bluuebunny/crossref_metadata_2025 huggingface dataset:
Intramolecular ionic interactions of lysine residues and a possible folding domain in fructose diphosphate aldolase
Authors: Lambert J M , Perham R N , Coggins J R
Published: August 2015

1. Treatment with methyl acetimidate was used to probe the topography of the tetrameric fructose 1,6-diphosphate aldolase from ox liver. A single treatment with imido ester in the presence or absence of 20mM-fructose 1,6-diphosphate caused the number of amino groups in the enzyme to fall to approx. 30% of the starting number (assumed to be 30 per subunit). The catalytic activity of the aldolase modified in the presence of fructose 1,6-diphosphate was unaffected, whereas that of the enzyme modified in the absence of substrate fell by about 20%. 2. Use of methyl [1-14C]acetimidate and small-scale methods of protein chemistry showed that the amino group of lysine-27 (the numbering is that of the highly homologous rabbit muscle enzyme) is essentially unavailable for amidination in the native enzyme and is therefore predicted to be buried in a hydrophobic environment, probably in the form of an ion-pair with a negatively charged side-chain carboxyl group. All the other lysine residues that reacted poorly with methyl acetimidate in the native enzyme (a total of 7) were found to be within the primary structure bounded by lysine-107 and lysine-227. An important member of this group of lysine residues displaying aberrant reactivity is lysine-227, which is known to form an imine with the substrate as part of the catalytic mechanism of the enzyme. 3. The results of the amidination experiments can be correlated in an interesting way with previous studies of thiol-group modification in the aldolases. Taken together, and arguing in part by analogy with the results of identical experiments with glyceraldehyde 3-phosphate dehydrogenases where the three-dimensional structure is known [Lambert & Perham (1977) Biochem. 4. 161. 49-62], they suggest that the region of primary structure from residues 107-227 may form the whole or part of a three-dimensional structural feature, perhaps a folding domain. A three-dimensional structure deduced from X-ray-crystallographic analysis will be needed to interpret these findings more closely. 4. The amino groups of lysine residues are commonly thought to reside at the ‘surface’ of protein structures. The patterns of specific lysine residues in glyceraldehyde 3-phosphate dehydrogenases and in aldolases that have been found to react poorly with methyl acetimidate in the native enzymes can be attributed to intramolecular ionic interactions deep in hydrophobic pockets and at the protein ‘surface’. Such ionic interactions may contribute significantly to the stability of a given protein.
Ionic-strength-dependent changes in the structure of the major protein of the human erythrocyte membrane
Authors: Jenkins R E , Tanner M J A
Published: August 2015

The effect of ionic strength on the proteolysis by trypsin of the major membrane-penetrating protein (polypeptide 3) in the erythrocyte membrane was studied. Both the intracellular and extracellular regions of the protein are susceptible to trypsin proteolysis under hypo-osmotic conditions, whereas under iso-osmotic conditions the extracellular region of the protein is resistant to trypsin, and the intracellular region yields only two cleavage products with trypsin. Studies of the fragments obtained from polypeptide 3 by trypsin digestion under iso-osmotic conditions of ‘ghosts’ radioiodinated with lactoperoxidase confirmed our earlier conclusions that the polypeptide chain of polypeptide 3 traverses the membrane twice. Ionic-strength-dependent changes were also observed in the incorporation of iodine by lactoperoxidase into the individual extracellular tyrosine sites of the protein. These results show that polypeptide 3 undergoes ionic-strength-dependent changes in structure.
Mechanism of anticholinesterase activities of cardiotoxin, protamine and polylysine
Authors: Lin S Y S , Liao C , Lee C Y
Published: August 2015

Cardiotoxin, protamine and polylysine are potent inhibitors of various cholinesterases. CaCl2 and MgCl2 overcome the inhibition. The order of addition of the inhibitor and the protecting agent (MgCl2) influences the final degree of the inhibition observed. These findings suggest that cardiotoxin, protamine and polylysine inhibit cholinesterases by the ionic binding of their basic groups with the anionic sites of cholinesterase molecules.
Inhibition of the reconstitution of the haemolytic activity of the first component of human complement by a pepsin-derived fragment of subcomponent C1q
Authors: Reid K B M , Sim R B , Faiers A P
Published: August 2015

1. A fragment of subcomponent C1q, which contained all the collagen-like features present in the intact molecule, was isolated by pepsin digestion as described by Reid [Biochem. J. (1976) 155, 5-17]. 2. The pepsin-derived fragment of subcomponent C1q did not bind to antibody-coated erythrocytes under conditions where complete binding of sub-component C1q took place. 3. The peptic fragment blocked the reconstitution of C1 haemolytic activity by competing with intact subcomponent C1q in the utilization of a mixture of the other two subcomponents, C1r and C1s. 4. Reduction and alkylation of the interchain disulphide bonds in the pepsin fragment did not markedly affect its inhibitory effect, whereas heating at 56 degrees C for 30min completely abolished the effect. 5. Lathyritic rat skin collagen and CNBr-derived peptides of pig type II collagen showed no ability to mimic the inhibitory effect of the pepsin fragment when tested over the same concentration range as used for the peptic fragment. 6. The peptic fragment was unable to block efficiently the reconstitution of C1 haemolytic activity unless it was added to the mixture of subcomponents C1r and C1s before the attempt to reconstitute C1 haemolytic activity, in solution, or on the surface of antibody-coated erythrocytes. 7. Evidence was obtained that suggested that subcomponent C1q bound the subcomponent C1r-C1s complex more efficiently when the subcomponent C1q was bound to antibody than when it was free in solution.
Purification of glycolytic enzymes by using affinity-elution chromatography
Authors: Scopes R K
Published: August 2015

