bluuebunny/crossref_metadata_2025 huggingface dataset:
The purification and properties of a ribonucleoenzyme, o-diphenol oxidase, from potatoes
Authors: Balasingam K. , Ferdinand W.
Published: August 2015

1. o-Diphenol oxidase was isolated from potato tubers by a new approach that avoids the browning due to autoxidation. 2. There are at least three forms of the enzyme, of different molecular weights. The major form, of highest molecular weight, was separated from the others in good yield and with high specific activity by gel filtration through Bio-Gel P-300. 3. The major form is homogeneous by disc electrophoresis but regenerates small amounts of the species of lower molecular weight, as shown by rechromatography on Bio-Gel P-300. 4. There is an equal amount of RNA and protein by weight in the fully active enzyme. The RNA cannot be removed without loss of activity, and is not attacked by ribonuclease. 5. The pH optimum of the enzyme is at pH5.0 when assayed with 4-methylcatechol as substrate. It is ten times more active with this substrate than with chlorogenic acid or catechol. The enzyme is fully active in 4m-urea. 6. A minimal molecular weight of 36000 is indicated by copper content and amino acid analysis of the protein component of the enzyme. 7. The protein contains five half-cystinyl residues per 36000 daltons, a value similar to that found in o-diphenol oxidase from mushrooms. It also contains tyrosine residues although, when pure, it does not turn brown by autoxidation.
Preferential alkylation of mitochondrial deoxyribonucleic acid by N-methyl-N-nitrosourea
Authors: Wunderlich V. , Schütt M. , Böttger M. , Graffi A.
Published: August 2015

The reaction of the carcinogen N-methyl-N-nitrosourea with mitochondrial DNA from various rat tissues was examined in vivo and in vitro. After incubation of isolated mitochondria or cell nuclei with N[14C]-methyl-N-nitrosourea in vitro and subsequent isolation and purification of the DNA the specific radioactivity of the mitochondrial DNA was 3–7 times that of the nuclear DNA. The incorporation of 14C into embryonic mitochondrial DNA in vitro was about twice that into the liver mitochondrial DNA. Identical incorporation rates, however, were found for the reaction of isolated mitochondrial DNA or nuclear DNA respectively with N[14C]-methyl-N-nitrosourea. After intraperitoneal injection of 43.3–58.5mg of N[14C]-methyl-N-nitrosourea/kg body wt. to adult rats the labelling of the mitochondrial DNA was on average 5 times that of the nuclear DNA. A smaller specific labelling was observed for the ribosomal RNA, transfer RNA, and mitochondrial RNA as well as for the mitochondrial protein as compared with the mitochondrial DNA. After hydrolysis of the alkylated nucleic acids with hydrochloric acid, fractionation was carried out on Dowex 50 cation-exchange columns. In most experiments 70–80% of the input 14C radioactivity was eluted in the 7-methylguanine fraction. The preferential alkylation of the mitochondrial DNA by N-methyl-N-nitrosourea in situ is discussed in connexion with the cytoplasmic-mutation hypothesis of carcinogenesis.
The characterization and stereochemistry of biosynthesis of dolichols in rat liver
Authors: Gough Doreen P. , Hemming F. W.
Published: August 2015

It was shown that rat liver contains a series of dolichols with chain lengths of from 17 (or possibly 16) to 21 isoprene residues, the main constituent of the mixture being dolichol-18. By using double-labelled radioactive mevalonates it was demonstrated that each of these dolichols possesses three biogenetically trans-isoprene residues and that the remaining residues are biogenetically cis, suggesting that these polyprenols are biosynthesized from all-trans-farnesyl pyrophosphate by the cis additions of isoprene residues, followed by saturation of the α-isoprene residue. The results obtained with these radioactive mevalonates also indicated that the activity of isopentenylpyrophosphate isomerase is low relative to the activity of prenyltransferase in rat liver.
Regulation of lipogenesis. Stimulation of fatty acid synthesis in vivo and in vitro in the liver of the newly hatched chick
Authors: Goodridge Alan G.
Published: August 2015

1. A single glucose meal stimulated the incorporation of acetate into fatty acids in liver slices. If the glucose was added in vitro, it had no effect. Fructose and glycerol in vitro markedly stimulated fatty acid synthesis from acetate. Fructose and glycerol probably by-passed a rate-controlling reaction between glucose and triose phosphate. This reaction may have been stimulated by glucose administered in vivo. 2. The stimulation of fatty acid synthesis caused by fructose did not require the synthesis of enzyme, thus indicating that fatty acid-synthesizing enzymes were present in a latent form in the livers from unfed chicks.
Differences between manganese and magnesium ions with regard to fatty acid biosynthesis, acetyl-coenzyme A carboxylase activity and malonyl-coenzyme A decarboxylation
Authors: Scorpio R. M. , Masoro E. J.
Published: August 2015

Fatty acid-biosynthetic activity in rat liver cytosol fractions is much greater when the bivalent cation in the assay system is Mn2+ than when it is Mg2+. This difference between bivalent cations can be abolished if the cytosol fractions are preincubated with isocitrate and the bivalent cation for 30min before assay of fatty acid-biosynthetic activity. In a search for the biochemical basis of this phenomenon, the following differences between Mg2+ and Mn2+ were established: (1) Mn2+ promotes acetyl-CoA carboxylase activity of the protomeric form of the enzyme under conditions in which Mg2+ does not; (2) Mn2++ATP have little inhibitory effect on the polymerization of acetyl-CoA carboxylase whereas Mg2++ATP are markedly inhibitory; (3) under conditions in which utilization of malonyl-CoA in condensation reactions is prevented, the steady-state concentration of malonyl-CoA formed by a cytosol fraction is much greater with Mn2+ than with Mg2+. The role that each of these specific differences between Mn2+ and Mg2+ might play in causing liver cytosol preparations to have greater fatty acid-biosynthetic activity in the presence of Mn2+ is discussed.
An electrophoretic study of the low-molecular-weight components of myosin
Authors: Perrie W. T. , Perry S. V.
Published: August 2015

