bluuebunny/crossref_metadata_2025 huggingface dataset:
The mechanism of action of xanthine oxidase. The relationship between the rapid and very rapid molybdenum electron-paramagnetic-resonance signals
Authors: Bray R C , Gutteridge S , Stotter D A , Tanner S J
Published: August 2015

On the basis of the work of Gutteridge, Tanner & Bray [Biochem. J. (1978) 175, 887-897] and of other data in the literature, a mechanism for the reaction of xanthine oxidase with reducing substrates is proposed. In the Michaelis complex, xanthine is bound to molybdenum via the N-9 nitrogen atom. Coupled transfer of two electrons to molybdenum and the C-8 proton to the enzyme yields (Enzyme)-Mo-SH. Concerted with this process, reaction of the xanthine residue with a nucleophile in the active centre yields a covalent intermediate that breaks down to give the product by alternative pathways at high and at low pH values.
Rapid aging of neurotoxic esterase after inhibition by di-isopropyl phosphorofluoridate
Authors: Clothier Bruce , Johnson Martin K.
Published: August 2015

1. It was proposed [Johnson (1974) J. Neurochem.23, 785–789] that an essential step in the genesis of delayed neuropathy caused by some organophosphorus esters was aging of phosphorylated neurotoxic esterase, involving generation of a charged monosubstituted phosphoric acid residue on the protein. 2. Neurotoxic esterase of hen brain was inhibited with di-isopropyl phosphorofluoridate either unlabelled or mixed-labelled with 3H and 32P. 3. Reactivation of inhibited enzyme by KF was possible only immediately after a brief inhibition:aging at pH8.0 and 37°C occurred with a half-life of about 2–4min. 4. When the radiolabelled enzyme was studied no loss of label was observed during the expected aging period, but a change in the nature of the bound radioisotopes occurred (half-life=3.25min). 5. Alkaline hydrolysis of labelled enzyme liberated di-isopropyl phosphate at early times after labelling, but increasing amounts of monoisopropyl phosphate plus a volatile tritiated compound (possibly propan-2-ol) at later times. 6. Treatment of labelled enzyme with KF released di-isopropyl phosphate and caused reactivation of enzyme to similar degrees. It is concluded that the chemical change from di-isopropyl phosphoryl-enzyme to mono-isopropyl phosphoryl-enzyme and the loss of reactivatibility are related. 7. The rate of aging is similar at pH5.2, 6.5 and 8. Aging is unaffected by addition of reduced glutathione and imidazole at pH5.2 or 8, and none of the transferred 3H is trapped by these reagents. The mechanism of aging must be different from the better-known dealkylation aging of the cholinesterases.
Low-temperature kinetics of the reactions of fully reduced membrane-bound cytochrome oxidase with oxygen in the Soret, α and near-infrared regions
Authors: Clore G M , Chance E M
Published: August 2015

The kinetics of the reaction of fully reduced membrane-bound cytochrome oxidase with O2 obtained in the Soret, alpha and near-i.r. regions were analysed, and the contributions of the three intermediates of the reaction [Clore & Chance (1978) Biochem. J. 173, 799–810] to seven wavelength pairs (430–463, 444–463, 590–630, 608–630, 740–940, 790–940 and 830–940 nm) were determined. The nature of the intermediates is discussed on the basis of the data in the present paper together with data in the literature from optical wavelength scanning, e.p.r., i.r. and magnetic-susceptibility studies.
Convolution analysis of transcription by yeast DNA-dependent ribonucleic acid polymerase A. A mathematical method for studying ribonucleic acid chain elongation
Authors: Cooper C S , Quincey R V
Published: August 2015

The rate of initiation of RNA synthesis catalysed by yeast RNA polymerase A on native calf thymus DNA decayed exponentially with a half-life of about 4.3 min. The rate constant for initiation was unaffected by preincubating the enzyme with DNA, or by decreasing the concentration of GTP 4-fold. The rate of RNA synthesis was constant for 15–20 min and then decreased. Each enzyme molecule made no more than one RNA molecule. In this situation, initiation, elongation and total RNA synthesis are related by a convolution integral. Solution of the convolution integral revealed that the rate of elongation was apparently biphasic. Analysis of the size of the RNA product showed that this biphasic profile arose because most but not all of the enzyme stopped RNA synthesis soon after initiation.
A possible role for dehydrodihydroxylysinonorleucine in collagen fibre and bundle formation
Authors: Boucek R J , Noble N L , Gunja-Smith Z
Published: August 2015

The concentrations of NaB3H4-reducible collagen cross-links were determined at the time when collagen fibres and bundles are observed in electron micrographs of connective tissue developing around the implanted Ivalon sponge in adult male rats. The highest radioactivity occurs with hydroxylysinonoreleucine and histidinohydroxymerodesmosine, and the lowest with lysinonorleucine, the reducible amounts of these cross-links remaining relatively constant as fibres and bundles appear. On the other hand, dihydroxylysinonorleucine amounts are low during the initial stages of connective-tissue formation and rise sharply as collagen fibres and bundles develop and collagen matures, as shown by increased resistance of insoluble collagen to digestion with bacterial collagenase. The bulk of hydroxylysinonorleucine and dihydroxylysinonorleucine is glycosylated, the former with galactosyl or glucosylgalactosyl residues and the latter with glucosylgalactosyl residues. The changing relationships between the amounts of 3H-labelled hydroxylysinonorleucine, glucosylgalactosyldihydroxylysinonorleucine and non-glycosylated dihydroxylysinonorleucine as fibres and bundles appear suggest three post-translational steps involving lysyl-derived cross-links in the organization of collagen into fibres and bundles.
An improved method of light-induced pigmentation
Authors: Kuan K N , Lee Y Y , Melius P
Published: August 2015

