or both (forms A and B) are absent, but glycosaminoglycans containing N-acetylhexosamines are not accumulated. The presence of NN′-diacetylchitobiase in livers from patients with these diseases raises the possibility that this new enzyme is involved in the metabolism of glycosaminoglycans rather than the enzymes previously described.
Kinetic studies of bovine liver carbamoyl phosphate synthetase
Authors: Elliott Keith R. F. , Tipton Keith F.
Published: August 2015

A through study of initial-rate data has been made on carbamoyl phosphate synthetase from bovine liver. On the basis of the results the order of substrate binding to the enzyme is ATPMg followed by HCO3−, ATPMg and NH4+. A model for the enzymic mechanism is proposed, and the rate equations describing it are presented. Details of the derivation of the initial-rate equation for the kinetic mechanism proposed have been deposited as Supplementary Publication SUP 50032 (6 pages) at the British Library, Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7QB, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1973), 131, 5.
Protein synthesis by synaptosomes from rat brain. Contribution by the intraterminal mitochondria
Authors: Hernández A. G.
Published: August 2015

(1) The characteristics of protein synthesis in microsomal and synaptosomal fractions from rat brain were examined. A high sensitivity to ribonuclease and to cycloheximide, and the need for the presence of pH5 enzymes distinguished protein synthesis in microsomal fractions from protein synthesis in synaptosomes. (2) Under various conditions of incubation synaptosomal fractions prepared in sucrose showed limited protein synthesis compared with synaptosomal fractions prepared by using Ficoll. Such discrepancies could not be attributed to: (i) animal age, (ii) the metabolic state of the synaptosomal fraction, (iii) the absence of bivalent cations in the incubation medium or (iv) the temperature. (3) Protein synthesis in synaptosomal fractions was inhibited 50–65% by cycloheximide, 38–50% by chloramphenicol, 95% by puromycin, 70% by azide and 40% by deoxyglucose; ribonuclease had only a negligible inhibitory effect. (4) As a first approximation to the localization of the protein-synthetic machinery present in the synaptosomal fraction, the distribution of enzymes and radioactivity in subfractions of prelabelled synaptosomes was determined after osmotic shock with water. Approximately 60% of the total protein synthesis in the synaptosomal fraction occurred in the intraterminal mitochondria. (5) Protein synthesis in the intraterminal mitochondria did not show any fundamental difference from synthesis in somatic mitochondria, with respect to inhibition by cycloheximide and chloramphenicol. (6) It was concluded that if extramitochondrial protein synthesis occurs in synaptosomes, it must be very low.
Folic acid metabolism in vitamin B12-deficient sheep. Effects of injected methionine on liver constituents associated with folate metabolism
Authors: Smith Richard M. , Osborne-White William S. , Gawthorne Jeffrey M.
Published: August 2015

1. The effects of injected l-methionine (2g every second day for 28 days) on liver folates and other constituents of liver associated with folate metabolism were studied in vitamin B12-deficient ewes and their pair-fed controls receiving vitamin B12. The dose rate of methionine used was sufficient to restore almost to normal the elevated excretion in the urine of formiminoglutamate in the deficient animals. 2. Liver folates active for Lactobacillus casei, Streptococcus faecalis R and Pediococcus cerevisiae were severely depressed in deficient livers and were partly restored by methionine. Analysis of the folates after ion-exchange chromatography showed that the major effect of methionine was to increase the concentrations of tetrahydrofolates and formyltetrahydrofolates. Methyltetrahydrofolates were also increased, but there was no effect of methionine on the small amounts of incompletely reduced folates present in deficient livers. The folates present were predominantly penta-, hexa- and hepta-glutamates whether or not animals received vitamin B12 or methionine. 3. Concentrations of ATP, NAD+, NADH and NADPH were lower in freeze-clamped liver from vitamin B12-deficient sheep than in liver from pair-fed, vitamin B12-treated sheep. These changes were not affected by methionine which was also without effect on the elevated K+/Na+ ratios found in deficient livers. 4. The livers of vitamin B12-deficient animals contained lower concentrations of choline and higher concentrations of lipid than their pair-fed controls. These effects were reversed by methionine.
The amounts and rates of export of polysaccharides found within the membrane system of maize root cells
Authors: Bowles Dianna J. , Northcote D. H.
Published: August 2015

1. Maize seedling roots were incubated in vivo with d-[U-14C]glucose for 2, 5, 10, 15, 30 and 45min. The total incorporation of radioactivity into polysaccharide components in isolated fractions was investigated, and the pattern of incorporation into different polysaccharide components within the rough endoplasmic reticulum, Golgi apparatus and exported material was analysed. 2. The membrane compartments reached a saturation value of radioactivity in polysaccharide components by 30min incubation. Radioactivity in exported polysaccharide continued to increase after that time. The latter was formed and maintained by a steady-state turnover of polysaccharide synthesis and transport from the membrane system. 3. If the only access of the slime polysaccharide to the cell surface is via dictyosome-derived vesicles, the amount of slime components in the Golgi apparatus would have to be displaced every 0.3min in order to maintain the observed rates of increase in slime. This is in contrast with a displacement time of about 2.5min that is necessary for polysaccharide components in the Golgi apparatus to produce the observed increase in cell-wall material. The activity of the membrane system in the production of maize root slime is 8 times as great as that of the membrane system involved in cell-wall synthesis. 4. If the amount of polysaccharide material in the Golgi apparatus is maintained only by inflow of polymeric material from the rough endoplasmic reticulum the total amount of slime components in the rough endoplasmic reticulum would have to be displaced every 7min to maintain a constant amount in the Golgi apparatus. If the endoplasmic reticulum contributed directly to the cell surface in the synthesis of cell-wall material, displacement times necessary to maintain the observed rate of polymer production would be very slow.
The effect of oral inorganic sulphate on the metabolism of 4-hydroxyphenethylamine (tyramine) in man: tyramine O-sulphate measurement in human urine (Short Communication)
Authors: Smith Ivor , Mitchell Paul D.
Published: August 2015

