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Showing papers in "Biochemical Journal in 1987"


Journal ArticleDOI
TL;DR: It is suggested that a component of protein glycosylation is dependent upon glucose autoxidation and subsequent covalent attachment of ketoaldehydes, and the chemical evidence for the currently accepted 'Amadori' product is consistent with the structure expected for the attachment of a glucose-derived ketoaldehyde to protein.
Abstract: Monosaccharide autoxidation (a transition metal-catalysed process that generates H2O2 and ketoaldehydes) appears to contribute to protein modification by glucose in vitro The metal-chelating agent diethylenetriaminepenta-acetic acid (DETAPAC), which inhibits glucose autoxidation, also reduces the covalent attachment of glucose to bovine serum albumin A maximal 45% inhibition of covalent attachment was observed, but this varied with glucose and DETAPAC concentrations in a complex fashion, suggesting at least two modes of attachment The extent of inhibition of the metal-catalysed pathway correlated with the extent of inhibition of glycosylation-associated chromo- and fluorophore development DETAPAC also inhibited tryptophan fluorescence quenching associated with glycosylation Conversely, ketoaldehydes analogous to those produced by glucose autoxidation, but generated by 60Co irradiation, bound avidly to albumin and accelerated browning reactions It is therefore suggested that a component of protein glycosylation is dependent upon glucose autoxidation and subsequent covalent attachment of ketoaldehydes The process of glucose autoxidation, or ketoaldehydes derived therefrom, appear to be important in chromophoric and fluorophoric alterations It is noted, consistent with these observations, that the chemical evidence for the currently accepted ‘Amadori’ product derived from the reaction of glucose with protein amino groups is consistent also with the structure expected for the attachment of a glucose-derived ketoaldehyde to protein The concept of ‘autoxidative glycosylation’ is briefly discussed in relation to oxidative stress in diabetes mellitus

1,294 citations




Journal ArticleDOI
TL;DR: The results indicate that TGF beta causes a marked enhancement of the production of types I and III collagens and fibronectin by cultured normal human dermal fibroblasts and may contribute to the development of pathological states of fibrosis.
Abstract: It has been previously shown that transforming growth factor beta (TGF beta) is capable of stimulating fibroblast collagen and fibronectin biosynthesis. The purpose of this study was to examine the mechanisms involved in TGF beta stimulation of fibroblast biosynthetic activity. Our results indicate that TGF beta causes a marked enhancement of the production of types I and III collagens and fibronectin by cultured normal human dermal fibroblasts. The rate of collagen production by fibroblasts exposed to TGF beta was 2-3-fold greater than that of control cells. These effects were associated with a 2-3-fold increase in the steady-state amounts of types I and III collagen mRNAs and a 5-8-fold increase in the amounts of fibronectin mRNAs as determined by dot-blot hybridization with specific cloned cDNA probes. In addition, the increased production of collagen and fibronectin and the increased amounts of their corresponding mRNAs remained elevated for at least 72 h after removal of TGF beta. These findings suggest that TGF beta may play a major role in the normal regulation of extracellular matrix production in vivo and may contribute to the development of pathological states of fibrosis.

561 citations


Journal ArticleDOI
TL;DR: The technique has been rapidly developed with the invention of the superior dyes, fura-2 and indo1, and technologies for monitoring [Ca2], in single cells, whole fields of identified cells, and even localized areas within cells, with improvements in time resolution down to the millisecond range.
Abstract: The first measurements of cytosolic free Ca2\", [Ca2\"]1, in living cells were made with aequorin in giant muscle cells (Ridgway & Ashley, 1967) in the late 1960s. During the 1970s two further methods were introduced, bis-azo absorbance dyes (mainly arsenazo III) and calciumselective microelectrodes (Ashley & Campbell, 1979). However, even as late as 1981, [Ca'1] had been reliably measured in very few cell types other than giant cells of invertebrates and the techniques were largely confined to the laboratories of those specialized in excitable cell physiology and familiar with microelectrodes or microinjection and advanced optical or electronic technologies. In 1982 a new generation of fluorescent dyes was introduced, with quin2 (Tsien et al., 1982a) and a chemical trick for loading it non-disruptively into populations of cells of any size (Tsien, 1981). This technique in its basic form is simple enough, and needs only basic laboratory instrumentation, so that it is now routinely used in hundreds oflaboratories. The technique has been rapidly developed with the invention of the superior dyes, fura-2 and indo1, and technologies for monitoring [Ca2\"], in single cells, whole fields ofidentified cells, and even localized areas within cells, and also improvements in time resolution down to the millisecond range. An outline of these developments forms part of this review. Clearly, with hundreds of papers already published we can only point out the major features, advantageous and problematic, of the technique and hope to provide a critical guide to the literature that details the important points. Another development has been the refinement of the use of the calcium sensitive photoproteins aequorin and obelin. Important novel data on microinjected small single cells are now coming from specialist laboratories and the techniques are not as fearsomely difficult as commonly thought. Part of this review is therefore devoted to an explanation of this method, its advantages and pitfalls, and its successes. We will not consider bisazo dyes or calcium-selective microelectrodes. There are several detailed accounts of these techniques and their place in relation to other approaches (e.g. Thomas, 1982; Blinks et al., 1982; Rink, 1983; Tsien & Rink, 1983). Nor will we review the fluorine derivatives of the fluorescent dyes that have provided n.m.r. probes (Metcalf et al., 1985). This review covers the technical considerations of using fluorescent and bioluminescent probes for measuring [Ca2+]1; we make no attempt to cover the extensive and proliferating data obtained from such measurements.

