bluuebunny/crossref_metadata_2025 huggingface dataset:
The purification and some properties of 3-hydroxy-3-methylglutaryl-coenzyme A synthase from baker's yeast
Authors: Middleton B. , Tubbs P. K.
Published: August 2015

1. A purification of 3-hydroxy-3-methylglutaryl-CoA synthase from baker's yeast is described. This yields a preparation of average specific activity 2.1 units (μmol/min)/mg in which contamination by acetoacetyl-CoA thiolase is less than 0.2%. 2. The molecular weights of 3-hydroxy-3-methylglutaryl-CoA synthase and acetoacetyl-CoA thiolase from baker's yeast were determined by gel filtration on Sephadex G-200. The values obtained were 130000 and 190000 respectively. 3. 3-Hydroxy-3-methylglutaryl-CoA synthase is susceptible to irreversible inhibition by a wide variety of alkylating and acylating agents. The time-course of inhibition of the enzyme by some of these, including the active-site-directed inhibitor bromoacetyl-CoA, was studied in the presence and absence of substrates, products and product analogues. Acetyl-CoA, even when present at concentrations as low as 5μm, gives almost complete protection. Other acyl-CoA derivatives give some protection, but only at concentrations 10–30-fold higher. 4. These results are discussed with reference to an ordered reaction pathway in which acetyl-CoA reacts to give a covalent acetyl-enzyme intermediate.
Studies on the particulate lactose synthetase of mouse mammary gland and the role of α-lactalbumin in the initiation of lactose synthesis
Authors: Jones E. A.
Published: August 2015

1. Some of the kinetic properties of the particulate lactose synthetase of mouse mammary gland were investigated and shown to resemble those that have been reported for the soluble enzyme. Typical values for intact preparations were 2.3μm for the apparent Km for α-lactalbumin at 40mm-glucose and 1.7mm for the apparent Km for glucose at the endogenous concentration of α-lactalbumin. 2. Digitonin treatment increased total assayable activity approximately twofold but almost eliminated the endogenous activity found in the absence of α-lactalbumin, these findings being consistent with the location of endogenous activity within Golgi vesicles. 3. From the properties of the particulate fraction from lactating mice it was deduced that the effective endogenous α-lactalbumin concentrations were in the range 1–10μm. 4. The concentration of α-lactalbumin was not significantly different in particles isolated at various stages of pregnancy and early lactation. 5. The implications of these results for the control of lactose synthetase in vivo are discussed.
The regulatory proteins of the myofibril. Characterization and biological activity of the calcium-sensitizing factor (troponin A)
Authors: Schaub M. C. , Perry S. V. , Häcker W.
Published: August 2015

1. Electrophoretically homogeneous calcium-sensitizing factor was prepared from the troponin complex by chromatography successively on sulphoethyl-Sephadex and on diethyl-(2-hydroxypropyl)aminoethyl-Sephadex in 6m-urea. It is a protein containing 53% of polar amino acids, of which a net excess consists of acidic residues. 2. On gel filtration the calcium-sensitizing factor was shown to be the only myofibrillar protein that bound 45Ca2+ tightly in the presence of 2–6m-urea. 3. Calcium-sensitizing factor effectively neutralized the effect of the inhibitory factor on the ATPase activities of actomyosin systems. Tropomyosin was essential for the regulation, by changes in the Ca2+ concentration, of the neutralizing effect of calcium-sensitizing factor on the inhibitory factor. 4. Prolonged exposure to chelators of Ca2+ produced an irreversibly modified form of calcium-sensitizing factor of higher electrophoretic mobility at pH8.6. The modified form neutralized the inhibitory factor action but this property could no longer be controlled by the Ca2+ concentration in the presence of tropomysin. 5. The calcium-sensitizing factor and tropomyosin could be replaced by their carboxymethylated derivatives in the relaxing-protein system.
The turnover of protein in discrete areas of rat brain
Authors: Austin Lawrence , Lowry Oliver H. , Brown Joseph G. , Carter Joyce G.
Published: August 2015

1. Rats were injected serially with [14C]glucose to obtain a constant specific radioactivity of brain amino acids. Measurements with this system for periods of up to 8h gave an apparent mean half-life for protein in whole brain of 85h (indicating the presence of a protein fraction with much more rapid turnover than this). 2. The half-lives of proteins in the granule-cell, molecular and white-matter layers of cerebellum were also determined. These had values of 33, 59 and 136h respectively. In addition, the incorporation into protein in six layers of the cerebral cortex, subjacent white matter and five layers of Ammon's horn was studied. All cell-body layers incorporated amino acids at about the same rate irrespective of location, and these rates were considerably higher than those for incorporation into proteins in areas rich in dendrites or fibre tracts. 3. A new method for measuring small amounts of glutamate with a cyclic enzyme system is presented.
Further studies of the chemical composition of the lipopolysaccharide of Pseudomonas aeruginosa
Authors: Chester I. R. , Gray G. W. , Wilkinson S. G.
Published: August 2015