1. Urinary excretion of tyramine O-sulphate in six normal human subjects after an oral dose of 125mg of tyramine hydrochloride alone, and with a supplement of Na2SO4, was determined by using chromatographic and electrophoretic separations followed by spectrophotofluorimetry. 2. In every case the excretion of tyramine O-sulphate was increased significantly after sulphate supplementation.
The effect of energization on the apparent Michaelis–Menten constant for oxyge in nmitochondrial respiration
Authors: Petersen Lars Christian , Nicholls Peter , Degn Hans
Published: August 2015

Lineweaver–Burk plots of 1/v against 1/[O2] for rat liver mitochondrial respiration with succinate or ascorbate+NNN′N′-tetramethyl-p-phenylenediamine as substrates are non-linear. In state 3u (uncoupled by trifluoromethoxycarbonyl cyanide phenylhydrazone) such plots tend to be concave upward, whereas in state 4 (energized) the plots were concave downward. The apparent Km for oxygen is larger in state 4 than in state 3u, despite the higher turnover in the latter system. It is postulated that at least one reversible reaction occurs between cytochrome c and cytochrome c oxidase, whose rate is increased on energization (reversed electron transfer); a model including such a reaction is proposed which accounts semiquantitatively for the observations.
Phytochrome-induced synthesis of ribonuclease de novo in lupin hypocotyl sections
Authors: Acton G. John , Schopfer Peter
Published: August 2015

1. Density-labelling with 99 atoms% of2H2O distinguished pre-existing from newly synthesized ribonuclease molecules in sections of developing hypocotyl tissue. 2. Activity profiles of enzyme extracted from the fraction pelletable at 100000g showed heterogeneity after isopycnic centrifugation in CsCl gradients. 3. Measurement of density shifts of the entire heterogeneous band shows that ribonuclease protein is synthesized de novo in both continuous far-red light and darkness. 4. A twofold increase in enzyme activity after irradiation was accompanied by band-broadening and a significantly faster rate of labelling than in darkness. 5. The conclusion is drawn from the experimental evidence and theoretical arguments presented that phytochrome regulates the synthesis of new enzyme molecules against a background of continuous (dark-rate) synthesis and degradation. 6. Further information has been deposited as Supplementary Publication SUP 50033 (3 pages) at the British Library Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973), 131, 5.
Lipogenesis in rabbit isolated fat-cells
Authors: Saggerson E. David
Published: August 2015

1. Fat-cells isolated from rabbit perirenal adipose tissue were incubated with the following U-14C-labelled substrates: 5mm-glucose (+insulin), 5mm-pyruvate, 5mm-lactate, 5mm-glucose+5mm-acetate (+insulin), and the relative rates of incorporation of these substrates into glyceride fatty acids determined. In general total rates of fatty acid synthesis were similar whatever substrate was supplied to the cells. 2. Rabbit fat-cells incorporated [U-14C]acetate into fatty acids and CO2 as well in the absence of glucose as in the presence of this substrate. 3. The disposition of the utilization of glucose-derived carbon through various metabolic pathways was determined. 4. Extramitochondrial and mitochondrial activities were determined for 11 enzymes. The cells contained a very low activity of pyruvate carboxylase, undetectable NADP–malate dehydrogenase activity and a high mitochondrial phosphoenolpyruvate carboxylase activity. 5. Various rabbit fat-cell metabolic parameters based on the measurement of14C incorporation and enzyme activity were compared with the same parameters previously measured in rat and guinea-pig fat-cells. In general guinea pig occupied a position between rat and rabbit with respect to these parameters. 6. The profiles of substrate incorporation into fatty acids and of relative enzyme activities in rabbit fat-cells indicated that the operation of a ‘citrate-cleavage’ pathway may not be obligatory for the supply of lipogenic acetyl units.
A study of the kinetics and mechanism of rabbit muscle l-glycerol 3-phosphate dehydrogenase
Authors: Bentley Philip , Dickinson F. Mark
Published: August 2015

