bluuebunny/crossref_metadata_2025 huggingface dataset:
An active-site peptide from pepsin C
Authors: Kay J. , Ryle A. P.
Published: August 2015

Porcine pepsin C is inactivated rapidly and irreversibly by diazoacetyl-dl-norleucine methyl ester in the presence of cupric ions at pH values above 4.5. The inactivation is specific in that complete inactivation accompanies the incorporation of 1mol of inhibitor residue/mol of enzyme and evidence has been obtained to suggest that the reaction occurs with an active site residue. The site of reaction is the β-carboxyl group of an aspartic acid residue in the sequence Ile-Val-Asp-Thr. This sequence is identical with the active-site sequence in pepsin and the significance of this in terms of the different activities of the two enzymes is discussed.
Location of aromatic amino acids and helix content in Escherichia coli ribonucleic acid polymerase
Authors: Nicholson B. H.
Published: August 2015

1. The perturbing effect of glycerol on the direct spectrum of Escherichia coli DNA-dependent RNA polymerase has been studied. 2. By comparison with model compounds and with the unfolded polymerase in 3.8m-urea it was possible to determine the ratio of tyrosine and tryptophan residues present. On reduction of the urea-treated enzyme with 2-mercaptoethanol, no further change in the difference spectrum occurred. 3. The amino acid composition of the enzyme is given. 4. In the intact protein approx. 30% of the tryptophan and 54% of the tyrosine residues were exposed. In conjunction with the extinction value and molecular weight this corresponded to 7 tryptophan residues and 57 tyrosine residues on the surface and 16 tryptophan residues and 48 tyrosine residues ‘buried’. 5. The optical rotatory dispersion of the enzyme was unaffected by 20% glycerol. 6. The helix content calculated from Moffit plots over 560–300nm was 13%, and from the 233nm trough 13%.
The utilization of prolyl peptides by Escherichia coli
Authors: Payne J. W.
Published: August 2015

Peptides that have an N-terminal proline residue are taken up by Escherichia coli and are degraded by intracellular peptidases. A mutant that is unable to transport oligopeptides with N-terminal α-amino acids is also unable to transport the peptides with N-terminal proline. Dipeptides and oligopeptides can prevent the uptake of the corresponding prolyl peptides and the converse competitive interactions are also observed. Although the peptide α-amino group is essential to the process of peptide transport, the results with the prolyl peptides indicate that the dipeptide and oligopeptide permeases can handle peptides with either an α-amino or α-imino group.
Metabolism of sodium oestrone [35S]sulphate in the rat
Authors: Dolly J. O. , Curtis C. G. , Dodgson K. S. , Rose F. A.
Published: August 2015

Intraperitoneal, intravenous or oral administration of sodium oestrone [35S]-sulphate to male and female Medical Research Council hooded rats is followed by the rapid excretion of the bulk of the radioactivity in urine in the form of inorganic [35S]sulphate. Pre-treatment of rats with an antibiotic regimen does not affect the results except in the case of oral administration, when relatively large amounts of the dose are recovered as ester [35S]sulphate in faeces. Intravenous administration of the labelled ester to male and female rats with cannulae in bile duct and ureter gave results similar to those obtained with free-range animals. Only small amounts of radioactivity appeared in bile and this was mainly in the form of ester sulphate, including both oestrone [35S]sulphate and oestradiol-17β 3[35S]-sulphate. Whole-body radioautography pinpointed the liver as the probable site of the desulphation of the sulphate ester and this was confirmed by liver and kidney perfusion experiments and by studies with rats in which kidney function had been eliminated by ligation of the renal pedicles.
Thiol-dependent changes in the properties of rat liver sulphotransferases
Authors: Barford D. J. , Jones J. G.
Published: August 2015

1. Two enzymes (A and B) which catalyse the sulphation of p-nitrophenol and l-tyrosine methyl ester have been isolated from female rat livers. One of these enzymes (A) also catalyses the sulphation of dehydroepiandrosterone. 2. The Km values for the sulphation of p-nitrophenol and l-tyrosine methyl ester by enzyme B at pH7.5 are 1.5μm and 2.9mm respectively. 3. Enzyme B is oxidized on keeping at 0°C when the Km and Vmax. values for the sulphation of p-nitrophenol are increased approx. 200-fold and fourfold respectively. This oxidized preparation of enzyme B fails to catalyse the sulphation of l-tyrosine methyl ester. 4. When the oxidized form of enzyme B is kept at 0°C and low ionic strength then further forms of p-nitrophenol sulphotransferase are produced having even lower affinities for the sulphate acceptor. 5. The Km value for adenosine 3′-phosphate 5′[35S]-sulphatophosphate is not affected during storage of the enzyme under these conditions. 6. Prolonged storage of enzyme B at low ionic strength leads to a considerable degree of polymerization of p-nitrophenol sulphotransferase and l-tyrosine methyl ester sulphotransferase. 7. The changes in the kinetic properties and molecular size of enzyme B during storage are reversed by dithiothreitol.
Ubiquinone biosynthesis in Escherichia coli K-12. Accumulation of an octaprenol, farnesylfarnesylgeraniol, by a multiple aromatic auxotroph
Authors: Hamilton J. A. , Cox G. B.
Published: August 2015

