bluuebunny/crossref_metadata_2025 huggingface dataset:
Lipid oxidation by heart mitochondria from young adult and senescent rats
Authors: Hansford Richard G.
Published: August 2015

1. State-3 (i.e. ADP-stimulated) rates of O2 uptake with palmitoylcarnitine, palmitoyl-CoA plus carnitine, pyruvate plus malonate plus carnitine and octanoate as respiratory substrate were all diminished in heart mitochondria isolated from senescent (24-month-old) rats compared with mitochondria from young adults (6 months old). By contrast, State-3 rates of O2 uptake with pyruvate plus malate or glutamate plus malate were the same for mitochondria from each age group. 2. Measurements of enzyme activities in disrupted mitochondria showed a decline with senescence in the activity of acyl-CoA synthetase (EC 6.2.1.2 and 6.2.1.3), carnitine acetyltransferase (EC 2.3.1.7) and 3-hydroxy-acyl-CoA dehydrogenase (EC 1.1.1.35), but no change in the activity of carnitine palmitoyltransferase (EC 2.3.1.21) or acyl-CoA dehydrogenase (EC 1.3.99.3). 3. Measurement of dl-[3H]carnitine (in)/acetyl-l-carnitine (out) exchange in intact mitochondria showed decreased rates when the animals used were senescent. However, this followed from a decreased intramitochondrial pool of exchangeable carnitine, such that calculated first-order rate constants for exchange were identical in mitochondria from the two age groups. 4. The decline in acyl-CoA synthetase activity is thought to be the reason for the diminished rate of O2 uptake with octanoate in senescence. The decline in carnitine acetyltransferase activity is considered to be the cause of the diminished rate of O2 uptake with acetylcarnitine or with pyruvate plus malonate plus carnitine as substrate. The mechanism of the diminished rate of O2 uptake with palmitoylcarnitine in senescence is discussed.
Calcitonin-mediated changes in plasma tryptophan and brain 5-hydroxytryptamine and acetylcholinesterase activity in rats
Authors: Nakhla A M , Nandi Majumdar A P
Published: August 2015

After a single injection of calcitonin (20 M.R.C. units/kg body wt.) marked decreases in both Ca2+ and free tryptophan in plasma were observed, during the initial period of the treatment (up to 1 h). However, 5-hydrotryptamine contents of the whole brain and the cerebral acetylcholinesterase activity were greatly enhanced. The cerebellar acetylcholinesterase activity was not influenced by calcitonin.
The cellulase of Trichoderma koningii. Purification and properties of some endoglucanase components with special reference to their action on cellulose when acting alone and in synergism with the cellobiohydrolase
Authors: Wood T M , McCrae S I
Published: August 2015

1. Four principal endoglucanase components of Trichoderma koningii cellulase were separated and purified by gel filtration on Sephadex G-75, ion-exchange chromatography on DEAE- and sulphoethyl-Sephadex and isoelectric focusing. 2. All four endoglucanases hydrolysed CM-cellulose, H3PO4-swollen cellulose, cellotetraose and cellopentaose, but differed in the rate and mode of attack. 3. Attack on cotton fibre by the endoglucanases was minimal, but resulted in changes that were manifested by an increased capacity for the uptake of alkali, and a decrease in tensile strength. 4. All four endoglucanases acted synergistically with the exoglucanase [cellobiohydrolase; Wood & McCrae (1972) Biochem. J. 128, 1183-1192] of T. koningii during the early stages of the breakdown of cotton fibre, but only two could produce extensive solubilization of cotton cellulose when acting in admixture with the exoglucanase component. 5. The mode of action of the enzymes is discussed in relation to these synergistic effects. It is suggested that the results are compatible with the interpretation that the ‘crystalline’ areas of cotton cellulose are hydrolysed only by those endoglucanases capable of forming of forming an enzyme-enzyme complex with the cellobiohydrolase on the surface of the cellulose chains.
The fate of bilirubin-IXα glucuronide in cholestasis and during storage in vitro. Intramolecular rearrangement to positional isomers of glucuronic acid
Authors: Blanckaert N , Compernolle F , Leroy P , Van Houtte R , Fevery J , Heirwegh K P M
Published: August 2015

1. In aqueous solution above pH7 bilirubin-IXalpha 1-O-acylglucuronide rapidly isomerizes to the non-C-1 glucuronides by sequential migration of the bilirubin acyl group from position 1 to positions 2, 3 and 4 of the sugar moiety. The transformations are enhanced by increasing the pH. Compared with the rates at 37 degrees C the transformations are rather slow at 0 degrees C. Virtually complete inhibition is observed at values below pH6. The isomerization at 25 degrees C and pH 7.4 is not affected by the presence in the solutions of a molar excess of human serum albumin. 2. Isomerization in bile kept at 37 degrees C at pH7.7-7.8 is probably non-enzymic, as the rates of change are similar to those observed under comparable conditions for aqueous solutions of glucuronides of bilirubin-1Xalpha and of azodipyrrole. 3. Analysis without delay of normal biles of man and rats collected at 0 degrees C over a maximum period of 10 min shows that the bilirubin-IXalpha mono- and di-glucuronides consist exclusively of the 1-O-acyl isomers. 4. The mixtures of the four positional isomers of bilirubin-IXalpha glucuronide found in freshly collected biles of man and rats with cholestasis probably originate from initially synthesized 1-O-acylglucuronide by the same mechanism of sequential migration as has been observed in aqueous solutions of conjugated bilirubin-IXalpha.
Kinetics of tetrahydrobiopterin synthesis by rabbit brain dihydrofolate reductase
Authors: Abelson H T , Spector R , Gorka C , Fosburg M
Published: August 2015