An improved procedure was developed whereby a primary light signal can be intensified and made visible by activation of a pre-tyrosinase (pre-phenoloxidase) enzyme [isolated from silkworm (Bombyx mori)] by alpha-chymotrypsin; this activation results from the light-activated conversion of the inactive cis-cinnamoyl-alpha-chymotrypsin.
The effects of barbiturates on the metabolism of phosphatidic acid and phosphatidylinositol in rat brain synaptosomes
Authors: Miller J C , Leung I
Published: August 2015

Barbiturates and diphenylhydantoin inhibit the carbamoylcholine-stimulated increase in 32P incorporation into phosphatidylinositol and phosphatidic acid, but have a relatively slight effect on the incorporation of 32P into these lipids in the absence of carbamoylcholine and no effect on 32P incorporation into phosphatidylcholine and phosphatidylethanolamine. Inhibition of the carbamoylcholine-stimulated increase was observed for pentobarbital, thiopental, phenobarbital, 5-(1,3-dimethylbutyl)-5-ethylbarbiturate, (+)- and (-)-5-ethyl-N-methyl-5-propylbarbituate and diphenylhydantoin. Similar concentrations of barbiturates and diphenylhydantoin were previously reported to inhibit the K+-stimulated Ca2+ influx, and therefore other agents that affect Ca2+ influx were tested to find whether they had any effect on 32P incorporation into these lipids. K+ (35 mM) increases 32P incorporation into phosphatidic acid, but to a smaller degree than 100 micrometer-carbamoylcholine, and its effect was inhibited by pentobarbital. Veratridine (75 micrometer) does not increase 32P incorporation into either phosphatidic acid or phosphatidylinositol, but did inhibit the carbamoylcholine-stimulated increase in 32P incorporation into phosphatidylinositol. The possible relationship between the phospholipid effect and stimulated Ca2+ influx is discussed.
The respiratory chain of Hydrogenomonas H16
Authors: Beatrice M C , Chappell J B
Published: August 2015

The respiratory chain of Hydrogenomonas H16 is shown to consist of two pathways, one of which has as its oxidase and only cytochrome a high-potential b-type cytochrome which is concluded to be cytochrome o. The Km values for the oxygen of the two pathways are consistent with the high-potential b-type cytochrome functioning as a relatively high-affinity oxidase.
Mechanism of activation of glycogen phosphorylase by fructose in the liver. Stimulation of phosphorylase kinase related to the consumption of adenosine triphosphate
Authors: Van de Werve G , Hers H G
Published: August 2015

1. A dose-dependent activation of phosphorylase and consumption of ATP was observed in isolated hepatocytes incubated in the presence of fructose; histone kinase and phosphorylase kinase activities were unchanged at doses of this sugar that were fully effective on phosphorylase. The activation of phosphorylase by fructose was also observed in cells incubated in a Ca2+-free medium as well as in the livers of rats in vivo. 2. In a liver high-speed supernatant, fructose, tagatose and sorbose stimulated the activity of phosphorylase kinase; this effect was dependent on the presence of K+ ions, which are required for the activity of fructokinase; it was accompanied by the transformation of ATP into ADP. In the presence of hexokinase, glucose also stimulated phosphorylase kinase, both in an Na+ or a K+ medium. 3. The activities of partially purified muscle or liver phosphorylase kinase were unchanged in the presence of fructose. 4. Some properties of liver phosphorylase kinase are described, including a high molecular weight and an inhibition at ATP/Mg ratios above 0.5, as well as an effect of ATP concentration on the hysteretic behaviour of this enzyme. 5. The effect of fructose on the activation of phosphorylase is discussed in relation to the comsumption of ATP.
Energy coupling to the transport of inorganic phosphate in Escherichia coli K12
Authors: Rosenberg H , Gerdes R G , Harold F M
Published: August 2015

The nature of the energy source for phosphate transport was studied in strains of Escherichia coli in which either one of the two major systems (PIT, PST) for phosphate transport was present. In the PIT system, phosphate transport is coupled to the proton-motive force. The energy source for the PST system appears to be phosphate-bond energy, as has been found in other systems involving binding proteins. High concentration gradients of phosphate (between 100 and 500) are established by both systems.
Methane synthesis without the addition of adenosine triphosphate by cell membranes isolated from Methanobacterium ruminantium
Authors: Sauer F D , Erfle J D , Mahadevan S
Published: August 2015

The membrane fraction isolated from broken cells of Methanobacterium ruminantium actively synthesized methane from CO2 and H2 without the addition of ATP or other cofactors. This activity was lost unless strictly anaerobic conditions were maintained throughout the isolation and incubation procedures. 3H2, but not 3H2O, was readily incorporated into methane. This indicates that hydrogen atoms are used in the formation of methane without the prior equilibration of protons with the water phase. Methylenetetrahydrofolate was shown to be converted into methane, but less efficiently than CO2. The evidence indicates that tetrahydrofolate derivatives may not be of primary importance in the formation of methane from CO2 and H2. No requirement for ATP in methanogenesis could be demonstrated. However, chemical reagents that can increase proton conductance in membranes and therby abolish the membrane electrical potential were also effective inhibitors of methanogenesis. It was postulated that, although the reduction of CO2 to methane by bacterial membranes may require energy derived from a transmembrane potential, this does not appear to be coupled to the intermediary synthesis of ATP.
Studies on the relationship between the degradative rates of proteins in vivo and their isoelectric points
Authors: Dice J. Fred , Hess Emma Jean , Goldberg Alfred L.
Published: August 2015

