bluuebunny/crossref_metadata_2025 huggingface dataset:
The reaction of aldolase with 2-methylmaleic anhydride
Authors: Gibbons I. , Perham R. N.
Published: August 2015

1. The reaction of rabbit muscle aldolase with 2-methylmaleic anhydride is described. All the protein amino groups can be reversibly blocked. 2. As the reaction proceeds, the enzyme activity decreases until, at about 50% citraconylation of amino groups, the enzyme is completely inhibited. At this stage, little or no dissociation of the enzyme tetramer is observed and 75% of the activity is recoverable on unblocking the amino groups. 3. At 80% blocking, the enzyme is completely dissociated but little enzymic activity is recoverable after unblocking. Inability to recover activity after citraconylation and unblocking correlates with the onset of dissociation of the citraconyl-aldolase seen on ultracentrifugation. 4. The only irreversible modification of the enzyme primary structure detectable after the citraconylation and unblocking reactions is the partial loss of thiol groups. It is probable that this is responsible for the inability to reform active enzyme from the citraconylated subunit. 5. Other reversible side reactions of maleic anhydride and citraconic anhydride that may occur with proteins are discussed.
The composition of milk xanthine oxidase
Authors: Hart L. I. , McGartoll Mary A. , Chapman Helen R. , Bray R. C.
Published: August 2015

The composition of milk xanthine oxidase has been reinvestigated. When the enzyme is prepared by methods that include a selective denaturation step in the presence of sodium salicylate the product is obtained very conveniently and in high yield, and is homogeneous in the ultracentrifuge and in recycling gel filtration. It has specific activity higher than previously reported preparations of the enzyme and its composition approximates closely to 2mol of FAD, 2g-atoms of Mo and 8g-atoms of Fe/mol of protein (molecular weight about 275000). In contrast, when purely conventional preparative methods are used the product is also homogeneous by the above criteria but has a lower specific activity and is generally comparable to the crystallized enzyme described previously. Such samples also contain 2mol of FAD/mol of protein but they have lower contents of Mo (e.g. 1.2g-atom/mol). Amino acid compositions for the two types of preparation are indistinguishable. These results confirm the previous conclusion that conventional methods give mixtures of xanthine oxidase with an inactive modification of the enzyme now termed ‘de-molybdo-xanthine oxidase’, and show that salicylate can selectively denature the latter. The origin of de-molybdo-xanthine oxidase was investigated. FAD/Mo ratios show that it is present not only in enzyme purified by conventional methods but also in ‘milk microsomes’ (Bailie & Morton, 1958) and in enzyme samples prepared without proteolytic digestion. We conclude that it is secreted by cows together with the active enzyme and we discuss its occurrence in the preparations of other workers. Studies on the milks of individual cows show that nutritional rather than genetic factors determine the relative amounts of xanthine oxidase and de-molybdo-xanthine oxidase. A second inactive modification of the enzyme, now termed ‘inactivated xanthine oxidase’, causes variability in activity relative to E450 or to Mo content and formation of it decreases these ratios during storage of enzyme samples including samples free from demolybdo-xanthine oxidase. We conclude that even the best purified xanthine oxidase samples described here and by other workers are contaminated by significant amounts of the inactivated form. This may complicate the interpretation of changes in the enzyme taking place during the slow phase of reduction by substrates. Attempts to remove iron from the enzyme by published methods were not successful.
Purification and properties of human serum cholinesterase
Authors: Das P. K. , Liddell J.
Published: August 2015

Cholinesterase was purified from human serum by a three-stage procedure involving chromatography on DEAE-cellulose at pH4.0, an electrofocusing technique and gel filtration on Sephadex G-200. The final product was purified 13000-fold with a yield of 54%, and only one protein and one cholinesterase band could be demonstrated by polyacrylamide-disc electrophoresis. The catalytic properties appeared to be unchanged by the purification procedure. The molecular weight was determined by both ultracentrifugation in a density gradient and gel filtration, and values close to 366000 were obtained. The isoelectric point of cholinesterase was estimated to be pH3.99. The method appears suitable for the preliminary purification of the rare genetic variants of human cholinesterase.
Metabolism of a glutathione conjugate of 2-hydroxyoestradiol by rat liver and kidney preparations in vitro
Authors: Elce John S.
Published: August 2015

Adult male rat liver and kidney preparations were incubated with (2-hydroxyoestradiol-1-yl)[35S]glutathione. The glutamic acid and glycine residues were removed by enzymes present in the kidney microsomal fraction; the liver preparations had no effect. The resulting 2-hydroxyoestradiol–cysteine conjugate was acetylated at the α-amino group by both liver and kidney homogenates fortified with acetyl-coenzyme A, but not significantly in the absence of this coenzyme, or by liver or kidney slices. These results suggest that an oestrogen–glutathione conjugate, if formed in vivo, would be converted into the corresponding mercapturic acid before excretion.
Effects of ischaemia on metabolite concentrations in rat liver
Authors: Brosnan J. T. , Krebs H. A. , Williamson D. H.
Published: August 2015