Product identification and kinetic data are presented for the conversion of 7,8-dihydrobiopterin into tetrahydrobiopterin by purified rabbit brain dihydrofolate reductase.
Sequence of the full-length immunoglobulin κ-chain of mouse myeloma MPC 11
Authors: Smith G P
Published: August 2015

MPC 11 mouse myeloma cells synthesize two immunoglobulin kappa light chains, coded by two separate genes. One of these Kappa-chains has no variable region and is degraded intracellularly. The other is a full-length kappa-chain contaning both variable and constant regions: this chain is secreted, both by itself and combined with heavy chains in molecules of immunoglobulin G. This paper reports the amino acid sequence of the myeloma MPC 11 full-length kappa-chain. The chain is unusual in having 12 extra residues at its N-terminus when its sequence is aligned with those of other mouse kappa-chains; no other anomalies were found in its sequence.
Effect of different compounds on 1-aspartamido-β-N-acetylglucosamine amidohydrolase from human liver
Authors: Dugal B
Published: August 2015

The effect of varous compounds on 1-aspartamido-beta-N-acetylglucosamine amidohydrolase (aspartylglucosylaminase, EC 3.5.1.26) was studied. N-Acetylcysteine inhibited the nezyme non-competitively (Ki 3.2 mM), whereas 3-hydroxybutanone inhibited competitively (Ki 4.1 mM). Methionine, isoleucine and cystathionine apparently enhanced the enzyme activity. The enzyme had a mol. wt. of 63000 as determined by gel filtration. The present studies differentiate between the aspartylglucosylaminase from human liver and that obtained from various other sources.
Purification and properties of a manganese-stimulated deoxyribonuclease produced during sporulation of Bacillus subtilis
Authors: Akrigg A
Published: August 2015

A DNAase (deoxyribonuclease) was isolated from culture supernatants of sporulating Bacillus subtilis 168. The purified enzyme migrated as a single band during polyacrylamide-gel electrophoresis. The enzyme differs from other DNAases of B. subtilis in molecular weight, metal-ion requirement and mode of action. The enzyme was inactive in the absence of metal ions, and exhibited optimum activity with 10 mM-Mn2+, although Mg2+, Cd2+ and Co2+ could also permit some activity. The pH optimum for the enzyme was pH 7.5, and it degraded linear-duplex DNA or closed-circular-duplex DNA to acid-soluble material. There was little or no activity on single-stranded DNA or rRNA. Sucrose-gradient analysis of the products of DNAase action on bacteriophage T7 DNA showed that endonucleolytic cleavage had occurred by the introduction of single-strand breaks in both strands of the duplex. The molecular weight of the enzyme was determined, by gel filtration on Sephadex G-75, to be 12000.
Evidence that the coproporphyrinogen oxidase activity of rat liver is situated in the intermembrane space of mitochondria
Authors: Elder G H , Evans J O
Published: August 2015

Preferential rupture of the outer membrane of mitochondria from rat liver releases coproporphyrinogen oxidase in parallel with components of the intermembrane space. Coproporphyrinogen III enters the mitochondrion through the freely-permeable outer membrane. Either protoporphyrinogen IX or protoporphyrin IX must then cross the inner membrane before haem synthesis can be completed.
Proline biosynthesis by cell-free extracts of Escherichia coli and potential errors arising from the use of a bioradiological assay procedure
Authors: Hayzer D J , Moses V
Published: August 2015

1. The growth of Escherichia coli proline auxotrophs on medium containing L-proline (50 microgram/ml) induces catabolic enzymes. A bioradiological assay system for proline, using proB cells of E. coli, might give erroneous results owing to proline catabolism by the proline auxotrophs on which the assay depends. 2. Differential utilization of proline and 1-pyrroline-5-carboxylate by the proB cells for the synthesis of protein, and failure of the method to distinguish between these two possible products of the proline-biosynthetic enzymes, might also give rise to error. 3. The proline-dependent incorporation of [14C]phenylalanine into the protein of proline-starved proB auxotrophs was to some degree directly influenced by the presence of crude cell extract from E. coli, even though this was not supplied with substrate and cofactors, and could thus not itself synthesize proline. 4. The kinetics of proline biosynthesis by cell-free extracts were linear and biphasic, only the last phase being affected by the concentrations of substrate and extract. This phenomenon is not understood. 5. Proline biosynthesis is inhibited, not only by high concentrations of ATP, but also by aspartate, glycine, alanine and serine, aspartate having the greatest effect. 6. Attempts at complementation in vitro between extracts of proline auxotrophic mutants were not successful, suggesting the possibility that strain X680 (proA) and/or X278 (proB) may be a double mutant. 7. The enzymes of proline biosyntehsis are co-eluted from a column of Bio-Gel A1.5M in a position corresponding to a mol.wt. of 350000. 8. Comparisons between rates of proline biosynthesis in vivo and in vitro were made.
Kinetics and mechanism of the rat brain phenol sulphotransferase reaction
Authors: Pennings E J M , Vrielink R , van Kempen G M J
Published: August 2015

Some properties of rat brain phenol sulphotransferase were investigated in in vitro at pH7.4. The enzyme was purified 10-fold by chromatography on DEAE-Sephadex −50. It can be assayed with 4-hydroxy-3-methoxyphenylethylene glycol or 4-methylumbelliferone as the sulphate acceptor. The partially purified enzyme is stable for at least 1 week when stored at 4 degrees C. It is, however, additionally activated (10–20%) and stabilized by 1 mM-dithiothreitol. The activity of the enzyme does not depend on the addition of exogenous Mg2+. The pH optima for the sulphation of 4-hydroxy-3-methoxyphenylethylene glycol and 4-methylumbelliferone are 7.8 and 7.4 respectively. Substrate inhibition by the sulphate acceptor is apparent at concentrations over 0.05mM. Initial-velocity studies in the absence and presence of product and dead-end inhibitors suggested that the mechanism of the rat brain sulphotransferase reaction is sequential ordered Bi Bi with a dead-end complex of enzyme with adenosine 3′,5′-biphosphate and sulphate acceptor. The sulphate donor adenosine 3′-phosphate 5′-sulphatophosphate is the first substrate that adds to the enzyme, and the sulphate acceptor is the second substrate. The dissociation constant for the complex of enzyme with sulphate donor is 21 micron. The sulphated substrate is the first product and adenosine 3′,5′-biphosphate is the second product that leaves the enzyme.
Regulation of pig heart pyruvate dehydrogenase by phosphorylation. Studies on the subunit and phosphorylation stoicheiometries
Authors: Sugden Peter H. , Randle Philip J.
Published: August 2015