1. A systematic procedure for the purification of enzymes by affinity-elution chromatography is described. Enzymes are adsorbed on a cation-exchanger, and eluted with ligands specific for the enzyme concerned. 2. All of the glycolytic and some related enzymes present in rabbit muscle can be purified by the affinity-elution technique. The pH range for adsorption and elution of each enzyme was found, and the effects of minor variations of conditions are described. 3. A description of experimental conditions suitable for affinity elution of each enzyme is given, together with special features relevant to each individual enzyme. 4. Theoretical considerations of affinity elution chromatography are discussed, including its limitations, advantages and disadvantages compared with affinity-adsorption chromatography. Possible developments are suggested to cover enzymes which because of their adsorption characteristics are not at present amenable to affinity-elution procedures.
Properties of the multiple forms of the soluble 17α-hydroxy steroid dehydrogenase of rabbit liver
Authors: Hasnain S , Williamson D G
Published: August 2015

The six forms of the 17alpha-hydroxy steroid dehydrogenase purified from rabbit liver cytosol have very similar physical properties. The molecular weights of all the enzymes were within 3% of the average mol.wt of 39 600. Only one of the six enzymes showed a significant difference in amino acid composition. All but one form of the 17alpha-hydroxy steroid dehydrogenases exhibited greater activities towards the androgen, epitestosterone, than towards oestrogen substrates. With oestrogen substrates one enzyme displayed a high specificity towards the substrate oestradiol-17alpha 3-glucuronide. This high activity was lost if the glucuronic acid moiety was removed or replaced by glucose or galacturonic acid. The other enzyme forms had approximately equal activity toward oestradiol-17alpha and its glucuronide or glucoside derivative. However, substitution of galacturonic acid at C-3 of oestradiol-17alpha substantially decreased the activity of all but one enzyme form.
Isolation and characterization of sheep pepsin
Authors: Fox P F , Whitaker J R
Published: August 2015

Sheep pepsin was isolated (approx. 120-fold purification) from aqueous abomasal homogenates by (1) pH fractionation, (2) chromatography on Sepharose 4B-poly-L-lysine columns and (3) gel filtration on Sephadex G-100. The enzyme had mol.wt. approx. 34000, N-terminal valine and C-terminal alanine. The amino acid composition of sheep pepsin was generally similar to that of pig and ox pepsins, with a very low content of basic residues and a high content of acidic and hydroxy-amino acids. The pH optimum for NN-dimethyl-casein and NN-dimethyl-haemoglobin as substrates was approx. 1.8. The Km and kcat. for NN-dimethyl-haemoglobin were 46micronM and 1100min-1 respectively, and for NN-dimethyl-casein the corresponding parameters were 50micronM and 420min-1. These values were generally similar to those for pig and ox pepsins. At the pH optimum of 4.6, the sheep pepsin was about 50% as active on benzyloxycarbonyl-L-histidyl-L-phenyl-alanyl-L-tryptophan ethyl ester as was pig pepsin. The pH optimum for the hydrolysis of N-acetyl-L-phenylalanyl-L-di-iodotyrosine by sheep, ox and pig pepsins was approx. 1.85.
The disulphide-bonded nature of procollagen and the role of the extension peptides in the assembly of the molecule
Authors: Harwood R , Merry A H , Woolley D E , Grant M E , Jackson D S
Published: August 2015

1. The molecular weights of chick tendon and cartilage procollagens, and their constituent polypeptides, were determined by gel filtration and gel electrophoresis. The values obtained are in good agreement and indicate that the mol.wts. of the secreted procollagens (types I and II) and their individual pro-alpha-chains are of the order of 405 000-445 000 and 137 000-145 000 respectively.2. Digestion of tendon procollagen with human rheumatoid synovial collagenase gave products consistent with the presence of large non-helical peptide extensions at both N-and C-termini. Electrophoretic analysis gave apparent mol.wts. of 17 500 and 36 000 for the respective N- and C-terminal extensions of pro-alpha1(I)-and pro-alpha2-chains, and inter-chain disulphide bonds were restricted to the C-terminal location. 3. During the biosynthesis of procollagen by tendon and cartilage cells a close correlation was observed between the extent of inter-chain disulphide bonding and the proportion of procollagen polypeptides having a triple-helical conformation. These processes appeared to commence in the rough endoplasmic reticulum and be completed in the smooth endoplasmic reticulum, but the rate at which they occur in cartilage cells is markedly slower than that found in tendon cells. 4. When the intracellular [14C]procollagen polypeptides present in the rough-endoplasmic-reticulum fractions of tendon and cartilage cells were analysed under non-reducing conditions on agarose/polyacrylamide composite gels, no significant pools of dimeric intermediates were detected. 5. In both cell types, inter-chain disulphide-bond formation occurred even when hydroxylation, and hence triple-helix formation, was inhibited. The presence of pro-alpha1- and pro-alpha2-components in a ratio of 2:1 in the disulphide-linked unhydroxylated procollagen isolated from tendon cells demonstrated that correct chain association occurs in the absence of hydroxylation. This observation is consistent with a model for the assembly of pro-gamma112-chains in which the recognition and selection of pro-alpha1-and pro-alpha2-chains in a 2:1 ratio are directed by the non-helical C-terminal extension peptides of tendon procollagen.
Molecular conformation of sodium heparan sulphate in the condensed phase
Authors: Elloway H F , Atkins E D T
Published: August 2015