506 citations


Journal ArticleDOI
TL;DR: A cascade mechanism is proposed in which collagenase is activated by stromelysin, which causes a further apparent decrease in the Mr of procollagenase.
Abstract: The latent forms of stromelysin and collagenase from human gingival fibroblasts were purified to homogeneity. These latent proenzymes underwent serial small reductions in Mr upon activation by treatment with either 4-aminophenylmercuric acetate or trypsin. Similar shifts in Mr and activation kinetics were observed upon identical treatments of either recombinant prostromelysin or procollagenase. Prostromelysin showed a lag between activation and Mr fall, suggesting an initial activation by conformational change. Collagenase activity was enhanced up to 12-fold by either natural or recombinant stromelysin in the presence of trypsin or 4-aminophenylmercuric acetate. Stromelysin caused a further apparent decrease in the Mr of procollagenase. Since these important connective-tissue-degrading enzymes are usually co-ordinately produced by cells, a cascade mechanism is proposed in which collagenase is activated by stromelysin.

458 citations


Journal ArticleDOI
TL;DR: Results indicate that depletion is due to an increased rate of degradation, with no change either in mRNA amounts or in rates of polypeptide synthesis.
Abstract: The phorbol-ester-induced loss of protein kinase C that has been documented in many cell types appears to be a critical event in the generation of a cellular refractory state. We have investigated here the synthesis and degradation of the protein kinase C polypeptide in order to determine why its steady-state amounts are depleted in response to phorbol esters. These results indicate that depletion is due to an increased rate of degradation, with no change either in mRNA amounts or in rates of polypeptide synthesis.

437 citations


Journal ArticleDOI
TL;DR: The purpose of this article is to specify the conditions under which Fru-2,6-P2 plays a role in the control of glycolysis, and to review recent studies dealing with Fru’s metabolism in mammalian tissues other than liver.
Abstract: Glycolysis is an ubiquitous metabolic pathway. In addition to its catabolic role, glycolysis also serves an anabolic function by providing C3 precursors for the synthesis of fatty acids, cholesterol and amino acids. It is therefore an amphibolic pathway. The difference in metabolic orientation is tissue-specific and depends on the hormonal and nutritional state. Therefore, it is not surprising that different and specific regulatory mechanisms exist to control glycolysis under these various conditions. Cancer cells are a special case in which glycolysis is abnormally high. Qualitative and quantitative approaches have been developed to elucidate the control mechanisms of glycolysis. On the one hand, so-called 'rate-limiting' steps in the pathway have been identified by qualitative analysis. Reactions displaced far from equilibrium, particularly when catalysed by an allosterically regulated enzyme, have been assumed to qualify for control [1]. One such reaction is catalysed by phosphofructokinase (PFK-1). The experimental evidence supporting the key role of PFK-1 stems from the changes in the concentration of PFK-1 effectors observed when glycolysis is stimulated, e.g. following anoxia [2,3]. However, this information remains qualitative and may lead to an oversimplified view. On the other hand, the quantitative analysis of control, developed by Kacser & Burns [4] and Heinrich & Rapoport [5], allows the distribution of control among all steps in a pathway to be calculated. Such an approach has been applied to glycolysis in erythrocytes [6] and yeast [7], and it indicates that control is mainly distributed between hexokinase and PFK1. The study of the mechanism of action of glucagon on liver gluconeogenesis led to the discovery of fructose 2,6-bisphosphate (Fru-2,6-P2) [8,9]. This sugar phosphate is a potent stimulator of PFK-1 [10-12] and is also an inhibitor of fructose 1,6-bisphosphatase (FBPase-l) [13,14]. Fru-2,6-P2 has been detected in all mammalian tissues studied so far, as well as in fungi and plants, but not in prokaryotes [15]. Earlier reviews have dealt with the effect of Fru-2,6-P2 on its two main targets, PFK-1 and FBPase-1, together with the regulation of its synthesis and breakdown in relation to the control of glycolysis/gluconeogenesis in liver [15-22]. Since Fru-2,6-P2 is present in all mammalian tissues, it is tempting to suppose that it plays the major role in the control of glycolysis. The purpose of this article is to specify the conditions under which Fru-2,6-P2 plays a role in the control of glycolysis, and to review recent studies dealing with Fru-2,6-P2 metabolism in mammalian tissues other than liver.