1. The molecular weights of the subunits of purified pig heart pyruvate dehydrogenase complex were determined by sodium dodecyl sulphate/polyacrylamide-disc-gel electrophoresis and were: pyruvate decarboxylase, α-subunit 40600, β-subunit 35100; dihydrolipoyl acetyltransferase 76100; dihydrolipoyl dehydrogenase 58200. 2. Inactivation of the pyruvate dehydrogenase complex by its integral kinase corresponded to the incorporation of 0.46nmol of P/unit of complex activity inactivated. 3. Further incorporation of phosphate into the complex occurred to a limit of 1.27nmol of P/unit of complex inactivated (approx. 3 times that required for inactivation). 4. Phosphate was incorporated only into the α-subunit of the decarboxylase. 5. The molar ratio of phosphate to α-subunits of the decarboxylase was estimated by radioamidination of amino groups of pyruvate dehydrogenase [32P]phosphate complex by using methyl [1-14C]acetimidate, followed by separation of α-subunits by sodium dodecyl sulphate/polyacrylamide-disc-gel electrophoresis. Inactivation of the complex (0.46nmol of P/unit of complex inactivated) corresponded to a molar ratio of one phosphate group per two α-chains (i.e. one phosphate group/α2β2 tetramer). Complete phosphorylation corresponded to three phosphate groups per α2β2 tetramer. 6. Subunit molar ratios in the complex were also estimated by the radioamidination technique. Results corresponded most closely to molar ratios of 4 α-subunits:4 β-subunits:2 dihydrolipoyl acetyltransferase subunits:1 dihydrolipoyl dehydrogenase subunit.
The oxidation of Pseudomonas cytochrome c-551 oxidase by potassium ferricyanide
Authors: Barber D , Parr S R , Greenwood C
Published: August 2015

Stopped-flow kinetics were made of the reaction between ascorbate-reduced Pseudomonas cytochrome oxidase and potassium ferricyanide under both N2 and CO atmospheres. Under N2 three kinetic processes were observed, two being dependent on ferricyanide concentration, with second-order rate constants of 9.6×10(4)M-1.s-1 and 1.5×10(4)M-1.s-1, whereas the other was concentration-independent, with a first-order rate constant of 0.17 +/- 0.03s-1. Measurements of their kinetic difference spectra have allowed the fastest and second-fastest phases of the reaction to be assigned to direct bimolecular reactions of ferricyanide with the haem c and haem d, moieties of the enzyme respectively. Under CO, the second-order rate constant for the reaction of the haem c was, at 1.3×10(5)M-1.s-1, slightly enhanced over the rate in a N2 atmosphere, but the reaction velocity of the haem d1 component was greatly decreased, being apparently limited to that of the rates of CO dissociation from the molecule (0.15s-1 and 0.03s-1). The results are compared with those obtained during a previous study of the reaction of reduced Pseudomonas cytochrome oxidase with oxidized azurin.
The oxygenation of [3-methyl-3H]desacetoxycephalosporin C [7β-(5-d-aminadipamido)-3-methylceph-3-em-4-carboxylic acid] to [3-hydroxymethyl-3H]desacetylcephalosporin C by 2-oxoglutarate-linked dioxygenases from Acremonium chrysogenum and Streptomyces clavuligerus
Authors: Turner M K , Farthing J E , Brewer S J
Published: August 2015

Cell-free extracts of Acremonium chrysogenum and Streptomyces clavuligerus oxidize the 3-methyl group of desacetoxycephalosporin C to a 3-hydroxymethyl group. The enzyme responsible for this reaction in these organisms was purified 20- and 30-fold respectively by chromatography on DEAE-cellulose. The enzymes, which were assayed with [3-methyl-3H]desacetoxycephalosporin C as substrate, have the properties expected of 2-oxoglutarate-linked dioxygenases. They require 2-oxoglutarate, Fe2+ cations and a mixture of reducing agents (dithiothreitol and ascorbate) for full activity. The enzyme from A. chrysogenum, but not that S. clavuligerus, is activated about 10-fold when it is preincubated with a reaction mixture from which either desacetoxycephalosporin C or 2-oxoglutarate is omitted. Fe2+ cations seem to play a key role in this activation. Both enzymes seem highly specific for cephalosporins with the natural 7beta-(5-D-aminoadipamido) side chain and are likely to be responsible for the oxidation of the 3-methylcephem nucleus in vivo.
A protein-glucan intermediate during paramylon synthesis
Authors: Tomos A D , Northcote D H
Published: August 2015