1. The kinetics of oxidation of l-glycerol 3-phosphate by NAD+and of reduction of dihydroxyacetone phosphate by NADH catalysed by rabbit muscle glycerol 3-phosphate dehydrogenase were studied over the range pH6–9. 2. The enzyme was found to catalyse the oxidation of glyoxylate by NAD+at pH8.0 and the kinetics of this reaction were also studied. 3. The results are consistent with a compulsory mechanism of catalysis for glycerol 3-phosphate oxidation and dihydroxyacetone phosphate reduction in the intermediate regions of pH, but modifications to the basic mechanism are required to fully explain results at the extremes of the pH range, with these substrates and for glyoxylate oxidation at pH8.0.
Activation of carbamoyl phosphate synthase by N-acetyl-l-aspartate
Authors: Forman Henry Jay , Waddell Ronald , Hamilton Paul B. , Grisolia Santiago
Published: August 2015

Carbamoyl phosphate synthase from liver of both rat and frog, normally dependent on N-acetyl-l-glutamate (on the basis of Km and physiological concentrations) as an activator, was shown to be activated by high concentrations of N-acetyl-l-aspartate. However, the high concentrations of N-acetyl-l-aspartate required for activation produce non-competitive inhibition. Similarly, high concentrations of N-acetyl-l-glutamate, in very large excess of the amount required to activate the enzyme, inhibit. The limit for N-acetyl-l-glutamate as an impurity in N-acetyl-l-aspartate was found to be less than 1 in 5000 parts, far below the 1 in 250 parts needed to produce the activation observed with N-acetyl-l-aspartate.
Mammalian chromatin substructure studies with the calcium–magnesium endonuclease and two-dimensional polyacrylamide-gel electrophoresis
Authors: Burgoyne Leigh A. , Hewish Dean R. , Mobbs Jennifer
Published: August 2015

The basic regularity of chromatin substructure that has been reported in rat liver chromatin (Hewish & Burgoyne, 1973b) was also detected in mouse chromatin. The regular series of DNA fragments produced by the action of Ca–Mg endonuclease on rat chromatin were studied further. The smallest single-stranded class has a molecular weight of approx. 45000–63000 and the smallest double-stranded class has a molecular weight of approx. 120000–150000. Studies of the substructure of the DNA fragments produced by the Ca–Mg endonuclease have shown that the regular series of double-stranded fragments have regular series of single-stranded fragments within them. It was concluded that the regular series of double-stranded fragments was probably a consequence of the regular series of single-stranded fragments. Digestion time-courses are presented for mouse and rat nuclear DNA.
A new method for determining the Michaelis constant
Authors: De Miguel Merino Felipe
Published: August 2015

A new method is described for evaluating the parameters Km and V in the Michaelis–Menten equation, and is illustrated with experimental data from the literature.
Ribonucleic acid polymerase activities in Jerusalem-artichoke tissue
Authors: Gore John R. , Ingle John
Published: August 2015

1. Artichoke tuber tissue contained RNA polymerase activity bound to the chromatin and in the supernatant after chromatin sedimentation. 2. The activity in the supernatant, the soluble polymerase, was fractionated into polymerases I and II by DEAE-cellulose chromatography, and the properties of each activity were determined. 3. The proportions of chromatin-bound and soluble activities varied with growth of the tissue, and there was a correlation between chromatin-bound activity and RNA accumulation. 4. The properties of the solubilized chromatin activity were compared with those of the soluble activity, and the relationship between these two activities is discussed.
Biological, proton-magnetic-resonance- and ultraviolet-spectroscopic evidence for a molecular complex of actinomycin D and 10,11-dihydro-3H-naphth[1,2-g]indazol-7-ol (Short Communication)
Authors: Chesnut Robert W. , Durham Norman N. , Mawdsley Elizabeth A. , Thyvelikakath George X. , Berlin K. Darrell , Fung B. M.
Published: August 2015

A proton-magnetic-resonance study of the complex formed between actinomycin D and 10,11-dihydro-3H-naphth[1,2-g]indazol-7-ol strongly suggests that the indazole lies above the phenoxazine ring of actinomycin D. The complex can be destroyed by addition of dimethyl sulphoxide or dimethylformamide. The actinomycin D–indazole complex inhibits growth of Pseudomonas fluorescens and raises the thermal denaturation response of DNA. These data support the hypothesis that a molecular complex is formed which readily inhibits cell growth and interacts with DNA.
Phenethylbiguanide and the inhibition of hepatic gluconeogenesis in the guinea pig
Authors: Ogata Korechika , Jomain-Baum Mireille , Hanson Richard W.
Published: August 2015

