bluuebunny/crossref_metadata_2025 huggingface dataset:
The formation of ferritin from apoferritin. Kinetics and mechanism of iron uptake
Authors: Macara I. G. , Hoy T. G. , Harrison Pauline M.
Published: August 2015

Ferritin has a high capacity as an iron store, incorporating some 4500 iron atoms as a microcrystalline ferric oxide hydrate. Starting from apoferritin, or ferritin of low iron content, Fe2+ and an oxidizing agent, the uptake of iron can be recorded spectrophotometrically. Progress curves were obtained and the reconstituted ferritin was shown by several physical methods to be similar to natural ferritin. The progress curves of iron uptake by apoferritin are sigmoidal; those for ferritins of low iron content are hyperbolic. The rate of iron uptake is dependent on the amount of iron already present in the molecule. The distribution of iron contents among reconstituted ferritin molecules is inhomogeneous. These findings are interpreted in terms of a crystal growth model. The surface area of the crystallites forming inside the protein increases until the molecule is half full, and then declines. This surface controls the rate at which new material is deposited. The experimental results can best be accounted for by a two-stage mechanism, an initial slow ‘nucleation’ stage, which is apparently zero order with respect to [Fe2+], followed by a more rapid ‘growth‘ stage. The rate of Fe2+ oxidation is increased in the presence of apoferritin as compared with controls. Ferritin can therefore be regarded as an enzyme to which the product remains firmly attached. The protein appears to increase the rate of ‘nucleation’ The apparent zero order of this stage suggests the presence of binding sites on the protein, which are saturated with respect to Fe2+. These sites are presumed also to be oxidation sites. The oxidation and subsequent formation of the ferric oxide hydrate may proceed according to one of three alternative models.
The release of collagenase as an inactive proenzyme by bone explants in culture
Authors: Vaes G.
Published: August 2015

1. A latent collagenase, activated only by limited proteolysis, was found in culture media of mouse bone explants. It could be activated by trypsin or, less efficiently, by chymo-trypsin. Skin explants also released latent collagenase. 2. Bone collagenase attacks native collagen at about neutral pH when it is in solution, in reconstituted fibrils or in insoluble fibres, producing two fragments representing 75 and 25% of the molecule. It requires calcium and is inhibited by EDTA, cysteine or serum. 3. Latent collagenase is not activated by trypsin-activated collagenase but by a distinct unidentified thermolabile agent present in a latent trypsin-activatable state in the culture media, or by purified liver lysosomes between pH5.5 and pH7.4. Trypsin activation decreases the molecular weight of latent collagenase from 105000 to 84000 as determined by gel filtration. 5. The latency of collagenase is unlikely to be due to an enzyme–inhibitor complex. Although some culture media contain a collagenase inhibitor, its presence is not constant and its molecular weight (at least 120000) is not compatible with the decrease in molecular weight accompanying activation; also combinations of collagenase with inhibitor are not reactivated by trypsin. Moreover, the latency remains after gel filtration, or treatment by high dilution, exposure to pH values between 2.5 and 10, or high ionic strength, urea or detergent. 6. It is proposed that latent collagenase represents an inactive precursor of the enzyme, a `procollagenase', and that the extracellular activity of collagenase is controlled by another protease that activates procollagenase by a limited proteolysis of its molecule.
The pentose cycle and insulin release in mouse pancreatic islets
Authors: Ashcroft S. J. H. , Weerasinghe L. C. C. , Bassett J. M. , Randle P. J.
Published: August 2015

1. Rates of insulin release, glucose utilization (measured as [3H]water formation from [5-3H]glucose) and glucose oxidation (measured as14CO2 formation from [1-14C]- or [6-14C]-glucose) were determined in mouse pancreatic islets incubated in vitro, and were used to estimate the rate of oxidation of glucose by the pentose cycle pathway under various conditions. Rates of oxidation of [U-14C]ribose and [U-14C]xylitol were also measured. 2. Insulin secretion was stimulated fivefold when the medium glucose concentration was raised from 3.3 to 16.7mm in the absence of caffeine; in the presence of caffeine (5mm) a similar increase in glucose concentration evoked a much larger (30-fold) increase in insulin release. Glucose utilization was also increased severalfold as the intracellular glucose concentration was raised over this range, particularly between 5 and 11mm, but the rate of oxidation of glucose via the pentose cycle was not increased. 3. Glucosamine (20mm) inhibited glucose-stimulated insulin release and glucose utilization but not glucose metabolism via the pentose cycle. No evidence was obtained for any selective effect on the metabolism of glucose via the pentose cycle of tolbutamide, glibenclamide, dibutyryl 3′:5′-cyclic AMP, glucagon, caffeine, theophylline, ouabain, adrenaline, colchicine, mannoheptulose or iodoacetamide. Phenazine methosulphate (5μm) increased pentose-cycle flux but inhibited glucose-stimulated insulin release. 4. No formation of14CO2 from [U-14C]ribose could be detected: [U-14C]xylitol gave rise to small amounts of14CO2. Ribose and xylitol had no effect on the rate of oxidation of glucose; ribitol and xylitol had no effect on the rate of glucose utilization. Ribose, ribitol and xylitol did not stimulate insulin release under conditions in which glucose produced a large stimulation. 5. It is concluded that in normal mouse islets glucose metabolism via the pentose cycle does not play a primary role in insulin-secretory responses.
Stimulation of the production of unesterified fatty acids in nerve endings of guinea-pig brain in vitro by noradrenaline and 5-hydroxytryptamine
Authors: Price C. J. , Rowe C. E.
Published: August 2015

