bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective elution of rodent glutathione S-transferases and glyoxalase I from the S-hexyglutathione-Sepharose affinity matrix
Authors: Hayes J D
Published: August 2015

1. The major hepatic glutathione S-transferases (GSTs) from gerbil, guinea-pig, hamster, mouse and rat comprise Ya- (Mr 25,500-25,800), Yb- (Mr 26,100-26,400), Yc- (Mr 27,000-27,500) and Yf- (Mr 24,800) type subunits. 2. In all rodent species the GST subunits possess characteristic affinities for S-hexyglutathione-Sepharose and are eluted at distinct positions when a gradient of counter-ligand is employed to develop this affinity gel. The enzymes that bind to this matrix can be eluted, according to their subunit composition, in the order Ya-, Yc-, Yf- and Yb-containing GST; glyoxalase I, also retained by S-hexylglutathione-Sepharose, is eluted after the major GST YbYb peak. 3. Conditions are also described for the isocratic affinity elution of S-hexylglutathione-Sepharose that allow rat GST to be divided into four separate fractions (pools 1-4). A further fraction (pool 5) can be prepared from material that does not bind S-hexylglutathione-Sepharose and is obtained by chromatography on glutathione-Sepharose. 4. The sequential use of S-hexylglutathione-Sepharose and glutathione-Sepharose has facilitated the isolation of novel GSTs by enriching the various affinity-purified fractions with different subunits. This strategy allowed the Yk (Mr 25,000) and Yo (Mr 26,500) subunits from rat testis as well as Y1 (Mr 25,700) from rat kidney to be rapidly purified. 5. The binding properties of GST subunits for S-hexylglutathione-Sepharose have been compared with their Km values for GSH. The elution order from this matrix is inversely related to the Km value. The GSTs that do not bind to S-hexylglutathione-Sepharose have considerably higher Km values for GSH (i.e. greater than 2.0 mM) than do those enzymes that readily bind to the affinity gel (i.e. 0.13-0.77 mM). GST YkYk and YoYo, which have weak affinities for S-hexylglutathione-Sepharose, possess intermediate Km values for GSH of 1.0 and 1.2 mM respectively.
Characterization and N-terminal sequence of human platelet proteoglycan
Authors: Périn J P , Bonnet F , Maillet P , Jollès P
Published: August 2015

Human platelet proteoglycan (P.PG) was prepared from a 4 M-guanidinium chloride platelet extract in the presence of proteinase inhibitors. The purification procedure included CsCl-density-gradient centrifugation, DEAE-Sepharose CL-6B ion-exchange chromatography and f.p.l.c. on a Mono Q HR 5/5 column. P.PG was recovered as a polydisperse molecule, but the protein core appeared to be at least 90% homogeneous. This observation could be due to partial proteolysis of the core protein during extraction. The N-terminal sequence of the human P.PG core protein was determined up to residue 66 and was shown to be highly homologous to the propeptide of an embryonic rat yolk-sac tumour proteoglycan (PG19); the significance of this homology is discussed.
The small dermatan sulphate proteoglycans synthesized by fibroblasts derived from skin, synovium and gingiva show tissue-related heterogeneity
Authors: Larjava H , Heino J , Krusius T , Vuorio E , Tammi M
Published: August 2015

Dermatan sulphate proteoglycans (DSPGs) synthesized in the presence of 35SO4 were characterized in culture media of fibroblast lines obtained from skin, synovium, and gingiva. The molecular mass of DSPG varied from 95-130 kDa as estimated by SDS/polyacrylamide-gel electrophoresis. Gingival fibroblasts constantly produced larger DSPGs than skin fibroblasts. This was due to the larger dermatan sulphate (DS) chains, which also showed tissue-related heterogeneity in the distribution of 4- and 6-sulphated disaccharide units. The N-glycosylated cores (44 and 47 kDa) obtained following chondroitinase ABC treatment were of identical size in all tissues. The cores from the different tissues were also of the same size (38 kDa) when addition of the N-linked oligosaccharides was inhibited by tunicamycin or when they were removed by N-glycanase treatment. No evidence for low-molecular-mass sulphated oligosaccharides was found. All tissues contained two mRNA species (1.6 and 1.9 kb) for the DSPG core protein. These data suggest that the pattern of transferase activities involved in the construction of DS chains differs from one tissue to another. This variation may modulate the functions of DSPG in the extracellular matrix.
Purification and characterization of the core-specific lectin from human serum and liver
Authors: Colley K J , Beranek M C , Baenziger J U
Published: August 2015

A lectin that displays specificity for the core region of asparagine-linked oligosaccharides (Man3GlcNAc2-Asn) was isolated from human serum and liver by affinity chromatography on mannan-Sepharose. The designation ‘core-specific lectin’ (CSL) is used to indicate its specificity. Selective elution of human CSL from mannan-Sepharose was accomplished with 50 mM-mannose. Two additional proteins that displayed Ca2+-dependent binding to mannan-Sepharose were eluted by mannose 6-phosphate or beta-glycerophosphate but not by mannose. The latter proteins were identified as C-reactive protein and serum amyloid protein. Human CSL isolated from liver was indistinguishable from serum CSL in its physicochemical properties, immunological properties and specificity. The N-terminal sequence of human CSL is homologous to that reported for ‘mannan-binding protein C’ (MBP-C) [Drickamer, Dordal & Reynolds (1986) J. Biol. Chem. 261, 6878-6887]. The amino acid composition of human CSL is similar to that of rat MBP-C, including the presence of hydroxyproline and hydroxylysine residues. Collagen-like sequences with hydroxylated proline and lysine residues appear to be present in human CSL as well as in rat CSL. The collagen-like regions of human and rat CSL may play a role in assembly of CSL subunits into complexes consisting of nine subunits that display Ca2+-dependent carbohydrate-binding activity.
Energetics of renal Na+ and H+/l-alanine co-transport systems
Authors: Jessen H , Vorum H , Jørgensen K E , Sheikh M I
Published: August 2015