1. Phenethylbiguanide inhibits the synthesis of phosphoenolpyruvate from malate or 2-oxoglutarate by isolated guinea-pig liver mitochondria. This inhibition is time- and concentration-dependent, with the maximum decrease in the rate of phosphoenolpyruvate synthesis (80%) evident after 10min of incubation with 1mm-phenethylbiguanide. 2. The phosphorylation of ADP by these mitochondria is also inhibited at increasing concentrations of phenethylbiguanide and there is a progressive increase in AMP formation. Guinea-pig liver mitochondria are more sensitive to this inhibition in oxidative phosphorylation caused by phenethylbiguanide than are rat liver mitochondria. 3. Simultaneous measurements of O2 consumption and ADP phosphorylation with guinea-pig liver mitochondria oxidizing malate plus glutamate in State 3 indicated that phenethylbiguanide at low concentrations (0.1mm) inhibits respiration at Site 1. At higher phenethylbiguanide concentrations Site 2 is also inhibited. 4. Gluconeogenesis from lactate, pyruvate, alanine and glycerol by isolated perfused guinea-pig liver is inhibited to various degrees by phenethylbiguanide. Alanine is the most sensitive to inhibition (60% inhibition of the maximum rate by 0.1mm-phenethylbiguanide), whereas glycerol is relatively insensitive (25% inhibition at 4mm). 5. Gluconeogenesis from lactate and pyruvate by perfused rat liver was also inhibited by phenethylbiguanide, but only at high concentrations (8mm). Unlike guinea-pig liver, the inhibitory effect of phenethylbiguanide on rat liver was reversible after the termination of phenethylbiguanide infusion. 6. The time-course of inhibition of gluconeogenesis from the various substrates used in this study indicated a time-dependency which was related in part to the concentration of infused phenethylbiguanidine. This time-course closely paralleled that noted for the inhibition by phenethylbiguanide of phosphoenolpyruvate synthesis in isolated guinea-pig liver mitochondria.
Metabolism of [14C]citrulline in the perfused sheep and goat udder
Authors: Roets E , Verbeke R , Massart-Leën A M , Peeters G
Published: August 2015

1. A lactating-sheep mammary gland was perfused for 12h in the presence of l-[2-14C]-citrulline and received adequate quantities of glucose, acetate and amino acids. Two lactating-goat udders were similarly perfused in the presence of either l-[carbamoyl-14C,-2-14C]citrulline or l-[carbamoyl-14C,1-14C]citrulline and l-[4-3H]arginine. 2. In these experiments, [14C]citrulline was substantially oxidized to CO2 and converted into arginine and proline of casein. 3. The specific radioactivities of arginine, ornithine and proline of the plasma increased after passage through the udders, demonstrating that [14C]citrulline is metabolized by the mammary gland. 4. The presence of two unknown radioactive metabolites of [14C]citrulline was detected in the perfusate. These substances were not found after incubation in vitro of oxygenated blood in the presence of the radioactive precursor. 5. From these experiments, it is concluded that citrulline is metabolized in mammary tissue by way of arginine to urea, ornithine and proline.
Studies on the role of calcium ions in the stimulation by adrenaline of amylase release from rat parotid
Authors: Dormer R L , Ashcroft S J H
Published: August 2015

1. Mitochondrial and microsomal fractions were prepared from rat parotid glands. Both fractions were able to take up45Ca. The mitochondrial45Ca-uptake system could be driven by ATP (energy-coupled Ca2+uptake) or by ADP+succinate (respiration-coupled Ca2+uptake). Energy-coupled Ca2+uptake was blocked by oligomycin but not by carbonyl cyanide m-chlorophenylhydrazone; respiration-coupled Ca2+uptake was blocked by carbonyl cyanide m-chlorophenylhydrazone but not by oligomycin. Microsomal Ca2+uptake was dependent on the presence of ATP; the ATP-dependent Ca2+uptake was not affected by oligomycin or carbonyl cyanide m-chlorophenylhydrazone. Ca2+uptake by both fractions was inhibited by Ni2+. 2. Incubation of parotid pieces with adrenaline increased the rate of release of amylase and the uptake of45Ca. The adrenaline-stimulated release of amylase was not dependent on the presence of extracellular Ca2+. 3. The effect of adrenaline on the subcellular distribution of45Ca in parotid pieces incubated with45Ca was studied. In parotid tissue incubated with45Ca, both mitochondrial and microsomal fractions contained45Ca. Incubation with adrenaline increased the amount of45Ca incorporated into the mitochondrial fraction but not the microsomal fraction. In parotid tissue preloaded with45Ca subsequent incubation with adrenaline caused a decrease in the amount of45Ca found in both the mitochondrial and microsomal fractions. 4. From these data we conclude that the regulation of the cytosolic Ca2+concentration in the parotid may involve both mitochondrial and microsomal Ca2+-uptake systems. We suggest that the action of adrenaline on the parotid may be to increase the movement of Ca2+to the cytosol by increasing the flux of Ca2+across mitochondrial, microsomal and plasma membranes.
Induction of cystathionase in human foetal liver
Authors: Heinonen Kirsti , Räihä Niels C. R.
Published: August 2015

Incubation of liver explants from second-trimester human foetuses with dexamethasone, glucagon or dibutyryl cyclic AMP (plus theophylline) increased the activity of liver cystathionase from unmeasurable or trace values to adult values. Simultaneous incubation with cycloheximide or actinomycin D inhibited this effect.
Properties of catechol O-methyltransferases from brain and liver of rat and human
Authors: White H L , Wu J C
Published: August 2015