1. Qualitative and quantitative analytical results for the lipopolysaccharide from acetone-dried cells of Pseudomonas aeruginosa (N.C.T.C. 1999) are presented and possible contamination of the material with nucleic acid was further examined. 2. Additional sugars detected (only in large-scale hydrolysates) were mannose and arabinose; traces of spermidine and putrescine were also found. 3. The heptose component is l-glycero-d-mannoheptose. 4. The thiobarbituric acid-positive component is a 3-deoxy-2-octulonic acid, of which only 35–40% links lipid A to the polysaccharide. This linkage is not broken by hydrolysis with acetic acid up to 0.08m. 5. Liberation of lipid A required hydrolysis with 0.1m-hydrochloric acid, which substantially degraded the polysaccharide moiety. 6. Fractions obtained from the degraded polysaccharide by high-voltage electrophoresis were examined; in these, the alanine/galactosamine molar ratio is approx. 1. 7. Hydrazinolysis of whole lipopolysaccharide showed that at least 40% of the alanine is in amide linkage, possibly with galactosamine. 8. Lipid A, solubilized by alkaline methanolysis was fractionated; most of the phosphorus of the higher-molecular-weight fractions was released as Pi by a phosphomonoesterase. 9. Hydrazinolysis of lipid A destroyed approx. 80% of the glucosamine, and glycosidically linked glucosamine oligosaccharides could not be isolated.
Cell migration and cortisone induction of sucrase activity in jejunum and ileum
Authors: Herbst J. J. , Koldovský O.
Published: August 2015

The increase of sucrase activity in homogenates of jejunum and ileum of suckling rats after cortisone administration has been investigated. Serial tissue sections of villi and crypts were also assayed for sucrase activity and these results were compared with the migration of cells labelled with [3H]thymidine along the villus. By using a low dose of cortisone (0.5mg/day per 100g body wt.) it was found that the sensitivity of the small intestine producing system to cortisone stimulation increased during the suckling period. On the other hand, 5mg of cortisone/day per 100g body wt. produced practically the same increase of sucrase during the entire suckling period. Sucrase activity in homogenates of the entire small-intestinal wall was first detected 24h after the first injection of cortisone (5mg/day per 100g body weight) to 9-day-old animals and maximum activity both in the jejunum and ileum was reached by 120h. Jejunal activity was greater than ileal activity, but the rate of the increase was similar. The half-time of the increase was 23–27h, whereas enterocytes migrate from the base to the tip of the villi in approximately 72h. Comparison of sucrase activity in serial tissue sections of villi and crypts at various times after cortisone treatment showed that the leading edge of sucrase activity proceeds toward the tip of the villi at the same rate as the advancing edge of newly formed cells. Sucrase activity increased in the newly induced cells as they migrated to the tip of the villi. It was concluded that the increase of sucrase activity in suckling rats after cortisone stimulation is due to at least three factors: (1) increase of activity in newly differentiating cells, (2) increased percentage of villus cells with sucrase activity and (3) continued production or activation of sucrase activity as the cells migrate along the villi.
The effect of cross-links on the mobility of proteins in dodecyl sulphate–polyacrylamide gels
Authors: Griffith I. P.
Published: August 2015

The effect of reduction of intramolecular disulphide bridges on the mobility of proteins in 5% (w/v) polyacrylamide gels in the presence of sodium dodecyl sulphate was investigated. A series of polypeptide polymers, containing up to 68 intramolecular disulphide bridges, was prepared by cross-linking proteins of known structure with glutaraldehyde. These model polypeptides were denatured with heat, sodium dodecyl sulphate and urea, and their mobilities in sodium dodecyl sulphate–polyacrylamide gels compared before and after reduction with dithiothreitol. The mobilities of polypeptides containing no cystine were unaffected by reduction. However, reduction generally decreased the mobilities of polypeptides containing cystine; the extent of this decrease depended on the number of cystine residues originally present in the polypeptide polymer, and on the protein from which the latter was derived. In contrast with their higher oligomers, the monomer of lysozyme and the dimer of ribonuclease increased in mobility after reduction. The reduced polypeptide oligomers formed by reaction with glutaraldehyde were generally found to migrate at a rate significantly faster than was expected from their calculated molecular weights. It was concluded that the use of unreduced proteins and protein aggregates for molecular-weight measurements by the sodium dodecyl sulphate–polyacrylamide-gel method may give erroneous estimates of the molecular weight of any protein being investigated.
Glycogen synthetase and the control of glycogen synthesis in the cellular slime mould Dictyostelium discoideum during the growth (myxamoebal) phase
Authors: Weeks G. , Ashworth J. M.
Published: August 2015

1. Myxamoebae of the cellular slime mould Dictyostelium discoideum Ax-2 that are grown in axenic medium containing 86mm-glucose have seven times the glycogen content of the same myxamoebae grown in the same medium but lacking added carbohydrate. 2. During the transition from the exponential to the stationary phase of growth in axenic medium containing glucose myxamoebae preferentially synthesize glycogen and can have as much as three times the glycogen content during the stationary phase as they have during the exponential phase of growth. 3. The rate of glycogen degradation by myxamoebae is, under all conditions of growth, small compared with the rate of glycogen accumulation and the changes in glycogen content thus reflect altered rates of glycogen synthesis. 4. There is no correlation between the rate of glycogen synthesis by myxamoebae and the glycogen synthetase content of the myxamoebae. 5. The activity of glycogen synthetase of D. discoideum is inhibited by a physiological concentration of ATP and this inhibition is overcome by glucose 6-phosphate. Both effects are especially marked at physiological concentrations of UDP-glucose. 6. The rate of glycogen accumulation by myxamoebae growing exponentially in axenic media can be satisfactorily accounted for in terms of the known intracellular concentrations of glucose 6-phosphate, UDP-glucose and glycogen synthetase. The rate-limiting factors controlling glycogen synthesis by the myxamoebae are apparently the substrate (UDP-glucose) and effector (glucose 6-phosphate and ATP) concentrations rather than the amount of the enzyme.
Comparative studies of the specificities of α-chymotrypsin and subtilisin BPN′. Studies with flexible substrates
Authors: Pattabiraman T. N. , Lawson W. B.
Published: August 2015