Cell extracts of a multiple aromatic auxotroph of Escherichia coli K-12, strain AB2830, grown in the absence of precursors of the quinone rings of the ubiquinone and menaquinone molecules, converted 4-hydroxy[U-14C]benzoate into a mixture of 3-octaprenyl-4-hydroxybenzoate and 2-octaprenylphenol. An octaprenol, farnesylfarnesylgeraniol, was isolated from such cell extracts and characterized by n.m.r. and mass spectroscopy. Neither the octaprenol, nor polyprenylation of 4-hydroxy[U-14C]benzoate, could be detected in cell extracts of strain AB2830 grown in the presence of 0.1mm-4-hydroxybenzoate. It was concluded that, in the biosynthesis of ubiquinone, the polyprenyl side chain is added to 4-hydroxybenzoate as a C40 unit, the active form of which is converted by cell extracts into farnesylfarnesylgeraniol. The multiple aromatic auxotroph, when grown in the absence of 4-hydroxybenzoate but in the presence of 4-aminobenzoate, converted the latter compound into 3-octaprenyl-4-aminobenzoate. This compound was isolated from whole cells and characterized by n.m.r. and mass spectroscopy.
Effects of glucose and other modifiers of insulin release on the oxidative metabolism of amino acids in micro-dissected pancreatic islets
Authors: Hellman B. , Sehlin J. , Täljedal I.-B.
Published: August 2015

The oxidation of alanine, arginine, leucine, glucose, and pyruvate was studied in microdissected pancreatic islets of obese–hyperglycaemic mice. The following main observations were made. The oxidation of glucose was enhanced severalfold when its concentration was raised from 3 to 20mm. At the latter concentration the rate was about 65mmol/h per kg dry wt. The oxidation of 17mm-pyruvate amounted to 20mmol/h per kg dry wt. indicating a significant entry of this compound into the β-cells. Leucine oxidation was little affected by concentration changes above 5mm, the rate at 20mm corresponding to about 25% of that obtained with 20mm-glucose. In the absence of glucose, the oxidation of alanine or arginine was barely significant. Glucose stimulated the oxidation of alanine but depressed that of leucine. These effects of glucose were blocked by mannoheptulose or iodoacetamide but were not influenced by adrenaline, diazoxide, dibutyryl 3′:5′-cyclic AMP, or glibenclamide. The rate of alanine oxidation was doubled in the presence of 17mm-pyruvate but was unaffected by citrate or succinate. Succinate depressed the oxidation of leucine. Neither alanine nor leucine significantly affected the oxidation of glucose. It is suggested that the effects of glucose on the oxidation of alanine and leucine were mediated by metabolism of the sugar, and that amino acids do not act as insulin secretagogues by serving as fuels for the β-cells. The results are consistent with the existence of mechanisms auxiliary to glucose metabolism for control of insulin release.
Characterization of a neutral protease from lysosomes of rabbit polymorphonuclear leucocytes
Authors: Davies Philip , Rita Giuseppe A. , Krakauer Kathrin , Weissmann Gerald
Published: August 2015

1. The subcellular distribution has been investigated of a protease from rabbit polymorphonuclear leucocytes, obtained from peritoneal exudates. The enzyme, optimally active between pH7.0 and 7.5, hydrolyses histone but not haemoglobin, sediments almost exclusively with a granule fraction rich in other lysosomal enzymes, and is latent until the granules are disrupted by various means. 2. Enzymic analysis of specific and azurophilic granules separated by zonal centrifugation showed that neutral protease activity was confined to fractions rich in enzymes characteristic of azurophile granules. 3. Recovery of neutral protease activity from subcellular fractions was several times greater than that found in whole cells. This finding was explained by the presence of a potent inhibitor of the enzyme activity in the cytoplasm. 4. The effect of the inhibitor was reversed by increasing ionic strength (up to 2.5m-potassium chloride) and by polyanions such as heparin and dextran sulphate, but not by an uncharged polymer, dextran. 5. The enzyme was also inhibited, to a lesser extent, by 1-chloro-4-phenyl-3-l-toluene-p-sulphonamidobutan-2-one, soya-bean trypsin inhibitor and ∈-aminohexanoate (∈-aminocaproate). 6. The granule fractions failed to hydrolyse artificial substrates for trypsin and chymotrypsin. 7. Partial separation of the enzyme was achieved by Sephadex gel filtration at high ionic strength and by isoelectric focusing. The partially separated, activated enzyme showed an approximately 300-fold increase in specific activity over that in whole cells.
Sporulation in Bacillus subtilis. The appearance of sulpholactic acid as a marker event for sporulation
Authors: Wood D. A.
Published: August 2015

1. The synthesis of sulpholactic acid in sporulating cultures of Bacillus subtilis was studied. 2. Sulpholactic acid was first detected about 4h after the initiation of sporulation and 1h before refractility. The rate of synthesis paralleled that of the other events of sporulation examined. 3. Sulpholactic acid accounted for 1.7% of the material of the spore. 4. Because the addition of chloramphenicol in the earlier stages of sporulation inhibited formation of the compound, it is likely that the enzymes concerned are synthesized de novo during sporulation. 5. In asporogenous mutants only those blocked at a late stage and showing partial refractility were able to produce sulpholactic acid. This correlation makes sulpholactic acid a useful marker event in sporulation.
Assembly of immunoglobulin M. Blocked thiol groups of intracellular 7S subunits
Authors: Askonas Brigitte A. , Parkhouse R. M. E.
Published: August 2015