398 citations


Journal ArticleDOI
TL;DR: The data suggest that Ca2+ and oxidative stress synergistically promote the reversible opening of an inner membrane pore and t-Butylhydroperoxide-induced permeabilization was reversed by EGTA.
Abstract: Rat heart mitochondria became permeabilized to sucrose when incubated with 100 nmol of Ca2+/mg of protein in the presence of Pi. Ca2+ chelation with EGTA restored impermeability to sucrose, which became entrapped in the matrix space. t-Butylhydroperoxide markedly promoted permeabilization in the presence of Ca2+ but not in its absence, and Ca2+-plus-t-butylhydroperoxide-induced permeabilization was reversed by EGTA. The data suggest that Ca2+ and oxidative stress synergistically promote the reversible opening of an inner membrane pore.

342 citations


Journal ArticleDOI
TL;DR: The membrane potential of cytoplasts, derived from human neutrophils, was depolarized by the activation of the superoxide-generating NADPH-dependent oxidase, indicating that the outward translocation of H+ eventually compensates for superoxide generation.
Abstract: The membrane potential of cytoplasts, derived from human neutrophils, was depolarized by the activation of the superoxide-generating NADPH-dependent oxidase. The extent of the depolarization was inhibited by diphenylene iodonium and was therefore due directly to the activity of the oxidase, which must be electrogenic. The extent of the depolarization was influenced by alteration of the delta pH across the cytoplast membrane, indicating that the outward translocation of H+ eventually compensates for superoxide generation. The depolarization of the potential is enhanced by Cd2+, a blocker of H+ currents, suggesting that the compensatory movement is via an H+ channel.

314 citations


Journal ArticleDOI
TL;DR: The enzymes required for these hydrolyses, namely alpha-rhamnosidase and beta-galactosidase, were produced by some strains of Bacteroides distasonis; other strains, however, synthesized beta-glucosid enzyme.
Abstract: Rutin and quercitrin are hydrolysed to quercetin, and robinin is hydrolysed to kaempferol, by faecal flora from healthy subjects. The enzymes required for these hydrolyses, namely alpha-rhamnosidase and beta-galactosidase, were produced by some strains of Bacteroides distasonis; other strains, however, synthesized beta-glucosidase. The last-named enzyme was also elaborated by Bacteroides uniformis and Bacteroides ovatus. All the enzymes were produced constitutively. A cell-free extract of B. distasonis containing beta-glucosidase displayed an enzymic activity of 1 mumol/10 min per 10 mg of protein.

Journal ArticleDOI
TL;DR: The findings suggest that metabolism of endogenous substrate can provide most, if not all, of the energy requirement of these cells, at least for a short period, and that long-chain fatty acids might provide an important fuel in situ.
Abstract: The concentrations of ATP and the ATP/AMP concentration ratios were maintained in thioglycollate-elicited mouse peritoneal macrophages incubated in vitro for 90 min in the presence or absence of added substrate: rates of glycolysis, lactate formation and glutamine utilization were approximately linear with time for at least 60 min of incubation. The rate of oxygen consumption by macrophages was only increased above the basal rate (i.e. that in the absence of added substrate) by addition of succinate or pyruvate, or by addition of the uncoupling agent carboxyl cyanide m-chlorophenylhydrazone ('CCCP'); it was decreased by 75% by the addition of KCN. These findings suggest that metabolism of endogenous substrate can provide most, if not all, of the energy requirement of these cells, at least for a short period. The rates of glucose and glutamine utilization by incubated macrophages were approx. 300 and 100 nmol/min per mg of protein respectively. A large proportion of the glutamine that is utilized is converted into glutamate and aspartate, and very little (perhaps less than 10%) is oxidized. Similarly almost all of the glucose that is utilized is converted into lactate and very little is oxidized. This characteristic is similar to that of resting lymphocytes and rapidly dividing cells; in non-proliferating macrophages it may be a mechanism to provide precision in control of the rate of biosynthetic processes that utilize intermediates of these pathways, e.g. purines and pyrimidines for mRNA for the synthesis of secretory proteins and glycerol 3-phosphate for phospholipid synthesis for membrane recycling. No utilization of acetoacetate or 3-hydroxybutyrate by macrophages was detected. In contrast, both butyrate and oleate were oxidized. The rate of [14C]oleate conversion into 14CO2 (1.3 nmol/h per mg of protein) could account for most of the oxygen consumption by incubated macrophages, suggesting that long-chain fatty acids might provide an important fuel in situ. This may be one explanation for the secretion of lipoprotein lipase by these cells, to provide fatty acids for oxidation from the degradation of local triacylglycerol.