The stoichiometric properties of Na+- and H+-dependent L-alanine transporters recently identified in luminal-membrane vesicles prepared from proximal convoluted tubules (pars convoluta) and proximal straight tubules (pars recta) of rabbit kidney were studied. We provide indirect evidence suggesting that one Na+ and one H+ ion are co-transported with the L-alanine molecule via Na+-dependent and H+-dependent transport systems located in vesicles from pars convoluta. Furthermore, our experimental data suggest that both the high-affinity and the low-affinity Na+-dependent L-alanine transport systems of pars recta vesicles operate with a 1:1 stoichiometry.
Inhibition of pancreatic lipase in vitro by the covalent inhibitor tetrahydrolipstatin
Authors: Hadváry P , Lengsfeld H , Wolfer H
Published: August 2015

Tetrahydrolipstatin inhibits pancreatic lipase from several species, including man, with comparable potency. The lipase is progressively inactivated through the formation of a long-lived covalent intermediate, probably with a 1:1 stoichiometry. The lipase substrate triolein and also a boronic acid derivative, which is presumed to be a transition-state-form inhibitor, retard the rate of inactivation. Therefore, in all probability, tetrahydrolipstatin reacts with pancreatic lipase at, or near, the substrate binding or active site. Tetrahydrolipstatin is a selective inhibitor of lipase; other hydrolases tested were at least a thousand times less potently inhibited.
Conserved region of the rat α-lactalbumin promoter is a target site for protein binding in vitro
Authors: Lubon H , Hennighausen L
Published: August 2015

Nuclear extracts from mammary glands of lactating rats contain DNA binding protein(s) with high affinity to the region -125 to -85 of the rat alpha-lactalbumin promoter. The protected sequence is part of a mammary conserved sequence described by Hall et al. [(1987) Biochem. J. 242, 735-742]. Comparison of the DNA protein-binding sites in the 5′ flanking sequence of the rat alpha-lactalbumin and mouse whey acidic protein genes suggests that the consensus sequence 5‘-TGGCARNNWCKKC-3’ is the protein(s) recognition site. This binding site is part of a highly conserved region [consensus sequence, TGGCAGSCTCGGCST(G)YTCTCTCT(NTG)TGGCARA] present in the promoter of four whey protein genes.
Proteolytic dissection as a probe of conformational changes in the human erythrocyte glucose transport protein
Authors: Gibbs A F , Chapman D , Baldwin S A
Published: August 2015

Tryptic digestion has been used to investigate the conformational changes associated with substrate translocation by the human erythrocyte glucose transporter. The effects of substrates and inhibitors of transport on the rates of tryptic cleavage at the cytoplasmic surface of the membrane have confirmed previous observations that this protein can adopt at least two conformations. In the presence of phloretin or 4,6-O-ethylidene-D-glucose, the rate of cleavage is slowed. Because these inhibitors bind preferentially at the extracellular surface of the transporter, their effects must result from a conformational change rather than from steric hindrance. A conformational change must also be responsible for the effect of the physiological substrate D-glucose, which is to increase the rate of cleavage. The regions of the protein involved in the conformational changes include both of the large cytoplasmic regions that are cleaved by trypsin: these are the central hydrophilic region of the sequence (residues 213-269) and the hydrophilic C-terminal region (residues 457-492).
The nucleotide sequence and deduced amino acid sequence of the cytochrome cL gene of Methylobacterium extorquens AM1, a novel class of c-type cytochrome
Authors: Nunn D N , Anthony C
Published: August 2015

The nucleotide sequence and deduced amino acid sequence of the cytochrome cL of Methylobacterium extorquens (Pseudomonas AM1; Methylobacterium AM1) shows that this cytochrome c is completely different, except for its haem-binding site, from all other cytochromes.
Myeloperoxidase oxidation states involved in myeloperoxidase-oxidase oxidation of thiols
Authors: Svensson B E
Published: August 2015

The changes in the oxidation state of the leucocyte enzyme myeloperoxidase, induced by buffer and thiols, were studied with visible-light-absorption spectroscopy. It was concluded that phosphate buffer contains small amounts of H2O2 and that thiols, when added to buffer, induce the generation of minute amounts of superoxide radical anion. These minute amounts of reduced oxygen species are suggested to account for the initiation of myeloperoxidase-oxidase oxidation of thiols. Myeloperoxidase was found to be in its Compound III oxidation state during myeloperoxidase-oxidase oxidation of thiols. However, myeloperoxidase-mediated oxidation of thiols with concomitant O2 consumption can also occur with myeloperoxidase in its Compound II oxidation state. These studies indicate that the ferro and Compound III oxidation states may not be essential intermediates in myeloperoxidase-oxidase oxidation of thiols, but rather that the formation of the Compound III oxidation state retards the reaction.
Circular-dichroism studies on two murine serum amyloid A proteins
Authors: McCubbin W D , Kay C M , Narindrasorasak S , Kisilevsky R
Published: August 2015