We have shown previously that immunoglobulin M (IgM) is present within IgM-forming cells mainly in its 7S subunit form (IgMs), whereas only fully assembled IgM pentamers are secreted. There is no spontaneous polymerization of intracellular IgMs in cell lysates, suggesting that the 7S subunits had blocked cysteine residues. This suggestion was explored and confirmed in the present paper. Radioactive IgM (secreted) and IgMs (intracellular) were prepared by sucrose-density-gradient centrifugation after incubation of cells of the IgM-producing mouse myeloma MOPC 104E with [3H]leucine. We investigated the susceptibility to reduction of fully assembled mouse IgM and its reconstitution from subunits by analysis by polyacrylamide-gel electrophoresis under dissociating conditions. With increasing concentrations of dithioerythritol, interchain disulphide bonds were cleaved in the following order: inter-IgMs subunit, intra-IgMs subunit H-H, intra-IgMs subunit H-L. Removal of the reducing agent from IgM-reduction mixtures by filtration through Sephadex G-25 caused partial reconstitution of IgM at low protein concentrations (5–100μg/ml) and total reconstitution at higher protein concentrations (300μg/ml or more). Isolated radioactive intracellular IgMs showed no tendency to polymerize unless first treated with a reducing agent; under optimum conditions removal of the reducing agent caused 70% of the subunits to be assembled into IgM. Similar assembly occurred when IgMs was isolated from cells that had been lysed in the presence of an irreversible alkylating reagent (iodoacetamide). The intracellular IgMs cysteine residues responsible for inter-IgMs linkage therefore appear to be reversibly blocked within the cells. Assembly into IgM is thus controlled by removal of this block during secretion.
An investigation of lipogenic and glycolytic enzyme activity in the liver of sexually immature and mature domestic fowl
Authors: Pearce J.
Published: August 2015

In the non-laying pullet and the cockerel it was observed that there was no significant variation in the activities of ATP citrate lyase and `malic' enzyme whereas in the laying hen there was a significantly greater activity of both these enzymes. Parallel increases in liver lipid content in the laying hen were also observed. Three glycolytic enzymes, phosphofructokinase, fructose diphosphate aldolase and pyruvate kinase, did not exhibit any significant variation in enzyme activity with the onset of egg laying. These results are discussed in relation to the hormonal status of the birds and also the demands of egg production for lipid.
The stimulatory effects of carbon tetrachloride on peroxidative reactions in rat liver fractions in vitro. Inhibitory effects of free-radical scavengers and other agents
Authors: Slater T. F. , Sawyer B. C.
Published: August 2015

1. The effects of a number of free-radical scavengers and other agents on the stimulation of malonaldehyde production due to low concentrations of carbon tetrachloride have been studied in rat liver microsome suspensions. 2. Promethazine, propyl gallate and NN′-diphenyl-p-phenylenediamine were extremely active in inhibiting the stimulation of malonaldehyde production due to carbon tetrachloride; inhibitory effects were demonstrable with these agents at 0.1μm. 3. Low concentrations (1–100nm) of vitamin E–polyethylene glycol 1000–succinate increased the stimulation of malonaldehyde production due to carbon tetrachloride, but higher concentrations of the vitamin E preparation decreased both the stimulation due to carbon tetrachloride and the endogenous peroxidation that occurs in the absence of carbon tetrachloride. 4. Other agents tested that were effective in the range 1–20μm in decreasing the stimulation of malonaldehyde production due to carbon tetrachloride were inosine, desferrioxamine and EDTA. Agents tested that were not effective, except at very high concentrations (100μm or greater), were Nupercaine, Cetab and sodium phenobarbitone. 5. The results are discussed in terms of the mechanisms responsible for the observed inhibitions of malonaldehyde production, and of the relevance of the in vitro system to the liver damage produced by carbon tetrachloride in vivo.
The binding of calcium ions by erythrocytes and ‘ghost’-cell membranes
Authors: Long C. , Mouat Barbara
Published: August 2015

1. Washed human erythrocytes, suspended in iso-osmotic sucrose containing 2.5mm-calcium chloride, bind about 400μg-atoms of calcium/litre of packed cells. Sucrose may be replaced by other sugars. 2. Partial replacement of sucrose by iso-osmotic potassium chloride diminishes the uptake of calcium, 50% inhibition occurring at about 50mm-potassium chloride. 3. Other univalent cations behave like potassium, whereas bivalent cations are much more inhibitory. The tervalent cations, yttrium and lanthanum, however, are the most effective inhibitors of calcium uptake. 4. An approximate correlation exists between the calcium uptake and the sialic acid content of erythrocytes of various species and of human erythrocytes that have been partially depleted of sialic acid by treatment with neuraminidase. However, even after complete removal of sialic acid, human erythrocytes still bind about 140μg-atoms of calcium/litre of packed cells. 5. A Scatchard (1949) plot of calcium uptake at various Ca2+ concentrations in the suspending media shows the presence of three different binding sites on the external surface of the human erythrocyte membrane. 6. Erythrocyte `ghost' cells, the membranes of which appear to be permeable to Ca2+ ions, can bind about 1000μg-atoms of calcium per `ghost'-cell equivalent of 1 litre of packed erythrocytes. This indicates that there are also binding sites for calcium on the internal surface of the erythrocyte membrane.
Importance of the pentose phosphate pathway for d-glucose catabolism in the obligatory aerobic yeast Rhodotorula gracilis
Authors: Höfer M. , Brand K. , Deckner K. , Becker J.-U.
Published: August 2015