1. Changes in the concentrations of ammonia, glutamine, glutamate, 2-oxoglutarate, 3-hydroxybutyrate, acetoacetate, alanine, aspartate, malate, lactate, pyruvate, NAD+, NADH and adenine nucleotides were measured in freeze-clamped rat liver during ischaemia. 2. Although the concentrations of most of the metabolites changed rapidly during ischaemia the ratios [glutamate]/[2-oxoglutarate][NH4+] and [3-hydroxybutyrate]/[acetoacetate] changed equally and the value of the expression [3-hydroxybutyrate][2-oxoglutarate][NH4+]/[acetoacetate][glutamate] remained approximately constant, indicating that the 3-hydroxybutyrate dehydrogenase and glutamate dehydrogenase systems were at near-equilibrium with the mitochondrial NAD+ couple. 3. The value of the expression [alanine][oxoglutarate]/[pyruvate][glutamate] was about 0.7 in vivo and remained fairly constant during the ischaemic period of 5min, although the concentrations of alanine and oxoglutarate changed substantially. No explanation can be offered why the value of the ratio differed from that of the equilibrium constant of the alanine aminotransferase reaction, which is 1.48. 4. Injection of l-cycloserine 60min before the rats were killed increased the concentration of alanine in the liver fourfold and decreased the concentration of the other metabolites measured, except that of pyruvate. During ischaemia the concentration of alanine did not change but that of aspartate almost doubled. 5. After treatment with l-cycloserine the value in vivo of the expression [alanine][oxoglutarate]/[pyruvate][glutamate] rose from 0.7 to 2.4. During ischaemia the value returned to 0.8. 6. The effects of l-cycloserine are consistent with the assumption that it specifically inhibits alanine aminotransferase. 7. Most of the alanine formed during ischaemia is probably derived from pyruvate and from ammonia released by the deamination of adenine nucleotides and glutamine. The alanine is presumably formed by the combined action of glutamate dehydrogenase and alanine aminotransferase. 8. The rate of anaerobic glycolysis, calculated from the increase in the lactate concentration, was 1.3μmol/min per g fresh wt. 9. Although the concentrations of the adenine nucleotides changed rapidly during ischaemia, the ratio [ATP][AMP]/[ADP]2 remained constant at 0.54, indicating that adenylate kinase established near-equilibrium under these conditions.
Biosynthesis of ubiquinone in non-photosynthetic Gram-negative bacteria
Authors: Whistance G. R. , Brown B. S. , Threlfall D. R.
Published: August 2015

1. The polyprenylphenol and quinone complements of the non-photosynthetic Gram-negative bacteria, Pseudomonas ovalis Chester, Proteus mirabilis and ‘Vibrio O1’ (Moraxella sp.), were investigated. 2. Ps. ovalis Chester and Prot. mirabilis were shown to contain 2-polyprenylphenols, 6-methoxy-2-polyprenylphenols, 6-methoxy-2-polyprenyl-1,4-benzoquinones, 5-demethoxyubiquinones, ubiquinones, an unidentified 1,4-benzoquinone [2-polyprenyl-1,4-benzoquinone (?)] and ‘epoxyubiquinones’. ‘Vibrio O1’ was shown to contain only 5-demethoxyubiquinones, ubiquinones and ‘epoxyubiquinones’. 3. It was established that in Ps. ovalis Chester 2-polyprenylphenols, 6-methoxy-2-polyprenylphenols, 6-methoxy-2-polyprenyl-1,4-benzoquinones, 5-demethoxyubiquinones and 2-polyprenyl-1,4-benzoquinones (?) are precursors of ubiquinones. 4. Intracellular distribution studies showed that in Ps. ovalis Chester ubiquinone and its prenylated precursors are localized entirely on the protoplast membrane. 5. Investigations into the oxygen requirements for ubiquinone biosynthesis by Ps. ovalis Chester showed that the organism could not convert p-hydroxybenzoic acid into ubiquinone in the absence of oxygen, although it could convert a limited amount into 2-polyprenylphenols. 6. Attempts were made to prepare cell-free preparations capable of synthesizing ubiquinone. Purified protoplast membranes of Ps. ovalis Chester were found to be incapable of carrying out this synthesis, even when supplemented with cytoplasm. With crushed-cell preparations of Ps. ovalis Chester, organism PC4 (Achromobacter sp.) and Escherichia coli, synthesis was observed, although this was attributable in part to a small number of intact cells present in the preparations.
The stereochemistry of betulaprenol biosynthesis
Authors: Gough Doreen P. , Hemming F. W.
Published: August 2015

By using stereospecifically double-labelled radioactive mevalonates it was shown that betulaprenols-6 to -9, found in the woody tissue of Betula verrucosa, each contain three biogenetically trans-isoprene residues and that the remaining residues are biogenetically cis. The results obtained with these radioactive mevalonates also indicated that the activity of isopentenyl pyrophosphate isomerase is low relative to the activity of prenyltransferase in this woody tissue. The incorporation of radioactive farnesyl pyrophosphate and radioactive geranylnerol and the lack of incorporation of radioactive geranylgeraniol into betulaprenols-6 to -9 demonstrated that they are formed by the cis-additions of isoprene residues to all-trans-farnesyl pyrophosphate.
Some structural features of the teichuronic acid of Bacillus licheniformis N.C.T.C. 6346 cell walls
Authors: Hughes R. C. , Thurman P. F.
Published: August 2015

A teichuronic acid, containing glucuronic acid and N-acetylgalactosamine, was purified from acid extracts of Bacillus licheniformis 6346 cell walls as described by Janczura, Perkins & Rogers (1961). After reduction of the carboxyl function of glucuronic acid residues in the polysaccharide the reduced polymer contains equimolar amounts of N-acetylgalactosamine and glucose. Methylation of the reduced polysaccharide by the Hakamori (1964) technique showed the glucose residues to be substituted on C-4. A disaccharide, 3-O-glucuronosylgalactosamine, was isolated from partial acid hydrolysates of teichuronic acid. After N-acetylation the disaccharide produces chromogen readily on heating at pH7, in agreement with C-3 substitution of the reducing N-acetylamino sugar. Teichuronic acid also produces chromogen under the same conditions, with concurrent elimination of a modified polysaccharide from C-3 of reducing terminal N-acetylgalactosamine residues of the teichuronic acid chains. The number-average chain lengths of several preparations of teichuronic acid were estimated from the amounts of chromogen produced in comparison with the N-acetylated disaccharide. The values obtained are in good agreement with the weight-average molecular weight determined by ultracentrifugal analysis. The reducing terminals of teichuronic acid are shown to be exclusively N-acetylgalactosamine by reduction with sodium boro[3H]hydride. The number-average chain lengths of the teichuronic acid preparations were estimated by the extent of in corporation of tritium and are in agreement with values obtained by the other methods.
The glycosaminoglycans of neonatal rat skin
Authors: Hardingham T. E. , Phelps C. F.
Published: August 2015