By using the X-ray-diffraction results reported previously for sodium heparan sulphate, a twofold helical conformation with an axially projected disaccharide repeat (h) equal to 0.93 nm has been examined in detail. On the basis of a repeating sequence of 1,4-alpha-D-glucosamine and 1,4-beta-D-glucuronic acid, trial and stereochemically feasible molecular models were computer-generated. An optimum twofold helical conformation is proposed, incorporating stabilizing intra-chain hydrogen bonds across both glycosidic linkages.
Human collagen ‘fingerprints’ produced by clostridopeptidase A digestion and high-pressure liquid chromatography
Authors: van Der Rest M , Cole W G , Glorieux F H
Published: August 2015

Samples (1-2mg) of purified human type I, II and III collagens and alpha1(I) and alpha2 chains were digested with clostridiopeptidase A and the released peptides analysed by ion-exchange high-pressure liquid chromatography. Specific ‘fingerprints’ were produced for each type of collagen. The reproducible nature of these ‘fingerprints’ and the reconstitution of the type I ‘fingerprint’ from the ‘fingerprints’ of the component alpha1(I) and alpha2 chains showed that the specificity of these ‘fingerprints’ was related to the primary structure of each type of collagen. In addition, some of the differences observed between the ‘fingerprints’ of the alpha1(I) and alpha2 chains of type I collagen were shown to be suitable for the quantitative analysis of these chains.
A latent form of collagenase in the involuting rat uterus and its activation by a serine proteinase
Authors: Woessner J F
Published: August 2015

1. The involuting rat uterus displays an extremely rapid breakdown of collagen. Collagenase activity can be assayed directly in the insoluble 6000g pellet of uterine homogenates. At 1 day post partum, about 85% of this collagenase activity is in a latent form. 2. This latent form can be activated by trypsin or by a serine proteinase present in the uterine pellets. 3. The activating enzyme of the tissue is inhibited by a wide spectrum of trypsin inhibitors, including Trasylol, soya-bean and lima-bean trypsin inhibitors, snail inhibitor and di-isopropyl phosphoro-fluoridate. Partial inhibition is produced by benzamidine, phenylmethanesulphonyl fluoride, epsilon-aminohexanoate, leupeptin, antipain and alpha1-antitrypsin. Ovomucoid, 7-amino-1-chloro-3-tosylamido-1-heptan-2-one and 1-chloro-4-phenyl-3-(N-benzyloxy-carbonyl)amino-L-butan-2-one are not inhibitory. 4. Extraction of uterine pellets with 0.1 M-CaCl2 at 60 degrees C releases both latent and active collagenase. Exclusion chromatography on Sephadex G-100 gives an apparent molecular weight of approx. 77000 for the latent form and 66000 for the active form. The latent form is suggested to be a zymogen of collagenase.
Studies on the mechanism and kinetics of the 2-oxoglutarate dehydrogenase system from pig heart
Authors: McMinn C L , Ottaway J H
Published: August 2015

1. The kinetic properties of the 2-oxoglutarate dehydrogenase system were investigated. To this end, initial-velocity studies were carried out by the method of Fromm [(1967) Biochim. Biophys. Acta 139, 221-230]. Reciprocal plots of the results did not agree with those expected for the Hexa Uni Ping Pong mechanism previously proposed for the system. 2. The measured initial velocities were fitted to initial-rate equations corresponding to several possible mechanisms by using a computer optimization technique. Statistical analyses performed on the results of the optimization studies indicated that one mechanism was a significantly better fit to the experimental data than the other mechanisms tested. This mechanism is one in which there is a random order of binding of NAD+ and CoA and release of succinyl-CoA, although the binding of 2-oxoglutarate and release of CO2 is still given a Ping Pong mechanism, which precedes the binding of the other substrates. These conclusions were supported by NADH-inhibition studies. 3. The usefulness of the method of fitting initial-rate data to rate equations and the applicability of the proposed enzymic mechanism to the enzyme complex are discussed.
Adenosine diphosphate ribosylated histones
Authors: Ord M G , Stocken L A
Published: August 2015

When rat liver nuclei were incubated with [adenine-3H]NAD, besides histone 1, histone 2A and especially histone 2B accepted 3H radioactivity. 3H radioactivity was also found on the non-histone proteins and on the small amounts of histones 1 and 3 released into the supernatant during incubation. [14C]Adenine uptake in vivo by liver and thymus nuclei showed radioactivity in histones 1 and 3. After digestion with Pronase and leucine aminopeptidase 14C- or 32P-labelled histone 3 released a serine phosphate-containing nucleotide, which on acid hydrolysis yielded ADP-ribose and serine phosphate. Serine phosphate was also found in the material from the nucleotide peaks from histones 2A and 2B. ADP-ribosylated histones 1 and 3 were more easily released from nuclei than their unmodified forms and showed higher [32P]Pi and [3H]lysine uptakes in vivo [Ord & Stocken (1975) FEBS Meet. Proc. 34, 113-125].
Self-association of α-chymotrypsin at low ionic strength in the vicinity of its pH optimum
Authors: Tellam R , Winzor D J
Published: August 2015