A sodium deoxycholate extract containing glucosyltransferase activity was obtained from a particulate preparation from Euglena gracilis. It transferred glucose from UDP-[14C]glucose into material that was precipitated by trichloroacetic acid. This material released beta-(1 leads to 3)-glucan oligosaccharides into solution on incubation with weak acid, weak alkali and beta-(1 leads to 3)-glucosidase. The products of the incubation of the deoxycholate extract with UDP-[14C]glucose were analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Radioactive bands were obtained that had the properties of beta-(1 leads to 3)-glucan covalently linked to protein by a bond labile to weak acid. High-molecular-weight material containing a beta-(1 leads to 3)-glucan was also shown to be present by gel filtration. The bond linking glucan to aglycone is possibly a pyrophosphate linkage. It is proposed that in Euglena gracilis beta-(1 leads to 3)-glucan (paramylon) is synthesized on a protein primer.
Oestrogen-induced cholesterol and fatty acid biosynthesis in Xenopus laevis liver during vitellogenic response
Authors: Smith Deborah F. , Penning Trevor M. , Ansari Abdul Quadar , Munday Kenneth A. , Akhtar Muhammad
Published: August 2015

1. Oestradiol-17β induces livers of Xenopus laevis (South African clawed toad) to synthesize and secrete into the serum large quantities of the egg-yolk-protein precursor, vitellogenin. The peak of this response occurs 9–16 days after hormone treatment [Dolphin, Ansari, Lazier, Munday & Akhtar (1971) Biochem. J.124, 751–758]. It is now shown that 6 days after hormone treatment a 120–160-fold stimulation of the synthesis of cholesterol and fatty acid compared with control values occurred. 2. A cell-free system, derived from Xenopus liver, which synthesizes squalene and fatty acid is described. By using this system, several hundredfold stimulation of incorporation of [14C]acetate into squalene was recorded 6 days after the administration of oestradiol-17β, compared with a 3–4-fold stimulation of incorporation of [3H]mevalonate compared with control values. It is argued that oestradiol-17β must affect enzyme(s) catalysing step(s) between acetate and mevalonate in the biosynthetic pathway to cholesterol. 3. In incubation of liver slices in vitro, most of the lipid and cholesterol synthesized in response to the steroid hormone was associated with those subcellular fractions that contained membranes. Moreover, pulse-labelling experiments in vivo showed that 70% of this lipid and cholesterol was retained in the liver. The remainder appeared in the serum, where it was equally distributed between vitellogenin and vitellogenin-free serum. 4. G.l.c. analyses of the cholesterol content of liver microsomal fractions of Xenopus laevis indicated that the cholesterol content was at least 50% higher in microsomal fractions obtained from livers that had been exposed to oestradiol-17β. Meanwhile, g.l.c. analysis of the lipid moiety of secreted vitellogenin showed that up to 35% of its lipid was cholesterol.
Stereospecific biosynthesis of triacylglycerols in mammary glands from lactating rats
Authors: Cooper S M , Grigor M R
Published: August 2015

Microsomal plus cytosol preparations from the mammary gland of lactating rats are capable of incorporating palmitic acid and oleic acid into triacylglycerols. These triacylglycerols are similar in structure to those found in rat milk, where palmitic acid tends to be confined to the sn-2-position of the glycerol. Both glycerol 3-phosphate and dihydroxyacetone phosphate function as acyl acceptors. The enzymic synthesis of triacylglycerols appears in late pregnancy, increases rapidly during early lactation, but disappears within 3 days of weaning.
Cerebral oxidized and reduced nicotinamide-adenine dinucleotide phosphate and glucose 6-phosphate dehydrogenase in mice during exposure to high oxygen pressure
Authors: Dodd D E , Faiman M D
Published: August 2015

NADP+, NADPH and glucose 6-phosphate dehydrogenase were determined in the cerebral cortex of mice exposed to high O2 pressure for 0, 8 and 16 min. These time intervals corresponded to 0, 50 and 100% of the CT50 (the time taken for 50% of the mice to convulse). Cerebral NADP+, NADPH and glucose 6-phosphate dehydrogenase also were determined in O2-exposed mice exhibiting hyperactivity, convulsions, and in mice killed 10s after convulsions. Similar increases in cortical NADP+ and decreases in NADPH were found in mice exposed to 610kPa (6 atm.) of 100% O2 for 0, 50 and 100% of the CT50, during hyperactivity, onset of seizure and 10s after convulsions. The NADP+/NADPH ratio increased approx. 25% at 0% of the CT50, and remained at this increased value at all O2-exposure periods including the hyperactive state, onset of seizure and 10s after convulsions. Identical changes in cerebral NADP+, NADPH and the NADP+/NADPH ratio were found in mice exposed for 16min to 100% O2 at 100, 350 or 610kPa. No change in cerebral glucose 6-phosphate dehydrogenase was found in mice exposed to 610kPa of 100% O2 during the various stages of O2 toxicity. Only in the 10s post-convulsive group was a statistically significant decrease in glucose 6-phosphate dehydrogenase observed. Disulfiram [bis(diethylthiocarbamoyl) disulphide], an effective O2-protective agent, did not prevent the O2-induced increase in cerebral NADP+ and the NADP+/NADPH ratio, or decrease in NADPH.
The interaction between mitochondrial NADH-ubiquinone oxidoreductase and ubiquinol-cytochrome c oxidoreductase. Restoration of ubiquinone-pool behaviour
Authors: Heron C , Ragan C I , Trumpower B L
Published: August 2015

1. In the inner mitochondrial membrane, dehydrogenases and cytochromes appear to act independently of each other, and electron transport has been proposed to occur through a mobile pool of ubiquinone-10 molecules [Kröger & Klingenberg (1973) Eur. J. Biochem. 34, 358–368]. 2. Such behaviour can be restored to the interaction between purified Complex I and Complex III by addition of phospholipid and ubiquinone-10 to a concentrated mixture of the Complexes before dilution. 3. A model is proposed for the interaction of Complex I with Complex III in the natural membrane that emphasizes relative mobility of the Complexes rather than ubiquinone-10. Electron transfer occurs only through stoicheiometric Complex I-Complex III units, which, however, are formed and re-formed at rates higher than the rate of electron transfer.
The adenylate cyclase-activating activity of cholera toxin is not associated with a nicotinamide—adenine dinucleotide glycohydrolase activity
Authors: Tait R M , van Heyningen S
Published: August 2015