Journal ArticleDOI
TL;DR: It is suggested that polyphosphoinositides are made inside the nucleus and that they have a role in chromatin function; either the phospholipids themselves play a role, or there is a possibility of intranuclear signalling by inositide-derived molecules.
Abstract: Previous work demonstrated the existence of phosphatidylinositol kinase and phosphatidylinositol phosphate kinase in rat liver nuclei, with the suggestion that these activities are in the nuclear membrane [Smith & Wells (1983) J. Biol. Chem. 258, 9368-9373]. Here we show that highly purified nuclei from Friend cells, washed free of nuclear membrane by Triton, can incorporate radiolabel from [gamma-32P]ATP into phosphatidic acid, phosphatidylinositol phosphate and phosphatidylinositol 4,5-bisphosphate. The degree of radiolabelling of phosphatidylinositol bisphosphate is highly dependent on the state of differentiation of the cells, being barely detectable in growing cells and much greater after dimethyl sulphoxide-induced differentiation; this difference is mostly due to different amounts of phosphatidylinositol phosphate in the isolated nuclei. We suggest that polyphosphoinositides are made inside the nucleus and that they have a role in chromatin function; either the phospholipids themselves play a role, or there is a possibility of intranuclear signalling by inositide-derived molecules.

Journal ArticleDOI
TL;DR: Four cyclic nucleotide phosphodiesterase activities were separated from low-speed supernatants of homogenates of human cardiac ventricle by DEAE-Sepharose chromatography, and their inhibition in all cases demonstrated simple hyperbolic competition.
Abstract: Four cyclic nucleotide phosphodiesterase (PDE) activities were separated from low-speed supernatants of homogenates of human cardiac ventricle by DEAE-Sepharose chromatography, and designated PDE I-PDE IV in order of elution with an increasing salt gradient PDE I was a Ca2+/calmodulin-stimulated activity, and PDE II was an activity with a high Km for cyclic AMP which was stimulated by low concentrations of cyclic GMP Human ventricle PDE III had Km values of 014 microM (cyclic AMP) and 4 microM (cyclic GMP), and showed simple Michaelis-Menten kinetics with both substrates PDE IV is a previously unrecognized activity in cardiac muscle, the human enzyme having Km values of 2 microM (cyclic AMP) and 50 microM (cyclic GMP) PDE III and PDE IV were not activated by cyclic nucleotides or calmodulin Four PDE activities were also isolated from guinea-pig ventricle, and had very similar kinetic properties By gel filtration, the Mr of PDE III was 60,000, and that of PDE IV 45,000 The drug SK&F 94120 selectively and competitively inhibited PDE III with a Ki value of 08 microM (human), showing simple hyperbolic inhibition kinetics Rolipram (Schering ZK 62711) and Ro 20-1724 (Roche), which have previously been reported to inhibit PDE III-like activities strongly, were shown to be weak inhibitors of human and guinea-pig PDE III enzymes (Ki values greater than 25 microM), but potent inhibitors of PDE IV [Ki values 24 microM (Rolipram) and 31 microM (Ro 20-1724) with human PDE IV] The inhibition in all cases demonstrated simple hyperbolic competition These observations suggest that the previously reported complex inhibition of PDE III-type activities from cardiac muscle was caused by incomplete separation of the PDE III from other enzymes, particularly PDE IV

Journal ArticleDOI
TL;DR: alpha-Human atrial natriuretic peptide was rapidly hydrolysed by pig kidney microvillar membranes in vitro, comparable with the rate observed with angiotensins II and III, with the pattern obtained with membranes being similar to that with purified endopeptidase-24.11 (EC 3.24.4.11).
Abstract: alpha-Human atrial natriuretic peptide, a 28-amino-acid-residue peptide, was rapidly hydrolysed by pig kidney microvillar membranes in vitro, with a t1/2 of 8 min, comparable with the rate observed with angiotensins II and III. The products of hydrolysis were analysed by h.p.l.c., the pattern obtained with membranes being similar to that with purified endopeptidase-24.11 (EC 3.4.24.11). No hydrolysis by peptidyl dipeptidase A (angiotensin I converting enzyme, EC 3.4.15.1) was observed. The contribution of the various microvillar membrane peptidases was assessed by including specific inhibitors. Phosphoramidon, an inhibitor of endopeptidase-24.11, caused 80-100% suppression of the products. Captopril and amastatin (inhibitors of peptidyl dipeptidase A and aminopeptidases respectively) had no significant effect. Hydrolysis at an undefined site within the disulphide-linked ring occurred rapidly, followed by hydrolysis at other sites, including the Ser25--Phe26 bond.