The self-association of alpha-chymotrypsin and its di-isopropyl phosphoryl derivative in in I0.03 sodium phophate buffer, pH7,9, was investigated by velocity sedimentation, equilibrium sedimentation and difference gel chromatography. No differences between the native and chemically modified enzyme were observed in the ultracentrifuge studies, and only a marginal (0.6%) difference in weight-average elution volume was detected by difference gel chromatography of 5g/litre solutions on Sephadex G-75. From quantitative analyses of sedimentation velocity and sedimentation-equilibrium distributions obtained with iPr2P (di-isopropylphosphoryl)-chymotrypsin, the polymerizing system is postulated to involve an indefinite association of dimer (with an isodesmic association constant of 0.68 litre/g) that is formed by a discrete dimerization step with equilibrium constant 0.25 litre/g. In addition to providing the best fit of the experimental results, this model of chymotrypsin polymerization at low ionic strength is also consistent with an earlier observation that dimer formation is a symmetrical head-to-head phenomenon under conditions of higher ionic strength (I0.29, pH7.9) where association is restricted to a monomer-dimer equilibrium. It is proposed that the dimerization process is essentially unchanged by variation in ionic strength at pH7.9, and that higher polymers are formed by an entirely different mechanism involving largely electrostatic interactions between dimeric species.
Factors controlling the activities of phosphatidate phosphohydrolase and phosphatidate cytidylyltransferase. The effects of chlorpromazine, demethylimipramine, cinchocaine, norfenfluramine, mepyramine and magnesium ions
Authors: Sturton R G , Brindley D N
Published: August 2015

1. Microsomal membranes from rat liver were incubated with ATP, CoA, Mg2+, [14C]palmitate, F- and sn-glycerol 3-phosphate in order to label them with [14C]phosphatidate. These membranes were isolated and used in a second incubation in which [3H]CTP was present, and the simultaneous synthesis of [14C]diacylglycerol and [3H]CDP-diacylglycerol was measured. 2. The addition of phosphatidate phosphohydrolase, which had been partially purified from the particle-free supernatant, supplemented the activity of the endogenous phosphohydrolase, but it did not alter the rate of CDP-diacylglycerol formation. 3. Adding EDTA inhibited phosphatidate cytidylyl-transferase activity and stimulated the activity of the phosphohydrolases by removing excess of Mg2+. 4. Increasing the concentration of Mg2+, norfenfluramine or chlorpromazine in the assay system stimulated cytidylyltransferase activity, but decreased the activities of both phosphohydrolases. 5. The mechanism for the stimulation of cytidylyl=transferase activity by the cationic drugs and Mg2+ was investigated with emulsions of phosphatidate and the microsomal fraction of rat liver. 6. There was a threshold concentration of about 5mM-MgCl2 below which no cytidylyltransferase activity was detected in the presence or absence of norfenfluramine. Just above this threshold concentration norfenfluramine stimulated cytidylyltransferase activity, but this stimulation disappeared as the Mg2+ concentration was raised to its optimum of 20mM. Norfenfluramine therefore partially replaced the bivalent-cation requirement. 7. At 30 mM-MgCl2 amphiphilic cationic drugs inhibited cytidylyltransferase activity at relatively high concentrations in a non-competitive manner with respect to phosphatidate. 8. The implications of these results are discussed with respect to the regulation of the synthesis of the acidic phospholipids compared with the synthesis of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol.
Mitochondrial adenosine triphosphatase of the fission yeast, Schizosaccharomyces pombe 972h−. Changes in activity and oligomycin-sensitivity during the cell cycle of catabolite-repressed and -de-repressed cells
Authors: Edwards S W , Lloyd D
Published: August 2015

1. Changes in activity of ATPase (adenosine triphosphatase) during the cell cycle of Schizosaccharomyces pombe were analysed in cell-free extracts of cells harvested from different stages of growth of synchronous cultures and also after cell-cycle fractionation. 2. Oligomycin-sensitive ATPase oscillates in both glucose-repressed synchronous cultures and shows four maxima of activity approximately equally spaced through the cell cycle. The amplitude of the oscillations accounts for between 13 and 80% of the total activity at different times in the cell cycle. 3. Oligomycin sensitivity varies over a fourfold range at different stages of the cell cycle. 4. The periodicity of maximum oligomycin sensitivity is one-quarter of a cell cycle. 5. These results were confirmed for the first three-quarters of the cell cycle by cell-cycle fractionation. 6. In cells growing synchronously with glycerol, ATPase activity increases in a stepwise pattern, with two steps per cell cycle; the first of these occurs at 0.54 of the cell cycle and the second at 0.95. 7. These results are discussed in relation to previously obtained data on the development of mitochondrial activities during the cell cycle.
Acetylcholine increases the breakdown of triphosphoinositide of rabbit iris muscle prelabelled with [32P] phosphate
Authors: Abdel-Latif A A , Akhtar R A , Hawthorne J N
Published: August 2015

1. Paired iris smooth muscles from rabbits were incubated for 30 min at 37 degrees C in an iso-osmotic salt medium containg glucose, inositol, cytidine and [32P]phosphate. 2. One of the pair was then incubated at 37 degrees C for 10 min in unlabelled medium containing 10mM-2-deoxyglucose and the other was incubated in the presence of acetylcholine plus eserine (0.05mM each). 2-Deoxyglucose, which was included in the incubation medium to minimize the biosynthesis of triphosphoinositide from ATP and diphosphoinositide, decreased the amount of labelled ATP by 71% and inhibited further 32P incorporation from ATP into triphosphoinositide by almost 30%. 3. Acetylcholine (0.05mM) increased significantly the loss of 32P from triphosphoinositide (the ‘triphosphoinositide effect’) in 32P-labelled iris muscle. This effect was measured both chemically and radiochemically. It was also observed when 32Pi was replaced by myo-[3H]inositol in the incubation medium. 4. The triphosphoinositide effect was blocked by atropine but not by D-tubocurarine. Further, muscarinic but not nicotinic agonists were found to provoke this effect. 5. Acetylcholine decreased by 28% the 32P incorporation into triphosphoinositide, presumably by stimulating its breakdown. This decrement in triphosphoinositide was blocked by atropine, but not by D-tubocurarine. 6. The triphosphoinositide effect was accompanied by a significant increase in 32P labelling, but not tissue concentration, of phosphatidylinositol and phosphatidic acid. The possible relationship between the loss of 32P label from triphosphoinositide in response to acetylcholine and the concomitant increase in that of phosphatidylinositol and phosphatidic acid is discussed. 7. The presence of triphosphoinositide phosphomonoesterase, the enzyme that might be stimulated in the iris smooth muscle by the neurotransmitter, was demonstrated, and, under our methods of homogenization and assay, more than 80% of its activity was localized in the particulate fraction.
The effect of insulin on the glycogenolytic cascade and on the activity of glycogen synthase in the liver of anaesthetized rabbits
Authors: van de Werve G , Stalmans W , Hers H G
Published: August 2015