1. Noradrenaline (1mm) and 5-hydroxytryptamine (1mm) stimulated the production of unesterified palmitate, oleate, stearate and arachidonate in nerve endings (synaptosomes) isolated from combined guinea-pig cerebral cortex and cerebellum. 2. Iproniazid phosphate (0.36mm) increased the concentrations of the same acids in osmotically ruptured synaptosomes. Further addition of 1mm-noradrenaline or 1mm-5-hydroxytryptamine reversed this increase. 3. Noradrenaline (0.01mm) stimulated the production of unesterified fatty acids in isolated synaptic membranes. 5-Hydroxytryptamine (0.01mm) stimulated the production of unesterified fatty acids in synaptic membranes and synaptic vesicles.
The effect of the coupled oxidation of substrate on the permeability of blowfly flight-muscle mitochondria to potassium and other cations
Authors: Hansford R. G. , Lehninger A. L.
Published: August 2015

1. Blowfly flight-muscle mitochondria respiring in the absence of phosphate acceptor (i.e. in state 4) take up greater amounts of K+, Na+, choline, phosphate and Cl-(but less NH4+) than non-respiring control mitochondria. 2. Uptake of cations is accompanied by an increase in the volume of the mitochondrial matrix, determined with the use of [14C]-sucrose and3H2O. The osmolarity of the salt solution taken up was approximately that of the suspending medium. 3. The [14C]sucrose-inaccessible space decreased with increasing osmolarity of potassium chloride in the suspending medium, confirming that the blowfly mitochondrion behaves as an osmometer. 4. Light-scattering studies showed that both respiratory substrate and a permeant anion such as phosphate or acetate are required for rapid and massive entry of K+, which occurs in an electrophoretic process rather than in exchange for H+. The increase in permeability to K+and other cations is probably the result of a large increase in the exposed area of inner membrane surface in these mitochondria, with no intrinsic increase in the permeability per unit area. 5. No increase in permeability to K+and other cations occurs during phosphorylation of ADP in state 3 respiration.
The presence of 5-hydroxymethylcytosine in animal deoxyribonucleic acid
Authors: Penn N. W. , Suwalski R. , O'Riley C. , Bojanowski K. , Yura R.
Published: August 2015

A method is given for small-scale preparation of DNA from 1.0–1.5g of adult rat tissues. The product from brain or liver is characterized by base ratios and phosphorus content which accord with reported values for rat tissue. It is reasonably free of RNA, protein and glycogen. It contains 5-hydroxymethylcytosine at a content of about 15% of the total cytosine bases present. 5-Hydroxymethylcytosine is also demonstrable in mouse and frog brain DNA and in the crude cytidylic acid fractions obtained from RNA hydrolysates of rat brain and liver. 5-Hydroxymethylcytosine is identified by paper chromatography, u.v. spectra in acid and alkaline solutions and by its conversion into 5-hydroxymethyluracil.
Identification of a group of tetracarboxylate porphyrins, containing one acetate and three propionate β-substituents, in faeces from patients with symptomatic cutaneous hepatic porphyria and from rats with porphyria due to hexachlorobenzene
Authors: Elder G. H.
Published: August 2015

1. Three tetracarboxylate porphyrins, apart from coproporphyrin, were isolated from the faeces of patients with symptomatic cutaneous hepatic porphyria and of rats with porphyria caused by hexachlorobenzene poisoning. The porphyrins were not present in the urine of the porphyric rats or in the faeces of control rats. 2. Two of the porphyrins were identified as tris-(2-carboxyethyl)-carboxymethyl ethyl trimethyl porphin (isocoproporphyrin) and tris-(2-carboxyethyl)-carboxymethyl trimethyl porphin (de-ethyliso-coproporphyrin). 3. The third porphyrin, previously reported to be an analogue of coproporphyrin in which one propionate group is replaced by an α- or β-hydroxypropionate group, was shown to be tris-(2-carboxyethyl)-carboxymethyl-(1-hydroxyethyl)-trimethyl porphin (hydroxyisocoproporphyrin). 4. The order of β-substituents around the porphin ring could not be determined for any of the compounds. 5. Evidence for the existence of a fourth porphyrin, tris-(2-carboxyethyl)-carboxymethyl trimethyl vinyl porphin (dehydroisocoproporphyrin), a postulated precursor of the others, is described. 6. It is suggested that dehydroisocoproporphyrinogen is produced by decarboxylation–dehydrogenation of one of the propionate groups of pentacarboxylate porphyrinogen III.
Purification and properties of a human seminal proteinase
Authors: Syner Frank N. , Moghissi Kamran S.
Published: August 2015

1. A method is described for the purification of a proteinase, present in human seminal plasma and previously shown to accelerate migration of spermatozoa through cervical mucus in vitro. A 25-fold purification was achieved in three steps, consisting of ammonium sulphate fractionation, chromatography on CM-cellulose and gel filtration. 2. The enzyme displays some properties similar to chymotrypsin: pH optimum 7.5–8.0; substrate preference of casein, haemoglobin and benzoyltyrosine ethyl ester but not benzoylarginine ethyl ester; mol.wt. 33000. However, it is unaffected by 1mm-di-isopropyl phosphofluoridate or 1mm metal cations, and in this respect differs from chymotrypsin. 3. The properties of the enzyme strongly resemble those of the ‘chymotrypsin-like’ enzyme discovered in seminal plasma by Lundquist et al. (1955). 4. The use of dimethyl-casein permitted the performance of enzyme assays at substrate concentrations five times higher (up to 50mg/ml) than could be achieved with ordinary casein (10mg/ml).
Peptides from a mycobacillin-synthesizing cell-free system
Authors: Sengupta S. , Bose S. K.
Published: August 2015