The acid glycosaminoglycans were extracted from the skins of young rats less than 1 day post partum. The isolated products were fractionated by a cetylpyridinium chloride–cellulose column technique and identified by chemical analysis, electrophoretic mobility and susceptibility to testicular hyaluronidase digestion. Hyaluronic acid (56%) dermatan sulphate (15.6%) and chondroitin 6-sulphate (9.1%) were the major components, but chondroitin 4-sulphate, heparan sulphate and heparin were also present, together with two further fractions tentatively suggested to be a heparan sulphate-like fraction and a dermatan sulphate fraction, both of short chain length or low degree of sulphation.
Studies on mammalian glucoamylases with special reference to monkey intestinal glucoamylase
Authors: Seetharam B. , Swaminathan N. , Radhakrishnan A. N.
Published: August 2015

1. Highly purified preparations of glucoamylase were obtained from liver, spleen and intestine of the monkey. The enrichment factor was lower for intestine (60-fold) compared with that of liver (1200-fold) and of spleen (2000-fold) but the final specific activities were of a similar magnitude. 2. The liver and spleen enzymes had maximum activity at pH4.8 whereas the intestinal enzyme showed an optimum at pH5.8. The Km values for both starch and maltose with spleen and liver enzymes were higher than for the intestinal enzyme. With the intestinal enzyme, the Vmax. values were higher for both starch and maltose than those of the spleen and liver enzymes. 3. Gel filtration on Sephadex G-200 under identical conditions revealed that liver and spleen enzymes emerge from the columns much later than the intestinal enzyme. 4. Evidence is presented that the glucoamylase activity of the intestinal mucosa is exhibited by the maltase II fraction. 5. Tris, pentaerythritol and turanose inhibited glucoamylase from all the three tissues, but turanose inhibited the spleen and liver enzymes to a higher degree than the intestinal enzyme.
Purification and general properties of glutamate decarboxylase from Clostridium perfringens
Authors: Cozzani I. , Misuri A. , Santoni C.
Published: August 2015

1. A seven-step procedure for preparing highly purified glutamate decarboxylase from Clostridium perfringens is described. 2. The homogeneity of the pure enzyme was established by sucrose-density-gradient centrifugation and starch-gel electrophoresis. 3. The isoelectric point of the pure enzyme is about pH4.5 and the molecular weight is 290000. 4. The pH optimum for activity is 4.7. The pure enzyme is specific for l-glutamate; β-hydroxyglutamate is decarboxylated at a lower rate. 5. Evidence is presented that each mol of enzyme contains 2mol of firmly bound pyridoxal 5-phosphate. 6. Resolution does not occur at acid pH; by dilution with neutral or alkaline buffers the enzyme is inactivated and the coenzyme is released. 7. Reconstitution of active enzyme was obtained by protecting the apoenzyme with thiol compounds.
Oxidative phosphorylation. The specific binding of trimethyltin and triethyltin to rat liver mitochondria
Authors: Aldridge W. N. , Street B. W.
Published: August 2015

1. The binding of trimethyltin and triethyltin to rat liver mitochondria was determined and the results were analysed by the method of Scatchard (1949). 2. One binding site (site 1) has the correct characteristics for the site to which trimethyltin and triethyltin are attached when they inhibit oxidative phosphorylation. For each compound the concentration of site 1 is 0.8nmol/mg of protein and the ratios of their affinity constants are the same as the ratio of the concentrations inhibiting oxidative phosphorylation. 3. Binding site 1 is present in a fraction derived from mitochondria containing only 15% of the original protein. In this preparation ultrasonication rapidly destroyed site 1. 4. Dimethyltin and diethyltin do not prevent binding of triethyltin to rat liver mitochondria, whereas triethyl-lead does. 5. Trimethyltin and triethyltin bind to mitochondria from brown adipose tissue and the results indicate a binding site 1 similar to that in rat liver mitochondria. 6. The advantages and limitations of this approach to the study of inhibitors are discussed.
Purification and properties of the nicotinamide–adenine dinucleotide phosphate-dependent isocitrate dehydrogenase from pig liver cytoplasm
Authors: Illingworth J. A. , Tipton K. F.
Published: August 2015

The NADP-dependent isocitrate dehydrogenase from pig liver soluble fraction was purified over 500-fold with an overall yield of 25%. The purified enzyme, which is homogeneous by all the usual criteria, has a molecular weight of about 75000 and is composed of two identical subunits. This has been demonstrated by ultracentrifugation, fluorescence titration and peptide `fingerprinting'. The maximal turnover number, extinction coefficients at 280nm and 260nm and amino acid analysis are described.
Factors that modify the metabolism of ethanol in rat liver and adaptive changes produced by its chronic administration
Authors: Videla L. , Israel Y.
Published: August 2015