1. The low-molecular-weight components of myosin freshly prepared by the standard procedure from adult rabbit skeletal muscle migrated as four main bands Ml1, Ml2, Ml3 and Ml4 on polyacrylamide-gel electrophoresis in 8m-urea. 2. The number of bands increased on storage. This change was accelerated by increasing the temperature and pH. 3. None of the bands had electrophoretic mobilities identical with those of the well-characterized proteins of the myofibril or with the sarcoplasmic proteins. 4. By varying the ionic conditions and concentration of muscle mince used for the initial extraction it was possible to change the relative proportions of the two electrophoretic bands of intermediate mobility, Ml2 and Ml3. 5. The four-band picture similar to that obtained with rabbit was observed with myosin isolated from skeletal muscle of the rat, mouse, hamster, pigeon and chicken. 6. Rabbit cardiac myosin gave only two bands on electrophoresis. Myosin from rabbit red muscle gave a pattern intermediate between cardiac and white-skeletal-muscle myosin, i.e. the two fastest bands were present in decreased relative amounts. 7. It is suggested that the differences in the low-molecular-weight components of myosin from different types of muscle are a consequence of differences in the isoenzyme composition of the myosins.
The action of o-dihydric phenols in the hydroxylation of p-coumaric acid by a phenolase from leaves of spinach beet (Beta vulgaris L.)
Authors: Vaughan P. F. T. , Butt V. S.
Published: August 2015

1. Under defined conditions, the hydroxylation of p-coumaric acid catalysed by a phenolase from leaves of spinach beet (Beta vulgaris L.) was observed to develop its maximum rate only after a lag period. 2. By decreasing the reaction rate with lower enzyme concentrations or by increasing it with higher concentrations of reductants, the length of the lag period was inversely related to the maximum rate subsequently developed. 3. Low concentrations of caffeic acid or other o-dihydric phenols abolished this lag period. With caffeic acid, the rate of hydroxylation was independent of the reductant employed. 4. Hydroxylation was inhibited by diethyldithiocarbamate, but with low inhibitor concentrations hydroxylation recovered after a lag period. This lag could again be abolished by the addition of high concentrations of caffeic acid or other o-dihydric phenols. 5. Catechol oxidase activity showed no lag period, and did not recover from diethyldithiocarbamate inhibition. 6. The purified enzyme contained 0.17–0.33% copper; preparations with the highest specific activity were found to have the highest copper content. 7. The results are interpreted to suggest that the oxidation of o-dihydric phenols converts the enzymic copper into a species catalytically active in hydroxylation. This may represent the primary function for the catechol oxidase activity of the phenolase complex. The electron donors are concerned mainly, but not entirely, in the reduction of o-quinones produced in this reaction.
A colorimetric determination of inositol monophosphates as an assay for d-glucose 6-phosphate–1l-myoinositol 1-phosphate cyclase
Authors: Barnett J. E. G. , Brice R. E. , Corina D. L.
Published: August 2015

A rapid and convenient chemical assay for the enzyme d-glucose 6-phosphate–1l-myoinositol 1-phosphate cyclase is described. The 1l-myoinositol 1-phosphate formed enzymically was oxidized with periodic acid liberating inorganic phosphate, which was assayed. myoInositol 2-phosphate can be assayed in the same way. Glucose 6-phosphate and other primary phosphate esters gave only very small quantities of inorganic phosphate under the conditions described. The Km of the enzyme for d-glucose 6-phosphate, 7.5±2.5×10−4m, was identical with that measured by the radiochemical method. 2-Deoxy-d-glucose 6-phosphate was a powerful competitive inhibitor, Ki 2.0±0.5×10−5m, but was not a substrate for the enzyme.
Metabolism of methyldecalins
Authors: Robertson J. S. , Champion D. I.
Published: August 2015

1. Oral administration of trans- and cis-2-methyldecalin to rabbits increased the urinary glucuronide content. 2. The metabolite of the trans isomer was isolated and shown to be either trans-cis-6- or -7-methyl-2-decalol. The metabolite of the cis isomer was shown to be a 1:1 mixture of cis-6-methyl-2-decalol and cis-7-methyl-2-decalols, but the conformations of the hydroxyl groups were not determined. 3. It was concluded that a specific hydroxylase was responsible for the oxidations observed, and that this hydroxylase could be responsible for the metabolic oxidation of other simple alicyclic compounds.
Turnover of mammalian phospholipids. Stable and unstable components in neoplastic mast cells
Authors: Pasternak C. A. , Bergeron J. J. M.
Published: August 2015

1. Choline- and inositol-labelled phospholipids of exponentially growing or static neoplastic mast cells turn over by degradation and resynthesis of the entire molecule. Turnover follows a biphasic pattern, the unstable rapidly turning-over component accounting for 60–80% of labelled phospholipid. The residual stable component does not turn over any more than does protein or DNA. 2. Subcellular fractions and surface membranes of choline-labelled P815Y cells contain the same proportion of stable and unstable components as do whole cells. The unstable component is largely phosphatidylcholine; the stable component is relatively richer in sphingomyelin. 3. It is concluded that the phospholipids of neoplastic mast cells are of two classes, one of which is susceptible to continual enzymic degradation and resynthesis, and the other of which is metabolically stable.
Phospholipid synthesis and degradation during the life-cycle of P815Y mast cells synchronized with excess of thymidine
Authors: Bergeron J. J. M. , Warmsley A. M. H. , Pasternak C. A.
Published: August 2015