1. The administration of insulin to anaesthetized rabbits caused the inactivation of liver phosphorylase and phosphorylase kinase, but did not change either the hepatic concentration of cyclic AMP or the activity of cyclic AMP-dependent histone kinase. All measured parameters were increased by the subsequent administration of glucagon. 2. Activation of glycogen synthase by insulin was only observed when phosphorylase had been strongly inactivated.
Labelling studies in vivo on the metabolism of the acyl and glycerol moieties of the glycerolipids in the developing maize leaf
Authors: Slack C R , Roughan P G , Balasingham N
Published: August 2015

1. When [2-3H]glycerol was supplied to developing maize-leaf laminae, label entered 3-sn-phosphatidycholine at a linear rate essentially from zero time, whereas other lipids were labelled at accelerating rates. On transfer of laminae from [3H]glycerol to unlabelled glycerol, radioactivity was rapidly lost from 3-sn-phosphatidylcholine and accumulated in other lipids, principally monogalactosyl diacyglycerol. 2. Degradation of these lipids showed that 3H was present only in the glycerol moiety of the lipids. 3. In double-labelling pulse-chase experiments with [14C]acetate, which labelled essentially only fatty acids and [3H]glycerol similar amounts of 14C and 3H radioactivity were lost from 3-sn-phosphatidylcholine and accumulated by monogalactosyl diacylglycerol. 4. The different molecular species of both lipids isolated from laminae during a double-labelled pulse-chase study were separated by argentation t.l.c., and the changes in the amount of radioactivity and the 14C/3H ratio in different species were compared. The greatest loss of radioactivity during the period in unlabelled substrates occurred from the 3-sn-phosphatidylcholine species containing oleate and from the dilinoleate species, and radioactivity accumulated by monogalactosyl diacyglycerol was mainly in the dilinolenate species. However, despite the considerable change in the radioactivity in these species during the chase, the 14C/3H ratio in each of them remained relatively unchanged. 5. It is proposed that 3-sn-phosphatidylcholine in the developing leaf may serve as a donor or linoleate-containing diacyl-glycerols which are incorporated into other lipids, principally monogalactosyl diacylglycerol.
Specific protein synthesis in isolated rat testis leydig cells. Influence of luteinizing hormone and cycloheximide
Authors: Janszen F H A , Cooke B A , van der Molen H J
Published: August 2015

The effect of luteinizing hormone (luteotropin) and cycloheximide on specific protein synthesis in rat testis Leydig cells has been investigated. Proteins were labelled with either I114C]leucine, [3H]leucine or [35S]methionine during incubation with Leydig-cell suspensions in vitro. Total protein was extracted from the cells and separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. No detectable increase in the synthesis of specific proteins could be observed after incubation of Leydig cells with luteinizing hormone for up to 1 h. However, after a 2h incubation period, an increase in [35S]methionine incorporation was observed in a protein with an apparent mol.wt. of 21000 (referred to as ‘protein 21’). When, after labelling of this protein with [35S]-methionine, Leydig cells were incubated for another 30min with cycloheximide, no decrease in radioactivity of this protein band was observed, indicating that it does not have a short half-life. However, another protein band was detected, which after incubation with cycloheximide disappeared rapidly, the reaction following first-order kinetics, with a half-life of about 11 min. This protein, with an apparent mol.wt. of 33000 (referred to as “protein 33”), was found to be located in the particulate fraction of the Leydig cell, and could not be demonstrated in other rat testis-cell types or blood cells. No effect of luteinizing hormone on molecular weight, subcellular localization or half-life of protein 33 was observed. A possible role for protein 33 and protein 21 in the mechanism of action of luteinizing hormone on testosterone production of Leydig cells is discussed.
The substrate specificity of adenosine 3′:5′-cyclic monophosphate-dependent protein kinase of rabbit skeletal muscle
Authors: Yeaman S J , Cohen P , Watson D C , Dixon G H
Published: August 2015