1. 2,4-Dinitrophenol (0.1mm) increases by 100–160% the rate of ethanol metabolism by rat liver slices incubated in a medium saturated with a gas mixture containing O2+CO2+N2 (18:5:77). Similar effects are produced by relatively low concentrations of arsenate (10mm). At higher concentrations (37.5 and 50mm) arsenate inhibits the rate of ethanol metabolism. 2. When liver slices are incubated under an atmosphere containing O2+CO2 (95:5) the metabolism of ethanol increases by about 100% over that obtained with O2+CO2+N2 (18:5:77). However, under these conditions the activating effect of dinitrophenol is no longer observed. 3. Chronic administration of ethanol to rats for 3–4 weeks, in doses from 3 to 8g/kg per day, increases by 70–90% the ability of the liver to metabolize ethanol. In the liver slices of these rats, although an O2+CO2+N2 (18:5:77) mixture was used, dinitrophenol does not further increase the metabolism of ethanol. If the chronic administration of ethanol is discontinued for two weeks, the rate of ethanol metabolism is lowered to control values and the activating effect of dinitrophenol is recovered. 4. No change in alcohol dehydrogenase activity was found in the liver of the rats in which the metabolism of ethanol had been increased as a result of the chronic ethanol treatment; a 40% increase in the activity of succinate dehydrogenase was observed.
Regulation of polyribosome formation and protein synthesis in the uterus. Effect of administration of graded doses of oestradiol-17β and actinomycin D on the amino acid-incorporation activity in vitro and the cytoplasmic concentration in vivo of polyribosomes
Authors: Teng Ching-Sung , Hamilton Terrell H.
Published: August 2015

1. The effects of graded doses of oestradiol-17β and actinomycin D, administered separately or together, on the amino acid-incorporation activity in vitro and the cytoplasmic concentration in vivo of uterine polyribosomes are described. Preparations of polyribosomes isolated from uteri of ovariectomized adult rats were determined for cytoplasmic concentration in vivo and assayed for [14C]leucine-incorporation activity in the cell-free system, exactly as described by Teng & Hamilton (1967b). 2. A minimal dose of 10μg of oestradiol-17β administered for 10h was found to increase, by about 100%, both the amino acid-incorporation activity in vitro and the cytoplasmic concentration in vivo of the polyribosomes. A minimal dose of 250μg of actinomycin D administered for 10h was found to inhibit, by about 50%, the incorporation activity in vitro of the polyribosomes. All doses of the inhibitor administered for 10h failed to alter the cytoplasmic concentration in vivo of the polyribosomes. 3. A dose of 10μg of oestradiol-17β restored to the control value the inhibitory effect of a dose of either 50 or 125μg of actinomycin D on the activity in vitro of the polyribosomes, at 10h after treatment with the inhibitor and the hormone. In these experiments, there was an increase of 60–100% in the cytoplasmic concentration in vivo of the polyribosomes. 4. A dose of 125μg of actinomycin D, administered to animals along with 10μg of oestradiol-17β for 6–36h, abolished the hormone-induced enhancement of the incorporation activity in vitro, but did not prevent an increase of about 200% in the cytoplasmic concentration in vivo of the polyribosomes. However, treatment with 750μg of the inhibitor abolished both stimulatory effects of the hormone. 5. The results reported indicate that the stimulatory effects of oestradiol-17β in vivo on the number and activity of the cytoplasmic polyribosomes in the uterus of the ovariectomized rat have different sensitivities to actinomycin D, but the primary molecular mechanisms responsible for the results are unknown. The major conclusion drawn is that the formation and appearance in the cytoplasm of newly formed polyribosomes are important features of the early action of oestrogen in the uterus.
Amino acid sequences around the disulphide bridges and methionine residues of porcine pepsin
Authors: Tang J. , Hartley B. S.
Published: August 2015

1. The amino acid sequences around three disulphide bridges and four methionine residues of porcine pepsin were studied by using diagonal electrophoresis methods. 2. Two of the three disulphide bridges were in small loops of five and six residues. The sequence around one of the two half-cystine residues of the third disulphide bridge had a large number of acidic residues. 3. The sequence of a tetrapeptide containing phosphoserine was also determined. 4. Four unique methionine-containing sequences were constructed. The information is sufficient for the determination of the overlaps in the cyanogen bromide fragments of pepsin. 5. The usefulness of diagonal methods in the study of protein structure, the relative positions of cystinyl and methionyl residues in porcine pepsin and the homology between pepsin and rennin are discussed.
Rat mammary-gland acetyl-coenzyme A carboxylase. Interaction with milk fatty acids
Authors: Miller A. L. , Geroch Mary E. , Levy H. Richard
Published: August 2015

1. Highly purified rat mammary-gland acetyl-CoA carboxylase was inhibited by milk obtained from rats 12h after their young were weaned. 2. All the inhibitory activity was found in the particulate fraction (R105) obtained on centrifuging the milk. It could be extracted from milk fraction R105 with acetone and identified as a complex mixture of non-esterified fatty acids, present in high concentration (nearly 10mm) in the milk. 3. Inhibition of acetyl-CoA carboxylase was observed at low concentrations (0.2–20μm) of several of these fatty acids when fresh fully active enzyme was used. Enzyme that had been partly inactivated by aging, or by storing in the absence of citrate, was stimulated by low concentrations but inhibited by high concentrations of fatty acids. 4. Various experiments suggested that fatty acids produce irreversible inactivation of acetyl-CoA carboxylase. 5. The effects of palmitoyl-CoA on mammary-gland acetyl-CoA carboxylase were found to resemble those of fatty acids, except that palmitoyl-CoA was effective at lower concentration. 6. The effect of milk fraction R105 was tested on six other enzymes previously shown to decline to various extents after weaning. Although several of these enzymes were affected by unfractionated milk fraction R105, none was significantly inhibited by the acetone extract or by low concentrations of lauric acid. 7. The findings are consistent, both qualitatively and quantitatively, with a regulatory mechanism whereby milk fatty acids shut off fatty acid synthesis in the mammary gland after weaning by inhibiting acetyl-CoA carboxylase.
The action of streptomycin in a mutant of Escherichia coli with increased sensitivity to the antibiotic
Authors: Turnock G.
Published: August 2015