Acidic proteins tend to be degraded more rapidly than neutral or basic proteins in rat liver, skeletal muscle, kidney and brain and in mouse liver and skeletal muscle. We now report a similar relationship among soluble proteins from rat lung, heart and testes, and from human fibroblasts and mouse-embryo cells grown in culture. These findings indicate that the correlation between protein net charge and degradative rate is a general characteristic of intracellular protein degradation in mammals. This relationship between isoelectric point and half-life appears to be distinct from the previously reported correlation between subunit molecular weight and protein half-lives. The more rapid degradation of acidic proteins does not result from their being of larger molecular weight than neutral or basic proteins. Furthermore, proteins within specific isoelectric point ranges still exhibit a relationship between subunit size and half-life. Finally, a group of membrane or organelle-associated proteins that are insoluble in phosphate-buffered saline and water but soluble in 1% Triton X-100 exhibit a correlation between size and half-life, but not between net charge and half-life. The biochemical reasons for the relationship between protein isoelectric point and half-life are unclear, although several possible explanations are presented. It is not due to a greater sensitivity of acidic proteins to proteolytic attack since experiments with a variety of endoproteinases, including trypsin, chymotrypsin, Pronase, papain, chymopapain, Staphylococcus aureus V8 proteinase, pepsin and lysosomal cathepsins from rat liver, have failed to demonstrate more rapid digestion of acidic proteins.
Leucine degradation in cell-free extracts of skeletal muscle
Authors: Odessey R , Goldberg A L
Published: August 2015

Since skeletal muscle is the major site in the body for oxidation of leucine, isoleucine and valine, the pathway and control of leucine oxidation were investigated in cell-free preparations of rat muscle. Leucine was found to be transaminated to 4-methyl-2-oxopentanoate, which was then oxidatively decarboxylated. On differential centrifugation 70–80% of the transaminase activity was recovered in the soluble fraction of the cell, and the remaining amount in the mitochondrial fraction. The transaminase, from both fractions had similar pH optima and both were markedly inhibited by Ca2+. Thus changes in cellular Ca2+ concentration may regulate transaminase activity. Both transaminases had a much higher affinity for 2-oxoglutarate than for pyruvate. Therefore the utilization of amino groups from leucine for the biosynthesis of alanine in muscle [Odessey, Khairallah & Goldberg (1974) J. Biol. Chem. 249, 7623–7629] in vivo involves transamination with 2-oxoglutarate to produce glutamate, which is then transaminated with pyruvate to produce alanine. The dehydrogenase activity assayed by the decarboxylation of methyl-2-oxo[1-14C]pentanoate was localized exclusively in the fraction containing mitochondria and required NAD+, CoA and thiamin pyrophosphate for optimal activity. Measurements of competitive inhibition suggested that the oxo acids of leucine, isoleucine and valine are all decarboxylated by the same enzyme. The enzyme activity was decreased by 90% upon freezing or sonication and was stimulated severalfold by Mg2+, K+ and phosphate ions. In addition, it was markedly inhibited by ATP, but not by non-metabolizable analogues. This observation suggests that splitting of ATP is required for inhibition. The oxidative decarboxylation of 4-methyl-2-oxopentanoate by the dehydrogenase appears to be the rate-limiting step for leucine oxidation in muscle homogenates and also in intact tissues. In fact, rat muscles incubated with [1-14C]leucine release 1-14C-labelled oxo acid into the medium at rates comparable with the rate of decarboxylation. Intact muscles also released the oxo acids of [1-14C]valine or [1-14C]isoleucine, but not of other amino acids. These findings suggest that muscle is the primary source of the branched-chain oxo acids found in the blood.
The distribution and chemical nature of radioactive folates in rat liver cells and rat liver mitochondria
Authors: Cook R J , Blair J A
Published: August 2015

Subcellular fractionation of rat liver cells revealed that a mixture of 14C- and 3H-labelled folic acid was distributed approximately equally between the mitochondria and cytosol 2, 24, 48 and 72 h after oral administration. Subfractionation of liver mitochondria 48 h after oral administration showed that the radioactivity was mainly associated with the inner membrane (27.7%) and matrix (51.5%). Hot-ascorbate extraction of the cell cytosol, mitochondrial inner membrane and matrix showed the majority of folates were present as polyglutamates. Acid treatment of isolated folates from cytosol, inner membrane and matrix produced breakdown products consistent with scission of tetrahydrofolates. The folates isolated in the mitochondrial matrix were bound to protein that had an estimated mol. wt. of 90,000.
The early development of white adipose tissue. Effects of litter size on the lipoprotein lipase activity of four adipose-tissue depots, serum immunoreactive insulin and tissue cellularity during the first four weeks of life in the rat
Authors: Cryer Anthony , Jones Heather M.
Published: August 2015

1. Newborn rats were reared in litters of either four or sixteen individuals. The animals from the small litters gained body weight more rapidly than those from large litters during the first 29 days of postnatal life studied. 2. The relative weights of the perigenital, perirenal, subcutaneous and intramuscular white-adipose-tissue sites in the animals from small litters indicated their relative obesity compared with controls. 3. The adipose depots from animals reared in small litters had a greater proportion of lipid present, by weight, and had a greater number of larger fat-cells present in them compared with the depots of animals reared in large litters. 4. Compared with both normal-sized litter controls and animals reared in sixteens, during the period of study the animals from small litters were hypertriacylglycerolaemic but normocholesterolaemic. 5. During suckling the blood glucose concentrations of animals reared in fours were increased, as were the concentrations of circulating immunoreactive insulin. 6. During the 29 days of life studied, in general, the lipoprotein lipase activity of adipose depots from animals reared in fours was greater than for animals in large litters when expressed as μmol of nonesterified fatty acid released from the substrate/h per g fresh weight of tissue, per depot, or per million fat-cells, but were similar per cm2 of fat-cell surface area. 7. The previously noted [Cryer & Jones (1978) Biochem. J.172, 319–325] pattern of mid-suckling elevation, late-suckling decline and post-weaning increase in the lipoprotein lipase activity of the four white-adipose depots studied was not obliterated by the nutritional manipulations employed. 8. The relation of the enzyme-activity changes and their hormonal stimuli to triacylglycerol accumulation in fat-cells of animals from large and small litters is discussed in relation to the possible significance they may have to our understanding of neonatally induced obesity.
4-pyridylmethyl, a thiol-protecting group suitable for the partial synthesis of proteins
Authors: Rüegg U T , Jarvis D , Rudinger J
Published: August 2015