The NAD+ glycohydrolase activity of cholera-toxin samples can be separated from their adenylate cyclase-activating activity by polyacrylamide-gel electrophoresis and is inhibited by sodium dodecyl sulphate (which does not inhibit the action of toxin on cells), but not by antibodies to pure toxin. It is therefore probably not a true property of the toxin.
The susceptibility of muscle phosphorylases a and b to digestion by a neutral proteinase from rat intestinal muscle. Comparison with the effects produced by pancreatic trypsin and chymotrypsin
Authors: Carney Ian T. , Beynon Robert J. , Kay John , Birket Nigel
Published: August 2015

1. Phosphorylase b was inactivated three times more rapidly than phosphorylase a by a neutral, trypsin-like proteinase from rat intestinal muscle. Digestion of phosphorylase a produced a modified form which was deactivated by AMP. Removal of the pyridoxal phosphate cofactor increased the rate of inactivation of the b form by about 3-fold but the subceptibility of apophosphorylase a was no different from the holo form. 2. The extent of proteolysis of both holoenzyme forms, as guaged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, was limited and similar digestion patterns were obtained in both cases. 3. With 32P-labelled phosphorylase a as substrate, the initial event in the inactivation was the release of a trichloroacetic acid-soluble peptide from the N-terminus of the enzyme, leaving the original 100000 subunit form essentially unchanged. Subsequent proteolysis was restricted, producing derivatives of mol.wt. 85000, 70000 and 65000, none of which contained any radioactive label. 4. By treatment of inactivated phosphorylase b with carboxypeptidase B, it was shown that the intestinal muscle proteinase had cleaved approximately 3 -Lys-X and 3 -Arg-X bonds in the polypeptide. 5. The protective effects of various allosteric modulators of phosphorylase on the inactivation of the a and b forms were generally in agreement with the known roles of the modifiers. Glucose increased the susceptibility of phosphorylase a. 6. Inactivation of phosphorylase b by trypsin and chymotrypsin also resulted in limited proteolysis but, in both cases, the digestion patterns obtained on sodium dodecyl sulphate/polyacrylamide gels were different from each other and from the pattern obtained with the intestinal muscle proteinase. 7. Inactivation of phosphorylase b by the muscle proteinase is about 100 times more rapid than the effects produced by trypsin or chymotrypsin when the activities are compared on an equimolar basis. 8. Consideration is given to regulation of the rate of enzyme degradation intracellularly by modulation of the conformation and susceptibility of the enzyme via factors such as covalent modification, allosteric ligands and state of aggregation.
A specific dimerization of rabbit β-globin messenger ribonucleic acid
Authors: Mezl V A , Hunt J A
Published: August 2015

Rabbit globin mRNA, when layered in low salt on 0.1 M-NaCl/sucrose gradients, separates into two peaks of material. Translation of these two RNA fractions in the wheat-germ cell-free system, hybridization against globin complementary DNA (cDNA) and cross-hybridization against cDNA species prepared from each fraction show that the first peak sedimenting at 10S is a alpha-globin mRNA and the second peak, sedimenting at approx. 15S, is beta-globin mRNA. The sedimentation rate of the beta-globin mRNA is concentration-dependent. By changing concentration and pH, it is indicated that in low-salt beta-globin mRNA adopts a conformation that leads to specific, but weak, self-dimerization during centrifugation in 0.1M-NaCl. This property permits rapid preparation of intact and relatively pure alpha- and beta-globin mRNA species.
α-l-iduronate ring conformations in heparin and heparin derivatives. 13C Nuclear-magnetic-resonance analysis and titration data for variously desulphated and periodate-oxidized heparins
Authors: Fransson L A , Huckerby T N , Nieduszynski I A
Published: August 2015

A heparin derivative that had been O/N-desulphated and re-N-acetylated was investigated by 13C n.m.r. spectroscopy and potentiometric titration. Three forms of uronic acid were observed, tentatively identified as beta-D-glucuronate, and two different forms of alpha-L-iduronate. A comparison of the n.m.r. spectra of heparin, an oligosaccharide (beta-D-glucuronate-2-acetamido-2-deoxy-alpha-D-glucose)n, and heparin that had been subjected to selective oxidation of beta-D-glucuronate, enabled the position of the anomeric carbon of the latter residue to be assigned [delta 102.9 (p.p.m.)]. Periodate oxidation of O/N-desulphated heparin destroyed in addition, approx. 40% of the alpha-L-iduronate content. The remainder of the alpha-L-iduronate residues displayed only one anomeric resonance, at delta 99.7 (p.p.m.). In another preparation, after sequential desulphation of heparin (N-desulphation, re-N-acetylation and O-desulphation) the anomeric resonance of the alpha-L-iduronate residue shifted downfield [from delta99.7 (p.p.m.) to delta 102.3]indicating a change in ring conformation. These data support the interpretation that the unsulphated alpha-L-iduronate residues may adopt two conformations. It was shown that the proportions of alpha-L-iduronate conformers are determined by the sequence of desulphation operations. Also minor components of heparin were assigned.
The aromatic alcohol dehydrogenases in Pseudomonas putida N.C.I.B. 9869 grown on 3,5-xylenol and p-cresol
Authors: Keat M J , Hopper D J
Published: August 2015