In a cell-free system from Bacillus subtilis B3, ATP–Pi exchange was catalysed by l-proline at a pH optimum of 7.2. Further stimulation by component amino acids of mycobacillin was inhibited by deprivation from the synthesizing system of even a single amino acid occurring at any point of the cyclic peptide. This inhibition, however, decreased with the distance in the molecule of the given amino acid from l-proline. Peptides containing respectively two, three, four, five and six amino acids were isolated from the mycobacillin-synthesizing system by an amino acid-deprivation technique. The amino acid composition of these peptides and also their N- and C-terminal amino acid residues were the same as those of peptides that would be obtained if mycobacillin synthesis occurred starting from l-proline and was interrupted at various points along the polypeptide chain.
The activities of fructose diphosphatase in flight muscles from the bumble-bee and role of this enzyme in heat generation
Authors: Newsholme E. A. , Crabtree B. , Higgins S. J. , Thornton S. D. , Start Carole
Published: August 2015

1. The maximum catalytic activities of fructose diphosphatase from flight muscles of bumble-bees (Bombus spp.) are at least 30-fold those reported for the enzyme from other tissues. The maximum activity of fructose diphosphatase in the flight muscle of any particular bee is similar to that of phosphofructokinase in the same muscle, and the activity of hexokinase is similar to or greater than the activity of phosphofructokinase. There is no detectable activity of glucose 6-phosphatase and only a very low activity of glucose 6-phosphate dehydrogenase in these muscles. The activities of both fructose diphosphatase and phosphofructokinase vary inversely with the body weight of the bee, whereas that of hexokinase is relatively constant. 2. There is no significant hydrolysis of fructose 1-phosphate, fructose 6-phosphate, glucose 1,6-diphosphate and glycerol 3-phosphate by extracts of bumble-bee flight muscle. 3. Fructose 1,6-diphosphatase from bumble-bee flight muscle and from other muscles is inhibited by Mn2+and univalent cations; the potency of inhibition by the latter varies in the order Li+>Na+>K+. However, the fructose diphosphatase from bumble-bee flight muscle is different from the enzyme from other tissues in that it is not inhibited by AMP. 4. The contents of ATP, hexose monophosphates, fructose diphosphate and triose phosphates in bumble-bee flight muscle showed no significant changes between rest and flight. 5. It is proposed that both fructose diphosphatase and phosphofructokinase are simultaneously active and catalyse a cycle between fructose 6-phosphate and fructose diphosphate in resting bumble-bee flight muscle. Such a cycle would produce continuous hydrolysis of ATP, with the release of energy as heat, which would help to maintain the thoracic temperature during rest periods at a level adequate for flight.
The location of spermine in bacterial ribosomes as indicated by 1,5-difluoro-2,4-dinitrobenzene and by ethidium bromide
Authors: Stevens L. , Pascoe Genevieve
Published: August 2015

1. When the binding of ethidium bromide to rRNA is measured both in the presence and in the absence of spermine, by spectrophotometric titrations, by gel filtration, or by the changes in fluorescence intensity, spermine competes with ethidium bromide for sites on the rRNA; under the conditions used in these experiments ethidium bromide is bound to the double-stranded regions of rRNA. 2. When an excess of ethidium bromide is added to ribosomes from Bacillus stearothermophilus approx. 80% of the endogenous spermine is displaced from the ribosomes. 3. [14C]Spermine is fixed to ribosomes by either formaldehyde or 1,5-difluoro-2,4-dinitrobenzene. Most of the [14C]spermine, fixed to ribosomes by 1,5-difluoro-2,4-dinitrobenzene, attaches to the ribosomal protein. 4. It is concluded that most of the endogenous spermine is bound to the double-stranded RNA in ribosomes, and that some of these double-stranded regions to which spermine is attached also have ribosomal proteins bound to them.
Lysosomal nature of hormonally induced enzymes in wheat aleurone cells
Authors: Gibson R. A. , Paleg L. G.
Published: August 2015

The subcellular distribution of the enzymes α-amylase, protease and ribonuclease in wheat aleurone layers after treatment with gibberellic acid was determined by differential centrifugation. Of the α-amylase 56% was precipitable from cell homogenates, indicating that it is a particulate enzyme. Similar results were recorded with protease. Particulate α-amylase showed distinct structural latency, and membrane-rupturing mechanical or chemical treatments were required to release the enzyme in an active form; the results were completely analogous to results with lysosomal enzymes found in animal tissues. The identification of the hormonally induced enzymes as lysosomal suggests that the hormonal mechanism may be more closely associated with extracellular enzyme synthesis rather than with nucleic acid metabolism.
The antibody binding site. Labelling of a specific antibody against the photo-precursor of an aryl nitrene
Authors: Fleet G. W. J. , Knowles J. R. , Porter R. R.
Published: August 2015

The isolation of specific rabbit antibodies for the haptenic group 4-azido-2-nitrophenyl, is described. These antibodies bind 1.8–2.0mol of hapten [∈-(4-azido-2-nitrophenyl)-l-lysine]/mol with an association constant of nearly 107m-1 at 4°C. On photolysis of the antibody–hapten complex, resulting in the formation of an aryl nitrene at the binding site, hapten was covalently bound to the antibody, and the antibody binding site was blocked. The ratio of labelling of heavy- and light-chains was 2.5:1. Two small peptides were isolated from digests of labelled heavy-chain, indicating that some 13% of the label in the antibody was attached to cysteine-92 and to alanine-93. These residues are adjacent to the major hypervariable region in rabbit heavy-chain (residues 95–105).
Effects of hypo- and hyper-thyroidism on liver composition, blood glucose, ketone bodies and insulin in the male rat
Authors: Aranda Ana , Montoya Eladio , Herrera Emilio
Published: August 2015