Cysteine is converted into S-4-pyridylmethylcysteine [Gosden, Stevenson & Young (1972) J. Chem. Soc. Chem Commun. 1123-1124] by 4-pyridylmethyl chloride in aqueous propanol at pH 8.3. The derivative is stable to the conditions of total acid hydrolysis. Reduction and alkylation of bovine insulin (pH 8.3, aq. 50% propanol) gives fully S-substituted derivatives in excellent yields. The S-pyridylmethylated A- and B-chains of insulin were separated by gel filtration: each of them has good solubility properties. The pyridylmethyl group is cleaved by electrolysis in a dilute acid medium, pH 2.6, to give reduced chains. They can be recombined to give insulin in the same yield and with the same degree of biological activity as chains which had not been subjected to the protection and de-protection steps. The results indicate that pyridylmethyl satisfactorily meets requirements for partial synthesis and suggest that it warrants more general use.
The specificity of some pig and human pepsins towards synthetic peptide substrates
Authors: Ryle A P , Auffret C A
Published: August 2015

1. The peptidase activities of pig pepsins A and C and human pepsin and gastricsin were compared. 2. The peptides studied had the general formula A Leu Val-His-B. Hydrolysis at 37 degrees C and pH 2.07 occurred at the amino side of the leucine residue for all the enzymes and all the peptides. 3. When A was Ac-Ala the peptides were hydrolysed under these conditions slowly by pig pepsin C only. 4. Pig pepsin A and human pepsin were unable to hydrolyse the tyrosine-containing peptides under the conditions tested. Gastricsin (human pepsin C) had about one-third of the activity of pig pepsin C with these substrates. 5. The increase in the rate of hydrolysis caused by the extension of the chain by a single alanine residue was most marked for pig pepsin A and human pepsin.
Intracellular serine proteinase of Bacillus subtilis strain Marburg 168. Comparison with the homologous enzyme from Bacillus subtilis strain A-50
Authors: Strongin A Y , Gorodetsky D I , Kuznetsova I A , Yanonis V V , Abramov Z T , Belyanova L P , Baratova L A , Stepanov V M
Published: August 2015

Intracellular serine proteinase was isolated from sporulating cells of Bacillus subtilis Marburg 168 by gramicidin S-Sepharose 4B affinity chromatography. The enzymological characteristics, the amino acid composition and the 19 residues of the N-terminal sequence of the enzyme are reported. The isolated proteinase was closely related to, but not completely identical with, the intracellular serine proteinase of B. subtilis A-50. The divergence between these two intracellular enzymes was less than that between the corresponding extracellular serine proteinases (subtilisins) of types Carlsberg and BPN′!, produced by these bacterial strains. This may be connected with the more strict selection constraints imposed in intracellular enzymes during evolution.
The β-glucosidase from Botryodiplodia theobromae. Mechanism of enzyme-catalysed reactions
Authors: Umezurike G M
Published: August 2015

The effects of pH and temperature on Michaelis constant (Km) and maximum velocity (Vmax.) and of NaCl on the activity of the high-molecular-weight beta-glucosidase (beta-D-glucoside glucohydrolase EC 3.2.1.21) from cultures of Botryodiplodia theobromae Pat. have been studied. 2. Donor binding and inhibition of activity by glucose were dependent on the ionization of a group (pK 6.0) that appeared to be an imidazole group. 3. Catalytic activity and the stimulation of activity by glycerol were dependent on the ionization of two groups, which appeared to be a carboxy group and an imidazole group. 4. The Arrhenius activation energy (Ea) calculated from results obtained at pH 4.0 and 5.0 was about 45–46kJ.mol-1. 5. The enthalpies (delta H0) calculated from results obtained at pH 4.0 and 5.0 were similar (about -4kJ.mol-1), whereas at pH 6.5 the value was about -33kJ.mol-1. 6. The entropies (delta S0) calculated from these results at 37 degrees C were -21, -22 and -118J.K-1.mol-1 at pH 4.0, 5.0 and 6.5 respectively. A low concentration of NaCl (16.6 mM) stimulated enzymic activity and decreased the Km for the donor, whereas high concentrations (up to 500 mM) inhibited enzymic activity, increased the Km and had no effect on Vmax. 8. Plots of initial velocity data obtained in the presence of dioxan as 1/v against the ratio of the molar concentration of dioxan to that of water were linear. 9. The results are discussed in terms of the enzyme mechanism.
The kinetics of rat liver and heart mitochondrial β-hydroxybutyrate dehydrogenase
Authors: Tucker G A , Dawson A P
Published: August 2015

The kinetic mechanisms of the beta-hydroxybutyrate dehydrogenase from rat heart and liver mitochondria were investigated. Both enzymes, show an Ordered Bi Bi mechanism and there are no major differences in the kinetic constants. In both cases, the solubilized enzyme, re-activated with phosphatidylcholine, shows kinetic properties very similar to those of the enzyme bound to the mitochondrial membrane.
Preparation of [32P]phosphatidylcholine and [32P]lysophosphatidylcholine by using soya beans
Authors: Hubmann F H
Published: August 2015