Whole cells of Pseudomonas putida N.C.I.B 9869, when grown on either 3,5-xylenol or p-cresol, oxidized both m- and p-hydroxybenzyl alcohols. Two distinct NAD+-dependent m-hydroxybenzyl alcohol dehydrogenases were purified from cells grown on 3,5-xylenol. Each is active with a range of aromatic alcohols, including both m- and p-hydroxybenzyl alcohol, but differ in their relative rates with the various substrates. An NAD+-dependent alcohol dehydrogenase was also partially purified from p-cresol grown cells. This too was active with m- and p-hydroxybenzyl alcohol and other aromatic alcohols, but was not identical with either of the other two dehydrogenases. All three enzymes were unstable, but were stabilized by dithiothreitol and all were inhibited with p-chloromercuribenzoate. All were specific for NAD+ and each was shown to catalyse conversion of alcohol into aldehyde.
An estimate of the enthalpic contribution to the interaction between dextran and albumin
Authors: Comper W D , Laurent T C
Published: August 2015

It is demonstrated that exclusion phenomena appear to dominate the interaction of dextran with albumin in aqueous solution. The enthalpic contribution to the interaction coefficient describing dextran/albumin mixtures is small, although its determination was subject to considerable error. These results support the earlier assumptions of the type of interaction between the two polymers. The conclusions are primarily based on the interpretation of the temperature-dependence of the interaction coefficient, as measured by light-scattering in the temperature range 6–33 degrees C. Enthalpy of dilution measurements of dextran/albumin mixtures by microcalorimetry were in qualitative agreement with the light-scattering data.
Mouse intracellular immunoglobulin M. Structure and identification of a free thiol group
Authors: Richardson Neil E. , Feinstein Arnold
Published: August 2015

Monomeric intracellular mouse immunoglobulin M (hereafter designated IgMs) was purified in milligram quantities from the plasma cells of mouse plasmacytoma MOPC 104E after lysis either in the presence or in the absence of iodoacetate. Peptide ‘mapping’ analysis of the IgMs after partial reduction and carboxy[14C]methylation to label the interchain disulphide bridges showed that the heavy–light bridge and the interheavy bridge present in the Cμ2 region were already formed at lysis. The cysteine residues in the C-terminal region of the heavy chains, which in pentameric IgM form an intersubunit bridge, had free thiol groups at lysis that were reversibly oxidized during isolation in the absence of iodoacetate, probably forming an intrasubunit inter-heavy-chain disulphide bridge. Isoelectric-focusing studies complemented the above findings, showing that all the intracellular IgMs carried free thiol groups that could be carboxymethylated at lysis, and that in non-alkylated preparations these had reversibly oxidized. On the basis of sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis intracellular μ-chains had a consistently lower apparent molecular weight than did secreted μ-chains, and the estimated difference could be accounted for by the known difference in carbohydrate content. We present evidence that in a position homologous to that of a complex oligosaccharide in the Cμ2 region of secreted human μ-chains there is a simple oligosaccharide in intracellular mouse μ-chains that becomes complex on secretion. On the basis of the above findings, we present a model for the mouse intracellular IgM subunit and suggest a mechanism for its assembly into secreted IgM pentamers.
Adenosine 3′:5′-cyclic monophosphate production and steroidogenesis by isolated rat adrenal glomerulosa cells. Effects of angiotensin II and [Sar1,Ala8]angiotensin II
Authors: Bing Robert F. , Schulster Dennis
Published: August 2015

Angiotensin II effects on cyclic AMP production and steroid output were studied in a sensitive preparation of isolated rat adrenal glomerulosa cells. With increasing concentrations of angiotensin II logarithmic dose–response curves for aldosterone and cyclic AMP production were similar. The minimum effective dose (0.2nm) for stimulation of aldosterone production also significantly (P<0.001) increased cyclic AMP output. For both aldosterone and cyclic AMP production, the peptide hormone concentration eliciting maximal response (0.2μm) and the ED50 (median effective dose) values (1nm) were the same; this is consistent with cyclic AMP acting as an intracellular mediator for angiotensin II-stimulated aldosterone production by glomerulosa cells. The angiotensin II antagonist [Sar1,Ala8]angiotensin II inhibited angiotensin II-stimulated corticosterone and aldosterone production in these cells. An equimolar concentration of antagonist halved the response to 20nm-angiotensin II, and complete inhibition was observed with 0.2μm-antagonist. In contrast, [Sar1,Ala8]angiotensin II had no effect on maximally stimulated steroidogenesis induced by serotonin and a raised extracellular K+ concentration. Increasing concentrations of [Sar1,Ala8]angiotensin II alone decreased corticosterone and aldosterone outputs significantly (P<0.05) at concentrations of 20nm and 2nm of antagonist respectively. A significant (P<0.001) decrease in cyclic AMP production occurred with 2μm antagonist and this was comparable with the decrease in aldosterone production. It is concluded that [Sar1,Ala8]angiotensin II can independently affect glomerulosa-cell steroidogenesis, possibly by modulating adenylate cyclase activity.
Cyanide-insensitive oxidation of ascorbate + NNN′N′-tetramethyl-p-phenylenediamine mixture by mung-bean (Phaseolus aureus) mitochondria. An energy-linked function
Authors: Wilson S B
Published: August 2015

Freshly prepared washed or purified mung-bean (Phaseolus aureus) mitochondria utilize oxygen with ascorbate/tetramethyl-p-phenylenediamine mixture as electron donor in the presence of KCN. ATP control of the oxygen uptake can be observed with very fresh mitochondria. The electron flow, which is inhibited by antimycin A, salicylhydroxamic acid or octylguanidine, takes place by reversed electron transport through phosphorylation site II and thence to oxygen through the cyanide-insensitive pathway. Oligomycin and low concentrations of uncoupler partially inhibit the oxygen uptake in a manner similar to that observed for other energy-linked functions of plant mitochondria. An antimycin A-insensitive oxygen uptake occurs if high concentrations of uncoupler are used, indicating that the pathway of electron flow has been altered. The process of cyanide-insensitive ascorbate oxidation is self-starting, and, since it occurs in the presence of oligomycin, it is concluded that the reaction can be energized by a single energy-conservation site associated with the cyanide-insensitive oxidase pathway.
Effect of corticotropin treatment in vivo on the synthesis of a specific adrenal cytosolic protein. Characterization by dual-labelling technique and polyacrylamide-gel electrophoresis
Authors: Dazord A , Gallet D , Saez J M
Published: August 2015