1. P815Y cells synchronized with excess of thymidine incorporate choline, proline and uridine throughout the cell cycle; the rate increases two- to four-fold during the S phase, when thymidine incorporation increases more than 15-fold. 2. Choline incorporated at any stage of the cell cycle turns over in a biphasic manner; stable and unstable components are each labelled maximally during the S phase. Total phospholipid also doubles predominantly during the S phase. 3. It is concluded that, despite turnover, choline incorporation is a useful measure of net phospholipid formation during the cell cycle.
The release of radioactive nucleic acids and mucoproteins by trypsin and ethylenediaminetetra-acetate treatment of baby-hamster cells in tissue culture
Authors: Snow Christine , Allen A.
Published: August 2015

Monolayers of baby-hamster kidney cells were grown on glass in tissue culture and harvested with trypsin or EDTA in order to investigate the cell surface macromolecules removed by these cell-disaggregating agents. The release of nucleic acids from the cells during the harvesting procedure was monitored by labelling the cellular RNA with [5-3H]uridine and the cellular DNA with [2-14C]thymidine. Treatment of the cells with EDTA was found to cause an increase in the permeability of the plasma membrane with 7.6% of the cellular RNA, but less than 1% of the cellular DNA, being released. Moreover, 61% of the cells harvested with EDTA were permeable to Trypan Blue. With crude trypsin, lysis of the cell occurred with the release of similar amounts of RNA and DNA amounting to about 11% of the total cellular nucleic acid. In contrast, crystalline trypsin released only 1% of the cellular nucleic acids. Since virtually all the cells (99%) after harvesting in crystalline trypsin were impermeable to Trypan Blue, this method was suitable for obtaining cell surface macromolecules without contamination by intracellular damage. [1-14C]Glucosamine was incorporated by the cells only into bound hexosamines and sialic acids. [By monitoring the release of radioactivity in high-molecular-weight material in such experiments a measure of the release of macromolecules containing amino sugars was obtained.] Of the total macromolecules containing amino sugars in the cells 33%, 24% and 13% were released when the cells were harvested with crude trypsin, crystalline trypsin or EDTA respectively. Crystalline trypsin also released 39% of the total sialic acid of the cell, whereas less than 1% of the cellular sialic acid was present in the EDTA-treated fraction. It is concluded that the macromolecules containing amino sugars released with crude trypsin and EDTA are likely to be heavily contaminated with intracellular material. However, the macromolecules released by crystalline trypsin appear to come from the cell surface.
Molecular hybridization between rat liver deoxyribonucleic acid and complementary ribonucleic acid
Authors: Melli Marialuisa , Bishop J. O.
Published: August 2015

RNA (cRNA) was synthesized in vitro on a template of rat liver DNA and its hybridization with rat liver DNA was studied by using the nitrocellulose-filter method. Sonication of the DNA diminished its apparent capacity to hybridize with RNA by about 50%. This is not due to cross-linkage of DNA molecules, because it could be shown that less than 2% of the sonicated DNA was cross-linked. The effect is due instead to the small size of the sonicated DNA molecules. Below a single-stranded molecular weight of 5×105 the DNA showed a progressive loss of capacity to hybridize with decrease in molecular weight. Evidence is presented suggesting that the apparently diminished capacity of the DNA to hybridize is due to loss of hybridized DNA from the membrane filters. When cRNA at concentrations of up to 25μg/ml is annealed with sonicated total DNA, an apparent hybridization saturation value is found at which about 2.5% of the DNA is hybridized with RNA. Increasing the cRNA concentration tenfold brought about the hybridization of a second component of the DNA approximately equal in amount to the first. The renaturation of rat liver DNA was studied by measuring the fall in the extinction at 260nm and two different components of renaturation were observed within the reiterated fraction of DNA. By hybridizing cRNA with different fractions of rat DNA the two components of the hybridization curve are shown to correspond to the two components of the renaturation curve. The conclusion is drawn that at a cRNA concentration of 250μg/ml most of the reiterated fraction of rat liver DNA is hybridized after annealing for 16h under standard conditions (0.30m-sodium chloride–30mm-sodium citrate at 65°C). Even with such a high cRNA concentration little or no hybridization of the slowly renaturing DNA fraction occurs. It is suggested that the most highly reiterated DNA component is poorly transcribed in vitro.
Effects of sex hormones on enzymic esterification of 2-(N-hydroxyacetamido)-fluorene by rat liver cytosol
Authors: Lotlikar Prabhakar D.
Published: August 2015

1. Enzymic esterifications of 2-(N-hydroxyacetamido)fluorene and several other hydroxamic acids by liver cytosol were studied. Determination of 2-acetamido-3-methylthiofluorene was used for the assay. 2. With rat liver cytosol, requirement for ATP, Mg2+ and SO42− suggested formation of phosphate and sulphate esters of 2-(N-hydroxyacetamido)fluorene. 3. Rats showed sex and age differences in their activity. Liver from adult male rat was at least twice as active as liver from adult female rat. No such sex differences were found in mice, hamsters and guinea pigs. 4. Administration of testosterone (300μg/day) subcutaneously for 8 days increased the activity in the female rat by 100%, whereas diethylstilboestrol (100μg/day) had no effect. In the male rat diethylstilboestrol treatment decreased the activity by 60%, whereas testosterone pretreatment was without any effect. 5. Among various endocrine ablations such as adrenalectomy, castration, adrenalectomy–castration and hypophysectomy in the adult male rat, hypophysectomy was found to be the most effective in decreasing the activity of the liver to about 50% of control values. 6. Like 2-(N-hydroxyacetamido)fluorene, various other N-hydroxy derivatives of 2-acetamido-7-fluorofluorene, 2-acetamidophenanthrene, 4-acetamidobiphenyl and 4-acetamidostilbene were also shown to be esterified to different extents by rat liver cytosol.
Flavin-photosensitized reactions of retinal and stilbene
Authors: Gordon-Walker Ann , Radda G. K.
Published: August 2015