1. Thyroidectomized rats injected daily with 0, 0.1, 2 or 25μg of l-thyroxine/100g body wt. were compared with intact controls. In plasma, the protein-bound iodine was decreased in the rats given the 0 or 0.1μg doses and increased in those given the 25μg dose. 2. Blood glucose decreased in those given 2μg and was augmented in those given 25μg, and ketone bodies were the same in all the groups. 3. Plasma insulin was lowest in the rats given the 0 or 0.1μg doses and was highest in those given the 2 or 25μg doses of thyroxine. 4. After 48h starvation, the decrease in blood glucose and increase in ketone bodies observed in all the groups was greatest in the group not supplemented with thyroxine. 5. Plasma insulin concentrations remained at the value for fed animals in the rats given the 25μg dose of thyroxine but decreased in the other groups. 6. In fed animals, concentrations of hepatic DNA P, citrate, total fatty acids and acetyl-CoA were similar in all the groups, and glycogen was low only in the rats given the 25μg dose of thyroxine. 7. After 48h starvation, liver DNA P, total fatty acids and acetyl-CoA increased in all the groups, except in the rats given the 25μg dose, where both total fatty acids and acetyl-CoA remained at the value for fed animals. Liver citrate did not change in the groups given the 0 or 25μg doses of thyroxine, but decreased in the other groups. 8. The results are discussed in relation to the regulation of intermediary metabolism in hypo- and hyper-thyroidism.
The cytochromes of mitochondria from Tetrahymena pyriformis strain ST
Authors: Lloyd D. , Chance B.
Published: August 2015

1. Mitochondria of the obligately aerobic ciliate protozoon, Tetrahymena pyriformis strain ST, are unusual in that they possess a cytochrome oxidase system that does not react with reduced mammalian cytochrome c; the presence of cytochromes a603+a3 is masked in the α-band region of spectra by the broad absorption band of cytochrome a620. 2. Other haemoproteins present include cytochromes b560, b556, c553 and c549. 3. The reaction of reduced cytochrome a3 with CO is reversed by flash photolysis, and in the presence of O2 the subsequent oxidation of this cytochrome is followed by that of cytochrome a603. 4. Cytochromes a620 and b560 also react with CO and with KCN; the latter cytochrome corresponds with that designated cytochrome o by other workers. 5. The contribution of cytochrome a603 to difference spectra is revealed by making use of the fact that it does not react with KCN. 6. Cytochrome a620 is unstable, and its α-absorption band is lost from spectra of mitochondria which have been aged or treated with ultrasound, detergents or organic solvents. 7. Possible pathways of electron transport via the several different terminal oxidases in Tetrahymena mitochondria are proposed.
Purification and properties of adenosine triphosphate–creatine phosphotransferase from muscle of the dogfish Scylliorhinus canicula
Authors: Simonarson B. , Watts D. C.
Published: August 2015

1. Creatine kinase occurs in high concentration in the soluble proteins of dogfish muscle. A fourfold purification gives essentially pure enzyme but with a low specific activity. This appears to be a property of the native enzyme and not a result of the isolation procedures used. 2. The amino acid composition is similar to that of other phosphagen kinases, but the enzyme differs from mammalian creatine kinases in having four thiol groups readily reactive towards 5,5′-dithiobis-(2-nitrobenzoic acid). Titration of two thiol groups is accompanied by almost complete loss of activity. The remaining two thiol groups react at different rates, suggesting that modifying the third thiol group affects the reactivity of the fourth thiol group. 3. The enzyme is markedly protected against inactivation by iodoacetamide by MgATP or MgADP. Addition of creatine to MgADP decreases protection, but the further addition of Cl− restores protection to the original value. The quaternary MgADP–creatine–enzyme–nitrate complex protects very strongly as is found for the rabbit enzyme. The involvement of the conformational state of the enzyme in such effects is discussed. 4. Creatine kinase from both dogfish and rabbit is equally sensitive to urea denaturation. Urea protects the dogfish enzyme by about 9% against inhibition by iodoacetamide. 5. The formation of a hybrid between the dogfish and rabbit enzymes in vitro has been demonstrated. 6. At high substrate concentrations the dogfish enzyme shows apparent ordered kinetics. The effect of temperature on Vmax. and the Michaelis constants for MgATP and creatine were determined. These and changes in the apparent activation energy suggest that limited adaptation has occurred commensurate with physiological need.
Biochemical effects of the hypoglycaemic compound diphenyleneiodonium. Catalysis of anion–hydroxyl ion exchange across the inner membrane of rat liver mitochondria and effects on oxygen uptake
Authors: Holland P. C. , Sherratt H. S. A.
Published: August 2015

1. The hypoglycaemic compound diphenyleneiodonium causes rapid and extensive swelling of rat liver mitochondria suspended in 150mm-NH4Cl, and in 150mm-KCl in the presence of 2,4-dinitrophenol and valinomycin. This indicates that diphenyleneiodonium catalyses a compulsory exchange of OH-for Cl-across the mitochondrial inner membrane. Br-and SCN-were the only other anions found whose exchange for OH-is catalysed by diphenyleneiodonium. 2. Diphenyleneiodonium inhibited state 3 respiration of mitochondria and slightly stimulated state 4 respiration with succinate or glutamate as substrate in a standard Cl--containing medium. 3. Diphenyleneiodonium did not inhibit state 3 respiration significantly in two Cl--free media (based on glycerol 2-phosphate or sucrose) but caused some stimulation of state 4. 4. In Cl--containing medium diphenyleneiodonium only slightly inhibited the 2,4-dinitrophenol-stimulated adenosine triphosphatase and it had little effect in the absence of Cl-. 5. The inhibition of respiration in the presence of Cl-is dependent on the Cl-–OH-exchange. 2,4-Dichlorodiphenyleneiodonium is ten times as active as diphenyleneiodonium both in causing swelling of mitochondria suspended in 150mm-NH4Cl and in inhibiting state 3 respiration in Cl--containing medium. Indirect evidence suggests that the Cl-–OH-exchange impairs the rate of uptake of substrate anions. 6. It is proposed that stimulation of state 4 respiration in the absence of Cl-depends, at least in part, on an electrogenic uptake of diphenyleneiodonium cations. 7. Tripropyl-lead acetate, methylmercuric iodide and nine substituted diphenyleneiodonium derivatives also catalyse Cl-–OH-exchange across the mitochondrial membrane. 8. Diphenyleneiodonium is compared with the trialkyltin compounds, which are also known to mediate Cl-–OH-exchange and which have in addition strong oligomycin-like effects on respiration. It is concluded that diphenyleneiodonium is specific for catalysing anion–OH-exchange and will be a useful reagent for investigating membrane-dependent systems.
The immobilization of enzymes on nylon structures and their use in automated analysis
Authors: Inman D. J. , Hornby W. E.
Published: August 2015