Journal ArticleDOI
TL;DR: The deoxyribose assay, although chemically complex, in the presence of EDTA appears to give a simple and cheap method of obtaining rate constants for OH.
Abstract: Hydroxyl radicals (OH.) in free solution react with scavengers at rates predictable from their known second-order rate constants. However, when OH. radicals are produced in biological systems by metal-ion-dependent Fenton-type reactions scavengers do not always appear to conform to these established rate constants. The detector molecules deoxyribose and benzoate were used to study damage by OH. involving a hydrogen-abstraction reaction and an aromatic hydroxylation. In the presence of EDTA the rate constant for the reaction of scavengers with OH. was generally higher than in the absence of EDTA. This radiomimetic effect of EDTA can be explained by the removal of iron from the detector molecule, where it brings about a site-specific reaction, by EDTA allowing more OH. radicals to escape into free solution to react with added scavengers. The deoxyribose assay, although chemically complex, in the presence of EDTA appears to give a simple and cheap method of obtaining rate constants for OH. reactions that compare well with those obtained by using pulse radiolysis.

Journal ArticleDOI
TL;DR: Comparison of the immunoprecipitated PtdIns kinase with the activities identified by ion-exchange chromatography indicates that it is the Type I enzyme which specifically associates with the middle-T/pp60c-src complex and appears to co-precipitate with partially purified platelet derived growth factor (PDGF) receptor.
Abstract: Phosphatidylinositol (PtdIns) kinase activities from non-transformed and polyoma-middle-T-transformed murine fibroblasts were examined. Both normal and transformed 3T3 fibroblasts have two PtdIns kinases, which can be separated by anion-exchange chromatography. One of these activities (Type I) has a Km for ATP of 10 microM, is resistant to inhibition by adenosine, AMP or ADP, and is inhibited by non-ionic detergents. The other activity (Type II) has a somewhat higher Km for ATP (35 microM) and is inhibited competitively by ADP, AMP and adenosine at concentrations suggesting regulation of this activity by the energy charge of the cell. The Type II PtdIns kinase is activated by non-ionic detergents. We have previously reported the specific association of a PtdIns kinase activity with polyoma-middle-T immunoprecipitates [Whitman, Kaplan, Schaffhausen, Cantley & Roberts (1985) Nature (London) 315, 239-242; Kaplan, Whitman, Schaffhausen, Raptis, Garcea, Pallas, Roberts & Cantley (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3624-3628]. Comparison of the immunoprecipitated PtdIns kinase with the activities identified by ion-exchange chromatography indicates that it is the Type I enzyme which specifically associates with the middle-T/pp60c-src complex. This PtdIns kinase activity is separable from both middle T and pp60c-src. Type I PtdIns kinase also associates with pp60v-src immunoprecipitates from Rous-sarcoma-virus-transformed cells. Furthermore, this PtdIns kinase appears to co-precipitate with partially purified platelet derived growth factor (PDGF) receptor. The amount of this activity found in anti-phosphotyrosine immunoprecipitates or in wheat-germ-lectin-agarose precipitates is increased 50-fold by stimulation of quiescent Balb/C 3T3 fibroblasts with PDGF. These results suggest that the Type I PtdIns kinase is regulated by agents which affect cell growth and transformation, whereas the Type II PtdIns kinase may be regulated by the local [ATP]/[ADP] ratio.

Journal ArticleDOI
TL;DR: At acidic pH values, the protective ability of the apoproteins is diminished, and the fully iron-loaded proteins can release some iron in a form able to accelerate .OH generation.
Abstract: Apo-lactoferrin and apo-transferrin protect against iron-ion-dependent hydroxyl-radical (.OH) generation from H2O2 in the presence of superoxide radicals or ascorbic acid at pH 7.4, whether the necessary iron is added as ionic iron or as ferritin. Iron-loaded transferrin and lactoferrin [2 mol of Fe(III)/mol] show no protective ability, but do not themselves accelerate .OH production unless chelating agents are present in the reaction mixture, especially if the proteins are incorrectly loaded with iron. At acidic pH values, the protective ability of the apoproteins is diminished, and the fully iron-loaded proteins can release some iron in a form able to accelerate .OH generation. The physiological significance of these observations is discussed.