The effect of corticotropin in vivo on total and specific protein synthesis in the adrenal was studied. Adrenal slices from control and corticotropin-treated animals were incubated with [14C]- and [3H]-leucine respectively, followed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of subcellular components. With this sensitive dual-labelling technique the following results were obtained. There was a general trophic effect on most adrenal proteins, but corticotropin produced a marked stimulation of a specific adrenal cytosolic protein. This protein has mol.wt. approx. 30 000 and pI 5.5. Corticotropin increased the incorporation of labelled leucine into proteins within 4 h, but no effect was observed before 2 h and after 16 h there was no further increase. These data suggest that this protein is not involved in the corticosteroidogenic action of corticotropin, but rather in the trophic action of this hormone.
New reaction sequences for the non-oxidative pentose phosphate pathway
Authors: Williams J F , Blackmore P F , Clark M G
Published: August 2015

1. Reactions leading to the formation of 14C-labelled volatile compounds and compounds volatile under acid conditions were investigated in a system actively synthesizing hexose 6-phosphates from [U-14C]ribose 5-phosphate by reactions catalysed by enzymes prepared from acetone-dried powder of rat liver; no reactions involving 14C-labelled volatile compounds were detected. Similarly the fixation of 14C-labelled volatile compounds into hexose 6-phosphate could not be detected. 2. A complete carbon balance was made for the reactants, intermediates and products of the reactions involved in the conversion of ribose 5-phosphate into hexose 6-phosphate by enzymes of rat liver. Five additional intermediates of pentose 5-phosphate metabolism in liver were detected, namely D-manno-heptulose 7-phosphate, D-altro-heptulose 1,7-bisphosphate, D-glycero-D-ido-octulose 1,8-bisphosphate, D-glycero-D-altro-octulose 1,8-bisphosphate and D-arabinose 5-phosphate. 3. D-Arabinose 5-phosphate was found to be utilized by a rat liver enzyme preparation to produce both hexose 6-phosphate and triose phosphate. 4. D-Arabinose 5-phosphate was reversibly converted into other pentose 5-phosphates. Paper chromatographic and enzymic evidence indicated that the conversion involved an enzyme tentatively named arabinose phosphate 2-epimerase, which catalyses the following reaction: D-arabinose 5-P in equilibrium D-ribose-5-P. 5. A variety of rat tissues also utilized D-arabinose 5-phosphate to produce both hexose 6-phosphate and triose phosphate and at a rate comparable with that obtained with D-ribose 5-phosphate. 6. A new reaction sequence for the non-oxidative pentose phosphate pathway in liver is proposed.
Opposite effects of starvation on oxidation of [14C]adenosine and adenosine-induced insulin release by isolated mouse pancreatic islets
Authors: Andersson A
Published: August 2015

To test further the hypothesis that ribonucleosides stimulate insulin secretion and biosynthesis by producing metabolic signals, the effects of starvation on adenosine-stimulated insulin production and the oxidation of adenosine by isolated mouse pancreatic islets were examined. No direct correlation was found between the metabolic flux and insulin secretion, since the starvation-induced impairment of the adenosine-stimulated insulin secretion was accompanied by an increased rate of adenosine oxidation. Adenosine-stimulated insulin biosynthesis was, however, unaffected by starvation.
Control of gluconeogenesis and of enzymes of glycogen metabolism in isolated rat hepatocytes. A parallel study of the effect of phenylephrine and of glucagon
Authors: Hue Louis , Felíu Juan Emilio , Hers Henri-Géry
Published: August 2015

Hepatocytes isolated from the livers of fed rats were used for a comparative study of the effects of phenylephrine, vasopressin and glucagon on gluconeogenesis and on enzymes of glycogen metabolism. When hepatocytes were incubated in the presence of Ca2+, phenylephrine stimulated gluconeogenesis from pyruvate less than did glucagon, but, in contrast with this hormone, it did not affect the activities of protein kinase and pyruvate kinase, nor the concentration of phosphoenolpyruvate, and it did not decrease the release of 3H2O from [6-3H]glucose. The effects of vasopressin were similar to those of phenylephrine. Gluconeogenesis from fructose was also stimulated by phenylephrine and, more markedly, by glucagon at the expense of the conversion of fructose into lactate. Insulin was able to antagonize the stimulatory effect of phenylephrine on gluconeogenesis from pyruvate. When Ca2+ was removed from the incubation medium, phenylephrine still stimulated gluconeogenesis from pyruvate, but it also caused an activation of protein kinase and an inactivation of pyruvate kinase; accordingly, the concentration of phosphoenolpyruvate was increased, and, in contrast, vasopressin had no effect on all these parameters. The property of phenylephrine to cause the activation of glycogen phosphorylase was decreased by glucose or by the absence of Ca2+; it was abolished when these two conditions were combined. Glycogen synthase was inactivated by phenylephrine in the presence or the absence of Ca2+, although presumably by different mechanisms.
Stimulation by vasopressin, angiotensin and oxytocin of gluconeogenesis in hepatocyte suspensions
Authors: Whitton P D , Rodrigues L M , Hems D A
Published: August 2015