1. Retinol and stilbene are both isomerized when they are illuminated anaerobically in the presence of flavins. 2. Triplet quenchers (e.g. oxygen, potassium iodide and paramagnetic ions) inhibit the reaction more efficiently than they quench flavin fluorescence. At 77°C in a diethyl ether–isopentane–ethanol (5:5:2) glass retinol quenches flavin phosphorescence, but not its fluorescence. 3. For the stilbene reaction cis/trans photostationary-state mixtures are obtained with different flavins and these are linearly related to the phosphorescence transition energies of the flavins used. 4. The reaction involves the triplet state of flavin and a scheme for the reaction is suggested. 5. The dependence of the rate of reaction on substrate concentration is explicable in terms of this scheme. 6. The photobleaching of rhodopsin sensitized by flavin is also demonstrated.
The reversible dissociation and activity of ribosomal subunits of liver and skeletal muscle from rats
Authors: von der Decken Alexandra , Ashby P. , McIlreavy Diana , Campbell P. N.
Published: August 2015

Ribosomal subunits were prepared from rat liver and skeletal muscle after incubation with puromycin and treatment at low concentrations of Mg2+. After isolation the resulting subunits could be recombined and the particles effected the synthesis of polyphenylalanine in the presence of polyuridylic acid. Hybrid particles formed from subunits of liver and muscle respectively were also active. The homogeneity of the isolated subunits was checked by polyacrylamide-gel–agarose electrophoresis. The method is shown to be a reliable and comparatively simple way of preparing active ribosomal subunits from skeletal muscle.
Activities of enzymes involved in acetoacetate utilization in adult mammalian tissues
Authors: Williamson D. H. , Bates Margaret W. , Page M. Ann , Krebs H. A.
Published: August 2015

1. The activities in rat tissues of 3-oxo acid CoA-transferase (the first enzyme involved in acetoacetate utilization) were found to be highest in kidney and heart. In submaxillary and adrenal glands the activities were about one-quarter of those in kidney and heart. In brain it was about one-tenth and was less in lung, spleen, skeletal muscle and epididymal fat. No activity was detectable in liver. 2. The activities of acetoacetyl-CoA thiolase were found roughly to parallel those of the transferase except for liver and adrenal glands. The high activity in the latter two tissues may be explained by additional roles of thiolase, namely, the production of acetyl-CoA from fatty acids. 3. The activities of the two enzymes in tissues of mouse, gerbil, golden hamster, guinea pig and sheep were similar to those of rat tissues. The notable exception was the low activity of the transferase and thiolase in sheep heart and brain. 4. The activities of the transferase in rat tissues did not change appreciably in starvation, alloxan-diabetes or on fat-feeding, where the rates of ketone-body utilization are increased. Thiolase activity increased in kidney and heart on fat-feeding. 5. The activity of 3-hydroxybutyrate dehydrogenase did not change in rat brain during starvation. 6. The factors controlling the rate of ketone-body utilization are discussed. It is concluded that the activities of the relevant enzymes in the adult rat do not control the variations in the rate of ketone-body utilization that occur in starvation or alloxan-diabetes. The controlling factor in these situations is the concentration of the ketone bodies in plasma and tissues.
The number of sex-factors per chromosome in Escherichia coli
Authors: Frame R. , Bishop J. O.
Published: August 2015

A substituted sex-factor of Escherichia coli, F′8 gal, was transferred to Proteus mirabilis by conjugation. The DNA of the episome was partially purified from Proteus DNA by preparative equilibrium centrifugation in caesium chloride, and by a bulk method using hydroxyapatite. The buoyant density of the episomal DNA is 1.707, corresponding to a (G+C) content of 47%. By optical renaturation the genetic complexity of the episomal DNA was found to be 76×106 daltons. RNA was synthesized in vitro by using the episomal DNA as template. By hybridizing this RNA with DNA extracted from E. coli carrying F′8 gal, it is shown that the number of copies of the episome per replicating chromosome is close to two during exponential growth. The episome makes up about 4.4% of the total DNA of the growing cells. The activities of galactokinase and galactose 1-phosphate uridylyl-transferase in cells with and without episomal and chromosomal gal genes were found to be proportional to the number of gal genes present, when the cells were induced with d-fucose, but not when they were induced with d-galactose.
Enzyme purification by electrodecantation
Authors: Winchester B. G. , Caffrey M. , Robinson D.
Published: August 2015

1. Electrodecantation has been applied to the initial fractionation from crude material of pig kidney β-d-glucosidase and sheep testicular N-acetylglucosaminidase. 2. The isoelectric points determined by isoelectric focusing were pH4.9 for the β-d-glucosidase and pH6.3 for the N-acetylglucosaminidase. 3. Electrodecantation of pig kidney extract and sheep testicular extract was carried out at pH4.9 and 6.3 respectively. 4. A six- to ten-fold increase in specific activity could be obtained with good recoveries after a single cycle of electrodecantation. 5. The technique has also been used to purify further an extracellular Bacillus subtilis protease preparation. 6. Attempts to use electrodecantation for the concentration of very dilute enzyme solutions resulted in considerable loss of activity. 7. The limitations and potential use of the technique in laboratory-scale enzyme preparation are discussed.
Carbohydrate content of insoluble elastins prepared from adult bovine and calf ligamentum nuchae and tropoelastin isolated from copper-deficient procine aorta
Authors: Grant M. E. , Steven F. S. , Jackson D. S. , Sandberg L. B.
Published: August 2015