Journal ArticleDOI
TL;DR: Car carbohydrate antigens which were previously the interest of specialist immunochemists are now of wide relevance to both biochemists and cell biologists.
Abstract: Antibodies are being used increasingly as reagents in biochemical and biological studies of glycoproteins, for example, in the search for glycoproteins with important roles in embryonic development, cell differentiation and oncogenesis, and as a means of monitoring the biosynthesis and subcellular location of specific glycoproteins such as enzymes and receptors. Crucial for the proper application of antibodies in such experiments is knowledge of the determinants they recognize. A glycoprotein may contain many antigenic determinants, each consisting ofup to seven monosaccharides or amino acids in consecutive sequence (Kabat, 1976) or, in the case of polypeptides, in non-continuous sequence brought together by chain folding (Lerner, 1984; Walter, 1986). Unravelling the specificities contained in conventional (polyclonal) antisera raised against glycoproteins often presents problems due to the mixture of antibodies present with different combining specificities. It is often difficult to distinguish specificities directed against peptide determinants from those directed against carbohydrate structures on a glycoprotein. Monoclonal antibodies have the advantage in this respect since each is a single antibody population with a narrow specificity. As a result ofwork with naturally-occurring monoclonal autoantibodies and the widespread use of hybridomaderived antibodies, there has been an increased awareness of carbohydrates as antigenic determinants of glycoproteins, since it has become apparent that many differentiation antigens ofnormal and neoplastic cells are oligosaccharide determinants of glycoproteins and glycolipids rather than peptide determinants (Feizi, 1985, 1987; Hakomori, 1985). Thus, carbohydrate antigens which were previously the interest of specialist immunochemists are now of wide relevance to both biochemists and cell biologists. We do not intend here to make an exhaustive review of antigens found to date on glycoprotein oligosaccharides; rather we shall discuss some examples of whole cells (haematopoietic and embryonic) and specific glycoproteins (a receptor and a glycosyltransferase) where carbohydrate structures have been found to be major antigenic determinants. We shall also discuss approaches to the study of antigenicity (reactivity with antibodies) and immunogenicity (ability to elicit antibodies) of the oligosaccharides of glycoproteins and the opportunities now available for exploiting antibodies with defined carbohydrate specificities as probes of the structure, tissue distribution and function of glycoprotein oligosaccharides. We also include in this article a hypothesis which considers the biological relevance and possible inter-relationships of three areas of knowledge: (a) the occurrence of the same carbohydrate antigens/structures on diverse and unrelated glycoproteins, (b) marked

Journal ArticleDOI
TL;DR: The concentrations of uric acid and allantoin in human serum and synovial fluid are reported and it is suggested that measurement of changes inAllantoin concentration may be a useful index of free-radical reactions taking place in vivo.
Abstract: Free-radical attack upon uric acid generates allantoin [Ames, Cathcart, Schwiers & Hochstein (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6858-6862]. Methods are described for the accurate measurement of uric acid and allantoin in human body fluids. The concentrations of uric acid and allantoin in human serum and synovial fluid are reported. It is suggested that measurement of changes in allantoin concentration may be a useful index of free-radical reactions taking place in vivo.

Journal ArticleDOI
TL;DR: The findings point to the existence of an equilibrium of EC-SOD fraction C between the plasma phase and endothelial-cell surfaces, which seems to be located on endothelial -cell surfaces in the vasculature.
Abstract: Extracellular superoxide dismutase (SOD) has previously been shown to be the major SOD isoenzyme in extracellular fluids. Upon chromatography on heparin-Sepharose it was separated into three fractions: A, without affinity; B, with intermediate affinity; and C, with relatively strong heparin affinity. Intravenous injection of heparin leads to a prompt increase in plasma extracellular-superoxide-dismutase (EC-SOD) activity. Heparin induces no release of EC-SOD from blood cells, nor does it activate EC-SOD in plasma, indicating that the source of the released enzyme is the endothelial-cell surfaces. No distinct saturation could be demonstrated in a dose-response curve up to 200 i.u. of heparin per kg body weight, showing that the releasing potency of heparin is lower for EC-SOD than for previously investigated heparin-released factors. Chromatography of human plasma on heparin-Sepharose shows nearly equal amounts of EC-SOD fractions A, B and C. Heparin induces specifically the release of fraction C. The findings point to the existence of an equilibrium of EC-SOD fraction C between the plasma phase and endothelial-cell surfaces. The major part of EC-SOD in the vasculature seems to be located on endothelial-cell surfaces.

Journal ArticleDOI
TL;DR: In this article, the authors analysed the skin secretion of the South African frog Xenopus laevis using fast atom bombardment mass spectrometry and h.p.c. in the mass range 500-3200 Da.
Abstract: Peptides present in the skin secretion of the South African frog, Xenopus laevis, have been analysed by fast atom bombardment mass spectrometry and h.p.l.c. in the mass range 500-3200 Da. We have investigated the effects of successive glandular secretions induced by noradrenaline injections on these peptide levels and have found that the replenishment of the whole range of peptides is complete within 2-6 days. Intact secretory vesicles free of cellular contaminants contain a relatively large number of peptides with molecular masses in the range 2400-2700 Da. We have termed these peptides primary products or spacer peptides, since they originate from spacer regions of the precursors to xenopsin and caerulein. However, if the secretory vesicles are disrupted during the collection procedure and the solution containing the secretion is kept at room temperature for up to 2 h, relatively little of the larger peptides remain. By comparing the relative levels of the various peptides present in these secretions we have found that the larger peptides are proteolytically cleaved into smaller fragments by a novel cleavage at the N-terminal side of a lysine residue (at Xaa-Lys bonds where Xaa is Leu, Gly, Ala or Lys). Preliminary evidence has been obtained suggesting that the larger intact peptides possess lytic activity whereas the smaller proteolytic fragments appear relatively inactive. This may represent a mechanism by which the secretions are rendered harmless to the frog itself, since prolonged exposure would be expected to result in toxic effects. The dorsal glands of X. laevis thus appear similar to endocrine glands, since they are involved in peptide biosynthesis, secretion and subsequent proteolytic degradation.