C.d. studies have shown that mouse SAA2 (serum amyloid A2) protein has about one-half of the alpha-helix content of the SAA1 (serum amyloid A1) analogue (15 as against 32%), although secondary-structure prediction analyses based on sequence data do not suggest such a large difference between the forms. The decreased helical content may be a reflection or indication of a stronger propensity to aggregation of the SAA2 form compared with SAA1. The main elements of secondary structure in both proteins are beta-sheets/turns. Interactions with Ca2+ are accompanied by small losses in alpha-helix content, whereas binding to chondroitin-6-sulphate in the presence of millimolar Ca2+ also decreases the amount of secondary structure. However, SAA2 binding to heparan sulphate increases its beta-sheet structure, whereas with SAA1 secondary structure is not apparently altered by its interaction with heparan sulphate. Computer-generated surface profiles show slight differences in accessibility, hydrophilicity and flexibility between the proteins. Understanding these differences may help to explain why SAA2 is found in amyloid fibrils whereas SAA1 is not. In particular, a stronger tendency to aggregation might be the reason why SAA2 is deposited exclusively in these fibrils.
The calcium ionophore A23187 increases the tight-junctional permeability in rat liver
Authors: Kan K S , Coleman R
Published: August 2015

The effect of an increase in intracellular Ca2+ concentration on tight-junctional permeability in rat liver was studied by using the calcium ionophore A23187. Infusion of 100 microliters of dimethyl sulphoxide containing various amounts of A23187 over 30 min into isolated perfused livers was followed by a pulse of horseradish peroxidase (HRP) under single-pass conditions. The first biliary HRP peak, a measure of junctional permeability, was increased 4-fold with 100 micrograms of A23187. There were, however, no significant effects on bile flow or on aspartate aminotransferase leakage as compared with the control at this dosage, and thus the increase in junctional permeability was occurring without evidence of appreciable cholestatic or hepatocellular damage. Higher dosages of A23187, however, caused not only an increase in HRP peak height but also changes in bile flow and increases in aminotransferase leakage, indicating more extensive effects at these higher dosages. A second peak of HRP secretion, occurring 20-25 min after the HRP pulse, was also elevated approx. 3.5-fold; this may indicate that pinocytosis and transcellular movement of HRP are also increased under these conditions.
Release of soluble resident as well as secretory proteins from HepG2 cells by partial permeabilization of rough-endoplasmic-reticulum membranes
Authors: Strous G J , Van Kerkhof P
Published: August 2015

Secretory proteins migrate from the rough endoplasmic reticulum (ER) to the Golgi complex at different rates. Selective retention of specific proteins to rough-ER membrane constituents could explain this phenomenon. We have permeabilized HepG2 cells with low concentrations of saponin. Release of newly synthesized proteins was studied after brief labelling in the presence of [35S]methionine. The efflux of several secretory proteins was studied at various saponin concentrations; a 2-fold higher saponin concentration was required to release transferrin compared with that required to release albumin and orosomucoid. Glucosidase II, a soluble resident protein of the ER, is released at the same saponin concentration as albumin. Saponin did not destroy the membrane skeleton structure; at the concentrations used, the integral membrane protein G of vesicular-stomatitis virus remained fully associated with the cells.
Transient-time analysis of substrate-channelling in interacting enzyme systems
Authors: Ovádi J , Tompa P , Vértessy B , Orosz F , Keleti T , Welch G R
Published: August 2015

The kinetics of dynamically interacting enzyme systems is examined, in the light of increasing evidence attesting to the widespread occurrence of this mode of organization in vivo. The transient time, a key phenomenological parameter for the coupled reaction, is expressed as a function of the lifetime of the intermediate substrate. The relationships between the transient time and the pseudo-first-order rate constants for the coupled reaction by the complexed and uncomplexed enzyme species are indicative of the mechanism of intermediate transfer (‘channelling’). In a dynamically interacting enzyme system these kinetic parameters are composite functions of those for the processes catalysed by the complex and by the separated enzymes. The mathematical paradigm can be extended to a linear sequence of N coupled reactions catalysed by dynamically (pair-wise) interacting enzymes.
Physiological significance of the marked increased branching of the glycans of human serotransferrin during pregnancy
Authors: Léger D , Campion B , Decottignies J P , Montreuil J , Spik G
Published: August 2015

Human serotransferrin (Tf) presents a microheterogeneity based on the existence of biantennary and triantennary glycans of the N-acetyl-lactosaminic type. By affinity chromatography on a concanavalin A-Sepharose column in well-defined conditions, human serotransferrin isolated from healthy donors was resolved into three carbohydrate molecular variants: Tf-I (less than 1%), Tf-II (17 +/- 2%) and Tf-III (82 +/- 3%) containing two triantennary glycans, one triantennary and one biantennary glycans and two biantennary glycans respectively. In addition, two ‘isomers’ of the triantennary glycans containing the third antenna beta-1,4-linked to the alpha-1,3-mannose residue or beta-1,6-linked to the alpha-1,6-mannose residue were characterized by methylation analysis in the ratio 1:1 in both Tf-I and Tf-II variants. On concanavalin A crossed immuno-affinity electrophoresis, the patterns exhibited by each of the three purified variants or by a mixture of these variants were compared with the patterns given by transferrin present in sera from nonpregnant and pregnant women. The results suggest that the relative proportions of transferrin carbohydrate variants was unchanged when the concentration of transferrin was increased in serum from normal donors, whereas in the serum of pregnant women, especially in the last 3 months of pregnancy, when the serum concentration of transferrin reached 4.5-5 g/l, the relative proportions of the carbohydrate variants Tf-I and Tf-II increased from 1 to 6 +/- 1% and from 17 +/- 2 to 26 +/- 3% respectively while that of Tf-III decreased from 82 +/- 3 to 67 +/- 3%. The binding of the three transferrin carbohydrate variants to the receptor of the syncytiotrophoblast plasma membranes was determined by using Scatchard-plot analysis. The number of binding sites remained constant with an increase in the number of triantennary glycans whereas a decrease up to 6-fold in the affinity constant was observed. Detection of the transferrin-receptor complex by immunoblotting in the presence of non-dissociating detergents revealed the existence of only one type of receptor or of a receptor possessing similar properties involved in the binding of each of the three serotransferrin carbohydrate variants.
Unexpected influence of ionic strength on branched-pathway interactions between β-lactamases and β-halogenopenicillanates
Authors: De Meester F , Matagne A , Dive G , Frère J M
Published: August 2015