1. Insoluble elastin has been prepared by several different methods from adult bovine and calf ligamentum nuchae. Highly purified tropoelastin has been prepared from copper-deficient porcine aorta. 2. Amino acid analyses indicated that all preparations, except that obtained from calf ligamentum nuchae by using an EDTA extraction followed by collagenase digestion (preparation E6), were typical of pure elastin having high concentrations of hydrophobic and low concentrations of hydrophilic amino acids. Preparation E6 was found to contain approx. 40% collagen. 3. The determination and composition of the carbohydrates associated with these preparations is reported. With the exception of preparation E6, the insoluble elastins contained only trace amounts of neutral sugars (0.13–0.35%, w/w) and amino sugars (0.01–0.06%, w/w). The porcine tropoelastin contained virtually no carbohydrate. 4. The results suggest that carbohydrate analyses can yield valuable information about the purity of elastin preparations.
Effect of osmotic shock on tetracycline resistance in Escherichia coli bearing an R-factor
Authors: Franklin T. J. , Foster S. J.
Published: August 2015

1. Escherichia coli with an R-factor conferring resistance to tetracycline was induced to high-degree resistance by pre-exposure to the antibiotic. The degree of resistance was drastically lowered by subjecting the cells to osmotic shock. 2. Resistance to tetracycline was rapidly restored by incubating the shocked cells in a glucose–salts medium containing shock proteins prepared from tetracycline-sensitive and -resistant cells. Resistance was also restored by incubating the cells in a complex medium without shock protein. 3. The initial recovery of resistance was followed by a secondary fall in resistance when the cells were cultured in complex medium; this secondary fall was largely prevented by the addition of a low concentration (10μg/ml) of tetracycline to cells. The secondary fall was significantly less in shocked E. coli cells harbouring a mutant R-factor in which tetracycline resistance is largely constitutive. 4. Tetracycline resistance was also transiently depressed by treating R-factor-bearing cells with EDTA in tris buffer. 5. The significance of these results in relation to the mechanism of tetracycline resistance in R-factor-bearing cells is discussed.
Sulphur utilization during growth of Pseudomonas fluorescens on potassium d-glucose 6-O-sulphate
Authors: Fitzgerald J. W. , Dodgson K. S.
Published: August 2015

Pseudomonas fluorescens N.C.I.B. 8248 was adapted to grow on potassium d-glucose 6-O-sulphate as the sole carbon and sulphur source. Adapted bacteria grew optimally at 37°C on 1.6% (w/v) sulphate ester and growth coincided with the disappearance of the ester from the culture medium at a rate of 2.4mg/h per ml. Three sulphated compounds were detected in the culture fluid at the termination of growth. One of these was present in traces only and has not been identified. The second was present in somewhat greater amounts and was identified as the 6-O-sulphate ester of d-gluconate, and the major metabolite was identified as d-glycerate 3-O-sulphate. Sulphur utilization by the organism was not associated with the appearance of a glycosulphatase enzyme in the cells. However, a novel enzyme system (or systems) was present that liberated inorganic 35SO42− ions from dipotassium d-gluconate 6[35S]-O-sulphate and from dipotassium dl-glycerate 3[35S]-O-sulphate. Activity towards the latter substrate could not be detected when the adapted or parent Pseudomonas strain was cultured on d-glucose and potassium sulphate as respective carbon and sulphur sources. Some properties of the enzyme acting on the glycerate ester are recorded.
Nicotinamide nucleotide synthesis in regenerating rat liver
Authors: Ferris G. M. , Clark J. B.
Published: August 2015

1. The concentrations and total content of the nicotinamide nucleotides were measured in the livers of rats at various times after partial hepatectomy and laparotomy (sham hepatectomy) and correlated with other events in the regeneration process. 2. The NAD content and concentration in rat liver were relatively unaffected by laparotomy, but fell to a minimum, 25 and 33% below control values respectively, 24h after partial hepatectomy. NADP content and concentration were affected similarly by both laparotomy and partial hepatectomy, falling rapidly and remaining depressed for up to 48h. 3. The effect of injecting various doses of nicotinamide on the liver DNA and NAD 18h after partial hepatectomy was studied and revealed an inverse correlation between NAD content and DNA content. 4. Injections of nicotinamide at various times after partial hepatectomy revealed that the ability to synthesize NAD from nicotinamide was impaired during the first 12h, rose to a peak at 26h and fell again by 48h after partial hepatectomy. 5. The total liver activity of NAD pyrophosphorylase (EC 2.7.7.1) remained at or slightly above the initial value for 12h after partial hepatectomy and then rose continuously until 48h after operation. The activity of NMN pyrophosphorylase (EC 2.4.2.12) showed a similar pattern of change after partial hepatectomy, but was at no time greater than 5% of the activity of NAD pyrophosphorylase. 6. The results are discussed with reference to the control of NAD synthesis in rapidly dividing tissue. It is suggested that the availability of cofactors and substrates for NAD synthesis is more important as a controlling factor than the maximum enzyme activities. It is concluded that the low concentrations of nicotinamide nucleotides in rapidly dividing tissues are the result of competition between NAD synthesis and nucleic acid synthesis for common precursor and cofactors.
The acetylation of insulin
Authors: Lindsay D. G. , Shall S.
Published: August 2015