Journal ArticleDOI
TL;DR: It is concluded that E. coli 'endonuclease III' is, after all, not an endonuclease, and that the cleavage is not a hydrolysis leaving a base-free deoxyribose at the 3' end as it has been so far assumed.
Abstract: The oligonucleotide [5′-32P]pdT8d(-)dTn, containing an apurinic/apyrimidinic (AP) site [d(-)], yields three radioactive products when incubated at alkaline pH: two of them, forming a doublet approximately at the level of pdT8dA when analysed by polyacrylamide-gel electrophoresis, are the result of the beta-elimination reaction, whereas the third is pdT8p resulting from beta delta-elimination. The incubation of [5′-32P]pdT8d(-)dTn, hybridized with poly(dA), with E. coli endonuclease III yields two radioactive products which have the same electrophoretic behaviour as the doublet obtained by alkaline beta-elimination. The oligonucleotide pdT8d(-) is degraded by the 3′-5′ exonuclease activity of T4 DNA polymerase as well as pdT8dA, showing that a base-free deoxyribose at the 3′ end is not an obstacle for this activity. The radioactive products from [5′-32P]pdT8d(-)dTn cleaved by alkaline beta-elimination or by E. coli endonuclease III are not degraded by the 3‘-5’ exonuclease activity of T4 DNA polymerase. When DNA containing AP sites labelled with 32P 5′ to the base-free deoxyribose labelled with 3H in the 1′ and 2′ positions is degraded by E. coli endonuclease VI (exonuclease III) and snake venom phosphodiesterase, the two radionuclides are found exclusively in deoxyribose 5-phosphate and the 3H/32P ratio in this sugar phosphate is the same as in the substrate DNA. When DNA containing these doubly-labelled AP sites is degraded by alkaline treatment or with Lys-Trp-Lys, followed by E. coli endonuclease VI (exonuclease III), some 3H is found in a volatile compound (probably 3H2O) whereas the 3H/32P ratio is decreased in the resulting sugar phosphate which has a chromatographic behaviour different from that of deoxyribose 5-phosphate. Treatment of the DNA containing doubly-labelled AP sites with E. coli endonuclease III, then with E. coli endonuclease VI (exonuclease III), also results in the loss of 3H and the formation of a sugar phosphate with a lower 3H/32P ratio that behaves chromatographically as the beta-elimination product digested with E. coli endonuclease VI (exonuclease III). From these data, we conclude that E. coli endonuclease III cleaves the phosphodiester bond 3′ to the AP site, but that the cleavage is not a hydrolysis leaving a base-free deoxyribose at the 3′ end as it has been so far assumed. The cleavage might be the result of a beta-elimination analogous to the one produced by an alkaline pH or Lys-Trp-Lys. Thus it would seem that E. coli ‘endonuclease III’ is, after all, not an endonuclease.

Journal ArticleDOI
TL;DR: Its sensitivity to DPI suggests that selective prevention of radical generation by macrophages in vivo is possible, and the O2(.-)-generating oxid enzyme appears to have components in common with the NADPH oxidase of neutrophils, despite differences in activity.
Abstract: Peritoneal macrophages were elicited in rats by using casein as a stimulus; when stimulated with phorbol 12-myristate 13-acetate (PMA) they produced O2.-. Nearly 60% of the total cytochrome b had a low Em,7.0 of -247 mV, typical of the cytochrome b component found in the NADPH-dependent O2(.-)-generating oxidase of neutrophils. The rate of O2.- generation by macrophages was 1.23 mol of O2.-/s per mol of cytochrome b. Treatment of intact macrophages with diphenyleniodonium (DPI) at 0.9 microM caused 50% inhibition of PMA-induced O2.- generation, with little effect on mitochondrial respiratory activity; KCN inhibited respiratory activity without affecting PMA-induced O2.- generation. A similar specificity of inhibition was found for di-2-thienyliodonium (50% inhibition of O2.- generation at 0.5 microM) and, at higher concentrations, for diphenyl iodonium. When macrophage suspensions were incubated with [125I]DPI followed by autoradiography of SDS/polyacrylamide-gel-electrophoresis-separated polypeptides, radioactivity was most strongly associated with a band of Mr 45,000, similar to that found in neutrophils [Cross & Jones (1986) Biochem. J. 237, 111-116]. The O2(.-)-generating oxidase of macrophages appears to have components in common with the NADPH oxidase of neutrophils, despite differences in activity. Its sensitivity to DPI suggests that selective prevention of radical generation by macrophages in vivo is possible.