Ionic strength strongly influenced the turnover/inactivation ratio in the interaction between beta-halogenopenicillanates and some class A beta-lactamases. This suggested the stabilization of a highly charged intermediate by solvation. Those data could be interpreted on the basis of a reaction pathway where an episulphonium ion was transiently formed. The various mechanisms proposed for explaining the formation of the dihydrothiazine chromophore are discussed.
A truncated v-abl-derived tyrosine-specific tyrosine kinase expressed in Escherichia coli
Authors: Pritchard M L , Rieman D , Feild J , Kruse C , Rosenberg M , Poste G , Greig R G , Ferguson B Q
Published: August 2015

Several biochemical properties of a 43 kDa v-abl-encoded tyrosine-specific protein kinase (p43v-abl) expressed in Escherichia coli were examined. p43v-abl is a fragment of a 60 kDa v-abl-encoded precursor, p60v-abl, and could be generated by limited proteolysis of a purified p60v-abl with trypsin. Tryptic cleavage of p60v-abl was prevented in the presence of ATP. These results suggest that the catalytic kinase domain of v-abl-derived protein can be separated from other (regulatory) domains by limited proteolysis. p43v-abl readily phosphorylated tyrosine residues on several different protein and peptide substrates, including peptides containing only two amino acid residues. However, the local sequence of the tyrosine-containing peptide substrate significantly affected its rate of phosphorylation. Thus the primary structure and local conformation at the tyrosine acceptor site can play an important role in determining the substrate specificity of v-abl-derived kinase. Phosphorylation by p43v-abl requires Mn2+, Co2+ or Mg2+ and exhibits a strong preference for ATP as phosphate donor. Analogues of ATP and the thiol-reactive reagent N-ethylmaleimide inhibited p43v-abl kinase activity. Purified p43v-abl is intrinsically thermolabile (t1/2 = 5 min at 40 degrees C) and phosphorylates glycerol inefficiently (Km = 1.4 M).
Effects of oestrogen on adenylate cyclase system and glucose output in rat liver
Authors: Shima S , Okeyama N , Akamatu N
Published: August 2015

Effects of chronic oestrogen treatment on catecholamine- and glucagon-sensitive adenylate cyclase activity and glucose output in hepatocytes of castrated male rats were studied. In hepatocytes from male intact or castrated rats, the beta-adrenergic agonist isoprenaline did not stimulate adenylate cyclase activity and glycogenolysis, but glucagon markedly stimulated all these activities. Treatment of castrated animals with 17 beta-oestradiol for 7 days led to the appearance of beta-adrenergic-stimulated increases in both cyclic AMP generation and glucose output. The basal, glucagon- or fluoride-stimulated activities of adenylate cyclase of hepatic membranes prepared from oestrogen-treated rats were similar to those of control animals. Treatment with oestrogen did not influence the number or affinity of beta-adrenergic receptors. In hepatic plasma membranes from control rats, GTP failed to decrease the affinity of beta-adrenergic receptors for agonists, whereas the GTP-induced shift was apparently observed in those from oestrogen-treated animals. These results suggest that oestrogen is able to facilitate the coupling of hepatic beta-adrenergic receptors to the enzyme by increasing the effectiveness of receptor-guanine nucleotide regulation.
Increased expression of the gene for the proα 1(IV) chain of basement-membrane procollagen in cultured skin fibroblasts from two variants of osteogenesis imperfecta
Authors: Pihlajaniemi T , McKeon J , Gay S , Gay R , de Wet W J , Myers J C , Prockop D J
Published: August 2015

Fibroblasts from two lethal variants of osteogenesis imperfecta were shown to synthesize increased amounts of type IV procollagen. Previous studies established that one of these variants had a non-functional allele for the pro alpha 2 chain of type I procollagen, whereas the other pro alpha 2(I) allele contained a mutation leading to synthesis of shortened pro alpha 2(I) chains. In the two variants, the relative level of mRNA for pro alpha 1(IV) was 31 and 42% of the level of mRNA for pro alpha 1(I) chains. A value of less than 2% was found for a third lethal and four non-lethal variants of osteogenesis imperfecta. Immunofluorescent staining of fibroblasts from the two variants synthesizing increased amounts of type IV procollagen indicated that a homogeneous population of cells synthesized both type IV and type I procollagen. The results suggest that mutations in the type I procollagen genes that result in osteogenesis imperfecta can be associated with increased expression of the genes for type IV procollagen.
Characteristics of butanol metabolism in alcohol dehydrogenase-deficient deermice
Authors: Alderman J A , Kato S , Lieber C S
Published: August 2015

Deermice lacking the low-Km alcohol dehydrogenase eliminated butan-1-ol, a substrate for microsomal oxidation but not for catalase, at 117 mumol/min per kg body wt. Microsomal fractions and hepatocytes metabolized butan-1-ol also (Vmax. = 6.7 nmol/min per nmol of cytochrome P-450, Km = 0.85 mM; Vmax. = 5.3 nmol/min per 10(6) cells, Km = 0.71 mM respectively). These results are consistent with alcohol oxidation by the microsomal system in these deermice.
The occurrence of disulphide bonds in purified clathrin light chains
Authors: Parham P , Brodsky F M , Drickamer K
Published: August 2015