The known amino acid sequences at the two sites on phosphorylase kinase that are phosphorylated by cyclic AMP-dependent protein kinase were extended. The sequences of 42 amino acids around the phosphorylation site on the alpha-subunit and of 14 amino acids around the phosphorylation site on the beta-subunit were shown to be: alpha-subunit Phe-Arg-Arg-Leu-Ser(P)-Ile-Ser-Thr-Glu-Ser-Glx-Pro-Asx-Gly-Gly-His-Ser-Leu-Gly-Ala-Asp-Leu-Met-Ser-Pro-Ser-Phe-Leu-Ser-Pro-Gly-Thr-Ser-Val-Phe(Ser,Pro,Gly)His-Thr-Ser-Lys; beta-subunit, Ala-Arg-Thr-Lys-Arg-Ser-Gly-Ser(P)-VALIle-Tyr-Glu-Pro-Leu-Lys. The sites on histone H2B which are phosphorylated by cyclic AMP-dependent protein kinase in vitro were identified as serine-36 and serine-32. The amino acid sequence in this region is: Lys-Lys-Arg-Lys-Arg-Ser32(P)-Arg-Lys-Glu-Ser36(P)-Tyr-Ser-Val-Tyr-Val- [Iwai, K., Ishikawa, K. & Hayashi, H. (1970) Nature (London) 226, 1056-1058]. Serine-36 was phosphorylated at 50% of the rate at which the beta-subunit of phosphorylase kinase was phosphorylated, and it was phosphorylated 6-7-fold more rapidly than was serine-32. The amino acid sequences when compared with those at the phosphorylation sites of other physiological substrates suggest that the presence of two adjacent basic amino acids on the N-terminal side of the susceptible serine residue may be critical for specific substrate recognition in vivo.
Co-ordinate changes in enzymes of fatty acid synthesis, activation and esterification in rabbit mammary gland during pregnancy and lactation
Authors: Short V J , Brindley D N , Dils R
Published: August 2015

1. The activities of fatty acid synthetase, acyl-CoA synthetase, glycerol phosphate acyltransferase and phosphatidate phosphatase were measured in the mammary glands of rabbits from day 16 of pregnancy to day 15 of post partum. 2. There were significant correlations between the increases in activities of these enzymes during this period. This was the case whether the activities were expressed per mg of homogenate protein, per g wet wt. of tissue or per total wet weight of the whole glands. The only exception was the lack of correlation between the activities of fatty acid synthetase and of phosphatidate phosphatase per g wet wt. of tissue. 3. These co-ordinate increases are discussed in relation to the changes which occur in fatty acid metabolism in the mammary gland during pregnancy and lactation.
Nature of the stimulation of biogenesis of cholesterol in the liver by noradrenaline
Authors: George R , Ramasarma T
Published: August 2015

1. Administration of noradrenaline increased the incorporation of [1-14C]acetate into hepatic sterols and the activity of liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase. 2. The stimulation was observed at short time-intervals with a maximum at 4h and was progressive with increasing concentrations of noradrenaline. 3. Protein synthesis de novo was a necessary factor for the effect. 4. The stimulatory effect was not mediated through the adrenergic receptors, but appears to involve a direct action of the hormone within the hepatocyte.
The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Phospholipid depletion and re-activation of guinea-pig liver microsomal enzyme
Authors: Graham A B , Pechey D T , Toogood K C , Thomas S B , Wood G C
Published: August 2015

More than 80% of the phospholipid component of guinea-pig liver microsomal membranes (prepared with 154mM-KCl) was removed by treatment with phospholipase A followed by extraction of the lysophosphatides and fatty acids produced with albumin. Delipidation strongly inactivated the highly active UDP-glucuronyltransferase of these preparations and activity was restored by mixtures of phosphatidylcholine and lysophosphatidylchlone. However, small quantities of lysophosphatides were still associated with the delipidated fractions after extraction with albumin and might have influenced the inactivation and re-activation observed. To eliminate these uncertainties, microsomal proteins and phospholipids were separated by gel filtration on Sephadex G-150 in the presence of cholate. This technique also strongly inactivated the enzyme but did not generate membrane-active phospholipid degradation products. High transferase activity was again restored to the delipidated protein by choline glycerophosphatides. These results confirm the view that the fully active form of microsomal UDP-glucuronyltransferase is phospholipid-dependent.
Possible multiple binding sites for o-aminophenol on uridine diphosphate glucuronyltransferase
Authors: Howland R D , Bohm L D
Published: August 2015

1. Hepatic microsomal UDP-glucuronyltransferase (EC 2.4.1.17) derived from either weanling or adult rats exhibits three pH optima, at pH 5.4, 7.2 and 9.2, when o-aminophenol is the acceptor substrate, whereas p-nitrophenol is the acceptor substrate only on pH optimum is observed, at pH 5.4.2. Prior treatment of rats of either age with 3-methylcholanthrene results in a 2-3-fold increase in o-aminophenol conjugation at pH 5.4 and a 6-9-fold increase at pH 9.2. At pH 7.2, the induced enzyme is 2 to 3 times more active towards o-aminophenol than the control enzyme, but no pH optimum is demonstrable. 3. o-Aminophenol conjugation at pH 5.4 and 9.2 is inhibited competitively by both p-nitrophenol and p-nitrophenyl glucuronide, suggesting that the two phenolic aglycones share the same binding site. At pH 7.2, however, p-nitrophenyl glucuronide does not inhibit o-aminophenol conjugation, suggesting that the binding site at this pH is not shared by the two phenols. These data are consistent with the existence of more than one binding site for o-aminophenol on UDP-glucuronyltransferase.
The proteoglycan content and the axial periodicity of collagen in tendon
Authors: Gillard G C , Merrilees M J , Bell-Booth P G , Reilly H C , Flint M H
Published: August 2015

The glycosaminoglycan content and the axial periodicity of collagen was determined in various regions of the rabbit flexor digitorum profundus tendon. This tendon, which passes from the calf to the toes round the inner side of the ankle, contains a thickened sesamoid-like pad where it is subjected to friction and pressure. Other regions of the tendon are subject only to longitudinal tension. In tensional areas the axial periodicity of collagen was of the order of 62 nm and the tissue contained less than 0.2% proteoglycan on a dry weight basis. In the sesamoid-like region, however, the axial periodicity was a significant 13-15% less, and the proteoglycan constituted about 3.5% of the dry weight. Also, in the tensional areas the predominant glycosaminoglycan was dermatan sulphate, whereas in the sesamoid the predominant glycosaminoglycan was chondroitin sulphate. The possible interrelationships between collagen axial peroidicity and proteoglycan content in this tissue are discussed.
The highly electrophilic character of 4-chloro-7-nitrobenzofurazan and possible consequences for its applications as a protein-labelling reagent
Authors: Baines B S , Allen G , Brocklehurst K
Published: August 2015