d-Glucose catabolism of a phosphofructokinase-deficient yeast Rhodotorula gracilis has been studied. By using d-glucose specifically 14C-labelled at different positions and measuring the distribution of the label in various fractions of cell metabolism, the following results were found. 1. The pentose phosphate pathway, being the main pathway of d-glucose catabolism, simultaneously converts glucose molecules into pentose phosphates oxidatively by using two NADP-linked dehydrogenases and via the non-oxidative transketolase–transaldolase pathway. 2. From the correlation of the 14CO2 liberation and the d-glucose consumption and from the fact that the pentose phosphate moiety in nucleic acids is almost equally labelled from d-[1-14C]- and d-[6-14C]-glucose, it is concluded that of the glucose utilized about 80% undergoes transformation via the non-oxidative pentose phosphate pathway. Only about 20% of glucose is directly decarboxylated to pentose phosphate. 3. For further degradation it is postulated that the pentose phosphates are split into C2 fragments and glyceraldehyde 3-phosphates. 4. All three loci of oxidative decarboxylation appear to be effective in Rh. gracilis, the oxidative part of the pentose phosphate pathway, the decarboxylation of pyruvate in the later part of the glycolytic pathway as well as the oxidation in the tricarboxylic acid cycle. 5. d-Glucose molecules taken up are only partially oxidized to CO2: about four-fifths of each glucose molecule metabolized is incorporated into cell constituents. 6. The quantitative interrelations of the fluxes of d-glucose subunits along the catabolic pathways have been estimated and are discussed.
Catalytic activity of α-chymotrypsin in which histidine-57 has been methylated
Authors: Henderson R.
Published: August 2015

The properties of a derivative of α-chymotrypsin in which histidine-57 has been methylated have been examined. Although the modified enzyme binds substrate with the same affinity as does native α-chymotrypsin, acylation and deacylation occur at much decreased rates. As for native α-chymotrypsin, a basic group of pKa approx. 7 is involved in both acylation and deacylation. The significance of these results is considered in relation to the normal function of histidine-57.
The binding of carbon dioxide by horse haemoglobin
Authors: Kilmartin J. V. , Rossi-Bernardi L.
Published: August 2015

1. Three modified horse haemoglobins have been prepared: (i) αc2βc2, in which both the α-amino groups of the α- and β-chains have reacted with cyanate, (ii) αc2β2, in which the α-amino groups of the α-chains have reacted with cyanate, and (iii) α2βc2, in which the two α-amino groups of the β-chain have reacted with cyanate. 2. The values of n (the Hill constant) for αc2βc2, α2βc2 and αc2β2 were (respectively) 2.5, 2.0 and 2.6, indicating the presence of co-operative interactions between the haem groups for all derivatives. 3. In the alkaline pH range (about pH8.0) all the derivatives show the same charge as normal haemoglobin whereas in the acid pH range (about pH6.0) αc2βc2 differs by four protonic charges and αc2β2, α2βc2 by two protonic charges from normal haemoglobin, indicating that the expected number of ionizing groups have been removed. 4. αc2β2 and αc2βc2 show a 25% decrease in the alkaline Bohr effect, in contrast with α2βc2, which has the same Bohr effect as normal haemoglobin. 5. The deoxy form of αc2βc2 does not bind more CO2 than the oxy form of αc2βc2, whereas αc2β2 and α2βc2 show intermediate binding. 6. The results reported confirm the hypothesis that, under physiological conditions, haemoglobin binds CO2 through the four terminal α-amino groups and that the two terminal α-amino groups of α-chains are involved in the Bohr effect.
Amino acid sequences around the cysteine residue of calf lens α-crystallin
Authors: Corran P. H. , Waley S. G.
Published: August 2015

1. Calf lens α-crystallin was carboxymethylated with radioactive sodium iodoacetate to label the thiol group. 2. The protein was then digested with trypsin or alternatively fractionated in urea to obtain the acidic (A) chains, which were then digested with trypsin. Either procedure gave two radioactive peptides containing carboxymethylcysteine. 3. These two peptides were closely related: the longer form contained 28 amino acid residues, and the shorter lacked two residues at the N-terminal end of the longer form. 4. The amino acid sequence of the peptides have been determined. 5. No evidence for the presence of more than one cysteine residue/chain was found. 6. The question of the molecular weight of the chains is discussed.
Acylation of carcinogenic hydroxamic acids by carbamoyl phosphate to form reactive esters
Authors: Lotlikar P D. , Luha L
Published: August 2015

1. Acylation of 2-(N-hydroxyacetamido)fluorene and several other aromatic hydroxamic acids by carbamoyl phosphate was studied and compared with the acylating activity of acetyl-CoA. Acetyl phosphate was also studied. 2. The carbamoylation reaction had a pH optimum of 4.5. The reaction had a lag period of 1h and was then linear for 4h. This linearity ranged between 0.5mm- and 8mm-carbamoyl phosphate concentration. 3. At pH7.5, acetyl-CoA was the most powerful acylating agent. Acetyl phosphate was a weaker acylating agent than either of the others. 4. Among the various hydroxamic acids tested with acetyl-CoA and carbamoyl phosphate at pH.7.5, 2-(N-hydroxyacetamido)fluorene was the most reactive. On the other hand the less reactive N-hydroxy derivatives of 2-acetamidonaphthalene, 2-acetamidophenanthrene and 4-acetamidostilbene reacted severalfold more with carbamoyl phosphate than with acetyl-CoA. 5. It is suggested that carbamoylation of aromatic hydroxamic acids might be one of the final activation steps in carcinogenesis by these compounds.
Kinetic specificity in papain-catalysed hydrolyses
Authors: Lowe G. , Yuthavong Y.
Published: August 2015