Kinetic and electrophoretic properties of catechol O-methyltransferases (EC 2.1.1.6) from brain and liver were studied. The enzyme of either rat or human tissues exhibited a single molecular form when subjected to electrophoresis at pH7.9. At pH9 a second, apparently oxidized, form was detected. Isoelectric-focusing experiments also indicated only one enzyme form, which was identical from extracts of brain and liver of each species (pI = 5.2 for rat, 5.5 for human). Similarities between brain and liver catechol O-methyltransferase of a given species were also demonstrated by kinetic parameters, meta/para ratios of products, and inhibitor potencies. Human catechol O-methyltransferase exhibited lower Km values than did the rat enzyme for S-adenosyl-L-methionine, dopamine and dihydroxybenzoic acid. Adrenochrome inhibited both rat and human enzyme. It was concluded (1) that only a single enzyme form could be demonstrated in the physiological pH region; (2) that catechol O-methyltransferase of brain could not be distinguished from the liver enzyme of the same species; and (3) that species differences exist between the enzymes of rat and human tissues.
Evidence for a histidine and a cysteine residue in the substrate-binding site of yeast alcohol dehydrogenase
Authors: Leskovac V , Pavkov-Peričin D
Published: August 2015

1. Yeast alcohol dehydrogenase (EC 1.1.1.1) is inhibited by stoicheiometric concentrations of diethyl pyrocarbonate. The inhibition is due to the acylation of a single histidine residue/monomer (mol.wt. 36000). 2. Alcohol dehydrogenase is also inhibited by stoicheiometric amounts of 5,5′-dithiobis-(2-nitrobenzoate), owing to the modification of a single cysteine residue/monomer. 3. Native alcohol dehydrogenase binds two molecules of reduced coenzyme/molecule of enzyme (mol.wt. 144000). 4. Modification of a single histidine residue/monomer by treatment with diethyl pyrocarbonate prevents the binding of acetamide in the ternary complex, enzyme-NADH-acetamede, but does not prevent the binding of NADH to the enzyme. 5. Modification of a single cysteine residue/monomer does not prevent the binding of acetamide to the ternary complex. After the modification of two thiol groups/monomer by treatment with 5,5′-dithiobis-(2-nitrobenzoate), the capacity of enzyme to bind coenzyme in the ternary complex was virtually abolished. 6. From the results presented in this paper we conclude that at least one histidine and one cysteine residue are closely associated in the substrate-binding site of alcohol dehydrogenase.
Metal-dependent proteinase of the lens. Assay, purification and properties of the bovine enzyme
Authors: Blow A M J , Heyningen R V , Barrett A J
Published: August 2015

1. Two new assay methods were developed for the lens proteinase. In both, the substrate was alpha2-crystallin (a major lens protein); in the first method, the products were detected by reaction with trinitrobenzenesulphonate in the presence of SO32-, whereas in the second method, 3H-labelled substrate was used, and the products were detected as radioactivity soluble in trichloroacetic acid. 2. The neutral proteinase from bovine lens was partially purified by extraction of the lens at pH5.0 and column chromatography on hydroxyapatite and Sepharose 6B gel. 3. The purified enzyme had no detectable activity against haemoglobin, azo-casein or gamma-crystallin under optimum conditions for alpha2-crystallin. 4. The enzyme showed greatest activity and stability at pH7.5. It was reversibly inhibited by EDTA and 1,10-phenanthroline, and activated by Ca2+ and Mg2+. 5. Molecular weights obtained for the enzyme by chromatography on Sepharose 6B were approx. 500,000 in buffer of I = 0.02, and 250,000 at I = 1.02. 6. The properties of the purified lens proteinase are such as to suggest that this enzyme could account for the entire endopeptidase activity of the lens.
Biosynthesis of caffeine by tea-leaf extracts. Enzymic formation of theobromine from 7-methylxanthine and of caffeine from theobromine
Authors: Suzuki T , Takahashi E
Published: August 2015

1. Extracts prepared from tea leaves with Polyclar AT (insoluble polyvinylpyrrolidine) contained two methyltransferase activities catalysing the transfer of methyl groups from S-adenosylmethionine to 7-methylxanthine, producing theobromine, and to theobromine, producing caffeine. 2. The methyltransferases exhibited the same pH optimum (8.4) and a similar pattern of effects by metal ions, thiol inhibitors and metal-chelating reagents, both for theobromine and caffeine synthesis. Mg2+, Mn2+ and Ca2+ slightly stimulated enzyme activity but they were not essential. Paraxanthine was shown to be most active among methylxanthines, as the methyl acceptor. However, the formation of paraxanthine from 1-methylxanthine was very low and that from 7-methylxanthine was nil, suggesting that the synthesis of caffeine from paraxanthine is of little importance in intact plants. Xanthine, xanthosine, XMP and hypoxanthine were all inactive as methyl acceptors, whereas [2(-14)C]xanthine and [8(-14)C]hypoxanthine were catabolized to allantoin and urea by tea-leaf extracts. The apparent Km values are as follows: 7-methylxanthine, 1.0 times 10(-14)M; theobromine, 1.0 times 10(-3)M; paraxanthine, 0.2 times 10(-3)M; S-adenosylmethionine, 0.25 times 10(-4)M (with each of the three substrates). 3. The results suggest that the pathway for caffeine biosynthesis is as follows: 7-methylxanthine leads to theobromine leads to caffeine. In contrast, it is suggested that theophylline is synthesized from 1-methylxanthine. The methyl groups of the purine ring of caffeine are all derived directly from the methyl group of S-adenosylmethionine. Little is known about the pathways leading to the formation of 7-methylxanthine. 4. A good correlation between caffeine synthesis and shoot formation or growth of tea seedlings was shown, suggesting that the methylating systems in caffeine synthesis are closely associated with purine nucleotide and nucleic acid metabolism in tea plants.
Inhibition by ricin of protein synthesis in vitro. Inhibition of the binding of elongation factor 2 and of adenosine diphosphate-ribosylated elongation factor 2 to ribosomes
Authors: Montanaro L , Sperti S , Mattioli A , Testoni G , Stirpe F
Published: August 2015