1. Glucose oxidase (EC 1.1.3.4) and urease (EC 3.5.1.5) were covalently attached through glutaraldehyde to low-molecular-weight nylon powder. 2. Immobilized derivatives of glucose oxidase and urease were prepared by cross-linking the respective enzymes within the matrix of a nylon membrane. 3. An improved process is described for the immobilization of glucose oxidase and urease on the inside surface of partially hydrolysed nylon tube. 4. Automated analytical procedures are described for the determination of glucose with each of the three immobilized glucose oxidase derivatives and for the determination of urea with each of the three immobilized urease derivatives. 5. The efficiencies of the three immobilized enzyme structures as reagents for the automated determination of their substrates were compared.
Separation and some properties of the major proteins of the human erythrocyte membrane
Authors: Tanner M. J. A. , Boxer D. H.
Published: August 2015

A fractionation procedure is described which allows the isolation of three major human erythrocyte membrane proteins. Their isolation involves three sequential extraction procedures followed by gel filtration in 1% sodium dodecyl sulphate and preparative gel electrophoresis. All three proteins can be isolated from a single preparation. One of the proteins is the erythrocyte sialoglycoprotein, for which no C- or N-terminal residues were found. The other two proteins, which have not previously been isolated, have subunit molecular weights of 74000 and 93000 and contain 9 and 7% carbohydrate respectively. These glycoproteins have blocked N-terminal residues and show similarities in their chemical properties. Preparations derived from blood-group O erythrocytes contain no N-acetylgalactosamine, but similar preparations from blood-group A erythrocytes do contain this sugar. These three proteins cannot easily be solubilized by gentle aqueous procedures and represent about half of the erythrocyte ‘ghost’ protein. They carry a large proportion of the cell-surface carbohydrate.
Clearing-factor lipase in obese–hyperglycaemic mice (ob/ob)
Authors: Enser M.
Published: August 2015

1. Clearing-factor lipase was assayed in acetone–ether-dried powders of heart and epididymal fat-pads of lean and genetically obese mice (ob/ob). In both tissues the enzyme activity in the adult was higher in the obese mice. 2. In heart the enzyme activity was unchanged from 8 to 48 weeks of age in lean mice, but in obese mice it increased between 8 and 12 weeks of age and remained elevated. 3. Starvation produced changes in the heart clearing-factor lipase activity in obese, but not lean, mice. 4. The clearing-factor lipase activity of epididymal fat-pads decreased rapidly during 24h starvation in both lean and obese mice, but the activity in the obese mice remained higher than that in lean mice. 5. Plasma triglyceride and cholesterol concentrations were determined in both lean and obese mice. Triglyceride concentrations were not greatly different, but the obese mice were hypercholesterolaemic. Plasma cholesterol concentrations were not correlated with changes in clearing-factor lipase activity.
Differences in the patterns of methylation in rat liver ribosomal ribonucleic acid after reaction in vivo with methyl methanesulphonate and NN-dimethylnitrosamine
Authors: O'Connor P. J. , Capps M. J. , Craig A. W. , Lawley P. D. , Shah S. A.
Published: August 2015

1. rRNA was isolated from rat liver at short intervals after the intraperitoneal injection of [14C]methyl methanesulphonate (50mg/kg) or NN-di[14C]methylnitrosamine (2mg/kg). These doses were chosen to minimize the effects of toxicity. 2. The following methods of hydrolysis of [14C]methylated rRNA were employed: enzymic digestion to nucleosides at pH8; alkaline hydrolysis and conversion into nucleosides; acid hydrolysis to bases. 3. The methylation products were analysed by chromatography on columns of Dowex-50 (H+ form) and Dowex-50 (NH4+ form). 4. With both methylating agents the principal product of methylation was 7-methylguanine. Differences were obtained, however, in the molar proportions of the minor bases 3-methylcytosine, 1-methyladenine and 7-methyladenine. Methylation at the O-6 position of guanine was a significant feature of rRNA obtained from the NN-di[14C]methylnitrosamine-treated animals but was not detected in rRNA after treatment with [14C]methyl methanesulphonate.
Isolation and properties of mesosomal membrane fractions from Micrococcus lysodeikticus
Authors: Owen Peter , Freer John H.
Published: August 2015