The acetylation of the free amino groups of insulin was studied by reaction of the hormone with N-hydroxysuccinimide acetate at pH6.9 and 8.5. The products formed were separated by chromatography on DEAE-Sephadex and were characterized by isoelectric focusing, by end-group analysis, by the incorporation of [3H]acetyl groups in the molecule, and by treatment with trypsin that had been treated with 1-chloro-4-phenyl-3-toluene-p-sulphonamidobutan-2-one (‘tosylphenylalanyl chloromethyl ketone’). Three monosubstituted products, two disubstituted products and one trisubstituted derivative were prepared. The α-amino groups of the terminal residues and the ∈-amino group of the lysine-B29 were the sites of reaction. Acetylation of any of the free amino groups did not affect the biological activity of insulin. It was demonstrated, however, that substitution at the glycine-A1 amino group by the larger residues, acetoacetyl or thiazolidinecarbonyl, produced a decrease in biological activity. Modification of the lysine-B29 or phenylalanine-B1 amino groups with these larger reagents did not affect the biological activity. Modification of the phenylalanine-B1 amino group by any of the three substituents resulted in a large decrease in the affinity of insulin for anti-insulin antibodies raised in the guinea pig. Modification of the other two amino groups did not affect the reaction with antibody. These observations are correlated with the tertiary structure of insulin.
Control of the redox state of the nicotinamide–adenine dinucleotide couple in rat liver cytoplasm
Authors: Stubbs Marion , Veech R. L. , Krebs H. A.
Published: August 2015

1. A study has been made of the ability of rat liver in vivo to maintain equilibrium in the combined glyceraldehyde 3-phosphate dehydrogenase, 3-phosphoglycerate kinase and lactate dehydrogenase reactions, i.e. in the system: [Formula: see text] Attempts were made to upset equilibrium. The [lactate]/[pyruvate] ratio was rapidly changed by injection of ethanol or crotyl alcohol, and the value of [ATP]/[ADP][HPO42-] was rapidly changed by injection of ethionine or carbonyl cyanide p-trifluoromethoxy-phenylhydrazone. 2. The concentrations of the metabolites occurring in the above equation were measured in freeze-clamped liver. 3. Although the injected agents caused large changes in the concentrations of the individual components, near-equilibrium in the system was maintained, as indicated by the fact that the value of [ATP]/[ADP][HPO42-], referred to as the phosphorylation state of the adenine nucleotides, measured directly agreed with the value calculated for equilibrium conditions from the above equation. 4. The results are discussed and taken to confirm that the order of magnitude of the value of the redox state of the cytoplasmic NAD couple in rat liver is controlled by the phosphorylation state of the adenine nucleotide system.
Effects of carbohydrate availability on lipogenesis in sheep
Authors: Ballard F. J. , Filsell O. H. , Jarrett I. G.
Published: August 2015

1. Lipogenesis in sheep liver and adipose tissue was investigated by incorporation studies in vitro with radioactive glucose and acetate and by assays of key enzymes. 2. Carbohydrate availability to sheep was increased by feeding on a diet containing 70% soluble carbohydrate, by infusing glucose into the abomasum or by direct intravenous infusion of glucose. 3. Under these conditions lipogenesis from glucose and acetate was increased from very low values in lìver and adipose tissue, especially in those animals where rumen fermentation was by-passed by glucose infusion. 4. Large increases in the activities of ATP citrate lyase (EC 4.1.3.8) and NADP–malate dehydrogenase (EC 1.1.1.40) occurred in both tissues when lipogenesis was increased. 5. No adaptations were found in the activities of pyruvate carboxylase (EC 6.4.1.1) in adipose tissue, glucokinase (EC 2.7.1.2) in liver or 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) in liver. It is proposed that the absence of these enzymes is not related to glucose availability. 6. The effect of glucose on liver lipogenesis was to increase conversion of acetate into lipid. 7. This effect also occurred in adipose tissue, but in this tissue glucose also became a quantitatively important precursor of triglyceride fatty acid.
1-(O-β-Glucosaminyl)-2,3-diglyceride in Bacillus megaterium
Authors: Phizackerley P. J. R. , MacDougall J. C. , Moore R. A.
Published: August 2015

1. A lipid that contains glucosamine but not phosphorus has been isolated from Bacillus megaterium. It constitutes about 5% of the total lipid glucosaminide in this organism and can be distinguished chromatographically from 2′-(O-β-glucosaminyl)phosphatidylglycerol and 3′-(O-β-glucosaminyl)phosphatidylglycerol, which are also present. 2. The lipid contains glycerol, fatty acids and glucosamine in the molar proportion 1:2:1. The fatty acids are bound by an ester linkage and are similar to those found in other lipids of this organism. Partial acid hydrolysis or alkaline hydrolysis of the lipid yields 1-(O-β-glucosaminyl)glycerol and degradation with nitrite yields 2,5-anhydromannose and diglyceride. 3. The lipid has been identified as 1-(O-β-glucosaminyl)-2,3-diglyceride.
Extraction of steroidal glucosiduronic acids from aqueous solutions by anionic liquid ion-exchangers
Authors: Mattox V R , Litwiller R. D. , Goodrich J E
Published: August 2015