The specificity of the proteolytic enzyme, papain, for the peptide bond of the substrate adjacent to that about to be cleaved and for the acyl residue of some N-acylglycine derivatives is manifest almost exclusively in the formation of the acyl-enzyme from the enzyme–substrate complex. Models for the enzyme–substrate complex and acyl-enzyme intermediate are suggested that account for these observations. In particular it is suggested that the peptide bond of the substrate adjacent to that about to be cleaved, is bound in the cleft of the enzyme between the NH group of glycine-66 and the backbone C=O group of aspartic acid-158, and provides a sensitive amplification mechanism through which the specificity of the enzyme for hydrophobic amino acids such as l-phenylalanine is relayed. It is also suggested that the distortion in the enzyme–substrate complex and the binding of the peptide bond adjacent to that about to be cleaved are also linked and behave co-operatively, the distortion of the protein facilitating binding and the stronger binding facilitating distortion. The results imply that between the enzyme–substrate complex and the acyl-enzyme a relaxation of the protein conformation must occur.
pH-dependence and structure–activity relationships in the papain-catalysed hydrolysis of anilides
Authors: Lowe G. , Yuthavong Y.
Published: August 2015

The pH-dependence of the Michaelis–Menten parameters for the papain-catalysed hydrolysis of N-acetyl-l-phenylalanylglycine p-nitroanilide was determined. The equilibrium binding constant, Ks, is independent of pH between 3.7 and 9.3, whereas the acylation constant, k+2, shows bell-shaped pH-dependence with apparent pKa values of 4.2 and 8.2. The effect of substituents in the leaving group on the acylation constant of the papain-catalysed hydrolysis of hippuryl anilides and N-acetyl-l-phenylalanylglycine anilides gives rise in both series to a Hammett ρ value of -1.04. This indicates that the enzyme provides electrophilic, probably general-acid, catalysis, as well as the nucleophilic or general-base catalysis previously found. A mechanism involving a tetrahedral intermediate whose formation is general-base-catalysed and whose breakdown is general-acid-catalysed seems most likely. The similarity of the Hammett ρ values appears to exclude facilitated proton transfer as a means through which the specificity of papain is expressed.
Cytochrome P-450 and hydroxylating activity of microsomal preparations from whole houseflies
Authors: Morello A. , Bleecker W. , Agosin M.
Published: August 2015

1. A new microsomal preparation, obtained from whole houseflies is described in terms of its cytochrome P-450 content and its hydroxylating activity. 2. Microsomes prepared from whole-fly brei, obtained with the aid of a mortar (a procedure that avoids the destruction of sarcosomes), contain 0.265nmol of cytochrome P-450 and hydroxylate naphthalene at a rate of 28.5nmol/mg of microsomal protein in 30min at 30°C. This corresponds to 104nmol of naphthalene hydroxylated/nmol of cytochrome P-450. This is the highest rate ever reported for housefly and rat liver microsomal preparations. 3. Microsomal fractions prepared by procedures that do not retain the integrity of sarcosomes show the presence in the CO-difference spectrum of a 428nm peak. This cytochrome is associated with sarcosomal microsomes and it may be involved in the inhibition of insect microsomal mixed-function oxidases, although other factors cannot be discarded at present. 4. The inability to show cytochrome P-450 in microsomal fractions isolated from whole houseflies by other procedures may be at least partially due to a masking effect brought about by contamination with the sarcosomal cytochrome.
Studies on the interactions between purified bovine caseins and alkaline-earth-metal ions
Authors: Dickson I. R. , Perkins D. J.
Published: August 2015

1. Alkaline-earth-metal cations at low concentrations form soluble complexes with bovine caseins. The relative order of binding capacities is: Mg2+>Ca2+>Ba2+>Sr2+. 2. The cations interact with both free ionized carboxyl groups of aspartic acid and glutamic acid and with monoester phosphate groups covalently bound to serine and threonine; at low concentrations of the cations interactions are predominantly with the phosphate groups. 3. The order of binding capacities for purified components of the casein complex is: αs1-casein>β-casein>κ-casein.
Changes in histone phosphorylation and associated early metabolic events in pig lymphocyte cultures transformed by phytohaemagglutinin or 6-N,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate
Authors: Cross M E. , Ord M G.
Published: August 2015

1. Pig lymphocytes were transformed by dibutyryl cyclic AMP (6-N,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate) at concentrations of 0.01–0.1μm. The pattern of incorporation of label from [5-3H]uridine and [6-3H]thymidine into RNA and DNA respectively was identical with that obtained with unpurified phytohaemagglutinin. 2. Chlorpromazine (0.1μm) prevented the stimulation of [5-3H]uridine incorporation into RNA by phytohaemagglutinin, but only slightly lowered the lymphocyte response to dibutyryl cyclic AMP. 3. An increase in the size and specific radioactivity of the intracellular Pi pool was found immediately after stimulation by both phytohaemagglutinin and dibutyryl cyclic AMP. This was followed after some 30min by a rise in the specific radioactivity and concentration of ATP. 4. There was an immediate increase in the specific radioactivity of phosphate groups of histones; by about 45min after stimulation only the histones remaining after extraction of histone fraction F1 continued to incorporate 32P from [32P]Pi. 5. Histone kinase activity increased in the first 30min after stimulation; subsequently histone F1 kinase activity decreased, but activity with the other histones as substrate continued to increase for a further 30min. Kinase activation was effected by cyclic AMP (adenosine 3′:5′-cyclic monophosphate). 6. Histone phosphatase activity behaved similarly to that of the kinase.
l-Tryptophan. A non-allosteric organspecific uncompetitive inhibitor of human placental alkaline phosphatase
Authors: Lin C W , Sie H G , Fishman W H.
Published: August 2015