1. A method is described for the isolation of pure mesosomal membrane fractions from Micrococcus lysodeikticus. 2. Plasmolysis of cells, before wall digestion, was necessary for effective mesosome release. 3. The effect of mild shearing forces, temperature and time upon the release of mesosomal membrane from protoplasts was investigated. 4. The optimum yield of mesosomal membranes from stable protoplasts was achieved at 10mm-Mg2+. 5. Mesosomal membrane vesicle fractions prepared at differing Mg2+ concentrations above 10mm were similar in chemical composition. 6. Comparison of the properties of peripheral and mesosomal membrane fractions revealed major differences in the distribution of protein components, membrane phosphorus, mannose and dehydrogenase activities between the two fractions. 7. Only cytochrome b556 was detected in mesosomal membranes, whereas peripheral membranes contained a full complement of cytochromes. 8. Preliminary investigations suggested the localization of an autolytic enzyme(s) in the mesosomal vesicles. 9. The anatomy of mesosomal and peripheral membrane have been compared by the negative-staining and freeze-fracture technique. 10. The results are discussed in relation to a plausible role for the mesosome.
The type-specific substance from Pneumococcus type 13
Authors: Watson M. J. , Tyler Jean M. , Buchanan J. G. , Baddiley J.
Published: August 2015

1. The type-specific substance, S.13, from Pneumococcus type 13 was subjected to hydrolysis with alkali, followed by enzymic dephosphorylation, to yield a pentasaccharide. 2. The pentasaccharide, corresponding to the dephosphorylated repeating unit of S.13, was shown to be O-β-d-galactopyranosyl-(1→4)-O-β- d-glucopyranosyl-(1→3)-O-β-d- galactofuranosyl-(1→4)-O-2-acetamido-2-deoxy-β-d- glucopyranosyl-(1→2)-ribitol. 3. The phosphodiester linkages in S.13 join the hydroxyl group at position 1 of ribitol and the hydroxyl group at position 4 of a galactopyranosyl residue in the next repeating unit. 4. Ester groups, presumably O-acetyl, are located on positions 2 or 3 of most glucopyranosyl residues in S.13. 5. A partial structure for S.13 is proposed.
The transfer of mannose from guanosine diphosphate mannose to dolichol phosphate and protein by pig liver endoplasmic reticulum
Authors: Richards J. B. , Hemming F. W.
Published: August 2015

When pig liver microsomal preparations were incubated with GDP-[14C]mannose, 10–40% of the 14C was transferred to mannolipid and 1–3% to mannoprotein. The transfer to mannolipid was readily reversible and GDP was one of the products of the reaction. It was possible to reverse the reaction by adding excess of GDP and to show the incorporation of [14C]GDP into GDP-mannose. When excess of unlabelled GDP-mannose was added to a partially completed incubation there was a rapid transfer back of [14C]mannose from the mannolipid to GDP-mannose. The other product of the reaction, the mannolipid, had the properties of a prenol phosphate mannose. This was illustrated by its lability to dilute acid but stability to dilute alkali, and by its chromatographic properties. Dolichol phosphate stimulated the incorporation of [14C]mannose into both mannolipid and into protein, although the former effect was larger and more consistent than the latter. The incorporation of exogenous [3H]dolichol phosphate into the mannolipid, and its release, accompanied by mannose, on treatment of the mannolipid with dilute acid, confirmed that exogenous dolichol phosphate can act as an acceptor of mannose in this system. It was shown that other exogenous polyprenol phosphates (but not farnesol phosphate or cetyl phosphate) can substitute for dolichol phosphate in this respect but that they are much less efficient than dolichol phosphate in stimulating the transfer of mannose to protein. Since pig liver contained substances with the chromatographic properties of both dolichol phosphate and dolichol phosphate mannose, which caused an increase in transfer of [14C]mannose from GDP-[14C]mannose to mannolipid, it was concluded that endogenous dolichol phosphate acts as an acceptor of mannose in the microsomal preparation. The results indicate that the mannolipid is an intermediate in the transfer of mannose from GDP-mannose to protein. Some 4% of the mannose of a sample of mannolipid added to an incubation was transferred to protein. A scheme is proposed to explain the variations with time in the production of radioactive mannolipid, mannoprotein, mannose 1-phosphate and mannose from GDP-[14C]mannose that takes account of the above observations. ATP, ADP, UTP, GDP, ADP-glucose and UDP-glucose markedly inhibited the transfer of mannose to the mannolipid.
The lactose and neuraminlactose content of rat milk and mammary tissue
Authors: Kuhn N. J.
Published: August 2015

Specific analyses of rat milk sugar revealed lactose and N-acetylneuraminyl-lactose. The latter showed multiple, interconvertible forms in certain paper-chromatographic systems. Mean lactose concentrations rose from 33.1μmol/ml at day 0 to 100μmol/ml at day 20 of lactation. Mean N-acetylneuraminyl-lactose concentrations rose from 7.1μmol/ml at day 0 to 25.9μmol/ml at day 4 and thereafter declined, decreasing to zero by day 20 of lactation. Similar data are given on the concentrations of these sugars in mammary tissue of rats. Neuraminyl-lactose was also detected in mouse mammary tissue.
The isocitrate dehydrogenases of Acinetobacter lwoffi. Separation and properties of two nicotinamide–adenine dinucleotide phosphate-linked isoenzymes
Authors: Self Colin H. , Weitzman P. David J.
Published: August 2015

Two isoenzymes of NADP-linked isocitrate dehydrogenase have been identified in Acinetobacter lwoffi and have been termed isoenzyme-I and isoenzyme-II. The isoenzymes may be separated by ion-exchange chromatography on DEAE-cellulose, by gel filtration on Sephadex G-200, or by zonal ultracentrifugation in a sucrose gradient. Low concentrations of glyoxylate or pyruvate effect considerable stimulation of the activity of isoenzyme-II. The isoenzymes also differ in pH-dependence of activity, kinetic parameters, stability to heat or urea and molecular size. Whereas isoenzyme-I resembles the NADP-linked isocitrate dehydrogenases from other organisms in having a molecular weight under 100000, isoenzyme-II is a much larger enzyme (molecular weight around 300000) resembling the NAD-linked isocitrate dehydrogenases of higher organisms.
Formation of compound I by the reaction of catalase with peroxoacetic acid
Authors: Jones Peter , Middlemiss D. N.
Published: August 2015

