bluuebunny/crossref_metadata_2025 huggingface dataset:
Ribonuclease-sensitivity of covalently closed rat liver mitochondrial deoxyribonucleic acid
Authors: Lonsdale David M. , Jones I. Gwyn
Published: August 2015

Preparations of covalently closed mitochondrial DNA of rat liver contain 10–30% of molecules that are converted into relaxed circular molecules after treatment with ribonuclease. Control experiments, with covalently closed bacteriophage PM2 DNA, indicate that ribonuclease-sensitivity cannot be induced either by depurination or by incubation with reducing agents.
A collagen-like amino acid sequence in a polypeptide chain of human C1q (a subcomponent of the first component of complement)
Authors: Reid Kenneth B. M.
Published: August 2015

1. A partial amino acid sequence of 95 residues of the 191 residues in the oxidized A chain of human subcomponent C1q was determined. The partial nature of the sequence is because one overlapping peptide is missing in the proposed sequence, also the proof of some of the overlapping peptides depends partly on their amino acid composition and not on their complete sequence. 2. This region of the A chain contained a repeating sequence of glycine-X-Y (where X is often proline and Y is often hydroxyproline) for 78 residues. 3. The five hydroxylysine residues and the five hydroxyproline residues present in the oxidized A chain were all in these 78 residues and only in the Y position of the repeating sequence. 4. Prolonged collagenase digestion of the oxidized A chain yielded a large, apparently C-terminal, peptide which contained most of the non-collagenous sequences present in the chain. 5. It is concluded that there is a collagen-like region in the A chain of subcomponent C1q which constitutes most of the N-terminal half of the chain and that similar collagen-like regions will be found in the B and C chains.
The kinetics of coupled enzyme reactions. Applications to the assay of glucokinase, with glucose 6-phosphate dehydrogenase as coupling enzyme
Authors: Storer Andrew C. , Cornish-Bowden Athel
Published: August 2015

1. A theory is developed to account for the kinetics of coupled-enzyme reactions without assuming that the second reaction follows first-order kinetics. 2. A simple procedure is described for applying the theory to the practical design of enzyme assays. 3. The validity of the theory is confirmed for the assay of glucokinase with glucose 6-phosphate dehydrogenase as coupling enzyme. 4. The possibility of extending the theory to three or more coupled reactions is discussed.
Amino acid sequence of the smaller basic protein from rat brain myelin
Authors: Dunkley Peter R. , Carnegie Patrick R.
Published: August 2015

1. The complete amino acid sequence of the smaller basic protein from rat brain myelin was determined. This protein differs from myelin basic proteins of other species in having a deletion of a polypeptide of 40 amino acid residues from the centre of the molecule. 2. A detailed comparison is made of the constant and variable regions in a group of myelin basic proteins from six species. 3. An arginine residue in the rat protein was found to be partially methylated. The ratio of methylated to unmethylated arginine at this position differed from that found for the human basic protein. 4. Three tryptic peptides were isolated in more than one form. The differences between the two forms of each peptide are discussed in relation to the electrophoretic heterogeneity of myelin basic proteins, which is known to occur at alkaline pH values. 5. Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50029 at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5.
The kinetics of oxidation of ferroperoxidase by molecular oxygen. A model of a terminal oxidase
Authors: Phelps Charles F. , Antonini Eraldo , Giacometti Giorgio , Brunori Maurizio
Published: August 2015

1. The decay of oxyferroperoxidase to ferriperoxidase was studied by rapidly mixing solutions of ferroperoxidase with various amounts of oxygen and following the time-course of appearance of oxyferroperoxidase and its subsequent decay to ferriperoxidase by reaction with ferroperoxidase. 2. The scheme can be accommodated by and [Formula: see text] and occurs without detectable intermediates being observed in the millisecond time-range. 3. Analogue-computer simulation of the reaction is in agreement with the initial rate-limiting reaction being an intermolecular, not intramolecular, electron-donating process, and analysis of the data leads to quantitative values for the rate constants of the overall process. 4. The reaction of oxyferroperoxidase with ferroperoxidase is a model of a terminal oxidase, and the results are discussed in terms of the possible importance of this reaction in peroxidase function, and also in the light it throws on autoxidation of haem compounds.
Catalytic properties of alkaline phosphatase from pig kidney
Authors: Hiwada Kunio , Wachsmuth Ernst D.
Published: August 2015

The enzymic properties of alkaline phosphatase (EC 3.1.3.1) from pig kidney brush-border membranes were studied. 1. It hydrolyses ortho- and pyro-phosphate esters, the rate limiting step (Vmax.) being independent of the substrate. It transphosphorylates to Tris at concentrations above 0.1m-Tris. 2. The pH optimum for hydrolysis was between 9.8 and 10. The pK of the enzyme–substrate complex is 8.7 for p-nitrophenyl phosphate and β-glycerophosphate. Excess of substrate inhibits the enzymic activity with decreasing pH. The pK of the substrate-inhibited enzyme–substrate complex, 8.7, is very similar to that for the enzyme–substrate complex. The pK values of the free enzyme appear to be 8.7 and 7.9. 3. Inactivation studies suggest that there is an essential tyrosine residue at the active centre of the enzyme. 4. The energy of activation (E) and the heat of activation (ΔH) at pH9.5 showed a transition at 24.8°C that was unaffected by Mg2+. 5. Kinetic and atomic-absorption analysis indicated the essential role of two Zn2+ ions/tetrameric enzyme for an ordered association of the monomers. Zn2+ in excess and other bivalent ions compete for a second site with Mg2+. Mg2+ enhances only the rate-limiting step of substrate hydrolysis. 6. Amino acid inhibition studies classified the pig kidney enzyme as an intermediate type of previously described alkaline phosphatases. It has more similarity with the enzyme from liver and bone than with that from placenta.
Specificity of glycerol kinase (Short Communication)
Authors: Eisenthal Robert , Harrison Roger , Lloyd William J.
Published: August 2015