A series of arylalkanoate esters and α-acetamidoarylalkanoate esters were tested as substrates for α-chymotrypsin and subtilisin BPN′. Chymotrypsin hydrolysed N-acetyl-l-phenylalanine methyl ester and methyl 4-phenylbutyrate faster than their respective higher and lower homologues, whereas methyl 2-acetamido-6-phenylhexanoate and methyl 6-phenylhexanoate were better substrates for subtilisin than their lower homologues. N-Acetyl-l-tryptophan methyl ester and its analogue, N-acetyl-3-(1-naphthyl)-alanine methyl ester, were hydrolysed 23 times faster by chymotrypsin than by subtilisin. These results indicate that the binding site of α-chymotrypsin is roughly 1.1nm (11Å) long and curved, whereas that of subtilisin is a longer system and less curved. The stereo-specificity during the hydrolysis of typical substrates by both enzymes was found to vary over a wide range. The enhancing effect of the α-acetamido group in the l-series of substrates and the detrimental effect in the d-series of substrates also varies considerably.
Exchange of phospholipids between brain membranes in vitro
Authors: Miller E. K. , Dawson R. M. C.
Published: August 2015

1. When unlabelled mitochondria from guinea-pig brain were incubated with a 32P-labelled microsomal fraction from brain there was a transfer of phospholipid to the mitochondria, which could not be accounted for by an aggregation of microsomes and mitochondria or an exchange with microsomes contaminating the mitochondria. Under similar circumstances there was a transfer of phospholipid from 32P-labelled mitochondria to microsomes, indicating that the process was one of exchange. 2. The transfer from microsomes was greatly stimulated by a non-dialysable heat-labile macromolecular component in the brain supernatant fraction but not by the concentration of the particulate fractions. 3. Phospholipid-exchange processes occurred most readily between pH7 and 7.5 and were inhibited by the presence of myelin and on the addition of lysophosphatidylcholine. 4. The rates of transfer of individual phospholipids from brain microsomes to mitochondria were similar. 5. 32P-labelled microsomes could slowly donate phospholipid to the isolated synaptosomal (nerve-ending) fraction but the phospholipids of the myelin fraction did not exchange. 6. Subfractionation of the synaptosomal fraction after [32P]phospholipid transfer showed that the mitochondria were most actively labelled during the incubation. All of the isolated individual synaptosomal membranes were capable of acquiring phospholipid on incubation with a 32P-labelled brain supernatant fraction although a greater percentage was again exchanged by the mitochondrial fraction.
The pattern of fatty acid synthesis in lactating rabbit mammary gland studied in vivo
Authors: Carey E. M. , Dils R.
Published: August 2015

The biosynthesis of fatty acids has been studied in lactating rabbits at 6h after intravenous injection of sodium [1-14C]acetate. The specific radioactivities of the individual fatty acids (C6:0 to C14:0) and the proportions of these fatty acids synthesized were similar in mammary tissue and milk. Hexanoic acid had the highest specific radioactivity, and the C8:0–C14:0 fatty acids had similar specific radioactivities, which were about five times those of C16 and C18 acids. No radioactivity was detected in fatty acids of chain length C14 in these tissues were similar to those of the long-chain fatty acids in the milk and mammary gland. The results show that the C4:0–C14:0 fatty acids are synthesized within the mammary gland rather than by fatty acid uptake from circulating blood or by oxidation of long-chain fatty acids within the gland. We conclude that de novo synthesis of esterified fatty acids in vivo by this tissue has a high degree of chain-length specificity.
Escherichia coli alkaline phosphatase. Kinetic studies with the tetrameric enzyme
Authors: Halford S. E. , Schlesinger M. J. , Gutfreund H.
Published: August 2015

1. The stability of the tetrameric form of Escherichia coli alkaline phosphatase was examined by analytical ultracentrifugation. 2. The stopped-flow technique was used to study the hydrolysis of nitrophenyl phosphates by the alkaline phosphatase tetramer at pH7.5 and 8.3. In both cases transient product formation was observed before the steady state was attained. Both transients consisted of the liberation of 1mol of nitrophenol/2mol of enzyme subunits within the dead-time of the apparatus. The steady-state rates were identical with those observed with the dimer under the same conditions. 3. The binding of 2-hydroxy-5-nitrobenzyl phosphonate to the alkaline phosphatase tetramer was studied by the temperature-jump technique. The self-association of two dimers to form the tetramer is linked to a conformation change within the dimer. This accounts for the differences between the transient phases in the reactions of the dimer and the tetramer with substrate. 4. Addition of Pi to the alkaline phosphatase tetramer caused it to dissociate into dimers. The tetramer is unable to bind this ligand. It is suggested that the tetramer undergoes a compulsory dissociation before the completion of its first turnover with substrate. 5. On the basis of these findings a mechanism is proposed for the involvement of the alkaline phosphatase tetramer in the physiology of E. coli.
Stimulation of liver 5-aminolaevulinate synthetase by drugs and its relevance to drug-induced accumulation of cytochrome P-450. Studies with phenylbutazone and 3,5-diethoxycarbonyl-1,4-dihydrocollidine
Authors: De Matteis F. , Gibbs A.
Published: August 2015