1. The formation of Compound I by the reactions of bacterial and ox liver catalases with peroxoacetic acid was examined. In both cases the process occurs almost entirely by reaction of catalase with un-ionized peroxoacetic acid molecules. The result suggests an important role for the bound peroxidic proton in the enzyme–substrate interaction. 2. The peroxidatic properties of the Compounds I formed when peroxoacetic acid was used were examined by studying the oxidations of ethanol and formate; the results closely resemble those previously reported when H2O2 and alkyl hydroperoxides were used. 3. Compound I formed with bacterial catalase and peroxoacetic acid is remarkably stable in the absence of added donor and the preparation has considerable potential for detailed studies of the nature of this intermediate.
Aromatic acids derived from phenylalanine in the tissues of rats with experimentally induced phenylketonuria-like characteristics
Authors: Edwards David J. , Blau Karl
Published: August 2015

1. Aromatic acids were extracted from brain and liver of rats with phenylketonuria-like characteristics produced by administration of phenylalanine, either alone or in combination with p-chlorophenylalanine. The metabolism of the aromatic acids in these tissues was measured by gas chromatography. 2. At 1h after an intraperitoneal injection of l-phenylalanine (1g/kg) in 23-day-old rats, the phenyl-lactate concentration was 2.2μg/g in the liver and 0.43μg/g in the brain, and the concentration of o-hydroxyphenylacetate was 0.26μg/g in the liver. 3. Phenylacetate concentrations in brain and liver were 0.26 and 0.14μg/g respectively. 4. Suckling rats produced phenyl-lactate less rapidly than weanling rats, but accumulated higher concentrations in longer-term experiments. 5. Intraperitoneal injections of phenyl-lactic acid showed that this compound could directly penetrate the blood–brain barrier, and could produce similar brain/liver ratios of phenyllactate to those found after phenylalanine injection. 6. Qualitative and quantitative similarities in urinary excretion of aromatic acids between the rats used in this study and human patients with uncontrolled phenylketonuria indicate that a patient with a circulating phenylalanine concentration of the order of those achieved in the experimental animal may have aromatic acid concentrations in brain and liver comparable with those found in the rats used in the present study.
The isolation, properties and amino acid sequence of erabutoxin c, a minor neurotoxic component of the venom of a sea snake Laticauda semifasciata
Authors: Tamiya N. , Abe H.
Published: August 2015

Erabutoxin c, a minor neurotoxic component of the venom of a sea snake Laticauda semifasciata, was isolated in pure form by repeated column chromatography on CM-cellulose columns. The toxin was crystallizable and monodisperse in rechromatography, disc electrophoresis and isoelectric focusing (isoelectric point, pH9.23–9.25). The molecular weight of the toxin, as estimated by gel filtration, was 7000. The toxin showed the same lethal activity to mice (0.13μg/g body wt., intramuscular injection) and the same effect on isolated frog muscle as erabutoxins a and b, the main toxic components of the venom. The toxin inhibited the acetylcholine contracture but not the potassium chloride contracture of muscle. Erabutoxin c consisted of 62 amino acid residues, containing one fewer lysine and one more histidine than erabutoxin a and one fewer lysine and one more aspartic acid (or asparagine) than erabutoxin b. Erabutoxin c was reduced, S-carboxymethylated and hydrolysed with trypsin. The only fragment different from the corresponding fragments from erabutoxin b was hydrolysed further with pepsin. One of the peptic fragments, which was assumed to have the aspartic acid (or asparagine) residue in question at the C-terminal end, was treated with carboxypeptidase A. The C-terminal residue was found to be an asparagine. It was therefore concluded that erabutoxin c was [51-asparagine]-erabutoxin b.
The effect of aflatoxin B1 on normal and cortisol-stimulated rat liver ribonucleic acid synthesis
Authors: Neal G. E.
Published: August 2015

1. Aflatoxin B1, administered in vivo, inhibits the incorporation of [14C]orotic acid in vivo into rat liver nuclei, and also inhibits both Mg2+- and Mn2+-dependent RNA polymerase activities in nuclei assayed in vitro. 2. Aflatoxin B1 inhibits the cortisol-induced increase in incorporation of [14C]leucine in vivo, but does not affect the control value of this activity. 3. Aflatoxin B1 administered in vivo inhibits the increase in nuclear Mg2+-dependent RNA polymerase activity, assayed in vitro, which results from the treatment with cortisol. 4. Adrenalectomy causes a decrease in Mg2+-dependent RNA polymerase activity. The effect on this enzymic activity of adrenalectomy plus treatment with aflatoxin B1 is no greater than that of treatment with aflatoxin B1 alone. 5. These results suggest that the inhibition of cortisol-stimulated biochemical pathways by aflatoxin B1 is due to an inhibition of cortisol-stimulated RNA synthesis. 6. The cytoplasmic action of aflatoxin is thought to be due to a competition for receptor sites on the endoplasmic reticulum between steroid hormones and aflatoxin B1. No evidence was obtained for a similar competition for nuclear receptor sites between [3H]cortisol and aflatoxin B1. 7. No differences were observed between the activities of RNA polymerase preparations solubilized from control or aflatoxin-inhibited nuclei. 8. No differences in ‘melting’ profiles were observed between DNA and chromatin preparations isolated from control nuclei or from aflatoxin-inhibited nuclei. 9. It is suggested that aflatoxin B1 exerts its effect on RNA polymerase by decreasing the template capacity of the chromatin and that the aflatoxin ‘target’ area of the chromatin includes that region which is stimulated by cortisol. This process, however, does not involve inhibiting the movement of cortisol from the outside of the hepatic cell to the nuclear chromatin.
The ionogenic nature of the secretory-granule membrane. Electrokinetic properties of isolated chromaffin granules
Authors: Matthews E. K. , Evans R. J. , Dean P. M.
Published: August 2015