The activity of a number of alcohols was examined as substrates or inhibitors of glycerol kinase (ATP–glycerol phosphotransferase; EC 2.7.1.30) from Candida mycoderma. On the basis of these and other results, a modified model is proposed to account for the substrate specificity of the enzyme.
Studies on the fractionation of total transfer ribonucleic acid and purification of an alanine transfer ribonucleic acid from chick embryo
Authors: Anandaraj M. P. Jacob Spurgeon , Cherayil Joseph D.
Published: August 2015

Chick embryo tRNA, prepared by a simple large-scale method, was fractionated on three different ion-exchange columns. In all cases simple chromatographic patterns for various tRNA species were observed, indicating the presence of only a few major species of tRNA for each amino acid. By repeated chromatography one species of alanine tRNA was purified to approx. 80% purity. T1 ribonuclease digest of this purified tRNA gave a simple chromatographic pattern. Because of the simplicity of the method of preparation of tRNA from this readily available source and the presence of only a few species of tRNA for each amino acid, chick embryo is suited for the study of tRNA and its various functions in higher systems.
Calcium and thiol reactivity of human plasma clotting factor XIII
Authors: Cooke Rodney D. , Pestell Timothy C. , Holbrook J. John
Published: August 2015

1. The reaction of iodoacetate, 2-chloromercuri-4-nitrophenol and 5,5′-dithiobis-(2-nitrobenzoate) with thrombin-cleaved Factor XIII (i.e. Factor XIIIa) was accompanied by enzyme inhibition. 2. The reaction with iodoacetate and 5,5′-dithiobis-(2-nitrobenzoate) was absolutely dependent on Ca2+, and the rate of reaction increased with the Ca2+concentration up to very high, non-physiological concentrations. 3. 2-Chloromercuri-4-nitrophenol reacted with Factor XIIIa in the absence of Ca2+, but at a much slower rate. 4. Stopped-flow methods were used to quantify the reaction with 5,5′-dithiobis-(2-nitro-benzoate) because of the Ca2+-dependent dissociation of Factor XIIIa (a′2b2) and subsequent aggregation of the a′ chains into turbid precipitates. 5. The 3-carboxy-4-nitrothio-phenolate released was consistent with the reaction of 2 thiol groups/molecule of Factor XIIIa. The isolated b chains of Factor XIII did not react with either of the chromophoric reagents. This indicated that the a′ chains of Factor XIIIa were responsible for the thiol reactivity of the enzyme. 6. The Ca2+dependence of the enzyme inhibition by these thiol reagents was very dependent on protein concentration. This is discussed in relation to the Ca2+-induced dissociation of Factor XIIIa. 7. The acceptor substrate, casein, decreased the Ca2+concentration required for enzyme inhibition by both the mercurial and the aromatic disulphide compounds. Dansylcadaverine did not affect Ca2+dependence of inhibition.
The interaction of the phosphonate analogue of 3-phospho-d-glycerate with phosphoglycerate kinase
Authors: Orr George A. , Knowles Jeremy R.
Published: August 2015

The methylene analogue of 3-phospho-d-glycerate, 2-hydroxy-4-phosphono-dl-butyric acid, is a substrate for phosphoglycerate kinase. The pKa values for the final dissociation of the natural substrate and its methylene isostere are 6.20 and 7.45 respectively. The kinetic parameters Km and kcat. for the enzyme-catalysed reaction were determined at pH6.9 and 8.5 by using low substrate concentrations. Although the kcat. values for the two substrates at each pH are similar, there is a 60-fold increase in the Km value for the methylene isostere on going to the lower pH. The results are most readily interpreted in terms of a dianionic group on C-3 being required for efficient substrate binding to the enzyme.
Analysis of the code relating sequence to conformation in globular proteins. The distribution of residue pairs in turns and kinks in the backbone chain
Authors: Robson Barry , Pain Roger H.
Published: August 2015

1. The residue pair is considered as the fundamental unit which differentiates α-helix, β-pleated sheet and the various turns and kink structures of the protein backbone. 2. The HPLG alphabet (Robson & Pain, 1974) is used to group pairs of residues, giving 16 possible conformational pairs, all of which are found with differing frequencies in the nine proteins examined. 3. The frequencies of occurrence of the 16 different types of turn or kink are analysed in relation to the constituent amino acids. Those containing the L or G conformation are of low frequency and are grouped for purposes of this analysis. 4. The distribution of amino acids within all the conformational pairs is non-random, with distinct preferences shown by certain residues. 5. All pairs containing an L or G conformation require the presence of a glycine or a proton-donor side chain. 6. The results are discussed in terms of the determination of these ‘random’ structures by local interactions.
Role of adenosine 3′:5′-cyclic monophosphate in the action of 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane (DDT) on hepatic and renal metabolism
Authors: Kacew Sam , Singhal Radhey L.
Published: August 2015