The relevance of the stimulation of 5-aminolaevulinate synthetase to the accumulation of cytochrome P-450 after administration of drugs was examined in rats treated with phenylbutazone and with 3,5-diethoxycarbonyl-1,4-dihydrocollidine. 3,5-Diethoxycarbonyl-1,4-dihydrocollidine alone stimulated 5-aminolaevulinate synthetase without increasing the concentration of cytochrome P-450, whereas phenylbutazone alone increased the microsomal cytochrome P-450 without significantly affecting the activity of the enzyme. When the two drugs were given together both effects were found. It is concluded that if an increased amount of 5-aminolaevulinate and haem must be made to provide for the accumulation of cytochrome P-450, it need only be a small amount. It is also concluded from these findings that stimulation of the drug-metabolizing system on the one hand and marked enhancement of 5-aminolaevulinate synthetase activity and porphyria on the other are likely to result from different actions of the drugs. Evidence is presented suggesting that porphyrogenic drugs stimulate markedly the activity of 5-aminolaevulinate synthetase by lowering the concentration of haem in the liver, thereby decreasing the normal feedback control. With 3,5-diethoxycarbonyl-1,4-dihydrocollidine a rapid inhibition of mitochondrial ferrochelatase and of liver haem synthesis may be the primary mechanism involved.
Comparative studies of bile salts. A new type of bile salt from Arapaima gigas (Cuvier) (family Osteoglossidae)
Authors: Haslewood G. A. D. , Tökés L.
Published: August 2015

1. Arapaima gigas bile salts were hydrolysed by alkali or cleaved with dioxan–trichloroacetic acid to give cholic acid, arapaimic acid, arapaimol-A and arapaimol-B. 2. I.r., n.m.r. and mass spectroscopy and [α]D measurements indicated that arapaimic acid and arapaimol-A and -B are respectively 2α,3α,7α,12α-tetrahydroxy−5β,25∈-cholestan-26-oic acid, 5β,25R-cholestane-2β,3α,7α,12α,26-pentol and 5β-cholestane-2β,3α,7α,12α,26,27-hexol. 3. Partial synthesis of 2β,3α,7α,12α-tetrahydroxy−5α- and −5β-cholan-24-oic acid and their spectral examination fully confirmed these conclusions. 4. A. gigas bile salts show primitive features in that they comprise alcohol sulphates and a C27 acid; they are also specialized in showing 2β-hydroxylation.
Deficiency of α-mannosidase in Angus cattle. An inherited lysosomal storage disease
Authors: Hocking J. D. , Jolly R. D. , Batt R. D.
Published: August 2015

A disease of Angus cattle previously known as pseudolipidosis has been shown to be an inherited lysosomal storage disease, in which an oligosaccharide containing mannose and glucosamine is the storage substance. Diseased animals have a near-absolute deficiency of the lysosomal enzyme, α-mannosidase, whereas heterozygotes have a partial deficiency of this enzyme. The condition is analogous to the human disease known as mannosidosis.
Inhibition of pig kidney diamine oxidase by substrate analogues
Authors: Bardsley W. G. , Ashford J. S.
Published: August 2015

1. The oxidation of p-dimethylaminomethylbenzylamine was followed spectrophotometrically by measuring the change in E250 caused by the p-dimethylaminomethylbenzaldehyde produced. 2. This reaction was inhibited by substrate analogues such as isothiouronium, guanidino, dimethylsulphonium and trimethylammonium compounds. 3. The inhibition by both mono- and bis-onium compounds has been studied and a comprehensive theory is developed to explain both the type and degree of inhibition produced.
Synthesis of ribonucleic acid by isolated rat liver mitochondria
Authors: Fukamachi S. , Bartoov B. , Freeman K. B.
Published: August 2015

Rat liver mitochondria isolated in sucrose–N-tris(hydroxymethyl)methyl-2-aminoethane-sulphonic acid (TES) incorporated [3H]UTP into RNA for 1h. Incorporation was inhibited 50% by 1μg of actinomycin D/ml, 1μg of acriflavine/ml and 0.5μg of ethidium bromide/ml but was insensitive to rifampicin, rifamycin SV, streptovarcin and deoxyribonuclease. After the first 10min of incubation, the synthesis was insensitive to ribonuclease. RNA synthesis by mitochondria isolated in sucrose–EDTA was insensitive to actinomycin D and sensitive to ribonuclease during the first 10min of the incubation but thereafter the sensitivities were the same as for mitochondria isolated in sucrose–TES. In a hypo-osmotic medium the relative extent of incorporation of the four ribonucleoside triphosphates into RNA was CTP>UTP=ATP»GTP. In an iso-osmotic medium the incorporation of CTP and GTP decreased. All four nucleotides were incorporated into RNA in a DNA-dependent process, as indicated by the inhibition by actinomycin D. In addition, CTP and ATP were incorporated into the CCA end of mitochondrial tRNA. ATP was also incorporated into an unidentified acid-insoluble compound, which hydrolysed in alkali to a product that was not ATP, ADP or 5′- or 2(3′)-AMP. Atractyloside inhibited the incorporation of ATP into RNA with 50% inhibition at 2–3nmol/mg of protein. The [3H]UTP-labelled RNA had peaks of 16S and 13S characteristic of mitochondrial rRNA. In addition a peak at 20–21S was observed as well as heterogeneous RNA sedimenting throughout the gradient. The synthesis of all these species was inhibited by actinomycin D, indicating that rat liver mitochondrial DNA codes for mitochondrial rRNA as well as other as yet unidentified species.
Metabolism of sodium oestrone [35S]sulphate in the guinea pig
Authors: Dolly J. O. , Curtis C. G. , Dodgson K. S. , Rose F. A.
Published: August 2015