1. In hepatocytes from starved rats, vasopressin, angiotensin (angiotensin II) and oxytocin stimulated gluconeogenesis from lactate by 25–50%; minimal effective concentrations were about 0.02pM, 1 nM and 0.2 nM respectively. 2. Vasopressin and angiotensin also stimulated gluconeogenesis from alanine, pyruvate, serine and glycerol. EGTA decreased gluconeogenesis from these substrates. 3. Hormonal stimulation of gluconeogenesis from lactate was abolished in the absence of extracellular Ca2+. 4. Insulin did not prevent stimulation of gluconeogenesis by vasopressin or angiotensin. 5. The potency of the stimulatory effects of vasopressin and angiotensin on hepatic gluconeogenesis suggests they are operative in vivo. Also, the data suggest that Ca2+ plays a role in the stimulation by these hormones.
The preparation of CMP-sialic acids by using CMP-acylneuraminate synthase from frog liver immobilized on sepharose 4B
Authors: Corfield A P , Schauer R , Wember M
Published: August 2015

A preparation of frog liver CMP-acylneuraminate synthase (2-10-fold enriched over the homogenate) obtained from DEAE-Sephadex A-50 chromatography of a 105,000g liver supernatant was bound to Sepharose 4B by the CNBr method. The enzyme retained 80-100% activity on binding and showed similar properties to the purified soluble enzyme from the same source with respect to Km, pH optimum and inhibition. The bound enzyme was stable to temperatures above 40 degrees C, in contrast with the soluble enzyme, and could be stored for 4 months at 2 degrees C with loss of 20% activity. The bound enzyme was used preparatively for the synthesis of radioactive and non-radioactive CMP-N-acetylneuraminic acid and CMP-N-glycolloylneuraminic acid. With suitable substrate concentrations and ratios, yields of 80% and over can be achieved.
The cellulolytic enzymes of Botryodiplodia theobromae Pat. Separation and characterization of cellulases and β-glucosidases
Authors: Umezurike Gabriel M.
Published: August 2015

1. Filtrates from cultures of different ages of Botryodiplodia theobromae Pat. were fractionated by gel filtration, ion-exchange chromatography and polyacrylamide-gel electrophoresis. 2. Five cellulases (C1, C2, C3, C4 and C5) were found, and their molecular weights, estimated by gel filtration, were 46000–48000 (C1), 30000–35000 (C2), 15000–18000 (C3), 10000–11000 (C4) and 4800–5500 (C5). 3. Cellulase C5 was absent from old culture filtrates. 4. Cellulase C1 had little or no activity on CM-cellulose (viscometric assay), but degraded cotton flock and Whatman cellulose powder to give cellobiose only. 5. The other components (C2–C5) produced cellobiose and smaller amounts of glucose and cellotriose from cellulosic substrates and were more active in lowering the viscosity of CM-cellulose. 6. The ratio of activities assayed by viscometry and by the release of reducing sugars from CM-cellulose increased with decrease in the molecular weights of cellulases C2–C5. 7. Cellobiose inhibited the activities of the cellulases, but glucose stimulated at low concentrations although it inhibited at high concentrations. 8. A high-molecular-weight β-glucosidase (component B1, mol.wt. 350000–380000) predominated in filtrates from young cultures, but a low-molecular-weight enzyme (B4, mol.wt. 45000–47000) predominated in older filtrates. 9. Intermediate molecular species of β-glucosidase (B2, mol.wt. 170000–180000; B3, mol.wt. 83000–87000) were also found. 10. Cellulases C2–C5 acted in synergism with C1, particularly in the presence of β-glucosidase.
Requirement for negatively charged dispersions of phospholipids for interaction with lipid-depleted adenosine triphosphatase
Authors: Isern de Caldentey M , Wheeler K P
Published: August 2015

The basis of the requirement for a net negative charge on phospholipid dispersions able to re-activate lipid-depleted (Na++K+)-dependent adenosine triphosphatase was studied. The origin and density of the charge in phospholipid dispersions were varied before interaction with the adenosine triphosphatase protein, and the charge density on restored phospholipid-adenosine triphosphatase complexes was changed after interaction. The results indicated that: (a) re-activation requires a lamellar arrangement of the lipid molecules with sufficient density of negative charge, but not necessarily negatively charged phospholipid molecules; (b) the net charge appears to be necessary for the correct interaction between the enzyme protein and the phospholipids, although the amount of phospholipid that binds to the protein is also a function of the nature of the acyl chains; (c) it is not possible on the basis of these findings and those in the literature to decide unequivocally if the charge is also required for the enzyme reaction itself. The possible relevance of the findings to the situation in vivo is discussed in terms of the charge being concerned only with lipid-protein interaction.
Evidence for the existence of isoenzymes of glycerol phosphate acyltransferase
Authors: Nimmo H G
Published: August 2015

Subcellular-fractionation studies confirmed previous findings that rat liver glycerol phosphate acyltransferase was located in both mitochondria and the microsomal fraction. Studies of the two activities revealed several differences between them. The mitochondrial enzyme had a lower Km for sn-glycerol 3-phosphate and was more resistant to heat inactivation than was the microsomal enzyme. Some preparations of the mitochondrial enzyme were inhibited by high concentrations of glycerol phosphate. The mitochondrial enzyme was not inactivated by thiol-group reagents, whereas the microsomal enzyme was very rapidly inactivated by these compounds. However, the microsomal enzyme could be specifically protected against this inactivation by low concentrations of palmitoyl-CoA. The results indicate the existence of distinct isoenzymes of glycerol phosphate acyltransferase with different intracellular locations.
The breakdown of 2,3-bis(phospho)-d-glycerate by Fe(III)-haemoglobin
Authors: El-Yassin D I , Fell D A , Lloyd B B , Fisher R B
Published: August 2015

Fe(III)-haemoglobin is shown to catalyse the hydrolysis of 2,3-bis(phospho)-D-glycerate to Pi and 3-phosphoglycerate, although the rate is slow, even when the protein is present at concentrations in the millimolar range. The rate of hydrolysis is proportional to the subtrate concentration up to at least 10 mM, so if the process is enzymic, the Km must be high.