The possibility whether alterations in the cyclic AMP–adenylate cyclase–phosphodiesterase system play a role in the action of 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane (DDT) on hepatic and renal carbohydrate metabolism was investigated. Administration of exogenous cyclic AMP (10mg/100g) was found to mimic the action of DDT which enhanced the activities of pyruvate carboxylase, phosphoenolpyruvate carboxylase, fructose 1,6-diphosphatase and glucose 6-phosphatase in both liver and kidney cortex, elevated the concentration of blood glucose and urea and decreased the amount of hepatic glycogen. Treatment with theophylline augmented the effects of a submaximal dose of this halogenated hydrocarbon on serum urea and glucose as well as the key gluconeogenic enzymes in liver and kidney cortex. Addition of DDT in vitro to liver and kidney homogenates resulted in a significant enhancement of adenylate cyclase activity. Hepatic and renal slices from rats already treated with DDT displayed an increased ability to convert [3H]adenosine into cyclic [3H]AMP. Whereas kidney-cortex slices excised from rats given caffeine and DDT produced an even greater amount of cyclic [3H]AMP, imidazole, propranolol and hydrazine prevented the insecticide-stimulated rise in cyclic nucleotide production. In contrast, prostaglandin E1 failed to exert any significant effect on DDT-induced increases in cyclic [3H]AMP synthesis from radioactive adenosine. The present study and our previous findings (Kacew & Singhal, 1973e) support the concept that the DDT-induced alterations in carbohydrate metabolism of liver and kidney cortex may be related to an initial stimulation of the cyclic AMP-adenylate cyclase system in these tissues.
Synthesis and maturation of ribosomal ribonucleic acids in isolated HeLa cell nuclei. A tracer study on the topology of the 45S precursor of ribosomal ribonucleic acids
Authors: Hadjiolov Asen A. , Milchev Georgui I.
Published: August 2015

The synthesis and processing of RNA by isolated HeLa cell nuclei was studied at low ionic strength in the presence of α-amanitin. The RNA polymerase reaction, with endogenous template and enzyme, rapidly reaches a plateau dependent on the amount of nuclei. Evidence is presented that incorporation of [3H]UMP proceeds only in growing RNA chains, whereas initiation of new RNA chains is arrested. The product formed contains all the main components of the 45S pre-rRNA (precursor of rRNA) maturation pathway (45S, 32S and 20S pre-rRNA; 28S and 18S rRNA). Most of the labelled material is in the mature rRNA components and their immediate precursors, even at very short times of incubation (2min). Small, but definite, 5S and 4S RNA peaks are also observed. At shorter incubation times a substantial amount of [3H]UMP is incorporated into RNA molecules in the 24S and 10–16S zones. This RNA material is considered to represent the non-conserved segments of 45S pre-rRNA in the process of nucleolytic degradation. A model for the tracer study of the topology of 45S pre-rRNA, on arrest of rRNA initiation, is discussed. The experimental evidence obtained supports the following structure of 45S pre-rRNA: 5′-end–28S rRNA unit–18S rRNA unit–nonconserved segment–3′-end.
Adaptive changes in the capacity of systems used for the synthesis of citrulline in rat liver mitochondria in response to high- and low-protein diets
Authors: McGivan J. D. , Bradford Norah M. , Chappell J. B.
Published: August 2015

1. Citrulline synthesis was measured in mitochondria from rats fed on a standard diet, a high-protein diet, or on glucose. 2. With NH4Cl as the nitrogen source the rate of citrulline synthesis was higher in mitochondria from rats fed on a high-protein diet than in those from rats fed on a standard diet. When rats were fed solely on glucose the rate of synthesis of citrulline from NH4Cl was very low. 3. With glutamate as the nitrogen source the relative rates of citrulline synthesis were much lower than when NH4Cl was present, but similar adaptive changes occurred. 4. The activity of the mitochondrial glutamate-transporting system increased two to three times on feeding rats on a high-protein diet, but the Km for glutamate was unchanged. 5. Adaptive changes in certain intramitochondrial enzymes were also measured. 6. The results were interpreted to indicate that when an excess of substrate was present, citrulline synthesis from NH4Cl was rate-limited by the intramitochondrial concentration of N-acetyl-glutamate, but citrulline synthesis from glutamate was rate-limited primarily by the activity of the glutamate-transporting system.
The influx of calcium ions into human erythrocytes during cold storage. The influences of extracellular pH, intracellular adenosine triphosphate and efflux of univalent cations
Authors: Mouat Barbara , Long Cyril
Published: August 2015

1. When human erythrocytes are stored at 3°C for several days as a suspension in iso-osmotic sucrose or KCl, containing CaCl2, the rates of cellular ATP degradation are similar. 2. During cold storage of erythrocytes in sucrose–CaCl2 medium, Ca2+influx and univalent-cation efflux occur, the pH value of the suspending medium rises and the intracellular pH falls. These pH changes correlate reasonably well with alterations in the membrane potential calculated from Cl−distribution. 3. The presence of Ca2+in the medium does not increase the rate of univalent-cation efflux from the cells. 4. When the pH of the medium is raised by addition of buffers, the rates of both Ca2+influx and univalent-cation efflux increase. 5. Replacement of sucrose by KCl as the main osmotic component of the medium completely suppresses Ca2+influx and univalent-cation efflux, although the pH of the KCl medium is higher than that of the sucrose medium. 6. When sucrose is replaced by choline chloride, Ca2+influx and univalent-cation efflux still occur, and the pH of the medium is similar to that found in iso-osmotic KCl. 7. When valinomycin, Pb2+or Cd2+are added to the iso-osmotic sucrose medium, the rate of efflux of univalent cations increases as also does the influx of Ca2+. 8. From these and other observations, it was concluded that it is univalent-cation efflux rather than ATP depletion or elevated extracellular pH which is the prerequisite for Ca2+influx during cold storage.
Model connective-tissue systems. A study of polyion–mobile ion and of excluded-volume interactions of proteoglycans
Authors: Comper Wayne D. , Preston Barry N.
Published: August 2015