Three forms of clathrin light chain contain two cysteine residues. These are the predominant brain-specific forms of LCa and LCb and the non-brain form of LCb. After purification in the absence of thiols they contain intramolecular disulphide bonds. The reduced and the oxidized forms show differences in electrophoretic mobility, explaining the variable and heterogeneous patterns observed on electrophoresis. Accessibility of the thiol groups in the free light chains is greater than when they are associated with the heavy chain. In contrast the cysteine residues of the clathrin heavy chain are completely inaccessible in the absence of denaturants and are not found in disulphide bonds. The antigenic properties of the oxidized and the reduced forms of the clathrin light chains are similar, as is their capacity to bind to the clathrin heavy chain. After isolation in the presence of 10 mM-iodoacetamide, the light-chain cysteine residues are fully alkylated. The results are consistent with the reduced form being the native state and the light-chain disulphide bonds an artifact of isolation.
Vanadium nitrogenase of Azotobacter chroococcum. MgATP-dependent electron transfer within the protein complex
Authors: Thorneley R N F , Bergström N H J , Eady R R , Lowe D J
Published: August 2015

The kinetics of MgATP-induced electron transfer from the Fe protein (Ac2V) to the VFe protein (AclV) of the vanadium-containing nitrogenase from Azotobacter chroococcum were studied by stopped-flow spectrophotometry at 23 degrees C at pH 7.2. They are very similar to those of the molybdenum nitrogenase of Klebsiella pneumoniae [Thorneley (1975) Biochem. J. 145, 391-396]. Extrapolation of the dependence of kobs. on [MgATP] to infinite MgATP concentration gave k = 46 s-1 for the first-order electron-transfer reaction that occurs with the Ac2V MgATPAclV complex. MgATP binds with an apparent KD = 230 +/- 10 microM and MgADP acts as a competitive inhibitor with Ki = 30 +/- 5 microM. The Fe protein and VFe protein associate with k greater than or equal to 3 x 10(7) M-1.s-1. A comparison of the dependences of kobs. for electron transfer on protein concentrations for the vanadium nitrogenase from A. chroococcum with those for the molybdenum nitrogenase from K. pneumoniae [Lowe & Thorneley (1984) Biochem. J. 224, 895-901] indicates that the proteins of the vanadium nitrogenase system form a weaker electron-transfer complex.
The 50 kDa protein-actin complex from unfertilized sea-urchin (Strongylocentrotus purpuratus) eggs. Interaction with actin
Authors: Golsteyn R M , Waisman D M
Published: August 2015

In the preceding paper [Golsteyn & Waisman (1989) Biochem. J. 257, 809-815] an EGTA-stable, Ca2+-binding heterodimer comprised of a 50 kDa protein and actin called ‘50K-A’ was identified in the unfertilized eggs of the sea urchin Strongylocentrotus purpuratus. In the present paper we have documented the binding of 50K-A to DNAase I and the effect of 50K-A on the kinetics of actin polymerization. When 50K-A was added to pyrene-labelled rabbit skeletal-muscle actin and the salt concentration increased, the initial rate of actin polymerization was inhibited by a very low molar ratio of 50K-A to actin. Furthermore, the steady-state level of G-actin was increased in the presence of 50K-A, suggesting that 50K-A caps the preferred end of actin polymer, shifting the steady-state concentration to that of the non-preferred end. Dilution of F-actin to below its critical concentration into 50K-A resulted in a much slower rate of depolymerization, consistent with capping of the preferred end. In contrast with the Ca2+-dependent binding to DNAase, the effect of 50K-A on the kinetics of actin assembly and disassembly was Ca2+-independent. These results suggest that 50K-A is a novel actin-binding protein with some similarities to the severin/fragmin/gelsolin family of F-actin-capping proteins.
Comparison of the role of tyrosine residues in human IgG and rabbit IgG in binding of complement subcomponent C1q
Authors: McCall M N , Easterbrook-Smith S B
Published: August 2015

Treatment of covalently cross-linked or heat-aggregated oligomers of human IgG with 4 mM-tetranitromethane abrogated their C1q-binding activity. In contrast, tetranitromethane modification of rabbit IgG oligomers, under identical conditions, had no effect upon their C1q-binding activity. The tetranitromethane treatment led to nitration of about ten tyrosine residues per IgG molecule in both species, and the modification was specific for tyrosine residues. Reduction of the nitrated protein with Na2S2O4 did not lead to recovery of C1q-binding activity in human IgG oligomers or to loss of activity in rabbit IgG oligomers. Tryptic peptides from the nitrated proteins were isolated and a peptide containing nitrotyrosine-319 was recovered from human IgG, as well as peptides from both species corresponding to the region around nitrotyrosine-278. These data are consistent with the inactivation of C1q-binding activity in human IgG being the result of nitration of tyrosine-319; the rabbit IgG is unaffected by nitration because position 319 is phenylalanine. The evidence supports the C1q-receptor site proposed by Burton, Boyd, Brampton, Easterbrook-Smith, Emanuel, Novotny, Rademacher, van Schravendijk, Sternberg & Dwek [(1980) Nature (London) 288, 338-344]: residues 316-338.
The interaction of xylosyltransferase and glucuronyltransferase involved in glucuronoxylan synthesis in pea (Pisum sativum) epicotyls
Authors: Baydoun E A H , Waldron K W , Brett C T
Published: August 2015