This method describes a procedure that can be carried out easily to obtain large amounts of [32P]phosphatidylcholine and [32P]lysophosphatidylcholine. The method involves germinating soya beans in the presence of [32P]Pi. The yield was 0.58% for [P]phosphatidylcholine and 0.52% for [32P]lysophosphatidylcholine, and the specific radioactivity of both was 10(7) d.p.m./mumol.
The cell-wall phosphatase of cotton (Gossypium) is inhibited by kelthane
Authors: Daley L S , Carroll P , Mussell H
Published: August 2015

Kelthane [4,4′-dichloro-alpha-(trichloromethyl)benzhydrol] was previously shown to decrease the limited tolerance of susceptible varieties of cotton (Gossypium) to Verticillium wilt. Kelthane was shown in the present study to inhibit the cell-wall p-nitrophenyl phosphatase of cotton. In view of information already establishing the cell wall as a primary site of action of Verticillium wilt, the data are interpreted as suggesting an as yet undefined interaction between Kelthane, cell-wall phosphatase and verticillium-resistance mechanisms of the cell wall.
The uncA gene codes for the α-subunit of the adenosine triphosphatase of Escherichia coli. Electrophoretic analysis of uncA mutant strains
Authors: Senior A E , Downie J A , Cox G B , Gibson F , Langman L , Fayle D R
Published: August 2015

Four mutant strains of Escherichia coli which lack membrane-bound adenosine triphosphatase activity were shown by genetic-complementation tests to carry mutations in the uncA gene. A soluble inactive F1-ATPase aggregate was released from the membranes of three of the uncA mutant strains by low-ionic-strength washing, and purified by procedures developed for the purification of F1-ATPase from normal strains. Analysis of the subunit structure by two-dimensional gel electrophoresis indicated that the F1-ATPase in strains carrying the uncA401 or uncA453 alleles had a subunit structure indistinguishable from normal F1-ATPase. In contrast, the F1-ATPase from the strain carrying the uncA447 allele contained an alpha-subunit of normal molecular weight, but abnormal net charge. Membranes from strains carrying the uncA450 allele did not have F1-ATPase aggregates that could be solubilized by low-ionic-strength washing. However, a partial dipolid strain carrying both the uncA+ and uncA450 alleles formed an active F1-ATPase aggregate which could be solubilized by low-ionic-strength washing of the membranes and which contained two types of alpha-subunit, one of which was normal and the other had abnormal net charge. It is concluded that the uncA gene codes for the alpha-subunit of the adenosine triphosphatase.
Properties of membranes from mutant strains of Escherichia coli in which the β-subunit of the adenosine triphosphatase is abnormal
Authors: Senior A E , Fayle D R , Downie J A , Gibson F , Cox G B
Published: August 2015

Five uncoupled mutant strains of Escherichia coli carrying mutations in the uncD gene have been studied. In each of these mutant strains the beta-subunit of the F1 portion of the membrane-bound adenosine triphosphatase is abnormal. In one of the mutant strains (carrying the uncD12 allele) in F1-ATPase aggregate was formed which was purified and found to have low ATPase activity. ATPase activity was absent in the other four strains and the abnormal beta-subunits were tightly bound to the membranes. However, membranes from these strains exhibited various proton permeabilities as indicated by NADH-dependent atebrin-fluorescence quenching and bound different amounts of normal F1-ATPase. The amounts of reconstitution of energy-linked reactions after the addition of normal F1-ATPase also varied depending on the mutant allele. It is apparent that considerable phenotypic variations can occur between strains carrying mutations in the same unc gene.
Sensitivity of glucose uptake and lipolysis of white adipocytes of the rat to insulin and effects of some metabolites
Authors: Green A , Newsholme E A
Published: August 2015

1. Insulin increased glucose uptake and inhibited lipolysis in white adipocytes of the rat over the same concentration range of the hormone: the half-maximal effects were observed at approx. 10 microunits of insulin/ml. Thus, contrary to previous reports, no difference in sensitivity of the two processes to insulin could be found, which suggests that both these effects of insulin are important in increasing the rate of glucose utilization after a meal. 2. Adenosine deaminase, which lowers the concentration of adenosine in the incubation medium, decreased the sensitivity of both processes (lipolysis and glucose uptake) to insulin: this suggests that adenosine increases the sensitivity of both processes. Similarly, lactate and 3-hydroxybutyrate increased the sensitivity of both processes (to the same extent) to insulin. It is suggested that this increased sensitivity will improve the response (of adipose tissue) to insulin on refeeding after a prolonged period of starvation (when the hydroxybutyrate concentration is high), and after a short burst of exercise, when the blood lactate concentration is high and when large amounts of glucose are produced from lactate via gluconeogenesis in the liver.
The separation of microsomal membranes and free polyribosomes by gel filtration
Authors: McCole N , Palmer D N , Williams D J
Published: August 2015

The integrity of polyribosome-membrane complexes in microsomal preparations is dependent on the centrifugal conditions used for their preparation. This paper describes techniques based on gel filtration that enable the rapid and gentle separation of microsomal membranes from both free polyribosomes and soluble protein.
Poly(adenosine diphosphate ribose) synthetase activity in nuclei of dividing and of non-dividing but differentiating intestinal epithelial cells
Authors: Porteous J W , Furneaux H M , Pearson C K , Lake C M , Morrison A
Published: August 2015

Poly(ADP-ribose) synthetase activity is found in nuclei of regenerating epithelial cells in the lower half of the crypts of guinea-pig small intestine. Nuclei from non-dividing but differentiating and maturing cells in the upper crypts and on the villi contain no more than about 10% of the synthetase activity of lower-crypt cell nuclei. The product in the active nuclei is shown to be 80% poly(ADP-ribosylated) protein and 20% mono(ADP-ribosylated) protein; 60% of thetotal labelled product was attached to acid-soluble proteins (including histones), and 40% to acid-insoluble (non-histone) proteins. The average number of ADP-ribosyl units in the oligomeric chains of the poly(ADP-ribosylated) proteins was 15 but the range of sizes of (ADP-ribose) oligomers attached to nuclear proteins was smaller in villus than in crypt cell nuclei.
Purification and properties of N-acetylgalactosamine 6-sulphate sulphatase from human placenta
Authors: Glössl Josef , Truppe Wolfgang , Kresse Hans
Published: August 2015