The binding of EF2 (elongation factor 2) and of ADP-ribosyl-EF 2 to rat liver ribosomes is inhibited by ricin. This result suggests that the native enzyme and its ADP-ribose derivative have the same or closely related binding sites on the ribosome. The inhibition by ricin of the binding of EF 2 to ribosomes is consistent with the previous observation that ricin affects EF 2-catalysed translocation during polypeptide chain elongation.
Role of glycerol 3-phosphate dehydrogenase in glyceride metabolism. Effect of diet on enzyme activities in chicken liver
Authors: Harding J W , Pyeritz E A , Copeland E S , White H B
Published: August 2015

1. The metabolic role of hepatic NAD-linked glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) was investigated vis-a-vis glyceride synthesis, glyceride degradation and the maintainence of the NAD redox state. 2. Five-week-old chickens were placed on five dietary regimes: a control group, a group on an increased-carbohydrate-lowered-fat diet, a group on a high-fat-lowered-carbohydrate diet, a starved group and a starved-refed group. In each group the specific activity (mumol/min per g wet wt. of tissue) of hepatic glycerol 3-phosphate dehydrogenase was compared with the activities of the β-oxoacyl-(acyl-carrier protein) reductase component of fatty acid synthetase, glycerol kinase (EC 2.7.1.30) and lactate dehydrogenase (EC 1.1.1.27). 3. During starvation, the activities of glycerol 3-phosphate dehydrogenase, glycerol kinase and lactate dehydrogenase rose significantly. After re-feeding these activities returned to near normal. All three activities rose slightly on the high-fat diet. Lactate dehydrogenase activity rose slightly, whereas those of the other two enzymes fell slightly on the increased-carbohydrate-lowered-fat diet. 4. The activity of the β-oxoacyl-(acyl-carrier protein) reductase component of fatty acid synthetase, a lipid-synthesizing enzyme, contrasted strikingly with the other three enzyme activities. Its activity was slightly elevated on the increased-carbohydrate diet and significantly diminished on the high-fat diet and during starvation. 5. The changes in activity of the chicken liver isoenzyme of glycerol 3-phosphate dehydrogenase in response to dietary stresses suggest that the enzyme has an important metabolic role other than or in addition to glyceride biosynthesis.
Glucose metabolism in perfused skeletal muscle. Interaction of insulin and exercise on glucose uptake
Authors: Berger M , Hagg S , Ruderman N B
Published: August 2015

1. The interaction of insulin and isometric exercise on glucose uptake by skeletal muscle was studied in the isolated perfused rat hindquarter. 2. Insulin, 10 m-i.u./ml, added to the perfusate, increased glucose uptake more than 10-fold, from 0.3-0.5 to 5.2-5.4 μmol/min per 30g of muscle in hindquarters of fed and 48h-starved rats respectively. In contrast, it did not stimulate glucose uptake in hindquarters from rats in diabetic ketoacidosis. 3. In the absence of added insulin, isometric exercise, induced by sciatic-nerve stimulation, increased glucose uptake to 4 and 3.4 μmol/min per 30g of muscle in fed and starved rats respectively. It had a similar effect in rats with moderately severe diabetes, but it did not increase glucose uptake in rats with diabetic ketoacidosis or in hindquarters of fed rats that had been “washed out” with an insulin-free perfusate. Insulin, at concentrations which did not stimulate glucose uptake in resting muscle, restored the stimulatory effect of exercise in these situations. 4. The stimulation of glucose uptake by exercise was independent of blood flow and the degree of tissue hypoxia; also it could not be reproduced by perfusing resting muscle with a medium previously used in an exercise experiment. 5. At rest glucose was not detectable in muscle cell water of fed and starved rats even when perfused with insulin. In the presence of insulin, a small accumulation of glucose, 0.25 mM, was noted in the muscle of ketoacidotic diabetic rats, suggesting inhibition of glucose phosphorylation, as well as of transport. 6. During exercise, the calculated intracellular concentration of glucose in the contracting muscle increased to 1.1-1.6mM in the fed, starved and moderately diabetic groups. Insulin significantly increased the already high rates of glucose uptake by the hindquarters of these animals but it did not alter the elevated intracellular concentration of glucose. 7. In severely diabetic rats, exercise did not cause glucose to accumulate in the cell in the absence of insulin. In the presence of insulin, it increased glucose uptake to 6.1 μmol/min per 30g of muscle and intracellular glucose to 0.72 mM. 8. The data indicate that the stimulatory effect of exercise on glucose uptake requires the presence of insulin. They suggest that in the absence of insulin, glucose uptake is not enhanced by exercise owing to inhibition of glucose transport into the cell.
Membrane-lipid unsaturation and mitochondrial function in Saacharomyces cerevisiae
Authors: Watson K , Houghton R L , Bertoli E , Griffiths D E
Published: August 2015