l-Tryptophan, but not d-tryptophan, inhibits human placental and intestinal alkaline phosphatases, but not those of liver and bone. The nature of this stereospecific organ-specific inhibition has been elucidated. Thus, from a study of the effect of substrate concentration on inhibition in which double-reciprocal plots of 1/v versus 1/s at various inhibitor concentrations were made, this inhibition is judged to be ‘uncompetitive’. That the inhibition is non-allosteric is an opinion based on (1) hyperbolic curves obtained from plotting the percentage inhibition against inhibitor concentration; (2) the independence of the inhibition to heat denaturation and urea treatment; (3) the relatively low value of entropy change; and (4) a value close to unity for n, the number of l-tryptophan molecules that combine with one molecule of enzyme. Finally, a homosteric mechanism is further postulated for the inhibition by l-tryptophan based on the increase of optimum temperature for maximum velocity and the decrease of this inhibition with increasing temperature. The mechanism of this inhibition is discussed.
Molecular-sieve chromatography and electrophoresis in polyacrylamide gels
Authors: Morris C. J. O. R. , Morris Peggy
Published: August 2015

1. The absolute electrophoretic mobilities of eight proteins have been measured at pH8.76, I 0.05, in polyacrylamide gels of 20 different compositions at 10°C. 2. The partition coefficients of these proteins have been determined chromatographically under the same conditions by using columns of granulated polyacrylamide gel prepared simultaneously. 3. The electrophoretic mobilities are an exponential function of the gel concentrations when the latter are corrected for water uptake. The constants of this function have been determined by curvefitting methods. They have been shown to be related to the free solution mobility and to the mean molecular radius respectively. 4. The reduced mobilities have been shown to be a linear function of the partition coefficients by statistical analyses. 5. The physical significance of the relation between electrophoretic mobility and chromatographic phase distribution in gel media is discussed in the context of these results.
Studies on the carbohydrate moiety of α1-acid glycoprotein (orosomucoid) by using alkaline hydrolysis and deamination by nitrous acid
Authors: Isemura M. , Schmid K.
Published: August 2015

Alkaline hydrolysis followed by deamination with nitrous acid was applied for the first time to a glycoprotein, human plasma α1-acid glycoprotein (orosomucoid). This procedure, which specifically cleaves the glycosaminidic bonds, yielded well-defined oligosaccharides. The trisaccharides, which were obtained from the native protein, consisted of a sialic acid derivative, galactose and 2,5-anhydromannose. The linkage between galactose and 2,5-anhydromannose is most probably a (1→4)-glycosidic bond. A hitherto unknown linkage between N-acetylneuraminic acid and galactose was also established, namely a (2→2)-linkage. The three linkages between sialic acid and galactose described in this paper appear to be about equally resistant to mild acid hydrolysis. The disaccharide that was derived from the desialized glycoprotein consisted of galactose and 2,5-anhydromannose. Evidence was obtained for the presence of a new terminal sialyl→N-acetylglucosamine disaccharide accounting for approximately 1mol/mol of protein. The presence of this disaccharide may explain the relatively severe requirements for the complete acid hydrolysis of the sialyl residues. The present study indicates that alkaline hydrolysis followed by nitrous acid deamination in conjunction with gas–liquid chromatography will afford relatively rapid determination of the partial structure of the complex carbohydrate moiety of glycoproteins.
The cell-free synthesis of cytochrome c by a microsomal fraction from rat liver
Authors: González-Cadavid Néstor F. , Ortega Juan P. , González Magally
Published: August 2015

Conditions were investigated for demonstrating the synthesis in vitro of the complete molecule of cytochrome c by isolated liver microsomal systems from partially hepatectomized rats. It was first found that in vivo the early labelled cytochrome c associated with the microsomal fraction required, by comparison with the mitochondrial pool, more drastic conditions of extraction and its binding was less affected by freezing and thawing of the subcellular particles. The procedure of extraction and purification of cytochrome c had to be modified accordingly, to assure the recovery of the recently synthesized molecule. Several subcellular fractions were isolated from regenerating liver with a homogenization medium containing either 5 or 10mm-Mg2+ and most of them were active in the synthesis of the cytochrome c apoprotein. The microsomal fraction, in the presence of either cell sap or pH5.0 fraction, was also able to incorporate [59Fe]haemin, δ-amino[3H]laevulic acid and 55Fe into the prosthetic group of cytochrome c. These experiments confirm firmly the conclusions of our previous results obtained in vivo showing that both the apoprotein and the haem moieties are made and linked together on cytoplasmic ribosomes and only then is the complete molecule transferred to the mitochondria.
The amino acid sequence of cytochrome c from Cucurbita maxima L. (pumpkin)
Authors: Thompson E. W. , Richardson M. , Boulter D.
Published: August 2015

The amino acid sequence of pumpkin cytochrome c was determined on 2μmol of protein. Some evidence was found for the occurrence of two forms of cytochrome c, whose sequences differed in three positions. Pumpkin cytochrome c consists of 111 residues and is homologous with mitochondrial cytochromes c from other plants. Experimental details are given in a supplementary paper that has been deposited as Supplementary Publication SUP 50005 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1971), 121, 7.
Metabolism of acetoin in mammalian liver slices and extracts. Interconversion with butane-2,3-diol and biacetyl
Authors: Gabriel M. A. , Jabara Haifa , Al-Khalidi U. A. S.
Published: August 2015