A mutant of Escherichia coli with increased sensitivity to streptomycin has been studied. This strain differed from a normal strs strain in that streptomycin produced inhibition of protein synthesis and loss of viability with almost no lag period. Chloramphenicol protected a normal strs strain but not the mutant against the bactericidal action of streptomycin. The results obtained support the idea that access of streptomycin to its site of action in a normal cell is restricted, and that this restriction, which is much less effective in the mutant, probably involves a permeability barrier. Comparison of the inhibition of protein synthesis by streptomycin with concomitant changes in the distribution of polyribosomes in both strains suggested that the antibiotic can directly inhibit the translation of mRNA.
Nucleotide ‘maps’ of digests of deoxyribonucleic acid
Authors: Murray K.
Published: August 2015

Various digests of 32P-labelled DNA were examined by two-dimensional ionophoresis on cellulose acetate and DEAE-cellulose paper. The products from digestion with pancreatic deoxyribonuclease and Neurospora crassa endonuclease were qualitatively closely similar, but very complex, and were used to investigate the mapping behaviour of nucleotides in various ionophoretic systems. Ionophoresis on DEAE-cellulose paper in triethylamine carbonate, pH 9.7, followed by ionophoresis in the second dimension at pH1.9 gave high resolution of nucleotides in very complex mixtures and permitted the fractionation of larger quantities than is possible on cellulose acetate. High resolution of nucleotides in compact spots was obtained with two-dimensional ionophoresis on cellulose acetate and AE-cellulose paper, a system that is a useful supplement to those based on DEAE-cellulose paper.
The influence of insulin on the metabolism of glucosamine in the isolated rat diaphragm muscle
Authors: Beloff-Chain Anne , Chain E. B. , Rookledge K. A.
Published: August 2015

1. The influence of insulin on the metabolism of [1-14C]glucosamine by diaphragm muscle from normal rats and rats rendered diabetic with streptozotocin has been studied. 2. The glucosamine was converted into glucosamine 1-phosphate, glucosamine 6-phosphate, glycogen, lactate and small amounts of other unidentified intermediates. 3. Insulin increased the incorporation of 14C into glycogen in both the normal and diabetic muscle, but did not increase the formation of the glucosamine phosphate esters. 4. The 14C content in the glycogen was present partly as glucose and partly as glucosamine; there was significantly more [14C]glucose in the glycogen of the diabetic muscle than in that of the normal muscle.
Ketone body and fatty acid metabolism in sheep tissues. 3-Hydroxybutyrate dehydrogenase, a cytoplasmic enzyme in sheep liver and kidney
Authors: Koundakjian Patricia P. , Snoswell A. M.
Published: August 2015

1. 3-Hydroxybutyrate dehydrogenase (EC 1.1.1.30) activities in sheep kidney cortex, rumen epithelium, skeletal muscle, brain, heart and liver were 177, 41, 38, 33, 27 and 17μmol/h per g of tissue respectively, and in rat liver and kidney cortex the values were 1150 and 170 respectively. 2. In sheep liver and kidney cortex the 3-hydroxybutyrate dehydrogenase was located predominantly in the cytosol fractions. In contrast, the enzyme was found in the mitochondria in rat liver and kidney cortex. 3. Laurate, myristate, palmitate and stearate were not oxidized by sheep liver mitochondria, whereas the l-carnitine esters were oxidized at appreciable rates. The free acids were readily oxidized by rat liver mitochondria. 4. During oxidation of palmitoyl-l-carnitine by sheep liver mitochondria, acetoacetate production accounted for 63% of the oxygen uptake. No 3-hydroxybutyrate was formed, even after 10min anaerobic incubation, except when sheep liver cytosol was added. With rat liver mitochondria, half of the preformed acetoacetate was converted into 3-hydroxybutyrate after anaerobic incubation. 5. Measurement of ketone bodies by using specific enzymic methods (Williamson, Mellanby & Krebs, 1962) showed that blood of normal sheep and cattle has a high [3-hydroxybutyrate]/[acetoacetate] ratio, in contrast with that of non-ruminants (rats and pigeons). This ratio in the blood of lambs was similar to that of non-ruminants. The ratio in sheep blood decreased on starvation and rose again on re-feeding. 6. The physiological implications of the low activity of 3-hydroxybutyrate dehydrogenase in sheep liver and the fact that it is found in the cytoplasm in sheep liver and kidney cortex are discussed.
Bilirubin conjugates of human bile. Isolation of phenylazo derivatives of bile bilirubin
Authors: Kuenzle Clive C.
Published: August 2015