A particulate enzyme preparation from etiolated pea (Pisum sativum) epicotyls was found to incorporate xylose from UDP-D-xylose into beta-(1----4)-xylan. The ability of this xylan to act as an acceptor for incorporation of [14C]glucuronic acid from UDP-D-[14C]glucuronic acid in a subsequent incubation was very limited, even though glucuronic acid incorporation was greatly prolonged when UDP-D-xylose was present in the same incubation as UDP-D-[14C]glucuronic acid. This indicated that glucuronic acid could not be added to preformed xylan. However, the presence of UDP-D-glucuronic acid inhibited incorporation of [14C]xylose from UDP-D-[14C]xylose into beta-(1----4)-xylan, and neither S-adenosylmethionine nor acetyl-CoA stimulated either the xylosyltransferase or the glucuronyltransferase.
Bovine milk-fat-globule membrane contains an enzymically inactive form of acetyl-CoA carboxylase
Authors: Shriver B J , Allred J B , Roman-Lopez C R
Published: August 2015

Enzymically inactive acetyl-CoA carboxylase [acetyl-CoA:carbon-dioxide ligase (ADP-forming), EC 6.4.1.2] was found as a component of bovine milk-fat-globule membrane (MFGM). Acetyl-CoA carboxylase was present in MFGM at a higher concentration than in cytosolic or mitochondrial fractions of bovine mammary tissue, which makes it unlikely that its presence was due to simple contamination by these subcellular constituents.
Clavulanate inactivation of Staphylococcus aureus β-lactamase
Authors: Rizwi I , Tan A K , Fink A L , Virden R
Published: August 2015

The interaction of clavulanic acid with beta-lactamase from Staphylococcus aureus was investigated, particularly with a view to determining whether conformational effects are involved. The inactivation at neutral pH is essentially stoichiometric, leading to an inactive species with an enamine chromophore. Two forms of the enamine were observed, the first-formed having a positive ellipticity with a maximum near 290 nm. This species slowly converted into the stable form of the inactivated enzyme that had a negative ellipticity with a minimum at 275 nm. This change in sign of the ellipticity of the enamine is consistent with the previously proposed cis-trans isomerization of the enamine [Cartwright & Coulson (1979) Nature (London) 278, 360-361). Both the far-u.v.c.d. and the intrinsic viscosity of the inactivated enzyme indicated that negligible change in conformation of the enzyme accompanied inactivation. The rates of inactivation and enamine formation were compared at low temperatures, where the initial rates were slow enough to be monitored. The rate of loss of 95% of the catalytic activity was almost 100-fold faster than the rate of formation of the first-formed enamine species. The remaining 5% activity was lost with a rate comparable with that for formation of the initial enamine. The simplest explanation of these results is that a relatively stable acyl-enzyme intermediate builds up initially and more slowly partitions between turnover (hydrolysis) and enamine formation. The initially formed enamine is in the cis conformation but slowly isomerizes to the more stable trans form.
Effects of oxidative stress on some physiochemical properties of caeruloplasmin
Authors: Winyard P G , Hider R C , Brailsford S , Drake A F , Lunec J , Blake D R
Published: August 2015

We report the effects of oxidative stress generated by low-intensity u.v. irradiation (366 and 254 nm), dialysis against ascorbate and isolated stimulated neutrophils on some physicochemical properties of caeruloplasmin. Low-intensity u.v. irradiation resulted in a loss of ferroxidase activity and 610 nm absorption, changes previously reported to occur during storage and manipulation of caeruloplasmin. These alterations were found to correspond to aggregation of the protein, induction of visible fluorescence (excitation, 360 nm; emission, 454 nm), changes in c.d. spectra which were indicative of alterations in protein conformation, loss of half-cystine, tryptophan and tyrosine content and loss immunoreactivity. The changes in the far-u.v. c.d. spectrum of caeruloplasmin were more pronounced than those observed for u.v.-irradiated IgG. Similar c.d. changes and induction of fluorescence were observed following dialysis of caeruloplasmin against ascorbate or exposure to stimulated neutrophils. It is concluded that the lability of caeruloplasmin may arise from oxidative modification, in addition to the previously described susceptibility of this protein to proteolysis.
Characterization of translation systems in vitro from three developmental stages of Strongylocentrotus purpuratus
Authors: Lopo A C , Lashbrook C C , Hershey J W B
Published: August 2015

We have developed and characterized cell-free systems active in translation from unfertilized eggs, 30-min zygotes and hatched blastulae of the sea urchin Strongylocentrotus purpuratus. The ion concentrations selected for preparation of the lysates were 150 mM-K+, 40 mM-Na+, 40 mM-Cl-, 5 x 10(-7) M free Ca2+ and 1 mM free Mg2+. It was necessary to include the ribonuclease inhibitor RNas in the preparations to obtain full activity consistently. The pH optimum was 7.2 and was extremely sharp for the three S. purpuratus lysates. The temperature optima of the three lysates were remarkably similar to those of the intact unfertilized egg and embryos. Lysates from unfertilized egg and 30-min zygotes showed a temperature optimum at 15 degrees C. The hatched blastula lysate showed a broader temperature optimum with a shift to about 20 degrees C. The optimized lysates incorporated radiolabelled amino acids into polypeptides for up to 90 min. The polypeptides synthesized ranged in Mr from 200,000 to 20,000, suggesting that the mRNA in the lysates was intact and capable of directing the synthesis of complete polypeptides. Furthermore, the three lysates were capable of initiation, as demonstrated by inhibition of initiation using the inhibitors edeine and 7-methylguanosine 5′-triphosphate (m7GTP). At 15 degrees C, the transit times for the three lysates were: unfertilized egg, 40 min; 30-min zygotes and hatched blastula lysates, 20 min. These transit times are similar to those of intact eggs and embryos, and significantly, reflect the two-fold increase in elongation rate seen following fertilization in intact embryos. Thus, these lysates display many features and characteristic responses typical of intact eggs and embryos, indicating that the lysates should be useful tools for the analysis of translation control in early embryogenesis.
Evidence for the involvement of a gastrointestinal peptide in the regulation of glucose uptake in the mammary gland of the lactating rat
Authors: Page T
Published: August 2015