1. [14C]Acetoin was enzymically synthesized from [14C]pyruvate with a pyruvate decarboxylase preparation. Its optical activity was [α]20d-78°. 2. Large amounts (1000-fold higher than physiological concentrations) of acetoin were incubated with rat liver mince. Acetoin disappeared but very little 14CO2 was evolved. A compound accumulated, which was purified and identified as butane-2,3-diol. Chromatography on borate-impregnated paper indicated the presence of both the erythro and threo forms. 3. Liver extracts capable of interconverting biacetyl, acetoin and butane-2,3-diol were obtained. These interconversions were catalysed by two different enzymes: acetoin dehydrogenase (EC 1.1.1.5) and butane-2,3-diol dehydrogenase (EC 1.1.1.4), previously identified in bacteria. Both required NAD+ or NADP+ as cofactors and were different from alcohol dehydrogenase. The equilibrium in both cases favoured the more reduced compound. 4. The activity of butane-2,3-diol dehydrogenase was decreased by dialysis against EDTA: the addition of Co2+, Cu2+, Zn2+ and other bivalent metal ions restored activity. 5. Biacetyl reductase was resolved into multiple forms by CM-Sephadex chromatography and electrophoresis.
Insolubilized enzymes. Kinetic behaviour of glucose oxidase bound to porous glass particles
Authors: Weibel Michael K. , Bright Harold J.
Published: August 2015

1. The spectrophotometric and steady-state kinetic properties of glucose oxidase (EC 1.1.3.4, from Aspergillus niger) that is covalently linked to porous glass beads have been examined. These properties have been compared with those of soluble glucose oxidase, for which the kinetic mechanism at pH5.5 and 25°C has been established previously by a combination of conventional and rapid-reaction techniques to be the following: [Formula: see text] where Eo and Er represent oxidized and reduced forms of the enzyme, respectively. 2. The ratio k+4/k+2 is unchanged after insolubilization, and evidence is presented which suggests that the absolute magnitudes of k+4 and k+2 are unchanged. 3. The kinetic efficiency of the insolubilized enzyme is greatly enhanced because of a 14-fold increase in the apparent affinity of glucose for Eo. This effect is attributed either to the binding of glucose to the glass surface or to a change in enzyme structure imposed by the insolubilization process. 4. Only 6% of the insolubilized enzyme which can be reduced by glucose is catalytically active. It is shown by calculation and direct experimental evidence that this fraction of catalytically active enzyme is bound to the exterior bead surface. The remaining 94% of the enzyme is bound within the pore network and may be subject to severe substrate diffusion control.
Dissociation of ribosomes from oocytes of Xenopus laevis into active subparticles
Authors: Pratt H , Cox R A
Published: August 2015

Ribosomes from oocytes of Xenopus laevis possess low endogenous activity in vivo and in vitro, yet are readily stimulated by poly(U). The ease with which these ribosomes dissociate into active subparticles under conditions where polyribosomes and active monoribosomes are stable supports the view that the majority are unprogrammed.
The preparation of an alkali-soluble collagen from demineralized bone
Authors: Kemp G. D. , Tristram G. R.
Published: August 2015

Ossein was solubilized by the action of alkali and a resulting high-molecular-weight fraction isolated. The chemical and physical properties of this fraction were studied and compared with those of an acid-soluble collagen prepared from calf skin by conventional techniques. From the results it is concluded that the alkali-soluble protein exhibits only minor differences from acid-soluble collagen, and that these differences can be ascribed for the most part to a decrease in the inter- and intramolecular cross-linking.
Comparison of the N-acetylhexosaminidase components in the ram testis and epididymis by isoelectric focusing
Authors: Winchester B. G.
Published: August 2015

1. Several enzymic components, with both N-acetyl-β-glucosaminidase and N-acetyl-β-galactosaminidase activity, have been demonstrated in the ram testis and epididymis by isoelectric focusing. 2. The component (I), which predominates in the testis and caput of the epididymis, is isoelectric at pH6.2±0.1, whereas the predominant component (II) in the epididymal isthmus and cauda is isoelectric at pH7.0±0.1. 3. The total activity and the relative proportions of the enzymic components vary in the different sections of the epididymis. 4. Although their pH optima are slightly different, the appropriate Km values for components I and II for the hydrolysis of 4-methylumbelliferyl-N-acetyl-β-glucosaminide and N-acetyl-β-galactosaminide and the ratios of the maximal velocities towards the two substrates are very similar.
The heterogeneity of uridine diphosphate glucuronyltransferase from rat liver
Authors: Mulder G. J.
Published: August 2015

1. The glucuronide conjugation of p-nitrophenol, phenolphthalein, o-aminophenol and 4-methylumbelliferone by rat liver microsomes has been studied. The detergent Triton X-100 activated UDP-glucuronyltransferase activity towards all these substrates, therefore the optimum activating concentration was added in all experiments. 2. Mg2+ enhanced the conjugation of the substrates. 3. With phenolphthalein substrate inhibition occurred but this could be relieved by adding albumin, which binds excess of phenolphthalein. 4. Kinetic constants of the substrates and UDP-glucuronate have been determined. Mutual inhibition was found with the substrates p-nitrophenol, 4-methylumbelliferone and phenolphthalein. p-Nitrophenol conjugation was inhibited competitively by phenolphthalein and 4-methylumbelliferone. 5. o-Aminophenol did not inhibit the conjugation of the other three substrates because these are conjugated preferentially to o-aminophenol. 6. It is concluded that the four substrates are conjugated by one enzyme at the same active site.
Comparative biochemistry of chondroitin sulphate–proteins of cartilage and notochord
Authors: Mathews Martin B.
Published: August 2015