4-Chloro-7-nitrobenzofurazan possesses at least one highly electrophilic centre (C-6) that is much more reactive towards nucleophiles than position C-4, the irreversible alkylating site of this reagent. Possible consequences of the electrophilic character of 4-chloro-7-nitrobenzofurazan for its application as a protein-labelling reagent are discussed.
The amino acid sequence of cytochrome c from the locust, Schistocerca gregaria Forskal
Authors: Lyddiatt A , Boulter D
Published: August 2015

The amino acid sequence of locust cytochrome c was determined, although the overlap between chymotryptic and tryptic peptides at residues tyrosine-97 and leucine-98 was not observed, owing to an anomalous tryptic break duplicating the chymotryptic digestion. The molecule consists of a single polypeptide chain of 107 residues, homologous with other mitochondrial cytochromes c. In common with other known insect cytochromes c, it possesses a non-acetylated, four-residue tail at the N-terminus relative to glycine-1 of the standard alignment. A molecular phylogeny for 17 species was constructed relating the cytochrome c molecules of Schistocerca gregaria and other invertebrates with those of representative taxonomic groups. Experimental details are given in a supplementary paper deposited as Supplementary Publication SUP 50077 (24 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can obtained on the terms indicated in Biochem. J. (1977) 161, 1.
An adenosine triphosphate-dependent deoxyribonuclease from Alcaligenes faecalis
Authors: Rosamond J D C , Lunt M R
Published: August 2015

A deoxyribonuclease was purified approx. 800-fold from crude extracts of the bacterium Alcaligenes faecalis. The enzyme requires ATP and Mn2+; ATP could be replaced by any other ribo- or deoxyribo-nucleoside triphosphate, and Mn2+ could be replaced by Mg2+ in 0.1 M-Tris/HCl, pH 8.0 at 37 degrees C. The enzyme could degrade linear duplex or denaturated DNA, but was inactive with closed-circular duplex DNA from bacteriophase PM-2. In the course of nucleolytic activity, ATP was hydrolysed. We have measured deoxyribonuclease and adenoxine triphosphatase activity in the presence of various salts, and found that the amount of ATP hydrolysis associated with a given amount of deoxyribonuclease activity was decreased in the presence of tetraethylammonium ions. Since these ions decrease the stability of the DNA helix, we conclude that one function of the ATP hydrolysis is to unwind the DNA.
Some factors affecting the production, by cultured baby-hamster kidney cells, of BHK glycoprotein I which cross-reacts immunologically with Tamm-Horsfall glycoprotein
Authors: Bloomfield F J , Dunstan D R , Foster C L , Serafini-Cessi F , Marshall R D
Published: August 2015

Cultured baby-hamster kidney cells (BHK-21/C13), which are adapted to grow in suspension (strain 2P), roduce a glycoprotein, termed BHK glycoprotein I, which cross-reacts immunologically with hamster urinary (Tamm-Horsfall glycoprotein. BHK glycoprotein I was isolated in an electrophoretically (sodium dodecyl sulphate/polyacrylamide gel) homogeneous form by application of affinity chromatography to the medium in which cells had been cultured. Insolubilized anti-(Tamm-Horsfall glycoprotein immunoglobulin G) was used as the adsorbent. The amount of BHK glycoprotein I associated with the cultured cells was found by both radioimmunoassay and immunofluorescence to be related to the amount of Ca2+ in the medium and to the particular stage of the cell cycle. 5'-Nucleotidase was also shed by the cells into the culture medium in amounts related to the stage of the cell cycle. The turnover of hamster Tamm-Horsfall glycoprotein in vivo appeared to be considerably more rapid than can be accounted for by cell turnover. Hamster Tamm-Horsfall glycoprotein was shown to be ineffective in inhibiting agglutination of chicken erythrocytes caused by influenza virus.
Catecholamine-sensitive adenylate cyclase of caudate nucleus and cerebral cortex. Effects of guanine nucleotides
Authors: Sulakhe P V , Leung N L , Arbus A T , Sulakhe S J , Jan S H , Narayanan N
Published: August 2015

1. GTP and GMP-P(NH)P (guanyl-5'-yl imidodiphosphate) were observed to increase the stimulation of neural adenylate cyclase by dopamine (3,4-dihydroxyphenethylamine) and noradrenaline. 2. GMP-P(NH)P had a biphasic effect on the enzyme activity. 3. Preincubation of membranes with GMP-P(NH)P activated the enzyme by a process dependent on time and temperature. Catecholamines increased the speed and the extent of this activation. 4. Membrane fractions contained high- and low-affinity sites for GMP-P(NH)P binding: this binding was due to protein(s) of the membrane preparations. 5. Low-affinity-site binding of GMP-P(NH)P appeared to be related to the stimulatory effect on the adenylate cyclase activity.
Heterogeneity of proteoglycans in developing chick limb cartilage
Authors: Vasan N S , Lash J W
Published: August 2015

Proteoglycan heterogeneity was studied during the maturation of embryonic-chick limb cartilage in vivo. The results suggest that during the differentiation of limb-bud cartilage the aggregated forms of proteoglycans increase between stages 24 and 35, whereas the non-aggregated or monomeric forms decrease. Only one link protein is found in stage-24 limb buds, whereas two are present at stage 35. Evidence suggests that the synthesis of link proteins may be a regulatory factor in limb chondrogenesis.
The effect of insulin on the washout of [45Ca]calcium from adipocytes and soleus muscle of the rat
Authors: Clausen T , Martin B R
Published: August 2015