1. A method of obtaining serial arterial and mammary-venous blood samples was used to identify possible factors involved in the regulation of glucose uptake in the gland of the lactating rat. 2. Administration of insulin alone increased the arteriovenous glucose difference across the mammary gland of starved rats, but the time course of the recovery could not account for the restoration of arteriovenous glucose difference observed during refeeding [Page & Kuhn (1986). Biochem. J. 239, 269-274]. 3. A crude extract of the gastrointestinal tract (stomach-ileum) from lactating rats enhanced the change in mammary glucose uptake observed with insulin, but only when large amounts (100 munits/rat) of insulin were used. To achieve a similar recovery of arteriovenous glucose difference using near-physiological amounts (5 munits/rat) of insulin it was necessary to sever the mammary nerves. 4. A peptide fraction (of less than 10 kDa) isolated from the gut extract enhanced the effect of insulin in a similar manner to the crude extract. 5. It is suggested that in addition to insulin at least another component, probably a gut peptide, is required for the restoration of mammary glucose uptake during refeeding. An inhibitory component may also contribute to the regulation of mammary glucose extraction in the lactating rat.
T-cell recognition and antigen presentation of myoglobin. Protein recognition by site-specific T-cell clones is influenced by amino acid substitutions outside the site
Authors: Yoshioka M , Atassi M Z
Published: August 2015

Six T-cell clones from SJL mice were prepared from T-cell lines that were obtained by passage with synthetic myoglobin (Mb) peptide 107-120. In addition, a T-cell clone, specific to this region of Mb, was isolated from a Mb-passaged T-cell culture. The proliferative responses of these clones to Mb variants from 14 different species were studied. It was found, as expected, that amino acid replacements within the site affected its recognition by the T-cell clones. In addition to these effects, the T-cell recognition site, like the sites recognized by antibodies, was also influenced by substitutions of residues that are close to site residues in three-dimensional structure but are otherwise distant in sequence. This is noteworthy in view of the fact that six of the clones were selected with a free peptide, and thus the environmental residues are clearly not part of the ‘contact’ residues of the site. These findings are discussed in relation to the presentation of the antigen and are interpreted as indicating that Mb is presented in its intact form to the T-cells in vitro.
A thiono-β-lactam substrate for the β-lactamase II of Bacillus cereus. Evidence for direct interaction between the essential metal ion and substrate
Authors: Murphy B P , Pratt R F
Published: August 2015

An 8-thionocephalosporin was shown to be a substrate of the beta-lactamase II of Bacillus cereus, a zinc metalloenzyme. Although it is a poorer substrate, as judged by the Kcat./Km parameter, than the corresponding 8-oxocephalosporin, the discrimination against sulphur decreased when the bivalent metal ion in the enzyme active site was varied in the order Mn2+ (the manganese enzyme catalysed the hydrolysis of the oxo compound but not that of the thiono compound), Zn2+, Co2+ and Cd2+. This result is taken as evidence for kinetically significant direct contact between the active-site metal ion of beta-lactamase II and the beta-lactam carbonyl heteroatom. No evidence was obtained, however, for accumulation of an intermediate with such co-ordination present.
Unconventional codon reading by Mycoplasma mycoides tRNAs as revealed by partial sequence analysis
Authors: Guindy Y S , Samuelsson T , Johansen T I
Published: August 2015

Continuing our investigation of the tRNA genes and gene products in Mycoplasma mycoides, we report the sequence of the gene for tRNALeu (CAA) as well as partial primary structures of the following tRNAs: Leu (CAA), Leu (UAG), Arg (UCU), Thr (AGU) and Ile (CAU). It is suggested that in M. mycoides, at least some of the family codon boxes are read by only one tRNA each, using an unconventional method which does not discriminate between the nucleotides in the third codon position. M. mycoides is the first free-living organism known to use an unconventional method of this kind.
Stability of the thrombin-thrombomodulin complex on the surface of endothelial cells from human saphenous vein or from the cell line EA.hy 926
Authors: Beretz A , Freyssinet J M , Gauchy J , Schmitt D A , Klein-Soyer C , Edgell C J S , Cazenave J P
Published: August 2015