Fragments that consisted mainly of two polysaccharide chains joined by a short polypeptide bridge (doublets) were prepared from chondroitin sulphate–proteins of lamprey, sturgeon, elasmobranch and ox connective tissues after hydrolysis with trypsin and chymotrypsin. Consideration of molecular parameters, compositions and behaviour on gel electrophoresis and density-gradient fractionation leads to a proposed parent structure for chondroitin sulphate–proteins. A single polypeptide chain of about 2000 amino acid residues contains alternating short and long repeating sequences. A short sequence consists of less than 10 amino acid residues with one N-terminal and one C-terminal serine residue, each of which carries a polysaccharide chain linked glycosidically to its hydroxyl group. This structure constitutes the doublet subunit. Some variation is introduced when the doublet subunit carries only a single polysaccharide chain. The long sequence contains about 35 amino acid residues and is subject to cleavage by trypsin and chymotrypsin. The main polypeptide is probably homologous in the vertebrate sub-phylum with strong conservation of structure suggested for the short sequence. However, polymorphism of polypeptide structures cannot be excluded.
Multiple forms of mammalian deoxyribonucleic acid polymerase. An attempt to relate their interactions with nuclei and free deoxyribonucleic acid in vitro with their possible functions in vivo
Authors: Wallace Patricia G. , Hewish D. R. , Venning M. M. , Burgoyne L. A.
Published: August 2015

The DNA polymerases of the following eukaryotic tissues were studied: regenerating rat liver, normal rat liver, rat thymus, normal mouse liver and Ehrlich ascites-tumour cells. In all cases two main polymerase forms are observed, one of mol.wt. 200000, preferring denatured DNA to native calf thymus DNA primer, designated type I, and the other, designated type II, of mol.wt. 100000, showing a variable and slight preference for native calf thymus DNA primer. Some catalytic properties of these polymerases are described. Nuclei have been isolated from some of these tissues by using two different buffer systems. The ionic composition of the isolation medium is found to affect greatly the amounts and types of polymerase that bind to the nuclei, and also affects the kinetic properties of the polymerases. The way the polymerases and nuclei change properties as the ionic composition of the buffers is changed suggests that ionic effects may be a significant factor in the control of DNA synthesis in vivo. These ionic effects also explain much of the previous confusion over the localization of specific DNA polymerases.
Poly-β-hydroxybutyrate biosynthesis and the regulation of glucose metabolism in Azotobacter beijerinckii
Authors: Senior P. J. , Dawes E. A.
Published: August 2015

Azotobacter beijerinckii possesses the enzymes of both the Entner–Doudoroff and the oxidative pentose phosphate cycle pathways of glucose catabolism and both pathways are subject to feedback inhibition by products of glucose oxidation. The allosteric glucose 6-phosphate dehydrogenase utilizes both NADP+ and NAD+ as electron acceptors and is inhibited by ATP, ADP, NADH and NADPH. 6-Phosphogluconate dehydrogenase (NADP-specific) is unaffected by adenosine nucleotides but is strongly inhibited by NADH and NADPH. The formation of pyruvate and glyceraldehyde 3-phosphate from 6-phosphogluconate by the action of the Entner–Doudoroff enzymes is inhibited by ATP, citrate, isocitrate and cis-aconitate. Glyceraldehyde 3-phosphate dehydrogenase is unaffected by adenosine and nicotinamide nucleotides but the enzyme is non-specific with respect to NADP and NAD. Citrate synthase is strongly inhibited by NADH and the inhibition is reversed by the addition of AMP. Isocitrate dehydrogenase, a highly active NADP-specific enzyme, is inhibited by NADPH, NADH, ATP and by high concentrations of NADP+. These findings are discussed in relation to the massive synthesis of poly-β-hydroxybutyrate that occurs under certain nutritional conditions. We propose that synthesis of this reserve material, to the extent of 70% of the dry weight of the organism, serves as an electron and carbon ‘sink’ when conditions prevail that would otherwise inhibit nitrogen fixation and growth.
The interaction of insulin with phospholipids
Authors: Perry M. C. , Tampion W. , Lucy J. A.
Published: August 2015

1. A simple two-phase chloroform–aqueous buffer system was used to investigate the interaction of insulin with phospholipids and other amphipathic substances. 2. The distribution of 125I-labelled insulin in this system was determined after incubation at 37°C. Phosphatidic acid, dicetylphosphoric acid and, to a lesser extent, phosphatidylcholine and cetyltrimethylammonium bromide solubilized 125I-labelled insulin in the chloroform phase, indicating the formation of chloroform-soluble insulin–phospholipid or insulin–amphipath complexes. Phosphatidylethanolamine, sphingomyelin, cholesterol, stearylamine and Triton X-100 were without effect. 3. Formation of insulin–phospholipid complex was confirmed by paper chromatography. 4. The two-phase system was adapted to act as a simple functional system with which to investigate possible effects of insulin on the structural and functional properties of phospholipid micelles in chloroform, by using the distribution of [14C]glucose between the two phases as a monitor of phospholipid–insulin interactions. The ability of phospholipids to solubilize [14C]glucose in chloroform increased in the order phosphatidylcholine<sphingomyelin<phosphatidylethanolamine<phosphatidic acid. Insulin decreased the [14C]glucose solubilized by phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid, but not by sphingomyelin. 5. The significance of these results and the molecular requirements for the formation of insulin–phospholipid complexes in chloroform are discussed.
Formation of N′-formylkynurenine in proteins from lens and other sources by exposure to sunlight
Authors: Pirie Antoinette
Published: August 2015

The photo-oxidative effect of sunlight on the tryptophan residues of proteins and on free tryptophan is described. Evidence is presented that the indole ring is split to yield N′-formylkynurenine. The possible relation of this photo-oxidative change to changes in the lens proteins of brown cataracts is discussed.