The lipid composition of yeast cells was manipulated by the use of an unsaturated fatty acid auxotroph of Saccharomyces cerevisiae. There was a 2-3-fold decrease in the concentration of cytochromes a+a3 when the unsaturated fatty acid content of the cells was decreased from 60-70% of the total fatty acid to 20-30%. The amounts of cytochromes b and c were also decreased under these conditions, but to a lesser extent. Further lipid depletion, to proportions of less than 20% unsaturated fatty acid, led to a dramatic decrease in the content of all cytochromes, particularly cytochromes a+a3. The ATPase (adenosine triphosphatase), succinate oxidase and NADH oxidase activities of the isolated mitochondria also varied with the degree of unsaturation of the membrane lipids. The lower the percentage of unsaturated fatty acid, the lower was the enzymic activity. Inhibition of mitochondrial ATPase by oligomycin, on the other hand, was not markedly influenced by the membrane-lipid unsaturation. Npn-linear Arrenius plots of mitochondrial membrane-bound enzymes showed transition temperatures that were dependent on the degree of membrane-lipid unsaturation. The greater the degree of lipid unsaturation, the lower was the transition temperature. It was concluded that the degree of unsaturation of the membrane lipids plays an important role in determining the properties of mitochondrial membrane-bound enzymes.
The fractionation of the fatty acid synthetase activities of avocado mesocarp plastids
Authors: Weaire P J , Kekwick R G O
Published: August 2015

1. The range of fatty acids formed by preparations of ultrasonically ruptured avocado mesocarp plastids was dependent on the substrate. Whereas [1-14C]palmitate and [14C]oleate were the major products obtained from [-14C]acetate and [1-14C]acetyl-CoA, the principal product from [2-14C]malonyl-CoA was [14-C]stearate. 2. Ultracentrifugation of the ruptured plastids at 105000g gave a supernatant that formed mainly stearate from [2-14C]malonyl-CoA and to a lesser extent from [1-14C]acetate. The incorporation of [1-14C]acetate into stearate by this fraction was inhibited by avidin. 3. The 105000g precipitate of the disrupted plastids incorporated [1-14C]acetate into a mixture of fatty acids that contained largely [14C]plamitate and [14C]oleate. The formation of [14C]palmitate and [14C]oleate by disrupted plastids was unaffected by avidin. 4. The soluble fatty acid synthetase was precipitated from the 105000g supernatant in the 35-65%-saturated-(NH4)2SO4 fraction and showed an absolute requirement for acyl-carrier protein. 5. Both fractions synthesized fatty acids de novo.
Metabolic interactions of dichloroacetate and insulin in experimental diabetic ketoacidosis. Studies on whole animals and after functional hepatectomy
Authors: Backshear P J , Holloway P A H , Alberti K G M M
Published: August 2015

1. The infusion of sodium dichloroacetate into rats with severe diabetic ketoacidosis over 4h caused a 2mM decrease in blood glucose, and small falls in blood lactate and pyruvate concentrations. Similar findings had been reported in normal rats (Blackshear et al., 1974). In contrast there was a marked decrease in blood ketone-body concentration in the diabetic ketoacidotic rats after dichloroacetate treatment. 2. The infusion of insulin alone rapidly decreased blood glucose and ketone bodies, but caused an increase in blood lactate and pyruvate. 3. Dichloroacetate did not affect the response to insulin of blood glucose and ketone bodies, but abolished the increase of lactate and pyruvate seen after insulin infusion. 4. Neither insulin nor dichloroacetate stimulated glucose disappearance after functional hepatectomy, but both agents decreased the accumulation in blood of lactate, pyruvate and alanine. 5. Dichloroacetate inhibited 3-hydroxybutyrate uptake by the extra-splachnic tissues; insulin reversed this effect. Ketone-body production must have decreased, as hepatic ketone-body content was unchanged by dicholoracetate yet blood concentrations decreased. 6. It was concluded that: (a) dichloroacetate had qualitatively similar effects on glucose metabolism in severely ketotic rats to those observed in non-diabetic starved animals; (b) insulin and dichloroacetate both separately and together, decreased the net release of lactate, pyruvate and alanine from the extra-splachnic tissues, possibly through a similar mechanism; (c) insulin reversed the inhibition of 3-hydroxybutyrate uptake caused by dichloroacetate; (d) dichloroacetate inhibited ketone-body production in severe ketoacidosis.
The control of tricarboxylate-cycle oxidations in blowfly flight muscle. The steady-state concentrations of citrate, isocitrate 2-oxoglutarate and malate in flight muscle and isolated mitochondria
Authors: Johnson R N , Hansford R G
Published: August 2015