The osmotic pressure of solutions of sulphated proteoglycans isolated from the intervertebral discs of animals of various ages was determined. The behaviour of the solutions in salt-added systems was investigated in terms of the Donnan distribution of the mobile ions. It is evident that this effect is the dominating factor in explaining the observed nonidealities. Although marked variations in the compositions of the proteoglycan, with regard to their chondroitin sulphate and keratan sulphate content and hence charge content, occur with increasing age of parent tissue, the osmotic activities of the various preparations are very similar. This is explained by the ‘fixation’ of the counterions in such a way as to counteract any change in the charge content of the polyion; an ‘osmotic buffering’ effect. The swelling behaviour of gelatin gels containing the proteoglycan preparations has been measured. In all cases pressures in excess of the sum of the osmotic pressures of the individual components are observed. However, the magnitude of the excess decreases with increasing age of the parent tissue. It is suggested that the age changes, as reflected by a decrease in water content of the gel system, are not the result of changes in the osmotic properties of the individual components but rather reflect changes in the entropic interaction of the proteoglycan with the gelatin matrix. The relevance of this observation to the situation in vivo is discussed.
The localization of a vitamin K-induced modification in an N-terminal fragment of human prothrombin
Authors: Skotland Tore , Holm Turid , Østerud Bjarne , Flengsrud Ragnar , Prydz Hans
Published: August 2015

1. The N-terminal fragment (PF-I) split off from prothrombin during coagulation was purified to homogeneity from human serum. 2. The apparent molecular weight is 27000±2000 in sodium dodecyl sulphate–polyacrylamide-gel electrophoresis, whereas a value of about 19600 is obtained by calculation based on amino acid and carbohydrate analyses. The N-terminal sequence is an Ala-Asx bond. The fragment contains about 16% carbohydrate, binds phospholipids in the presence of Ca2+and is adsorbed to BaSO4. The pKa of its BaSO4-binding group(s) is 3.1–3.5. 3. By CNBr cleavage of fragment PF-I two peptides (C-1 and C-2) were obtained with molecular weights of about 5900 (C-2) and 12400 (C-1) on the basis of amino acid and carbohydrate analyses. Only the smaller (N-terminal) peptide is adsorbed to BaSO4 and, since the ability of the whole protein to bind to BaSO4 is known to be absent in samples obtained from patients treated with vitamin K antagonists, this peptide probably contains the site of a modification to the structure of the protein which occurs during biosynthesis and depends on vitamin K. This peptide does not contain hexosamine or sialic acid.
Membrane-bound dd-carboxypeptidases from Bacillus megaterium KM. General properties, substrate specificity and sensitivity to penicillins, cephalosporins and peptide inhibitors of the activity at pH5
Authors: Diaz-Mauriño Teresa , Nieto Manuel , Perkins Harold R.
Published: August 2015

1. The membrane from Bacillus megaterium KM contained a dd-carboxypeptidase with optimum activity under the following conditions: pH5.2, bivalent cation, 3mm; ionic strength, 40mm; temperature, 35°C. It was inactivated by treatment with p-chloromercuribenzoate but was fairly insensitive to 2-mercaptoethanol. 2. The enzyme was inhibited by penicillins and cephalosporins. The inhibition of this enzyme was partially reversed on dialysis but 0.2m-2-mercaptoethanol could neither prevent nor reverse the inhibition. 3. The enzyme was extremely sensitive to changes in the configuration and size of the side chain of the C-terminal dipeptide of the substrate. An aliphatic side chain of a well-defined length and polarity was required in the residue that precedes the C-terminal dipeptide. 4. The enzyme was inhibited by a wide range of analogues of the peptidic portion of the natural substrate.
The role of ribosomal ribonucleic acid in the structure and function of mammalian brain ribosomes
Authors: Grove Barbara Kay , Johnson Terry C.
Published: August 2015

In order to resolve the functional role of intact rRNA in polypeptide chain elongation mouse brain ribosomes were treated with dilute pancreatic or T1 RNAase (ribonuclease). After RNAase treatment, several physical–chemical properties as well as the functional activity of the ribosomes were measured. RNAase treatment resulted in the extensive hydrolysis of both 18S and 28S rRNA; however, the sedimentation properties of mono-ribosomes were unaltered and more than 90% of the relatively low-molecular-weight RNA fragments remained associated with ribosome particles. Analysis of the ability of RNAase-treated ribosomes to participate in cell-free protein synthesis showed that ribosomes with less than 2% intact rRNA retained more than 85% of their activity in polyphenylalanine incorporation. Proof that the incorporation of phenylalanine by ribosomes with hydrolysed rRNA actually represented active translocation was obtained by the effective inhibition of incorporation by diphtheria toxin. In addition, the oligopeptide products of protein synthesis could be identified by BD (benzoylated diethylaminoethyl)-cellulose column chromatography. Analysis of the size distribution of oligopeptides synthesized by normal and RNAase-treated ribosomes showed no significant differences which indicated that there was no change in the proportion of ribosomes engaged in protein synthesis. Thus strong RNA–protein and protein–protein interactions must serve to maintain the functional integrity of ribosomes even when the rRNA is extensively degraded. The ability of the enzyme-treated ribosomes to efficiently incorporate amino acids clearly demonstrated that ‘intact’ rRNA is not required for protein-synthetic activity.
Topography of Escherichia coli ribosomal proteins. The order of reactivity of thiol groups
Authors: Bakardjieva Anastasia , Crichton Robert R.
Published: August 2015