A pilot study on the extraction of three steroidal glucosiduronic acids from water into organic solutions of liquid ion-exchangers is reported. A single extraction of a 0.5mm aqueous solution of either 11-deoxycorticosterone 21-glucosiduronic acid or cortisone 21-glucosiduronic acid with 0.1m-tetraheptylammonium chloride in chloroform took more than 99% of the conjugate into the organic phase; under the same conditions, the very polar conjugate, β-cortol 3-glucosiduronic acid, was extracted to the extent of 43%. The presence of a small amount of chloride, acetate, or sulphate ion in the aqueous phase inhibited extraction, but making the aqueous phase 4.0m with ammonium sulphate promoted extraction strongly. An increase in the concentration of ion-exchanger in the organic phase also promoted extraction. The amount of cortisone 21-glucosiduronic acid extracted by tetraheptylammonium chloride over the pH range of 3.9 to 10.7 was essentially constant. Chloroform solutions of a tertiary, a secondary, or a primary amine hydrochloride also will extract cortisone 21-glucosiduronic acid from water. The various liquid ion exchangers will extract steroidal glucosiduronic acid methyl esters from water into chloroform, although less completely than the corresponding free acids. The extraction of the glucosiduronic acids from water by tetraheptylammonium chloride occurs by an ion-exchange process; extraction of the esters does not involve ion exchange.
The specificity of dipeptidyl aminopeptidase I (cathepsin C) and its use in peptide sequence studies
Authors: Lindley H.
Published: August 2015

1. The characterization of cathepsin C as an aminodipeptidase has been confirmed. 2. An extra limitation on its specificity, namely that peptides involving proline as the third residue are immune to the enzyme, has been found. 3. A novel technique of using the enzyme for amino acid sequence determination has been shown to be feasible.
The binding of oxidized coenzymes by glutamate dehydrogenase and the effects of glutarate and purine nucleotides
Authors: Dalziel K. , Egan R. R.
Published: August 2015

1. The binding of NAD+ and NADP+ to glutamate dehydrogenase has been studied in sodium phosphate buffer, pH7.0, by equilibrium dialysis. Approximate values for the dissociation constants are 0.47 and 2.5mm respectively. For NAD+ the value agrees with that estimated from initial-rate results. 2. In the presence of the substrate analogue glutarate both coenzymes are bound more firmly, and there is one active centre per enzyme subunit. The binding results cannot be described in terms of independent and identical active centres, and binding is stronger at low coenzyme concentrations than at high concentrations. Either the six subunits of the oligomer are not identical or there are negative interactions between them in the binding of coenzymes in ternary complexes with glutarate. The latter explanation is favoured. 3. The binding studies support the conclusions drawn from earlier kinetic studies of the glutamate reaction. 4. ADP and GTP respectively decrease and increase the affinity of the enzyme for NAD+ and NADP+, in both the presence and absence of glutarate. The negative binding interactions in the presence of glutarate are abolished by ADP, which decreases the affinity for the coenzymes at low concentrations of the latter. 5. In the presence of glutarate, GTP and NAD+ or NADP+, the association of enzyme oligomers is prevented, and the solubility of the enzyme is decreased; the complex of enzyme and ligands readily crystallizes. 6. The results are discussed in relation to earlier kinetic studies.
Identification of the sources of nitrous oxide produced by oxidative and reductive processes in Nitrosomonas europaea
Authors: Ritchie G. A. F. , Nicholas D. J. D.
Published: August 2015

1. Cells of Nitrosomonas europaea produced N2O during the oxidation of ammonia and hydroxylamine. 2. The end-product of ammonia oxidation, nitrite, was the predominant source of N2O in cells. 3. Cells also produced N2O, but not N2 gas, by the reduction of nitrite under anaerobic conditions. 4. Hydroxylamine was oxidized by cell-free extracts to yield nitrite and N2O aerobically, but to yield N2O and NO anaerobically. 5. Cell extracts reduced nitrite both aerobically and anaerobically to NO and N2O with hydroxylamine as an electron donor. 6. The relative amounts of NO and N2O produced during hydroxylamine oxidation and/or nitrite reduction are dependent on the type of artificial electron acceptor utilized. 7. Partially purified hydroxylamine oxidase retained nitrite reductase activity but cytochrome oxidase was absent. 8. There is a close association of hydroxylamine oxidase and nitrite reductase activities in purified preparations.
Microbial metabolism of C1 and C2 compounds. The involvement of glycollate in the metabolism of ethanol and of acetate by Pseudomonas AM1
Authors: Dunstan Patricia M. , Anthony C. , Drabble W. T.
Published: August 2015

Pseudomonas AM1 grows on ethanol with a mean generation time of about 10h. A single alcohol dehydrogenase is responsible for oxidation of both methanol and ethanol. It is proposed that the glyoxylate bypass does not operate in Pseudomonas AM1 during growth on ethanol. Although malate synthase is present in extracts of ethanol-grown Pseudomonas AM1, the activity of isocitrate lyase is negligible. Short-term incubation experiments with [14C]ethanol and [14C]acetate indicate that a novel pathway operates during growth of Pseudomonas AM1 on ethanol. Glycollate, glyoxylate and malate are probably intermediates in this pathway.
The biosynthesis in vitro of keratan sulphate in bovine cornea
Authors: Handley C. J. , Phelps C. F.
Published: August 2015

1. Bovine corneas were incubated in vitro with [U-14C]glucose. 2. The glycosaminoglycans of corneal stroma were isolated and fractionated on cetylpyridinium chloride–cellulose columns. The major components were keratan sulphate (71%), chondroitin 4-sulphate (17%) and chondroitin 6-sulphate (4%). 3. The acid-soluble nucleotides and intermediates of glycosaminoglycan biosynthesis of corneal stroma were separated on Dowex 1 (formate form) and the tissue content and cellular concentrations were determined. 4. The rates of synthesis of the intermediates of glycosaminoglycan biosynthesis in corneal stroma were determined. 5. The incorporation of radioactivity from [U-14C]glucose into the uronic acid and hexosamine components of the glycosaminoglycans present in corneal stroma were measured and the turnover rates of these polymers were calculated. It was found that keratan sulphate was turning over in about 723h and chondroitin 6-sulphate in 251h.
Solubilization of a mannose-polymerizing enzyme from Phaseolus aureus
Authors: Heller J. S. , Villemez C. L.
Published: August 2015