1. N-Acetylgalactosamine 6-sulphate sulphatase was purified about 20000-fold from the soluble extract of human placenta with N-acetylgalactosamine 6-sulphate–glucuronic acid–N-acetyl[1-3H]galactosaminitol 6-sulphate as substrate in the activity assay. The enzyme appears to be a glycoprotein with a mol.wt. of about 100000 as determined by gel filtration. On gel electrophoresis in the presence of sodium dodecyl sulphate the major protein band had a mol.wt. of 78000. Variable charge heterogeneity was observed in several enzyme preparations. 2. The purified enzyme released up to one sulphate molecule from the disulphated trisaccharide. It was active towards N-acetylgalactosamine 6-sulphate and exhibited no measurable N-acetylglucosamine 6-sulphate sulphatase or any other known lysosomal sulphatase activity. Hydrolysis of [1-3H]galactitol 6-sulphate was achieved by incubation neither with a crude nor with a purified enzyme preparation. Chondroitin 6-sulphate and keratan sulphate, as well as heparin and heparan sulphate, served as competitive inhibitors of the enzyme. 3. Purified N-acetylgalactosamine 6-sulphate sulphatase activity was optimal at pH4.9 and 4.4 when assayed in 0.02m-sodium acetate buffer and at pH4.2 and 5.2 in 0.1m-sodium acetate buffer. A single pH-optimum at pH4.8 was observed for the crude enzyme and for the purified enzyme after mild periodate treatment. The sulphatase activity was inhibited by a variety of anions and cations and activated by thiol-specific and thiol reagents.
The electrophoretically ‘slow’ and ‘fast’ forms of the α2-macroglobulin molecule
Authors: Barrett A J , Brown M A , Sayers C A
Published: August 2015

alpha 2-Macroglobulin (alpha 2M) was isolated from human plasma by a four-step procedure: poly(ethylene glyco) fractionation, gel chromatography, euglobulin precipitation and immunoadsorption. No contaminants were detected in the final preparations by electrophoresis or immunoprecipitation. The protein ran as a single slow band in gel electrophoresis, and was designated ‘S-alpha 2M’. S-alpha 2M bound about 2 mol of trypsin/mol. Treatment of S-alpha 2M with a proteinase or ammonium salts produced a form of the molecule more mobile in electrophoresis, and lacking proteinase-binding activity (F-alpha 2M). The electrophoretic mobility of the F-alpha 2M resulting from reaction with NH4+ salts was identical with that of proteinase complexes. We attribute the change in electrophoretic mobility of the alpha 2M to a conformation change, but there was no evidence of a change in pI or Strokes radius. Electrophoresis of S-alpha 2M in the presence of sodium dodecylsulphate gave results consistent with the view that the alpha 2M molecule is a tetramer of identical subunits, assembled as a non-covalent pair of disulphide-linked dimers. Some of the subunits seemed to be ‘nicked’ into two-thires-length and one-third-length chains, however. This was not apparent with F-alpha 2M produced by ammonium salts. F-alpha 2M produced by trypsin showed two new bands attributable to cleavage of the subunit polypeptide chain near the middle. Immunoassays of F-alpha 2M gave ‘rockets’ 12-29% lower than those with S-alpha 2M. The nature of the interactions between subunits in S-alpha 2M and F-alpha 2M was investigated by treating each form with glutaraldehyde before electrophoresis in the presence of sodium dodecyl sulphate. A much greater degree of cross-linking was observed with the F-alpha 2M, indicating that the subunits interact most closely in this form of the molecule. Exposure of S-alpha 2M to 3 M-urea or pH3 resulted in dissociation to the disulphide-bonded half-molecules; these did not show the proteinase-binding activity characteristic of the intact alpha 2M. F-alpha 2M was less easily dissociated than was S-alpha 2M. S-alpha 2M was readily dissociated to the quarter-subunits by mild reduction, with the formation of 3-4 new thiol groups per subunit. Inact reactive alpha 2M could then be regenerated in high yield by reoxidation of the subunits. F-alpha 2M formed by reaction with a proteinase or ammonium salts was not dissociated under the same conditions, although the interchain disulphide bonds were reduced. If the thiol groups of the quarter-subunits of S-alpha 2M were blocked by carboxymethylation, oxidative reassociation did not occur. Nevertheless treatment of these subunits with methylammonium salts or a proteinase caused the reassembly of half-molecules and intact (F-) tetramers. It is emphasized that F-alpha 2M does not have the properties of a denatured form of the protein…
Studies on the intracellular segregation of polyribosome-associated messenger ribonucleic acid species in the lactating guinea-pig mammary gland
Authors: Craig R K , Boulton A P , Harrison O S , Parker D , Campbell P N
Published: August 2015