1. 30S and 50S ribosomal subunits of Escherichia coli were treated with N-[2,3-14C]-ethylmaleimide and iodo[14C]acetamide. 2. The proteins in the native subunits which reacted with the reagents were S1,‡ S2, S12, S13, S18, S21, L2, L5, L6, L10, L11, L15, L17, L20, L26+28 and L27. 3. Several proteins, such as S1, S12, S14, S18, L2, L6, L10, L11 and either L26 or 28, had thiol groups in an oxidized form and reacted to a greater extent after reduction with β-mercaptoethanol or dithiothreitol. 4. The total number of thiol groups in 30S and 50S subunits was determined as 16–17 and 26–27 respectively. The total number of thiol groups in each ribosomal protein was also determined. 5. The reaction of 30S and 50S subunits with iodoacetamide under several different conditions established the order of reactivity of thiol groups.
The amino acid sequence of plastocyanin from French bean (Phaseolus vulgaris)
Authors: Milne P. R. , Wells J. R. E. , Ambler R. P.
Published: August 2015

The amino acid sequence of the plastocyanin from French bean (Phaseolus vulgaris) was determined. The protein consists of a single polypeptide chain of 99 residues, and the sequence was determined by characterization of CNBr, tryptic, chymotryptic and thermolysin peptides. When the sequence is compared with that from the plastocyanin of the unicellular green alga Chlorella fusca, the French-bean protein shows the deletion of the N-terminal residue, a two residue insertion and 53 identical residues. Detailed evidence for the sequence of the protein has been deposited as Supplementary Publication SUP 50037 (16pp., 1 microfiche) at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5.
Completion of the analysis of the primary structure of the variable domain of a homogeneous rabbit antibody to type III pneumococcal polysaccharide
Authors: Jaton Jean-Claude
Published: August 2015

The amino acid sequence between residues 70 and 116 of the V (variable) region of the H (heavy) chain derived from rabbit antibody BS-5, specific for type III pneumococcal polysaccharide, was determined. The sequence of this section of the H chain which includes the hypervariable residues 94 to about 112 was unique, although minor variant sequences present in the H chain preparation would not have been detected by the techniques used in this work. Taken together with the known sequences of the N-terminal 69 residues of H chain BS-5 (Jaton & Braun, 1972) and of the V region of the light chain (Jaton, 1974b), the data establish the complete sequence of the V domain of a rabbit immunoglobulin G. The V region of H chain BS-5 is compared with the basic sequences of the three human V region subgroups known to date, with one mouse H chain, and with guinea-pig pooled H chains. Even though chains from guinea pig and mouse clearly belong to the subgroup III of variability (VHIII), rabbit H chain BS-5 (allotypic variant a1) appears more closely related to the subgroup VHII than to the subgroups VHIII or VHI. The homology between VL and VH regions of antibody BS-5 (28%) is not greater than that observed between the VH region of antibody BS-5 and the VL regions of different rabbit antibodies.
Androgen responsiveness of the liver of the developing rat
Authors: Gustafsson J-Å
Published: August 2015

The activities of the hepatic microsomal 2α-, 2β-, 7α- and 18-hydroxylase systems active on 5α-[4-14C]androstane-3α,17β-diol were studied in male and female rats which had been castrated at birth and at the age of 7, 13, 21, 27, 34, 43 and 55 days, treated for 5 days with 2mg of testosterone propionate/kg body weight and killed 6 days after castration. The 7α-hydroxylase system was affected very little by androgen treatment at all stages during development. On the other hand it was found that the rat liver passed through three phases during development with respect to androgen responsiveness as judged by changes in the activities of the 2α, 2β- and 18-hydroxylase systems: a first phase (from the neonatal period up to about 19 days of age) with a relative androgen unresponsiveness in both male and female rats, a second phase (from about 27 to about 33 days of age) when male and female rats responded equally well to androgens and a final phase (from about 40 days of age) with a successively decreasing androgen responsiveness in female rats but with a retained responsiveness in male rats. The hypothesis is presented that neonatal imprinting of the liver by testicular androgen(s) determines the development and degree of androgen responsiveness of liver tissue in the rat.
Isolation of microvillus plasma membranes from suckling-rat intestine. The influence of premature induction of digestive enzymes by injection of cortisol acetate
Authors: Galand G , Forstner G G
Published: August 2015

1. Cortisone administration to suckling rats leads prematurely to induction of enzymes of the intestinal microvillus plasma membrane and lengthening of the intestinal microvilli. To investigate the membrane changes that might be involved, a method for the isolation of a fraction enriched with microvillus plasma membrane was developed in suckling rats. Plasma-membrane fractions were compared from 13-day-old control rats and from 13-day-old rats given cortisol acetate by subcutaneous injection for 3 days. 2. After cortisol injection, the activity of maltase, trehalase, sucrase and leucyl β-naphthylamidase increased markedly, and to the same extent, in intestinal homogenates and plasma-membrane preparations. Purification, and recovery of five marker enzymes with respect to homogenate activity, and recovery of protein, were similar for both membrane preparations, particularly after correction for non-membrane activity, which was high in suckling rats and affected by cortisol. 3. In material released from the plasma membrane by digestion with papain, maltase protein was increased after cortisol injection at least as much as maltase activity. Sucrase activity increased at least 200-fold, and this increase was associated with the appearance of a new sucrase band on polyacrylamide-gel electrophoresis. 4. Sodium dodecyl sulphate electrophoresis of plasma-membrane proteins revealed at least four additional macromolecules after cortisol injection. Concurrently several proteins disappeared from the plasma membrane. The added proteins appeared in the main to be removed from the plasma membrane by papain, whereas the deleted proteins were in the papain-resistant fraction. 5. Enzymic stimulation induced by cortisol acetate in the suckling-rat plasma membrane therefore appears to involve the addition of new proteins, rather than activation of proteins in situ. Deletion of proteins from the membrane during induction of hydrolytic enzymes may reflect other phenomena such as protein reorganization associated with the change in microvillus shape.
The effect of adrenocorticotrophin on protein degradation in rat adrenal gland and liver
Authors: Canick J A , Villee D B
Published: August 2015