Intraperitoneal administration of sodium oestrone [35S]sulphate to male and female free-ranging guinea pigs is followed by excretion of most of the radioactivity mainly as inorganic [35S]sulphate in the urine within 72h. The remainder of the radioactivity in the urine was found in oestrone [35S]sulphate, two unidentified metabolites (A and B) and traces of oestradiol-17β 3-[35S]sulphate. When injected intraperitoneally into animals with bile-duct and bladder cannulae, most of the dose was excreted in the bile. Unchanged oestrone [35S]sulphate was the main biliary component excreted in males and females, but the latter also excreted appreciable amounts of oestradiol-17β 3-[35S]sulphate and metabolites A and B. The urine from these animals also contained these metabolites, inorganic [35S]sulphate and also oestrone [35S]sulphate, but in small amounts. Metabolite A was present only in samples from males. Whole body radioautography pinpointed the liver and kidney as the possible sites of metabolism of the ester. The ester underwent little desulphation in the isolated perfused female guinea-pig liver and in animals in which kidney function had been eliminated, and was excreted unchanged in the bile. These results and the observed low oestrogen sulphatase and arylsulphatase C activities found in guinea-pig liver and kidney support the view that the two enzymes are identical.
The statistical estimation of molecular weights from normal and difference ultracentrifuge boundaries
Authors: McCallum Margaret A. , Spragg S. P.
Published: August 2015

1. To determine molecular weights from boundary data taken from a sedimentation velocity experiment in an ultracentrifuge, the parameter s/D must be estimated. This can be obtained by using non-linear statistical methods to fit a mathematical model [the Fujita & MacCosham (1959) equation] to the results. 2. The statistical method chosen was the simplex method of Nelder & Mead (1965), which was found to be ideal for this problem. Internal errors were calculated at the end of the search for the minimum in the residuals, but in general these errors were found to not represent the overall true error of the experiment. 3. Calculations of molecular weights of myoglobin showed that instabilities at low concentrations of protein (less than 0.8mg/ml) disturbed the calculation of s/D. If 1% (w/v) sucrose was included in the solvent, these instabilities were decreased, and extrapolating to infinite time the linear function of s versus 1/(time) gave an acceptable value for s with an error of ±4.8%. The estimates of the molecular weights were less well-defined and the mean value was low by 8%, with an estimated error of the mean of ±3%. The conclusion was that vibration was responsible for the instabilities without sucrose. 4. The Fujita–MacCosham equation can be extended to make it possible to estimate ratios of sedimentation and molecular weights for difference boundaries. Tests using two solutions of orosomucoid in which a 2% decrease in velocity of one boundary was achieved by adding a calculated quantity of sucrose showed that the analysis gave realistic values for the two ratios, and the error for the ratio of sedimentation coefficients was ±10%. The error was larger for the estimated ratio of the molecular weights, but the analysis gave the expected value for the ratio.
The effects of hormones on milk-fat synthesis in mammary explants from pseudopregnant rabbits
Authors: Strong Christopher R. , Forsyth Isabel , Dils Raymond
Published: August 2015

1. When freshly prepared explants from pseudopregnant-rabbit mammary gland were incubated with sodium [1-14C]acetate plus glucose, they synthesized triglyceride and phospholipid containing long-chain fatty acids. Explants cultured with insulin and corticosterone also synthesized these products. The addition of prolactin to this culture medium increased the rate of fatty acid synthesis up to 40-fold and the explants synthesized predominantly triglyceride enriched with C8:0 and C10:0 fatty acids characteristic of rabbit milk. 2. The maximum rates of fatty acid synthesis obtained by explants from pseudopregnant-rabbit mammary gland after culture with insulin, corticosterone and prolactin were similar to those observed with freshly prepared explants from lactating-rabbit mammary gland. The time in culture required to attain these maximum rates varied between animals, and did not appear to be connected with the time required (6–7 days) to synthesize the maximum proportions of C8:0 and C10:0 acids. 3. As the pattern of short- and medium-chain milk fatty acids is characteristic for many species, the techniques described to determine the time-course for the development of this pattern can be used to investigate hormonal response.
Microbial metabolism of pyridinium compounds. Radioisotope studies of the metabolic fate of 4-carboxy-1-methylpyridinium chloride
Authors: Wright K. A. , Cain R. B.
Published: August 2015

Extracts of Achromobacter D formed CO2, methylamine, succinate and formate as metabolic end-products from N-methylisonicotinic acid (4-carboxy-1-methylpyridinium chloride). The origin of the CO2 in the 4-carboxyl group and of the methylamine in the N-methyl group of N-methylisonicotinate was demonstrated with carboxyl-14C- and N-Me-14C-labelled substrates respectively. The carbon skeletons of formate and succinate were shown to arise from the C-2 and the C-3–C-6 atoms of the heterocyclic ring respectively by using N-methyl[2,3-14C2]isonicotinate. This result is consistent with ring cleavage by the organism between C-2 and C-3.
Characterization of a long-chain 14C-labelled alcohol resulting from a plant enzyme-catalysed reaction with S-adenosyl[methyl-14C]methionine
Authors: Ludwig S. A. , Villemez C. L.
Published: August 2015

A particulate enzyme from Phaseolus aureus seedlings catalyses the incorporation of radioactivity from S-adenosyl[Me-14C]methionine into several lipid acceptors present in the enzyme preparation. The structure of one of the radioactive lipids has been elucidated by using mass, n.m.r. and i.r. spectral data. This compound has a molecular formula C28H57OH, is a saturated primary alcohol and has a single branch of one methyl group in the interior of the hydrocarbon chain. The results of a Kuhn–Roth degradation demonstrate that the branch methyl group is the one derived from S-adenosylmethionine.
The effect of phenylpyruvate on pyruvate metabolism in rat brain
Authors: Patel Mulchand S.
Published: August 2015