1. Free and membrane-bound polyribosomes were isolated and the associated mRNA species characterized by cell-free protein synthesis, RNA-complexity analysis and polyribosome run-off in vitro. 2. Of the recovered polyribosomal RNA 85% was associated with membrane-bound polyribosomes and contained 87–93% of the total milk-protein mRNA species as assessed by cell-free protein synthesis or RNA-complexity analysis. 3. RNA-complexity analysis showed that the abundant (milk-protein mRNA assumed) species constituted 55% of the post-nuclear poly(A)-containing RNA population, the remainder consisting of a moderately abundant population (18%) and a low abundance population (27%). Calculations suggest that each population contained up to 2, 48 and 5000 different species respectively. 4. RNA-complexity analysis of the free polyribosomal poly(A)-containing RNA demonstrated that all the species in the post-nuclear fraction were present, though in different proportions, the abundant, moderately abundant and low-abundance groups representing 38, 30 and 32% of this population. 5. RNA-complexity analysis of the membrane-bound polyribosomal poly(A)-containing RNA revealed a more limited population, 72% consisting of the abundant (milk-protein mRNA) species, and 28% a population of up to 900 RNA species. 6. Polyribosome run-off confirmed that milk-protein mRNA was associated with the membrane-bound and free polyribosomes, but represented only a small fraction of the total protein synthesized by the latter. 7. Comparative analysis of milk proteins synthesized in mRNA-directed cell-free systems, or by run-off of free and of membrane-bound polyribosomes, is consistent with the interpretation that in vivo the initiation of protein synthesis occurs on free polyribosomes, followed by the attachment of a limited population to the endoplasmic reticulum. After attachment, but before completion of peptide synthesis, the detachable N-terminal peptide sequence of one of these(pre-alpha-lactalbumin) is removed. 8. The results are discussed in terms of the mechanisms involved in the intracellular segregation of mRNA species in the lactating guinea-pig mammary gland.
Photoactivated covalent binding of [3H]bilirubin to human serum albumin
Authors: Hutchinson D W , Mutopo D S
Published: August 2015

A one-step procedure has been developed for the preparation of [3H]bilirubin IX-alpha in good yield from unlabelled bilirubin. Irradiation of an aqueous solution of [3H]bilirubin IX-alpha in the presence of human serum albumin results in the covalent attachment of the bilirubin to the protein. Preliminary degradation studies have been carried out to locate the site of attachment of the bilirubin to the albumin.
Determination of vitamin D and its metabolites in plasma from normal and anephric man
Authors: Shepard Richard M. , Horst Ronald L. , Hamstra Alan J. , DeLuca Hector F.
Published: August 2015

A multiple assay capable of reliably determining vitamins D2 and D3 (ergocalciferol and cholecalciferol), 25(OH)D2 (25-hydroxyvitamin D2) and 25(OH)D3 (25-hydroxyvitamin D3), 24,25(OH)2D (24,25-dihydroxyvitamin D), 25,26(OH)2D (25,26-dihydroxyvitamin D) and 1,25(OH)2D (1,25-dihydroxyvitamin D) in a single 3–5ml sample of human plasma was developed. The procedure involves methanol/methylene chloride extraction of plasma lipids followed by separation of the metabolites and purification from interfering contaminants by batch elution chromatography on Sephadex LH-20 and Lipidex 5000 and by h.p.l.c. (high-pressure liquid chromatography). Vitamins D2 and D3 and 25(OH)D2 and 25(OH)D3 are quantified by h.p.l.c. by using u.v. detection, comparing their peak heights with those of standards. 24,25(OH)2D and 25,26(OH)2D are measured by competitive protein-binding assay with diluted plasma from vitamin D-deficient rats. 1,25(OH)2D is measured by competitive protein-binding assay with diluted cytosol from vitamin D-deficient chick intestine. Values in normal human plasma samples taken in February are: vitamin D 3.5±2.5ng/ml; 25(OH)D 31.6±9.3ng/ml; 24,25(OH)2D 3.5±1.4ng/ml; 25,26(OH)2D 0.7±0.5ng/ml; 1,25(OH)2D 31±9pg/ml (means±s.d.). Values in two normal human plasma samples taken in February after 1 week of high sun exposure are: vitamin D 27.1±7.9ng/ml; 25(OH)D 56.8±4.2ng/ml; 24,25(OH)2D 4.3±1.6ng/ml; 25,26(OH)2D 0.5±0.2ng/ml. Values in anephric-human plasma are: vitamin D 2.7±0.8ng/ml; 25(OH)D 36.4±16.5ng/ml; 24,25(OH)2D 1.9±1.3ng/ml; 25,26(OH)2D 0.6±0.3ng/ml; 1,25(OH)2D was undetectable.
The subcellular distribution and some properties of the cytochrome b component of the microbicidal oxidase system of human neutrophils
Authors: Segal A W , Jones O T G
Published: August 2015

A novel cytochrome b has recently been described in human neutrophils. The subcellular distribution of this cytochrome was investigated by analytical fractionation on continuous sucrose gradients and it was found to have a dual localization, the major component having a similar distribution to the plasma-membrane marker [3H]concanavalin A, and a denser peak located with the specific granules. The two components were separated on discontinuous gradients and studied independently.
Effect of vitamin D deficiency on bone formation in the chick
Authors: Dickson I R , Kodicek E
Published: August 2015

1. The process of diaphyseal bone formation can be investigated by studying the rate of incorporation of radioactive precursors, administered in vivo into bone fractions of increasing density. 2. In the 4-week-old vitamin D-treated chick most of the osteoid becomes calcified within 12h and almost all within 2 days. The low-density calcified phase that is formed is converted into a higher density form and within 7 days the greater proportion of the calcified tissue is in the higher density form. 3. In the vitamin D-deficient chick of similar age the rate of calcification of osteoid is decreased, as is the rate of conversion into the higher density phase with the resultant accumulation of the lower density calcified form. 4. The higher density phase probably corresponds to hydroxyapatite and the lower density one to the ACP-pase described by Termine & Posner [(1967) Calcif. Tissue Res. 1, 8–23]. 5. The disorder in the process of calcification seems to be unrelated to the alteration in blood Ca2+ and phosphate concentrations, but related to the presence or absence of cholecalciferol.
Characterization and phenotypic control of the cytochrome content of Escherichia coli
Authors: Reid Graeme A. , Ingledew W. John
Published: August 2015