The rate of adrenal protein degradation appears to be slower in rats to which ACTH (adrenocorticotrophin) has been chronically administered. As measured by the exponential decay of radioactively labelled adrenal protein in vivo, the mean half-lives of total protein and of mitochondrial, microsomal and 18000g-supernatant protein were significantly longer in ACTH-treated animals. Experiments in which either [3H]leucine or NaH14CO3 was used to label proteins showed that of the fractions studied, the effect on mitochondrial protein degradation was most pronounced. The half-lives of the same subcellular fractions in rat liver were not affected by ACTH. The possibility that the results might have been caused by changes in radioisotope reutilization and pool size is discussed.
The effect of storage on enzyme activities in tissues
Authors: Cryer A , Bartley W
Published: August 2015

The stability to storage at −20°C of 29 enzymes of rat tissues was measured for periods of up to 100 days. The enzymes could be divided into four groups: (1) those that increased in activity with storage; (2) those that showed transient rises subsequently declining to the original activity or below; (3) those that showed no significant change; (4) those that declined in activity. Details of methods used and of results obtained have been deposited as Supplementary Publication SUP 50038 (19 pages) at the British Library (Lending Division) (formerly 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. (1973), 131, 5.
Mobilization of granulose in Clostridium pasteurianum. Purification and properties of granulose phosphorylase
Authors: Robson R L , Morris J G
Published: August 2015

1. The granulose of Clostridium pasteurianum ATCC 6013 is degraded when the organism is incubated in a medium containing no utilizable source of carbon and energy. 2. Mobilization of the polyglucan does not occur in the presence of exogenous glucose. 3. Breakdown of granulose is effected by a constitutively synthesized α-1,4-polyglucan phosphorylase. 4. Partial (530-fold) purification of this granulose phosphorylase was facilitated by its being loosely bound to the native granules of its substrate polyglucan. 5. The enzyme (pH optimum 6.4) was assayed both (a) in the degradative direction, Km for Pi=2.2mm, and (b) in the synthetic direction, Km for glucose 1-phosphate=0.05mm. No requirement for bivalent cations was evidenced. 6. Granulose phosphorylase was inhibited by various nucleotide sugars; GDP-glucose, ADP-glucose (Ki=20μm) and UDP-glucose (Ki=60μm) were particularly potent competitive inhibitors. ATP, NADP+and NADPH (at 1mm) were less effective inhibitors, whereas AMP was slightly stimulatory. 7. It would appear that granulose mobilization is favoured under conditions of low adenylate energy charge, but is prevented under conditions of ‘glucose excess’ chiefly by ADP-glucose-mediated inhibition of granulose phosphorylase.
The binding of 8-anilinonaphthalene-1-sulphonate to cytoplasmic aspartate aminotransferase from pig heart
Authors: Harris H E , Bayley P M
Published: August 2015

Anilinonaphthalenesulphonate binds to cytoplasmic aspartate aminotransferase with high affinity (Kd about 10 muM) and with a stoicheiometry of one molecule per dimer. It is not displaced by aliphatic or aromatic dicarboxylate substrate analogues. The enzyme is believed to be a symmetrical dimer with identical subunits; it can evidently function asymmetrically in binding anilinonaphthalenesulphonate.
The nature of inactivation of rat muscle 5'-adenylate aminohydrolase by fluorodinitrobenzene
Authors: Raggi A , Bergamini C , Ronca G
Published: August 2015

1. The inactivation of rat skeletal muscle AMP deaminase by Dnp-F (1-fluoro-2,4-dinitrobenzene) is accompanied by the arylation of thiol, amino and phenolic hydroxyl groups. 2. The number of thiol groups that react with Dnp-F is about 12; this is the number that reacts with Nbs2 [5,5′-dithiobis-(2-nitrobenzoic acid)] and N-ethylmaleimide without loss of enzyme activity, and it appears to be the same thiol groups that all three reagents attack. 3. Dinitrophenylation of these reactive SH groups is not the cause of inactivation, since active N-ethylmaleimide-substituted enzyme is also inactivated by Dnp-F.4. Complete inactivation of the N-ethylmaleimide-treated AMP deaminase occurs when about six tyrosine and two lysine residues are dinitrophenylated. 5. Since the treatment of Dnp-enzyme with 2-mercaptoethanol restores much of the enzyme activity, inactivation of AMP deaminase by Dnp-F is probably largely due to modification of tyrosine residues. 6. The kinetic properties of the Dnp-enzyme indicate that a marked decrease in V occurs only after extensive enzyme modification. The decreased activity after slight inactivation results from modification of Km.
Isolation and chemical characterization of collagen in bovine pulmonary tissues
Authors: Francis G , Thomas J
Published: August 2015