1. The effect of phenylalanine and phenylpyruvate on the metabolism of pyruvate by isolated mitochondria from rat brain was investigated. 2. Phenylpyruvate inhibited the fixation of H14CO3- in the presence of pyruvate by intact rat brain mitochondria, whereas phenylalanine and other metabolites of this amino acid had no inhibitory effect on this process. 3. Pyruvate carboxylase activity in freeze-dried rat brain mitochondrial preparations was also inhibited only by phenylpyruvate, and a ‘mixed type’ inhibition was observed. 4. The Km for pyruvate of rat brain pyruvate carboxylase was about 0.2mm. 5. The concentration of phenylpyruvate required for a 50% inhibition of H14CO3- fixation by the intact mitochondria and of pyruvate carboxylase activity was dependent on the concentration of pyruvate used in the incubation medium. 6. The possible significance of inhibition of pyruvate carboxylase activity by phenylpyruvate in the brains of phenylketonuric patients is discussed.
The utilization of iron and its complexes by mammalian mitochondria
Authors: Barnes R. , Connelly J. L. , Jones O. T. G.
Published: August 2015

Sonicated mitochondria catalyse the reduction of ferric salts, and the subsequent incorporation of Fe2+ into haem, when provided with a reducing substrate such as succinate or NADH. The rate of haem synthesis was low under aerobic conditions and, after a short lag period, accelerated once anaerobic conditions were achieved; it was insensitive to antimycin A. The lag period was decreased by preincubating the mitochondria with NADH and Fe3+. Newly formed Fe2+ was autoxidized rapidly and the consequent O2 uptake was measured with an oxygen electrode to determine the rate of enzymic formation of Fe2+ from FeCl3; this reaction was rapid in sonicated mitochondria provided with NADH or succinate and was insensitive to antimycin A. The reaction was very slow in intact mitochondria, suggesting a permeability barrier to Fe3+ ions. This system was used to test the permeability of the mitochondrial membrane to various iron complexes of biological importance. Of the compounds tested only ferrioxamine G appeared to penetrate readily and the iron of this complex was reduced when intact mitochondria were supplied with succinate or NADH-linked substrates. The reduction was insensitive to rotenone or antimycin A. Both ferrioxamine G and ferrioxamine B were, however, reduced by particles. The membrane fraction of sonicated mitochondria was necessary for the reduction. The rate of ferrioxamine B reduction by sonicated mitochondria was measured by a dual-wavelength spectrophotometric assay and was found to be stimulated in conditions where the Fe2+ produced was utilized for haem synthesis. The addition of FeCl3 to anaerobic particles caused an oxidation of cytochrome b when this region of the respiratory chain was isolated by treatment with rotenone and antimycin A. These results suggest that the reduction of ferric iron and its complexes occurs inside the inner mitochondrial membrane in proximity to ferrochelatase. Possible sites for this reduction are the flavoproteins, succinate and NADH dehydrogenase.
The role of glucose in the survival and ‘recovery’ of the anoxic isolated perfused rat heart
Authors: Hearse D. J. , Chain E. B.
Published: August 2015

Studies with the isolated perfused working rat heart were carried out to investigate factors that may enable the heart to recover after periods of anoxia. It was found that the presence of glucose in the perfusion fluid during anoxia was essential for complete post-anoxic recovery and the presence of a high concentration of K+ increased not only the rate of recovery but also the final extent of recovery. In an attempt to clarify the roles played by glucose and K+ in aiding the survival and recovery of the anoxic myocardium the concentrations of parameters associated with energy liberation and anaerobic glycolysis (ATP, ADP, AMP, Pi, creatine phosphate, glycogen and lactate) were measured in the presence and absence of glucose during the anoxic phase. Determinations of these parameters were carried out during the working aerobic control period, the anoxic period (K+ arrest) and the recovery period. The results demonstrated that glucose acted as an energy source during anoxia and thus maintained myocardial concentrations of high-energy phosphates, particularly ATP. These studies have also shown a direct relationship between the ability of the heart to recover and the concentration of myocardial ATP at the time of reoxygenation.
Photosynthesis by isolated chloroplasts. Inhibition by dl-glyceraldehyde of carbon dioxide assimilation
Authors: Stokes D. M. , Walker D. A.
Published: August 2015

Photosynthetic carbon assimilation and associated CO2-dependent O2 evolution by chloroplasts isolated from pea shoots and spinach leaves is almost completely inhibited by 10mm-dl-glyceraldehyde. The inhibitor is without appreciable effect on photosynthetic electron transport, photophosphorylation, the carboxylation of ribulose 1,5-diphosphate or the reduction of 3-phosphoglycerate, but apparently blocks the conversion of triose phosphate into ribulose 1,5-diphosphate.
Fatty acid biosynthesis in rabbit mammary gland during pregnancy and early lactation
Authors: Strong Christopher R. , Dils Raymond
Published: August 2015

The pattern of fatty acids synthesized by mammary-gland explants from rabbits during pregnancy and early lactation has been studied. From day 12 to day 18 of pregnancy, long-chain (C14:0–C18:1) fatty acids were the major products. From day 18 to day 21 of pregnancy there was an increase of up to 12-fold in the rate of fatty acid synthesis per unit wet weight of tissue that was almost exclusively caused by the synthesis of octanoic fatty acid and decanoic fatty acid, which are characteristic of rabbit milk. These medium-chain fatty acids were mainly incorporated into triglycerides. From day 22 to day 27 of pregnancy there was little change in the rate of fatty acid synthesis and the proportions of fatty acids synthesized were essentially the same as those synthesized by the lactating gland, i.e. 80–90% octanoic acid plus decanoic acid. About 2–4 days before parturition a second lipogenic stimulus occurred, although the pattern of fatty acids synthesized did not change.
Isolation and characterization of rabbit kidney brush borders
Authors: Quirk S. J. , Robinson G. B.
Published: August 2015