1. Electron-transport particles derived from Escherichia coli grown aerobically contain three b-type cytochromes with mid-point oxidation–reduction potentials at pH7 of +260mV, +80mV and −50mV, with n=1 for each. The variation of these values with pH was determined. 2. E. coli develops a different set of b-type cytochromes when grown anaerobically on glycerol with fumarate or nitrate as terminal electron acceptor. Electron-transport particles of fumarate-grown cells contain b-type cytochromes with mid-point potentials at pH7 of +140mV and +250mV (n=1). These two cytochromes are also present in cells grown with nitrate as terminal acceptor, where an additional cytochrome b with a mid-point potential of +10mV (n=1) is developed. 3. The wavelengths of the α-absorption-band maxima of the b-type cytochromes at 77K were: (a) for aerobically grown cells, cytochrome b (Em7 +260mV), 556nm and 563nm, cytochrome b (Em7 +80mV), 556nm and cytochrome b (Em7−50mV), 558nm; (b) for anaerobically grown cells, cytochrome b (Em7 +250mV), 558nm, cytochrome b (Em7 +40mV), 555nm and cytochrome b (Em7 +10mV), 556nm. 4. Cytochrome d was found to have a mid-point potential at pH7 of +280mV (n=1). 5. Cytochrome a1 was resolved as two components of equal magnitude with mid-point potentials of +260mV and +160mV (n=1). 6. Redox titrations performed in the presence of CO showed that one of the b-type cytochromes in the aerobically grown cultures was reduced, even at the upper limits of our range of electrode potentials (above +400mV). Cytochrome d was also not oxidizable in the presence of CO. Neither of the cytochromes a1 was affected by the presence of CO.
Kinetics of 3-O-methyl-D-glucose transport in isolated rat hepatocytes
Authors: Craik J D , Elliott K R F
Published: August 2015

3-O-Methyl-D-glucose transport across the plasma membrane of isolated rat hepatocytes was followed for net entry of the sugar into sugar-free cells (zero trans entry), net exit of sugar into sugar-free medium (zero trans exit) and for unidirectional entry and exit fluxes when cells had been equilibrated with sugar in the extracellular medium (equilibrium exchange entry and exit). These measurements were performed at 20 degrees C and pH 7.4 by the use of simple manual methods. Initial rates of transport showed a Michaelis–Menten dependency on the sugar concentration at the cis side of the membrane over the range of concentrations tested (100 microM to 100 mM). Transport was found to be symmetrical with no evidence of substrate stimulation of transport from the trans side of the membrane. Parameters (mean values +/- S.E.M.) of transport were estimated as Vmax. 86.2 +/- 9.7 mmol/litre of cell water per min and Km 18.1 +/- 5.9 mM for exchange entry, Vmax. 78.8 +/- 5.3 mmol/litre of cell water per min and Km 17.6 +/- 3.5 mM for exchange exit, Vmax. 84.1 +/- 8.4 mmol/litre of cell water per min and Km 16.8 +/- 4.6 mM for zero trans exit.
Detection of peroxisomal fatty acyl-coenzyme A oxidase activity
Authors: Inestrosa N C , Bronfman M , Leighton F
Published: August 2015

It has been postulated that the peroxisomal fatty acid-oxidizing system [Lazarow & de Duve (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2043–2046; Lazarow (1978) J. Biol. Chem. 253, 1522–1528] resembles that of mitochondria, except for the first oxidative reaction. In this step, O2 would be directly reduced to H2O2 by an oxidase. Two specific procedures developed to detect the activity of the characteristic enzyme fatty acyl-CoA oxidase are presented, namely polarographic detection of palmitoyl-CoA-dependent cyanide-insensitive O2 consumption and palmitoyl-CoA-dependent H2O2 generation coupled to the peroxidation of methanol in an antimycin A-insensitive reaction. Fatty acyl-CoA oxidase activity is stimulated by FAD, which supports the flavoprotein nature postulated for this enzyme. Its activity increases 7-fold per g wet wt. of liver in rats treated with nafenopin, a hypolipidaemic drug. Subcellular fractionation of livers from normal and nafenopin-treated animals provides evidence for its peroxisomal localization. The stoicheiometry for palmitoyl-CoA-dependent O2 consumption, H2O2 generation and NAD+ reduction is 1 : 1 : 1. This suggests that fatty acyl-CoA oxidase is the rate-limiting enzyme of the peroxisomal fatty acid-oxidizing system.
Binding of radioiodinated propylthiouracil to rat liver microsomal fractions. Stimulation by substrates for iodothyronine 5′-deiodinase
Authors: Visser T J , Van Overmeeren E
Published: August 2015

Previous studies have shown that 2-thiouracil derivatives are uncompetitive inhibitors of iodothyronine 5′-deiodinase activity of rat liver microsomal fraction. Therefore the interaction of radioiodinated 6-propyl-2-thiouracil with rat liver microsomal fraction and the effect of substrate, cofactor and other inhibitors of 5′-deiodinase activity activity were investigated. It was found that micromolar concentrations of, in order of increasing potency, 3,5-diiodotyrosine, thyroxine, 3,3′,5′-tri-iodothyronine and 3′,5′-di-iodothyronine significantly enhanced binding of 5-[125I]iodo-6-propyl-2-thiouracil to the enzyme preparation. This stimulation was not seen in the presence of 1 mM dithiothreitol, 0.1 mM-6-propyl-2-thiouracil, 0.1 mM-6-propyl-2-thiouracil, 0.1 M-2-mercapto-1-methylimidazole or 1 mM-sodium sulphite. These results support the hypothesis that thiouracil derivatives inhibit 5′-deiodinase activity by forming a mixed disulphide with an intermediate enzyme complex, probably a sulphenyl iodide.