1. The contents of the fibrous proteins collagen and elastin in the pleural and parenchymal regions of bovine lungs were determined. The collagen content was approx. 70% (g/100g of salt-extracted defatted powder) in each tissue, and the elastin content was 28% in pleura and 13.5% in parenchyma. 2. Purification of the insoluble collagen from the pleura and parenchyma of bovine lungs by various methods was attempted. The collagen fractions isolated after incubation of the pulmonary tissues with the proteolytic enzymes collagenase (“collagenase-soluble” fraction) or pancreatic elastase (“elastase-insoluble” fraction) each contained approx. 87% of the total collagen initially present. 3. Both collagen fractions were chemically analysed for their amino acid and carbohydrate contents and were found to be similar to those of the intact interstitial collagens isolated from skin, bone and tendon. 4. The contents of the two aldimine cross-linking compounds, dehydrohydroxylysinonorleucine and dehydrodihydroxylysinonorleucine, were determined in the bovine pulmonary collagen fractions, and were found to decrease with increasing age of the animals, and were similar to the values found in intact collagens from bone and tendon.
Fluorine as a hydroxy analogue. Stereospecific phosphorylation of 2-deosy-2-fluoroglycerol by glucerol kinase
Authors: Briley P A , Eisenthal R , Harrison R
Published: August 2015

Glycerol kinase catalyses the phosphorylation of the symmetrical substrate, 2-dexoy-2-flurooglycerol, by ATP to an asymmetric product, 2-deoxy-2-fluoro-sn-glycerol 3-phosphate. The stereospecificity of the enzymic reaction was extablished by unambiguous chemical synthesis of 2-deoxy-2-fluoro-sn-glycerol labelled with 2H at C-1, followed by glycerol kinase-catalysed phosphorylation and isolation of the labelled phosphate. The configuration of the 2H-labelled phosphate was determined by n.m.r. spectroscopy. This enzymic phosphorylation of 2-dexoy-2-fluoroglycerol is absolutely stereospecific in the same sence as that of glycerol, with fluorine replacing the C-2 hydroxy group. The behaviour of fluorine as a hydroxy analogue in directing the stereospecific course of the enzyme reaction is relevant to the use of the fluorine atom of fluoro analogues of substrate as a reporter group for hydroxy-binding sites of enzymes.
Evolutionary variation between a monomer and a dimer arginine kinase. Purification of the enzyme from Holothuria forskali and a comparison of some properties with that from Homarus vulgaris
Authors: Anosike E O , Moreland B H , Watts D C
Published: August 2015

1. A purification procedure for the dimeric arginine kinase of the sea cucumber Holothuria forskali is described. 2. The enzyme has a mean molecular weight of 77250 and is composed of two equal, dissociable subunits. 3. It also shows co-operativity between substrate binding at one catalytic site to a much greater extent than the nomomeric lobster arginine kinase for which such co-operativity could not be detected unambiguously. The constants for substrate binding are reported assuming that the enzyme follows rapid-equilibrium random kinetics. From a comparison with other species, the development of co-operativity between the nucleotide- and guanidine-binding sites on one subunit is suggested to have occurred more than once in the evolution of the phosphagen kinases and is not dependent on subunit aggregation. 4. Both enzymes show similar pH profiles for thermal inactivation at 22 degrees C and have very similar stabilities. Above 40 degrees C the dimeric enzyme is much more stable than the monomer. Rate constants for heat inactivation and Arrhenius activation energies are reported. 5. The dimeric enzyme is also more stable to urea inactivation. Substrates and argininic acid all improve the stability of both enzymes. The effects of individual substrates are more distincitive with the dimeric enzymes and increase its stability to an extent that makes it about as stable as dogfish creatine kinase. In the physiological range dimerization does not seem to confer any particular advantage with respect to stability over the monomer form.
Purification and properties of the pyruvate kinase of sturgeon muscle
Authors: Randall R F , Anderson P J
Published: August 2015

Pyruvate kinase was purified from sturgeon muscle in yeilds comparable with those obtained from the muscles of other species. In contrast with mammalian muscle pyruvate kinase the enzyme from sturgeon muscle gives a sigmoidal velocity curve with respect to phosphoenolpuruvate saturation, is activated by fructose 1.6-diphosphate, and is inhibited by bivalent copper ions. In these respects it is similar to the enzyme isolated from mammalian liver. The degree of interaction between phosphoenolpyruvate-binding sites is dependent on temperature.
Sterol biosynthesis in the echinoderm Asterias rubens
Authors: Smith A G , Goad L J
Published: August 2015

1. [2(-14)C]Mevalonic acid injected into the echinoderm Asterias rubens (Class Asteroidea) was effectively incorporated into the non-saponifiable lipid. 2. The most extensively labelled compounds were squalene and the 4,4-dimethyl sterols with much lower incorporations into the 4α-monomethyl and 4-demethyl sterol fractions. 3. Labelled compounds identified were squalene, lanosterol, 4,4-dimethyl-5α-cholesta-8,24-dien-3β-ol and 4α-methyl-5α-cholest-7-en-3β-ol; these are all intermediates in sterol biosynthesis. 4. The major sterol in A. rubens, 5α-cholest-7-en-3β-ol, was also labelled showing that this echinoderm is capable of sterol biosynthesis de novo. 5. No evidence was obtained for the incorporation of [2(-14)C]mevalonic acid into the C28 and C29 components of the 4-demethyl sterols or 9β,19-cyclopropane sterols found in A. rubens and it is assumed that these sterols are of dietary origin. 6. Another starfish Henricia sanguinolenta also incorporated [2(-14)C]mevalonic acid into squalene and lanosterol. 7. Various isolated tissues of A. rubens were all capable of incorporation of [2(-14)C]mevalonic acid into the nonsaponifiable lipid. With the body-wall and stomach tissues radioactivity accumulated in squalene and the 4,4-dimethyl sterols, but with the gonads and pyloric caecae there was a more efficient incorporation of radioactivity into the 4-demethyl sterols, principally 5α-cholest-7-en-3β-ol.
Induction of dopa (3,4-dihydroxyphenylalanine) decarboxylase in blowfly integument by ecdysone. A demonstration of synthesis of the enzyme de novo
Authors: Fragoulis E G , Sekeris C E
Published: August 2015