A method is presented that allows the isolation of eight different phenylazo derivatives of bile bilirubin. In step I of the isolation procedure, three bilirubin fractions (bilirubin fractions 1, 2 and 3) from human hepatic bile are separated by reverse-phase partition chromatography on silicone-treated Celite with the use of a solvent system prepared from butan-1-ol and 5mm-phosphate buffer, pH6.0. Azo coupling is then performed with diazotized aniline. The three azo pigment mixtures are subjected to step II, in which the above chromatography system is used again. With each azo pigment mixture this step brings about the separation of a non-polar and a polar azo pigment fraction (azo 1A and azo 1B, azo 2A and azo 2B, and azo 3A and azo 3B from bilirubin fractions 1, 2 and 3 respectively). Approximately equal amounts of non-polar and polar pigments are obtained from bilirubin fractions 1 and 2, whereas bilirubin fraction 3 yields azo 3B almost exclusively. In step IIIA the non-polar azo pigment fractions are fractionated further by adsorption chromatography on anhydrous sodium sulphate with the use of chloroform followed by a gradient of ethyl acetate in chloroform. Three azo pigments are thus obtained from both azo 2A (azo 2A1, azo 2A2 and azo 2A3) and azo 3A (azo 3A1, azo 3A2 and azo 3A3). The 2A pigments occur in approximately the following proportions: azo 2A1, 90%; azo 2A2, 10%; azo 2A3, traces. The pigments are purified by crystallization, except for the A3 pigments, which are probably degradation products arising from the corresponding A2 pigments. In step IIIB the polar azo pigment fractions are subjected to reverse-phase partition chromatography on silicone-treated Celite with the use of a solvent system prepared from octan-1-ol–di-isopropyl ether–ethyl acetate–methanol–0.2m-acetic acid (1:2:2:3:4, by vol.). Azo pigment fractions 2B and 3B each yield six azo pigments (azo 2B1 to azo 2B6 and azo 3B1 to azo 3B6 respectively) together with small amounts of products of hydrolysis (azo 2AB and azo 3AB). Only one azo B pigment is obtained from bilirubin fraction 1, and this azo pigment is probably of the B2 type. The yields of the azo 3B pigments suggest that these pigments are present in approximately the following proportions: azo 3B1, 0–0.4%; azo 3B2, traces; azo 3B3, traces; azo 3B4, 10%; azo 3B5, 50%; azo 3B6, 40%. Azo pigments 2B1 to 2B6 are estimated to occur in similar proportions. Since pairs of correspondingly numbered azo pigments from bilirubin fractions 1, 2 and 3 do not separate on rechromatography together (e.g. azo 2A1 co-chromatographs with azo 3A1, and azo 2B6 co-chromatographs with azo 3B6), it is concluded that such pigments are chemically identical. The structures of the isolated phenylazo derivatives are discussed in an accompanying paper (Kuenzle 1970c).
Turnover of mammalian phospholipids. Rates of turnover and metabolic heterogeneity in cultured human lymphocytes and in tissues of healthy, starved and vitamin A-deficient rats
Authors: Pasternak C. A. , Friedrichs Beverly
Published: August 2015

1. Choline- and inositol-labelled phospholipids of human cultured lymphocytes turn over in a biphasic manner; phytohaemagglutinin activation stimulates turnover. 2. Choline-labelled phospholipids of rat liver and kidney, but not of blood, turn over in vivo as fast as those of duodenum, ileum or colon. Turnover in the intestinal tissues is greater in fed than in starved or vitamin A-deficient rats. In each case phosphatidylcholine turns over relatively faster than sphingomyelin or lyso-phosphatidylcholine. 3. It is concluded that phospholipid turnover of the type described is a common feature of viable cells, and that metabolically favourable conditions increase, rather than decrease, turnover.
The use of conventional and zonal centrifugation to study the life cycle of mammalian cells. Phospholipid and macromolecular synthesis in neoplastic mast cells
Authors: Warmsley A. M. H. , Pasternak C. A.
Published: August 2015

1. Conventional gradient centrifugation has been used to separate cells according to their position in the cell cycle, and to obtain synchronously growing cells. Analysis of prelabelled cells by gradient centrifugation confirms that phospholipid, protein and RNA synthesis is continuous throughout the cell cycle and shows that the rate of synthesis begins to increase already during the G1 phase. The pattern of phospholipid degradation follows that of synthesis. 2. The limitations of conventional gradient centrifugation have been overcome by use of a zonal rotor. Analysis of prelabelled cells confirms the results obtained by conventional centrifugation and in addition shows that the rates of phospholipid, protein and RNA synthesis decrease during the G2 phase. The mean cell volume and the net amount of phospholipid, protein and RNA, unlike that of DNA, are found to increase continuously throughout the intermitotic period. 3. These results show that the synthesis of macromolecules, and probably that of membranes also, is controlled by a mechanism other than that of gene dosage.
Partial purification and properties of guinea-pig liver polynucleotide phosphorylase
Authors: See Yew Phew , Fitt P. S.
Published: August 2015

1. Polynucleotide phosphorylase has been isolated and partially purified from crude preparations of guinea-pig liver nuclei. 2. The enzyme is particulate and associated with RNA and lipids characteristic of membranes. 3. It has phosphorolysis and exchange activities, but the latter may be due to a contaminating enzyme. 4. The phosphorolysis activity is dependent on bivalent cations, preferably Mg2+, has a pH optimum between 8.6 and 9.2 and is inhibited by potassium chloride and sodium chloride. 5. The enzyme catalyses phosphorolysis of poly A, poly C, poly U, rRNA and tRNA. Poly G is only phosphorolysed to a very small extent and DNA is not a substrate. 6. The enzyme appears to lack nucleoside diphosphate polymerization activity.
The amino acid sequence of Helianthus annuus L (sunflower) cytochrome c deduced from chymotrypic peptides
Authors: Ramshaw J. A. M. , Thompson E. W. , Boulter D.
Published: August 2015