1. Blowfly (Phormia regina) flight-muscle mitochondria were allowed to oxidize pyruvate under a variety of experimental conditions, and determinations of the citrate, isocitrate, 2-oxoglutarate and malate contents of both the mitochondria and the incubation medium were made. For each intermediate a substantial portion of the total was present within the mitochondria. 2. Activation of respiration by either ADP or uncoupling agent resulted in a decreased content of citrate and isocitrate and an increased content of 2-oxoglutarate and malate when the substrate was pyruvate, APT and HCO3 minus. Such a decrease in citrate content was obscured when the substrate was pyruvate and proline owing to a large rise in the total content of tricarboxylate-cycle intermediates in the presence of proline and ADP. 3. An experiment involving oligomycin and uncoupling agent demonstrated that the ATP/ADP ratio is the main determinant of flux through the tricarboxylate cycle, with the redox state of nicotinamide nucleotide being of lesser importance. 4. Addition of ADP and Ca-2+ to activate the oxidation of both glycerol 3-phosphate and pyruvate, simulating conditions on initiation of flight, gave a decrease in citrate and isocitrate and an increase in 2-oxoglutarate and malate content. 5. There was a good correlation between these results with isolated flight-muscle mitochondria and the changes found in fly thoraces after 30s and 2 mihorax. 6. It is concluded that NAD-isocitrate dehydrogenase (EC 1.1.1.41) controls the rate of pyruvate oxidation in both resting fly flight muscle in vivo and isolated mitochondria in state 4 (nomenclature of Change & Williams, 1955).
The ribonucleic acids of the mammary gland of the guinea pig
Authors: Ashby P , Campbell P N
Published: August 2015

1. 32-P-labelled polyribosome preparations were made from the mammary glands of lactating and late-pregnant guinea pigs after injection of (32-P)i into the animals. 2. The RNA of polyribosomes, ribosomal subunits and that released from polyribosomes by EDTA were analysed by zone velocity centrifugation and by polyacrylamide-gel electrophoresis. 3. RNA species which have the physical properties expected for the milk protein mRNA were detected. RNA species of a size which could code for the caseins were present in lactating but not in pre-lactating mammary-gland polyribosomes.
The stoicheiometry of the absorption of protons with phosphate and L-glutamate by yeasts of the genus Saccharomyces
Authors: Cockburn M , Earnshaw P , Eddy A A
Published: August 2015

1. A study was made of the pH changes occurring when 0.1-4 mumol of glutamate, phosphate and certain phosphate esters was added at about pH 4.8 to washed cell preparations (50 mg dry wt.) of strains of Saccharomyces. The system also contained deoxyglucose and antimycin to inhibit energy metabolism and so prevent proton ejection from the yeast. 2. A strain of Sacc. carlsbergensis was grown in a chemostat with a limiting supply of phosphate in order to enhance the subsequent rate of phosphate transfer into the yeast. These preparations absorbed 0.2 mumol of phosphate with about 3 equiv. of protons/mol of phosphate. The charge balance was maintained by the efflux of 2 equiv. of K-+ from the yeast. 3. Larger amounts of phosphate were absorbed with fewer proton equivalents. 4. Arsenate and phosphate caused similar pH changes. 5. Glucose 6-phosphate, ATP and certain order phosphate esters each initiated a rise in pH, possibly because hydrolytic extracellular enzymes released phosphate that was subsequently absorbed. 6. Four strains of yeast were grown with glutamate as principal source of nitrogen. Each absorbed extra protons in the presence of L-glutamate. 7. One of them, a strain of Sacc. cerevisiae, absorbed 0.2 mumol of glutamate with 3equiv. of protons/mol of glutamate, and in these circumstances 1-2 equiv. of K-+ left the yeast cells. 8. The role of ionic gradients in the transport of these anions is discussed.
Aspergillus oryzae acid proteinase. Purification and properties, and formation of π-chymotrypsin
Authors: Davidson R , Gertler A , Hofmann T
Published: August 2015

An acid proteinase from Aspergillus oryzae was isolated from a commercial powder by successive (NH4)2SO4 fractionation, acetone precipitation, and ion-exchange chromatography on phosphate- and DEAE-cellulose columns. The purified enzyme was found to be homogeneous by ultracentrifuge-sedimentation analysis (S20, W equal 3.63S), but electrofocusing in polyacrylamide gels and electrophoresis at pH 3.2 revealed that it consists of two very closely migrating bands. No difference in the amino acid composition and enzymic activities of the two partially separated bands could be detected, and it was concluded that the acid proteinase exists in two molecular forms. The enzyme activates bovine trypsinogen and chymotrypsinogen at pH 3.5 (the kappacat. and Km values at 35degrees C are 11.3S- minus 1, 0.10mM and 1.14S- minus 1, 0.18mM respectively). It hydrolyses the Phe-Phe bond of the synthetic pepsin substrates Z-His-Phe-Phe-OEt (kappacat. equal 1.65S- minus 1, Km equal 0.640mM at pH 3.5, 30degrees C) and Z-Ala-Ala-Phe-Phe-OPy4Pr (kappacat. equal 0.37S- minus 1, Km equal 0.037 mM at pH2.9, 39degrees C), where Z represents benzyloxycarbonyl and OPy4Pr represents 3-(4-pyridyl)-propyl 1-ester. Activation of bovine chymotrypsinogen results from the cleavage of the Arg(15)-Ile(16) bond in the zymogen. No other cleavages were observed. The use of A. oryzae proteinase provides a simple tool for the production of pi-chymotrypsin in good yield and purity.