Insulin stimulates the washout of 45Ca from preloaded isolated fat-cells, whole epididymal fat-pads and isolated soleus muscles of the rat. This effect occurs within 10 min after the addition of the hormone, and it can be detected at concentrations down to those measured in rat plasma. When K+ is omitted from the washout medium, the effect on soleus muscles is more pronounced and increases with the time of exposure.
Development of mitochondrial energy metabolism in rat brain
Authors: Land John M. , Booth Robert F. G. , Berger Ruud , Clark John B.
Published: August 2015

1. The development of pyruvate dehydrogenase and citrate synthase activity in rat brain mitochondria was studied. Whereas the citrate synthase activity starts to increase at about 8 days after birth, that of pyruvate dehydrogenase starts to increase at about 15 days. Measurements of the active proportion of pyruvate dehydrogenase during development were also made. 2. The ability of rat brain mitochondria to oxidize pyruvate follows a similar developmental pattern to that of the pyruvate dehydrogenase. However, the ability to oxidize 3-hydroxybutyrate shows a different developmental pattern (maximal at 20 days and declining by half in the adult), which is compatible with the developmental pattern of the ketone-body-utilizing enzymes. 3. The developmental pattern of both the soluble and the mitochondrially bound hexokinase of rat brain was studied. The total brain hexokinase activity increases markedly at about 15 days, which is mainly due to an increase in activity of the mitochondrially bound form, and reaches the adult situation (approx. 70% being mitochondrial) at about 30 days after birth. 4. The release of the mitochondrially bound hexokinase under different conditions by glucose 6-phosphate was studied. There was insignificant release of the bound hexokinase in media containing high KCl concentrations by glucose 6-phosphate, but in sucrose media half-maximal release of hexokinase was achieved by 70μm-glucose 6-phosphate 5. The production of glucose 6-phosphate by brain mitochondria in the presence of Mg2++glucose was demonstrated, together with the inhibition of this by atractyloside. 6. The results are discussed with respect to the possible biological significance of the similar developmental patterns of pyruvate dehydrogenase and the mitochondrially bound kinases, particularly hexokinase, in the brain. It is suggested that this association may be a mechanism for maintaining an efficient and active aerobic glycolysis which is necessary for full neural expression.
Adenosine and the regulation of insulin secretion by isolated rat islets of Langerhans
Authors: Ismail N A , El Denshary E E S M , Montague W
Published: August 2015

The effect of adenosine in insulin secretion and adenylate cyclase activity of rat islets of Langerhans was investigated. Adenosine inhibited insulin secretion stimulated by glucose, glucagon, prostaglandin E2, tolbutamine and theophylline. Adenosine decreased basal adenylate cyclase activity of the islets as well as that stimulated by glucagon prostaglandin E2 and GTP, although fluoride-stimulated activity was not affected. Neither insulin secretion nor adenylate cyclase activity of the islets was affected by adenine, AMP or ADP. The inhibitory effect of adenosine on adenylate cyclase activity was not altered by either phenoxybenzamine (alpha-adrenergic blocker) or propranolol (beta-adrenergic blocker), suggesting that the effect is not mediated through the adrenergic receptors of the islet cells. These results suggest that the intracellular concentration of adenosine in the beta-cell may play a role in regulating insulin secretion and that this effect may be mediated via alterations in the activity of adenylate cyclase in the beta-cell.
Self-digestion of human erythrocyte membranes. Role of adenosine triphosphate and glutathione
Authors: Brovelli A , Suhail M , Pallavicini G , Sinigaglia F , Balduini C
Published: August 2015

Intact human erythrocytes incubated at 37 degrees C, pH7.4, release a sialoglycopeptide similar in its chemical composition, immunological and aggregation properties to the glycopeptide released by isolated ‘ghost’ membranes. The presence of ATP or reduced glutathione at physiological concentrations in the incubation medium of ‘ghost’ membranes inhibits this self-digestion process.
The effects of antioxidants on the metabolism and mutagenicity of benzo[a]pyrene in vitro
Authors: Rahimtula A D , Zachariah P K , O'Brien P J
Published: August 2015

Antioxidants inhibit the rat liver microsomal mixed-function-oxidase-catalysed hydroxylation of benzo[a]pyrene. These antioxidants also decrease the formation of mutagenic products from benzo[a]pyrene as judged by the Ames bacterial-mutagenicity assay [B.N. Ames, J. McCann & E. Yamazaki (1975) Mutat. Res. 31, 347-364]. It is suggested that antioxidants exert their protective effect against cancer by inhibiting the formation of carcinogenic metabolites.
Adaptation of protein metabolism to endurance training. Increased amino acid oxidation in response to training
Authors: Dohm G L , Hecker A L , Brown W E , Klain G J , Puente F R , Askew E W , Beecher G R
Published: August 2015

This study was conducted to investigate alterations in excretion of urea and total nitrogen after6-8 weeks of daily exercise and to establish if the capacity for amino acid oxidation in muscle is influenced by endurance training. Urea nitrogen excretion was increased in trained compared with untrained rats and nitrogen balance was less positive in trained than in untrained rats. Increased [14C]leucine oxidation with training was observed both in vivo and in vitro. The results of this study demonstrate that amino acid catabolism is increased during exercise training and that the muscle enzymes involved in leucine oxidation adapt to endurance training in a manner similar to the enzymes of carbohydrate and fat catabolism.