Peptides derived from digestion of 1 μmol of sunflower cytochrome c with chymotrypsin were separated by paper electrophoresis. The sequences of these peptides were determined by using the dansyl–Edman method (Gray & Hartley, 1963) and confirmed by analysis of their amino acid composition. Comparison of the set of peptides with the chymotryptic peptides of mung-bean (Thompson, Laycock, Ramshaw & Boulter, 1970) and wheat germ (Stevens, Glazer & Smith, 1967) cytochrome c shows a clear homology. The complete sequence of sunflower cytochrome c was established by alignment of the sunflower peptides with the sequences of mung bean cytochrome c and wheat germ cytochrome c.
Immunochemical studies with soluble and mitochondrial pyruvate carboxylase activities from rat tissues
Authors: Ballard F. J. , Hanson R. W. , Reshef Lea
Published: August 2015

1. Pyruvate carboxylase (EC 6.4.1.1), purified from rat liver mitochondria to a specific activity of 14 units/mg, was used for the preparation of antibodies in rabbits. 2. Tissue distribution studies showed that pyruvate carboxylase was present in all rat tissues that were tested, with considerable activities both in gluconeogenic tissues such as liver and kidney and in tissues with high rates of lipogenesis such as white adipose tissue, brown adipose tissue, adrenal gland and lactating mammary gland. 3. Immunochemical titration experiments with the specific antibodies showed no differences between the inactivation of pyruvate carboxylase from mitochondrial or soluble fractions of liver, kidney, mammary gland, brown adipose tissue or white adipose tissue. 4. The antibodies were relatively less effective in reactions against pyruvate carboxylase from sheep liver than against the enzyme from rat tissues. 5. Pyruvate carboxylase antibodies did not inactivate either propionyl-CoA carboxylase or acetyl-CoA carboxylase from rat liver. 6. It is concluded that pyruvate carboxylase in lipogenic tissues is similar antigenically to the enzyme in gluconeogenic tissues and that the soluble activities of pyruvate carboxylase detected in many rat tissues do not represent discrete enzymes but are the result of mitochondrial damage during tissue homogenization.
Characteristics of deoxyribonucleic acid polymerase activity in nuclear and supernatant fractions of cultured mouse cells
Authors: Lindsay J. G. , Berryman S. , Adams R. L. P.
Published: August 2015

1. DNA polymerase activity is present in both nuclear and supernatant fractions prepared from rapidly dividing L929 mouse cells. 2. Nuclear preparations are 2–5 times more active with added native DNA as template and the supernatant fractions show an equivalent preference for heat-denatured DNA. 3. Isolated nuclei can carry on limited DNA synthesis in the absence of added template but are stimulated five- to ten-fold by addition of 50μg of native DNA per assay. 4. DNA polymerase activity can be released from intact nuclei by ultrasonic treatment or by extraction with 1.5m-potassium chloride. 5. The activities in nuclear and supernatant fractions, with their preferred templates, respond similarly to changes in pH and Mg2+ and K+ concentrations. 6. Maximal enzyme activity is approached with 40μg of DNA per assay and activation of the DNA template by treatment with deoxyribonuclease does not decrease the amount of DNA required to reach saturation. 7. The nuclear enzyme, incubated with native DNA, is markedly inhibited by the addition of heat-denatured DNA to the assay. In contrast, the supernatant DNA polymerase activity on denatured templates is not affected by the presence of native DNA. 8. The nuclear enzyme exhibits high activity in the absence of one or more deoxyribonucleoside triphosphates but this is much diminished after partial purification of the enzyme by precipitation at pH5 and fractionation on Sephadex G-200 columns. 9. The 3H-labelled DNA products formed by Sephadex-purified nuclear and supernatant fractions, with their preferred templates, were found to be resistant to treatment with exonuclease I. Alkali-denaturation of the 3H-labelled DNA products rendered them susceptible to attack by exonuclease I. 10. Analysis of the products on alkaline sucrose density gradients suggests that the newly synthesized material may not be covalently bound to the original DNA template. 11. By using their preferred templates the specific activity of supernatant fractions varies markedly with the position of the cells in the cell-cycle, but the specific activity of nuclear fractions varies only slightly.
The role of bound potassium ions in the hydrolysis of low concentrations of adenosine triphosphate by preparations of membrane fragments from ox brain cerebral cortex
Authors: Goldfarb P. S. G. , Rodnight R.
Published: August 2015

1. The intrinsic Na+, K+, Mg2+ and Ca2+ contents of a preparation of membrane fragments from ox brain were determined by emission flame photometry. 2. Centrifugal washing of the preparation with imidazole-buffered EDTA solutions decreased the bound Na+ from 90±20 to 24±12, the bound K+ from 27±3 to 7±2, the bound Mg2+ from 20±2 to 3±1 and the bound calcium from 8±1 to <1nmol/mg of protein. 3. The activities of the Na++K++Mg2+-stimulated adenosine triphosphatase and the Na+-dependent reaction forming bound phosphate were compared in the unwashed and washed preparations at an ATP concentration of 2.5μm (ATP/protein ratio 12.5pmol/μg). 4. The Na+-dependent hydrolysis of ATP as well as the plateau concentration of bound phosphate and the rate of dephosphorylation were decreased in the washed preparation. The time-course of formation and decline of bound phosphate was fully restored by the addition of 2.5μm-magnesium chloride and 2μm-potassium chloride. Addition of 2.5μm-magnesium chloride alone fully restored the plateau concentration of bound phosphate, but the rate of dephosphorylation was only slightly increased. Na+-dependent ATP hydrolysis was partly restored with 2.5μm-magnesium chloride; addition of K+ in the range 2–10μm-potassium chloride then further restored hydrolysis but not to the control rate. 5. Pretreatment of the washed preparation at 0°C with 0.5nmol of K+/mg of protein so that the final added K+ in the reaction mixture was 0.1μm restored the Na+-dependent hydrolysis of ATP and the time-course of the reaction forming bound phosphate. 6. The binding of [42K]potassium chloride by the washed membrane preparation was examined. Binding in a solution containing 10nmol of K+/mg of protein was linear over a period of 20min and was inhibited by Na+. Half-maximal inhibition of 42K+-binding required a 100-fold excess of sodium chloride. 7. It was concluded (a) that a significant fraction of the apparent Na+-dependent hydrolysis of ATP observed in the unwashed preparation is due to activation by bound K+ and Mg2+ of the Na++K++Mg2+-stimulated adenosine triphosphatase system and (b) that the enzyme system is able to bind K+ from a solution of 0.5μm-potassium chloride.
Nucleic acid synthesis and nucleotide pools in purine-deficient Escherichia coli
Authors: Thomas Gillian A. , Varney N. F. , Burton K.
Published: August 2015