1. Brush borders were isolated from rabbit kidney-cortex homogenates by rate-zonal centrifugation through a sucrose density gradient in a B-XIV zonal rotor, followed by differential centrifugation. 2. The method of preparation gave brush borders of high purity with a reasonable yield. The morphological appearance supported the evidence from enzymic and chemical investigations, that the brush borders were only slightly contaminated with endoplasmic reticulum, mitochondria, lysosomes and nuclei. 3. The molar ratio of cholesterol to phospholipid lay within the range found in other plasma membranes, but the carbohydrate content was double that found in liver plasma membranes. 4. Alkaline phosphatase, maltase, trehalase and aminopeptidase were major enzymic constituents of the brush borders, and had an approximately equal yield and enrichment, but none of these enzymes fulfilled the criteria for marker enzymes. 5. Mg2+-dependent and Na+,K+-dependent adenosine triphosphatases, although found in brush borders, had low yields and low enrichments.
Dolichols, ubiquinones, geranylgeraniol and farnesol as the major metabolites of mevalonate in Phytophthora cactorum
Authors: Richards J. B. , Hemming F. W.
Published: August 2015

Farnesol, geranylgeraniol, dolichols and ubiquinones were the main radioactive components of the unsaponifiable lipid recovered from Phytophthora cactorum grown in aerated cultures containing [2-14C]mevalonate. The 14C recovered in each of these components was in the approximate proportion 2:4:3:5. When the culture was not aerated no radioactive ubiquinone was recovered. Most of the 14C recovered in the dolichols was found in dolichol−15 (37%), with decreasing amounts in dolichol−14 (30%) and −13 (14%) and only a little (5%) in dolichol−16, whereas the major components, by weight, of the mixture (13μg/g of damp-dry tissue) were dolichol−14, −15 and −16 in the approximate proportion of 1:3:1. Radioautography of appropriate chromatograms indicated the presence also of traces of radioactivity in dolichol-9, −10, −11, −12 and −17. Most (80%) of the 14C recovered in the ubiquinones was associated with ubiquinone-9, the rest being in ubiquinone-8. Most (80%) of the weight of ubiquinones (19μg/g of damp-dry tissue) was also ubiquinone-9. The identification of these compounds was by chromatographic methods and, for the ubiquinones and dolichols, was confirmed by mass spectrometry. In addition, the incorporation of 4R- and/or 4S-3H from [4-3H]-mevalonates showed the expected stereochemistry of biosynthesis, namely that farnesol, geranylgeraniol and ubiquinones were biogenetically all trans and the dolichols each contained three biogenetically trans isoprene residues, the remaining residues being biogenetically cis. The distribution of 14C in the components of the whole lipid of the fungus was consistent with 97% of both the farnesol and geranylgeraniol being present as the fatty acid ester. The corresponding value for dolichols was 37%. The observation by other workers, that this fungus does not form either squalene or sterol, was confirmed.
Conformational changes in human lens proteins in cataract
Authors: Harding John J.
Published: August 2015

The reactivity of protein thiol groups in human lens and the susceptibility of the proteins to tryptic digestion were investigated. Both were found to be greater in some cataractous lenses, indicating that lens proteins have unfolded during cataractogenesis. Almost all the tyrosine in the proteins of the normal human lens reacts with tetranitromethane and is therefore probably on the outside of the major lens proteins.
The structure of a glycerylphosphoryldiglucosyl diglyceride from the lipids of Acholeplasma laidlawii strain B
Authors: Shaw N. , Smith P. F. , Verheij H. M.
Published: August 2015

1. The phosphatidylglucose structure proposed previously (Smith & Henrikson, 1965) for the glucose-containing phospholipid from Acholeplasma laidlawii is incorrect. 2. The structure now proposed is 3-(sn-glycerol-3-phosphoryl-6′-[O-α-d-glucopyranosyl-(1&2)-O-α-d-glucopyranosyl])- sn-1,2-diglyceride, a new type of bacterial lipid. 3. Deacylation of the lipid gave a single water-soluble phosphate ester which could be distinguished on chromatography from synthetic samples of glucosylphosphorylglycerols. 4. Hydrolysis of the lipid with alkali gave a mixture of fatty acids, glycerol 2-phosphate, sn-glycerol 3-phosphate and O-α-d-glucopyranosyl-(1&2)-O-α- d-glucopyranosyl-(1&1)-d-glycerol. 5. The lipid was unaffected on incubation with phospholipases A, C and D. 6. Diglucosyl diglyceride was isolated after treatment of the lipid with 60% HF, establishing the location of the fatty acid residues. 7. Periodate oxidation studies showed that the sn-glycerol 3-phosphate was esterified to the 6-hydroxyl group of one of the glucose residues in diglucosyl diglyceride.
The graphical determination of Km and Ki
Authors: Dixon M.
Published: August 2015

The principle of Dixon (1965) has been extended to give rapid graphical methods for determining enzyme constants for substrates (Km) and inhibitors (Ki). It does away with the sometimes questionable assumption that the amounts of substrate or inhibitor bound by the enzyme are negligible in comparison with the total amount added, and is therefore valid even for cases of high affinity, where the usual methods fail. Besides doing away with the need for calculation, it enables the concentrations of the various components of the system to be read off directly for any point of the velocity curve.
Effects of organic mercurials on mammalian pancreatic β-cells. Insulin release, glucose transport, glucose oxidation, membrane permeability and ultrastructure
Authors: Bloom Gunnar D. , Hellman Bo , Idahl Lars-Åke , Lernmark Åke , Sehlin Janove , Täljedal Inge-Bert
Published: August 2015