The activity of the enzyme dopa (3,4-dihydroxyphenylalanine) decarboxylase, present in the epidermis cells of blowfly larvae, increases during the late third instar under the influence of the steroid hormone, ecdysone. By using the double-labelling technique and immune precipitation with univalent antibody to dopa decarboxylase, we demonstrated that the increase in enzyme activity was due to a stimulation of synthesis of enzyme molecules de novo. In this respect, the action of ecdysone is similar to the action of other steroid hormones.
Albumin, fibrinogen and transferrin synthesis in isolated rat hepatocyte suspensions. A model for the study of plasma protein synthesis
Authors: Jeejeebhoy K N , Ho J , Greenberg G R , Phillips M J , Bruce-Robertson A , Sodtke U
Published: August 2015

A system using hepatocyte suspensions in vitro was developed for studying the synthesis of albumin, fibrinogen and transferrin. Conditions for optimum survival of the hepatocyte and for synthesis of these plasma proteins were defined for this system. These conditions included the use of horse serum (17.5 percent, v/v, heat-inactivated), an enriched medium (Waymouth's MB 752/1), an O2 tension of between 18.7 times 10(3) and 26.7 times 10(3) Pa and constant stirring. Albumin, fibrinogen and transferrin synthesis rates were obtained of 0.32 p 0.094(10), 0.12 p 0.030(11) and 0.097 p 0.017(10) [mean p S.D. (n)]mg/h per g of hepatocytes respectively. These rates were maintained for the first 12h of study and synthesis continued at a diminished rate up to 48h. The synthesis of albumin was decreased in a medium containing less amino acids and glucose, but that of fibrinogen was substantially unaffected. ATP concentrations up to 12h and RNA/DNA ratios up to 24h were comparable with values in vivo. The ability to study cells up to 48h permitted us to find that the addition of a mixture of hormones consisting of glucagon, cortisol, tri-iodothyronine and growth hormone enhanced fibrinogen synthesis. Addition of insulin to the above mixture resulted in increased synthesis for albumin and transferrin but not for fibrinogen.
Electrophoretic and centrifugation behaviour of mitochondrial ribonucleic acid from Walker 256 carcinosarcoma
Authors: González-Cadavid N F , Pérez J L
Published: August 2015

To investigate the possibility that mitochondrial transcription could be altered in tumours we started by characterizing the RNA obtained from mitochondria, isolated from Walker carcinosarcoma and purified by a procedure devised to compensate for the lower size and density of these organelles in 10-day tumours. The RNA was extracted by the ‘hot phenol’ technique and analysed by electrophoresis in 2.7 and 2.5% polyacrylamide gels at different running times, identifying the usual cytoplasmic contaminants 28 and 18S peaks plus the other five major peaks at 40, 20.5, 16.3, 15.4, and 4Se. The 28 and 18Se peaks were not eliminated by digitonin treatment of the mitochondria, indicating that they arise from cytoplasmic ribosomes tightly associated with the mitochondria. From its sensitivity to DNAase (deoxyribonuclease), resistance to RNAase (ribonuclease) and coincidence with external marker DNA, the 40Se peak was identified as containing mainly DNA. Sucrosegradient centrifugation for different periods showed a major component at 16.2S, the 28 and 18S cytoplasmic RNA species, peaks at 13.8, 6.4 and 4S and a small 19.5S peak. By polyacrylamide-gel electrophoresis of the purified RNA classes separated by one or two cycles of centrifugation, the following correlation were established: 20.5Se19.5S; 16.3Se16.2S; 15.4Se13.8S. The 6.4S RNA ran as a mixture of 4 and 4.7Se species. When the 20.5Se and 15.4Se RNA species were centrifuged, they behaved as 16.2S and 13.8S respectively, thus suggesting that the 16.2S (16.3Se) arises by cleavage from the 19.5S(20.5Se), the 13.8S (15.4Se) being the other RNA from mitochondrial ribosomes.
Biosynthesis of serum albumin in rat liver. Isolation and probable structure of ‘proalbumin’ from rat liver
Authors: Quinn P S , Gamble M , Judah J D
Published: August 2015

1. Two methods are described for the preparation of ‘proalbumin’ in good yields from rat liver. 2. One of the methods does not depend on the use of specific antisera. 3. The product from both methods is identical as judged by electrophoresis on polyacrylamide gel, isoelectric focusing on pH gradients, ion-exchange chromatography and quantitative immunoelectrophoresis. The protein also appears to be radiochemically pure by these criteria. 4. The protein is free from serum albumin as judged by isoelectric focusing and co-chromatography on ion-exchange columns. It is judged to be free from other proteins by these same criteria and by specific precipitation with antibody. 5. It is converted into serum albumin by limited tryptic hydrolysis. The albumin so produced has the same N-terminal (glutamic acid) and C-terminal (alanine) amino acids as reported for rat serum albumin. 6. A hexapeptide is liberated from the N-terminal end of ‘proalbumin’ simultaneously. It contains three arginine, one phenylalanine, one valine and one glycine residues.