1. The synthesis of nucleic acids and the content of purine nucleotides have been studied in selected purine-requiring strains of Escherichia coli including a purB− strain and a purB−guaA− strain. 2. When the exogenous purines can be converted into GTP but not into ATP, RNA is synthesized at the expense of intracellular ATP, ADP and AMP. 3. Net synthesis of RNA as measured by the incorporation of uracil can be correlated with the availability of GTP except when ATP falls to a very low concentration. 4. Nicotinamide nucleotides are not an important reservoir of adenine nucleotides for RNA synthesis.
The interaction of triethyltin with a component of guinea-pig liver supernatant. Evidence for histidine in the binding sites
Authors: Rose M. S. , Lock E. A.
Published: August 2015

A protein fraction was isolated from guinea-pig liver that binds triethyltin with an affinity of approx. 2×106m−1 at pH8.0. It was shown that the protein responsible for binding 70% of the triethyltin found in guinea-pig liver after injection of radioactively labelled triethyltin is at most a few per cent of the total liver protein. Evidence is presented from the kinetics of loss of binding and loss of certain amino acids on photo-oxidation with either Methylene Blue or Rose Bengal that each binding site consists of two histidine residues.
The composition of triglycerides from liver, egg yolk and adipose tissue of the laying hen
Authors: Husbands D. R.
Published: August 2015

The composition of the triglycerides of liver, egg yolk and adipose tissue of laying hens fed on a standard diet were investigated by using argentation thin-layer chromatography to separate the triglycerides according to their degree of unsaturation. About 40% of liver triglycerides consisted of one saturated and two monoenoic fatty acids. Triglycerides containing linoleate were more abundant in adipose tissue than in either yolk or liver. Hydrolysis by pancreatic lipase of the tissue triglycerides and fractions obtained from these triglycerides showed that the triglycerides of adipose tissue had a less ordered arrangement of fatty acids at the 2-position than did either yolk or liver triglycerides. The labelling patterns of triglycerides formed in liver slices incubated in the presence of [1-(3)14C]glycerol indicated that triglycerides containing four or more double bonds are formed to a greater extent than are other triglyceride fractions. This is evidence for the concept that the type of triglyceride formed depends on the availability of fatty acids to the liver cells.
The development of gluconeogenesis in rat liver. Effects of glucagon and ether
Authors: Philippidis Helen , Ballard F. J.
Published: August 2015

1. Administration of glucagon to foetal rats produced a 10–15-fold increase in hepatic phosphoenolpyruvate carboxykinase activity together with a similar increase in the overall pathway of pyruvate conversion into glycogen in liver slices. 2. Glucagon was without effect on gluconeogenesis in vivo, which remained at approx. 0.1% of the incorporation as measured in newborn animals. 3. The apparent discrepancy between these results was due to the ether anaesthesia that was required for experimentation in vivo. Under conditions when minimal ether was used, the rates of labelling of glycogen from [3-14C]pyruvate in vivo were increased 10–20-fold and there was an additional stimulus by glucagon. 4. Ether anaesthesia produced a more reduced redox state of the foetal liver cytosol and lowered the ATP/ADP concentration ratio. 5. It is proposed that these effects are significant in the limitation of gluconeogenesis in the foetal rat liver, so that only with high phosphoenolpyruvate carboxykinase activity, high ATP concentration and a relatively oxidized cytosol redox state will a functional gluconeogenic pathway be present.
Bacteriolytic enzymes from Staphylococcus aureus. Specificity of action of endo-β-N-acetylglucosaminidase
Authors: Wadström T. , Hisatsune K.
Published: August 2015

The bacteriolytic enzyme with an isoelectric point of 9.5 that is produced by all strains of Staphylococcus aureus investigated was purified from strain M18 (Wadström & Hisatsune, 1970). This enzyme released reducing groups from cell walls of Micrococcus lysodeikticus and was thus shown to be a bacteriolytic hexosaminidase. Although dinitrophenylation and acid hydrolysis of cell walls hydrolysed by a partially purified enzyme gave DNP-alanine and DNP-glycine from staphylococcal peptidoglycan, which indicated the presence of a peptidase and probably also an N-acetylmuramyl-l-alanine amidase, hydrolysis of cell walls by the extensively purified enzyme did not give any DNP-amino acids. The enzyme digest was purified by Amberlite CG-120 and Sephadex G-10 chromatography. Reduction by sodium borohydride of the disaccharide obtained was followed by acid hydrolysis and paper chromatography. Glucosamine completely disappeared after this treatment and a new spot identical with glucosaminitol appeared. The muramic acid spot remained unchanged. The purified enzyme was found to be devoid of exo-β-N-acetylglucosaminidase activity. These results are compatible with the action of a bacteriolytic endo-β-N-acetylglucosaminidase. It is also proposed that this enzyme is probably identical with the staphylococcal lysozyme. The mode of action of this has not previously been investigated.