The effects of p-chloromercuribenzoic acid and chloromercuribenzene-p-sulphonic acid on pancreatic islets were studied in vitro. Obese–hyperglycaemic mice were used as the source of microdissected islets containing more than 90% β-cells. p-Chloromercuribenzoic acid and chloromercuribenzene-p-sulphonic acid stimulated insulin release at concentrations of 0.01mm or above. This stimulation was significantly inhibited by the omission of Ca2+or the addition of adrenaline, diazoxide or 2,4-dinitrophenol. p-Chloromercuribenzoic acid or chloromercuribenzene-p-sulphonic acid did not interfere with the insulin-releasing ability of glucose. Micro-perifusion experiments revealed that the release of insulin in response to organic mercurial occurred almost instantaneously, was reversible, and was biphasic. The two mercurials inhibited glucose transport as well as glucose oxidation, and increased the mannitol and sucrose spaces of isolated islets. Compared with the effects on insulin release, those on glucose transport and membrane permeability were characterized by a longer latency and/or required higher concentrations of organic mercurial. Apart from a seemingly higher proportion of β-cells exhibiting certain degenerative features, in islets exposed to 0.1mm-chloromercuribenzene-p-sulphonic acid for 60min, no significant differences with respect to β-cell fine structure were noted between non-incubated islets and islets incubated with chloromercuribenzene-p-sulphonic acid or glucose or both. It is suggested that insulin release may be regulated by relatively superficial thiol groups in the β-cell plasma membrane.
The effects of acute and chronic morphine treatment and of morphine withdrawal on rat brain in vivo
Authors: Miller A. L. , Hawkins R. A. , Harris R. L. , Veech R. L.
Published: August 2015

1. The effects of morphine, nalorphine, acetazolamide, and 10% CO2 on brain metabolite concentrations of 24h-starved rats were studied. 2. A single dose of morphine (20mg/kg body wt.) caused an increase in brain glucose concentration (42%) and decreased concentrations of lactate (24%), pyruvate (29%), citrate (20%), α-oxoglutarate (16%), malate (14%) and creatine phosphate (10%) after 30min. No changes were found in adenine nucleotide concentrations. 3. The same dose of morphine increased arterial CO2 from 5.07 to 7.60 kN/m2(38 to 57 Torr), decreased the pH from 7.41 to 7.31 and decreased O2 from 14.1 to 10.8kN/m2(106 to 81 Torr) at 30min. 4. Rats injected with morphine three times daily (20mg/kg body wt.) for 2 weeks had no changes in brain metabolite concentrations or in blood gases 30min after their last injection. 5. Nalorphine (an antagonist of morphine) caused essentially no changes in brain metabolite concentrations in normal rats. When nalorphine (20mg/kg) was administered to rats previously treated with morphine three times daily for 2 weeks, there was an increase in brain glucose (100%), lactate (23%), pyruvate (18%) and citrate (10%) concentrations. 6. Acetazolamide (an inhibitor of carbonic anhydrase) and 10% CO2 increased the arterial CO2 from 4.79 to 6.78kN/m2(36 to 51 Torr) and from 5.32 to 10.8kN/m2(40 to 81 Torr) respectively. 7. Both acetazolamide and 10% CO2 caused changes in brain metabolite concentrations similar to those for acutely administered morphine. Thus 10% CO2 caused increased brain glucose concentration (123%) and decreased brain lactate (46%), pyruvate (34%), citrate (26%), α-oxoglutarate (33%), malate (45%) and creatine phosphate (7%) concentrations. No changes in adenine nucleotide concentrations were found. 8. The results indicate that the effect of morphine on brain metabolite concentrations may be accounted for by the increased [CO2]. 9. These findings constitute a consistent pattern of metabolic changes after acute morphine administration, morphine addiction, and withdrawal from morphine addiction.
Relation between the oxidation state of nicotinamide–adenine dinucleotide and the metabolism of spermatozoa
Authors: Brooks D. E. , Mann T.
Published: August 2015

1. The existing procedures for extraction of oxidized and reduced nicotinamide coenzymes were adapted to spermatozoa to overcome the coenzyme-degrading activity of seminal plasma. 2. The content of total NAD+and NADH was determined in the spermatozoa of ram, bull, boar, stallion and cock. NADP+and NADPH were not detected in ram spermatozoa. 3. The oxidation state of sperm NAD depended on the seminal plasma, the removal of which produced a change in the percentage oxidation state of the coenzyme, 100×[NAD+/(NAD++NADH)], without altering the total content of NAD++NADH. 4. In suspensions of washed ram spermatozoa, incubated anaerobically at 25°C, the percentage oxidation state of NAD declined with increasing spermatozoa concentration. 5. When ram or boar spermatozoa that had been previously washed and resuspended in Ringer phosphate medium, were incubated anaerobically at 25°C with various substances, pronounced effects on the percentage oxidation state of NAD could be observed with l-lactate, pyruvate, oxaloacetate, dihydroxyacetone, formaldehyde and glyceraldehyde; sorbitol and acetoacetate acted only on ram spermatozoa; fructose, glucose, mannose and acetaldehyde acted predominantly on boar spermatozoa. Formaldehyde lowered the (NAD++NADH) content of ram spermatozoa, but none of the other substances had a comparable effect. 6. The percentage oxidation state of sperm NAD was not influenced by exogenous cysteine, cystine, ergothioneine or ascorbate. 7. A highly active sorbitol dehydrogenase could be prepared from ram, but not from boar, spermatozoa. 8. Sorbitol, acetoacetate and 3-hydroxybutyrate effectively supported the respiration of ram, but not boar, spermatozoa. 9. ‘Cold shock’, resulting from sudden cooling of spermatozoa, abolished motility completely and irreversibly but produced only a slow and partial decrease in the total NAD content. Slight over-heating, sufficient to produce loss of motility, had no adverse effect on the total NAD content. 10. Storage of ram sperm at 14°C produced only a small decrease of NAD after 2 days, but subsequently the loss became greater.