1. Chromaffin granules isolated from the bovine adrenal medulla possess an electrophoretic mobility of -1.12μm·s-1·cm·V-1, corresponding to a surface ζ potential of -14.4mV and surface charge density of 1.38×10-6C·cm-2. 2. The mobility of chromaffin granules is pH-dependent, indicating an amphoteric surface with an isoelectric point at pH3.0 and acidic groups with a pKa of 3.11. 3. Addition of bi- and ter-valent cations decreased the mobility of chromaffin granules in a dose-dependent fashion with a relative potency of La3+»Mn2+>Ca2+ >Sr2+>Mg2+>Ba2+. 4. Treatment with neuraminidase decreased the mobility of erythrocytes by 84%, whereas chromaffin-granule mobility was decreased by only 14%. This correlates well with the small complement of neuraminic acid present in the granule membrane. 5. The nature, origin and significance of the anionic surface charge of the chromaffin granule is discussed. It is concluded that the net negative charge at the surface of shear derives chiefly from a single type of chemical group, namely -CO2-, contributed by the α-carboxyl group of constituent proteins, the phospholipid phosphatidylserine and, to a lesser extent, the sialic acid component of glycoproteins.
Development of the activities of enzymes of the isoprenoid pathway during early stages of pea-seed germination
Authors: Green T. R. , Baisted D. J.
Published: August 2015

The activities of individual enzymes of the isoprenoid pathway from mevalonate kinase to squalene synthetase in homogenates of seeds germinated up to 32h were assayed. Changes in the activity of each enzyme were observed and compared with the activity at the 2h germination stage. Activities of alkaline phosphatase and fructose 1,6-diphosphate aldolase were similarly measured to provide a reference for changes in the general metabolic activity of seeds during imbibition of water. Water uptake reached a plateau after 12h. The reference enzymes almost doubled in activity between 2 and 8h and thereafter their activities steadily declined. All of the enzymes of the isoprenoid pathway increased in activity between 2 and 6h and, thereafter, with the exception of the prenyltransferase, their activities remained relatively constant. With the prenyltransferase activity the initial increase was followed by a short plateau between 6 and 9h and then a second increase to a maximum between 14 and 16h. After 16h the activity declined. The relative activities of the isoprenoid enzymes at 16h of germination were mevalonate kinase>phosphomevalonate kinase>pyrophosphomevalonate decarboxylase≈isopentenyl pyrophosphate isomerase>squalene synthetase>isopentenyl pyrophosphate/dimethylallyl pyrophosphate prenyltransferase. The finding that the prenyltransferase may be the rate-limiting enzyme in squalene synthesis from mevalonate is discussed in relation to regulation of isoprenoid synthesis during pea-seed germination.
Choline metabolism in the cerebral cortex of guinea pigs. Stable-bound acetylcholine
Authors: Barker L. A. , Dowdall M. J. , Whittaker V. P.
Published: August 2015

1. The turnover of synaptosomal (vesicular-cytoplasmic) and stable-bound (vesicular) acetylcholine isolated from cortical tissue was investigated after the administration, under local anaesthesia, of [N-Me-3H]choline into the lateral ventricles of guinea pigs. 2. Radioactive acetylcholine and choline present in acid extracts of subcellular fractions were separated by a combination of liquid and column ion-exchange and thin-layer chromatography. 3. The specific radioactivity and pattern of labelling of acetylcholine present in a fraction of monodisperse synaptic vesicles was found to be essentially the same as that of synaptosomal acetylcholine. 4. The specific radioactivity of stable-bound acetylcholine present in partially disrupted synaptosomes (fraction H) at short times (10–20min) after the injection of [N-Me-3H]choline was very variable and inversely related to the yield of acetylcholine in that fraction. 5. Evidence was found for the existence of two small, but highly labelled pools of acetylcholine, one which could be isolated in fraction H and the other which was lost when synaptosomes, after isolation by gradient centrifugation, were left at 0°C or pelleted. 6. It is concluded that the results are best explained by metabolic differences among the nerve-ending compartments (thought to be vesicles) which contain stable-bound acetylcholine. Computer simulation of our experiments supports this possibility and suggests that the highly labelled pool in fraction H is present in vesicles close to the external membrane.
The significance of abrupt transitions in Lineweaver–Burk plots with particular reference to glutamate dehydrogenase. Negative and positive co-operativity in catalytic rate constants
Authors: Engel Paul C. , Ferdinand William
Published: August 2015

1. Lineweaver–Burk plots for glutamate dehydrogenase, glucose 6-phosphate dehydrogenase and several other enzymes show one or more abrupt transitions between apparently linear sections. These transitions correspond to abrupt increases in the apparent Km and Vmax. with increasing concentration of the varied substrate. 2. The generalized reciprocal initial-rate equation for a multi-site enzyme requires several restrictions to be put on it in order to generate such plots. These mathematical conditions are explored. 3. It is shown that the effective omission of a term in the denominator of the reciprocal initial-rate equation represents a minimal requirement for generation of abrupt transitions. This corresponds in physical terms to negative co-operativity followed by positive co-operativity affecting the catalytic rate constant for the reaction. 4. Previous models for glutamate dehydrogenase cannot adequately account for the results. On the other hand, the model based on both negative and positive co-operativity gives a good fit to the experimental points. 5. The conclusions are discussed in relation to current knowledge of the structure and mechanism of glutamate dehydrogenase.