A soluble enzyme preparation, which catalyses the polymerization of mannose, was obtained by Triton X-100 extraction of a particulate fraction derived from Phaseolus aureus hypocotyls. The product that resulted when GDP-α-d-mannose was used as a substrate was a β-(1→4)-linked mannan, about three-quarters of which was alkali-insoluble. The mannose-polymerizing enzyme activity was at least as great in the soluble preparation as in the particulate preparation, and the specific activity of the solubilized enzyme was greater by a factor of at least 3.5. Kinetic studies of the soluble enzyme indicate that the apparent Km is 55–62μm, and a disproportionate increase in rate is observed at high concentrations. GDP-α-d-glucose is a strong competitive inhibitor of the mannose-polymerizing reaction, with an apparent Ki of 6.2μm. The soluble enzyme is relatively unstable, losing about two-thirds of its original activity in 5h at 0°C or in 24h at −20°C. A solvent (acetone, butanol, diethyl ether)-extracted particulate preparation, which also exhibits the same enzyme activity, is more stable, retaining full activity for at least 5 days at −20°C. There was no polymerizing-enzyme activity in the soluble enzyme preparation when UDP-d-glucose, UDP-d-galactose, UDP-d-xylose, UDP-l-arabinose or UDP-d-glucuronic acid were used as substrates. However, the soluble enzyme preparation would catalyse the polymerization of glucose, with GDP-d-glucose as substrate.
Purification and properties of Penicillium glucose 6-phosphate dehydrogenase
Authors: Malcolm A. Anne , Shepherd M. G.
Published: August 2015

1. Glucose 6-phosphate dehydrogenase was isolated and partially purified from a thermophilic fungus, Penicillium duponti, and a mesophilic fungus, Penicillium notatum. 2. The molecular weight of the P. duponti enzyme was found to be 120000±10000 by gelfiltration and sucrose-density-gradient-centrifugation techniques. No NADP+- or glucose 6-phosphate-induced change in molecular weight could be demonstrated. 3. Glucose 6-phosphate dehydrogenase from the thermophilic fungus was more heat-stable than that from the mesophile. Glucose 6-phosphate, but not NADP+, protected the enzyme from both the thermophile and the mesophile from thermal inactivation. 4. The Km values determined for glucose 6-phosphate dehydrogenase from the thermophile P. duponti were 4.3×10−5m-NADP+ and 1.6×10−4m-glucose 6-phosphate; for the enzyme from the mesophile P. notatum the values were 6.2×10−5m-NADP+ and 2.5×10−4m-glucose 6-phosphate. 5. Inhibition by NADPH was competitive with respect to both NADP+ and glucose 6-phosphate for both the P. duponti and P. notatum enzymes. The inhibition pattern indicated a rapid-equilibrium random mechanism, which may or may not involve a dead-end enzyme–NADP+–6-phosphogluconolactone complex; however, a compulsory-order mechanism that is consistent with all the results is proposed. 6. The activation energies for the P. duponti and P. notatum glucose 6-phosphate dehydrogenases were 40.2 and 41.4kJ·mol−1 (9.6 and 9.9kcal·mol−1) respectively. 7. Palmitoyl-CoA inhibited P. duponti glucose 6-phosphate dehydrogenase and gave an inhibition constant of 5×10−6m. 8. Penicillium glucose 6-phosphate dehydrogenase had a high degree of substrate and coenzyme specificity.
Pentose cycle and reducing equivalents in rat mammary-gland slices
Authors: Katz Joseph , Wals P. A.
Published: August 2015

1. Slices of mammary gland of lactating rats were incubated with glucose labelled uniformly with 14C and in positions 1, 2, 3 and 6, and with 3H in all six positions. Glucose carbon atoms are incorporated into CO2, fatty acids, lipid glycerol, the glucose and galactose moieties of lactose, lactate, soluble amino acids and proteins. C-3 of glucose appears in fatty acids. The incorporation of 3H into fatty acids is greatest from [3-3H]glucose. 3H from [5-3H]glucose appears, apart from in lactose, nearly all in water. 2. The specific radioactivity of the galactose moiety of lactose from [1-14C]- and [6-14C]-glucose was less, and that from [2-14C]- and [3-14C]-glucose more, than that of the glucose moiety. There was no randomization of carbon atoms in the glucose moiety, but it was extensive in galactose. 3. The pentose cycle was calculated from 14C yields in CO2 and fatty acids, and from the degradation of galactose from [2-14C]glucose. A method for the quantitative determination of the contribution of the pentose cycle, from incorporation into fatty acids from [3-14C]glucose, is derived. The rate of the reaction catalysed by hexose 6-phosphate isomerase was calculated from the randomization pattern in galactose. 4. Of the utilized glucose, 10–20% is converted into lactose, 20–30% is metabolized via the pentose cycle and the rest is metabolized via the Embden–Meyerhof pathway. About 10–15% of the triose phosphates and pyruvate is derived via the pentose cycle. 5. The pentose cycle is sufficient to provide 80–100% of the NADPH requirement for fatty acid synthesis. 6. The formation of reducing equivalents in the cytoplasm exceeds that required for reductive biosynthesis. About half of the cytoplasmic reducing equivalents are probably transferred into mitochondria. 7. In the Appendix a concise derivation of the randomization of C-1, C-2 and C-3 as a function of the pentose cycle is described.