Protein C activation by alpha-thrombin on the surface of endothelial cells depends on an essential membrane-glycoprotein cofactor, thrombomodulin. In the present study we have monitored the activity of thrombin-thrombomodulin complexes on human saphenous-vein endothelial cells (HSVEC) or on the endothelial cell line EA.hy 926. Cell monolayers were exposed for 5 min to 8.5 nM human alpha-thrombin and then washed to remove unbound thrombin. The cells were then incubated at 37 degrees C for 5-180 min. At the end of the respective incubation periods, purified human protein C (120 nM) was added in order to assay the activity of the thrombin-thrombomodulin complexes present on the cell surface. HSVEC pre-exposed to thrombin retained their full capacity to promote protein C activation up to 90 min after free thrombin was removed. This capacity then decreased slowly to reach 56% of control value after 180 min of incubation. Original activity was 3.8 +/- 0.9 pmol of activated protein C formed/min per ml per 10(6) cells (mean +/- S.E.M., n = 5). The capacity of protein C activation of EA.hy 926 cells remained constant for 120 min after free thrombin was removed, then decreased to 76% of control after 180 min. Original activity was 2.0 +/- 0.4 pmol of activated protein C formed/min per ml per 10(6) cells (mean +/- S.E.M., n = 3). Similar results were obtained with cells fixed with 3% paraformaldehyde. However, during the 5-180 min incubation period, non-fixed cells of both types were capable of significantly internalizing fluorescent acetylated low-density lipoprotein. In the experimental protocol used here, an eventual inhibition of thrombin internalization by protein C can be excluded, as protein C is only added at the end of the incubation period. We conclude that there is no evidence of rapid internalization of thrombin-thrombomodulin complexes on HSVEC or the EA.hy 926 cell line, as assessed by the ability of membrane-bound thrombin to activate protein C.
Involvement of thiol transferase- and thioredoxin-dependent systems in the protection of ‘essential’ thiol groups of ornithine decarboxylase
Authors: Flamigni F , Marmiroli S , Caldarera C M , Guarnieri C
Published: August 2015

Ornithine decarboxylase (ODC), an enzyme with ‘essential’ thiol group(s), may be inactivated in vitro by removal of thiol reducing agents and re-activated by soluble factors from rat liver in the presence of NADPH or GSH. The NADPH- and GSH-dependent reducing systems were separated and resolved into three components, called factors A, B1 and B2, by chromatographic techniques. Factor B1 (Mr 12,000) could reactivate ODC in the presence of GSH and co-purified with thiol transferase activity. Factor B2 (Mr 12,000) and factor A (Mr approx. 110,000) were both needed to re-activate ODC in the presence of NADPH, and co-purified with thioredoxin and thioredoxin reductase activity respectively. In an attempt to investigate the physiological role of the ‘essential’ thiol group(s) of ODC, erythroleukaemia cells were incubated with NN-bis-(2-chloroethyl)-N'-nitrosourea, t-butyl hydroperoxide and vinblastine, which are known to increase the cellular GSSG/GSH ratio, azelaic acid, an inhibitor of thioredoxin reductase, and sodium arsenite, a strong inhibitor of the ODC-re-activating factors. All these compounds were able to decrease significantly the ODC activity induced in these cells. These results suggest that the thiol transferase- and thioredoxin-dependent systems may be physiologically relevant in maintaining ODC in the active, reduced, state.
Proteolysis of N-ethylmaleimide-modified aldolase loaded into erythrocyte ghosts: prevention by inhibitors of calpain
Authors: Hopgood M F , Knowles S E , Ballard F J
Published: August 2015

1. When rabbit muscle aldolase labelled with tritium and inactivated by N-ethylmaleimide (NEM) was loaded into erythrocyte ghosts, significant proteolysis of the loaded protein occurred. The major product of this proteolysis, separated by electrophoresis under dissociating conditions, was found to be approx. 2 kDa smaller than the parent protein. 2. Proteolysis was detectable during erythrocyte ghost loading at 0 degrees C, reaching a plateau after approx. 12 min. Subsequent incubation at 37 degrees C to allow resealing of the ghosts resulted in additional proteolysis, and up to 20% of the loaded protein was converted to the smaller 38 kDa derivative. 3. EDTA, EGTA, leupeptin and chymostatin, each inhibitors of calcium-activated neutral proteinases (calpains), were the most effective inhibitors of the proteolysis of NEM-inactivated aldolase in ghosts. Other proteinase inhibitors were ineffective, while phenylmethanesulphonyl fluoride was only partially effective. 4. Inhibition of the proteolysis by EGTA was prevented by CaCl2, supporting the involvement of erythrocyte calpain. 5. Pretreatment of ghosts with EGTA prior to loading of NEM-modified aldolase followed by microinjection of the protein into HeLa cells did not result in a different rate of its overall breakdown to acid-soluble products. EGTA is suggested as a useful agent for the erythrocyte ghost-mediated microinjection of calpain-sensitive proteins.
Metabolic and structural evidence for the existence of a third species of polyphosphoinositide in cells: d-phosphatidyl-myo-inositol 3-phosphate
Authors: Stephens L , Hawkins P T , Downes C P
Published: August 2015

When human 1321 N1 astrocytoma cells were labelled to steady state with [3H]inositol and briefly with [32P]orthophosphate, a compound which contained both radiotracers and which co-migrated with phosphatidylinositol-myo-inositol 4-phosphate during t.l.c. could be extracted in acidic chloroform/methanol. Treatment with methylamine under conditions which lead to deacylation of conventional glycerophospholipids yielded a water-soluble moiety which was labelled with both radioisotopes and was eluted from an anion-exchange h.p.l.c. column with a retention time similar to, but distinct from, that of glycerophosphoinositol 4-phosphate. Experiments using sodium periodate and selective phosphatase enzymes to degrade this compound systematically generated a series of products which suggested the structure of the parent phospholipid was phosphatidyl-myo-inositol 3-phosphate (PtdIns3P). PtdIns3P is metabolically closely related to the pool(s) of inositol phospholipid(s) that serves as substrate(s) for an agonist-sensitive phosphoinositidase C, as the levels of PtdIns3P fell significantly when 1321 N1 cells were stimulated with carbachol. The relative rate of turnover of the inositol moiety of PtdIns3P is similar to that of both of the major polyphosphoinositides and significantly higher than that of total cellular phosphatidyl-myo-inositol. This suggests that all three polyphosphoinositides are synthesized from a common, rapidly metabolized, pool of phosphatidyl-myo-inositol.