Journal ArticleDOI
TL;DR: The findings suggest that calpactin I may be bound to phospholipids in vivo at Ca2+ concentrations of about 1.5 x 10(-7) M, typical of resting unstimulated cells, and that this interaction may be modulated by light-chain binding and phosphorylation by p60v-src.
Abstract: Calpactins I and II are proteins that bind Ca2+, phospholipids, actin and spectrin; they are also major substrates of oncogene and growth-factor-receptor tyrosine kinases. Since calpactins have been proposed to provide a link between membrane lipids and the cytoskeleton, we examined in detail the interactions between purified calpactin I and phospholipid liposomes. We focused on the Ca2+-dependence, the effects of phosphorylation of calpactin I by p60v-src (the protein kinase coded for by the Rous-sarcoma-virus oncogene), and the effects of the binding of calpactin I light chain to calpactin I heavy chain. Binding of the light chain to the heavy chain increased the affinity of calpactin I for phosphatidylserine (PS) liposomes. The opposite effect was observed for phosphorylation by p60v-src; phosphorylation decreased the affinity of calpactin I for PS liposomes. These two opposite effects appeared to be independent, since phosphorylation did not prevent light-chain binding to the heavy chain. Calpactin I was found, by the use of three different techniques, to bind to phospholipid liposomes at less than 10(-8) M free Ca2+. This result is in contrast with those of previous studies, which indicated that greater than 10(-6) M free Ca2+ was required. Our findings suggest that calpactin I may be bound to phospholipids in vivo at Ca2+ concentrations of about 1.5 x 10(-7) M, typical of resting unstimulated cells, and that this interaction may be modulated by light-chain binding and phosphorylation by p60v-src.

Journal ArticleDOI
TL;DR: Three major acidic proteins of bovine seminal plasma were purified to homogeneity, by employing fast protein liquid chromatography, gel filtration and h.p.l.c. electrophoresis, and it was concluded that BSP-A1, B SP-A2 and BSP -A3 are the same as the gonadostatins.
Abstract: Three major acidic proteins of bovine seminal plasma, BSP-A1, BSP-A2 and BSP-A3, were purified to homogeneity, by employing fast protein liquid chromatography, gel filtration and h.p.l.c. The proteins were purified on the basis of their stimulatory effect on the basal release of gonadotropins by rat anterior-pituitary cells in culture. All three proteins migrated as distinct single bands in the presence or absence of 2-mercaptoethanol in SDS/polyacrylamide-gel electrophoresis. Their Mr values were estimated to be between 15,000 and 16,500 by SDS/polyacrylamide-gel electrophoresis. Similar Mr estimates were obtained when they were subjected to gel filtration on a calibrated column of Sephadex G-75 equilibrated in 0.05 M-acetic acid, pH 3.0. However, BSP-A1 and BSP-A2 were eluted as aggregated molecules (Mr 60,000-120,000) during gel filtration on Sephadex G-200 equilibrated in 0.05 M-NH4HCO3, pH 8.5, or phosphate buffer, pH 7.0, containing 0.15 M-NaCl. In the presence of 8 M-urea both BSP-A1 and BSP-A2 were eluted at positions corresponding to Mr values of 17,000-20,000. BSP-A1 and BSP-A2 had an identical amino acid composition, which differed largely from that of BSP-A3. All three proteins contained aspartic acid as the N-terminal residue, and cysteine was identified as the C-terminal residue. BSP-A1 and BSP-A2 are glycoproteins containing galactosamine, sialic acid and neutral sugars, but BSP-A3 did not contain any covalently attached sugars. Whereas BSP-A2 and BSP-A3 were eluted unadsorbed, BSP-A1 bound to wheat-germ lectin-Sepharose 6MB and could be eluted by the competing sugar N-acetyl-D-glucosamine. Treatment of BSP-A1 and BSP-A2 with trypsin resulted in complete loss of gonadotropin-release activity, but BSP-A3 retained full activity. Antibody raised against BSP-A1 did not cross-react with BSP-A3, or vice versa. All these properties indicated marked structural differences between BSP-A3 and BSP-A1 (or BSP-A2). On the basis of amino acid composition it was concluded that BSP-A1, BSP-A2 and BSP-A3 are the same as the gonadostatins [Esch, Ling, Bohlen, Ying & Guillemin (1983) Biochem. Biophys. Res. Commun. 113, 861-867].

Journal ArticleDOI
TL;DR: It appears that ACE, unlike endopeptidase-24.11, does not have the general capacity to hydrolyse and inactivate the tachykinin peptides at a significant rate in brain.
Abstract: Peptidyl-dipeptidase A (angiotensin converting enzyme; ACE, EC 3.4.15.1), has been purified from pig kidney and striatum by affinity chromatography employing the selective inhibitor lisinopril as ligand. The inclusion of a 2.8 nm spacer arm improved the yield of the enzyme compared with the 1.4 nm spacer arm described in previous work. Two forms of striatal ACE (Mr 180,000 and 170,000), but only a single form of kidney ACE (Mr 180,000), were isolated by this procedure. Both forms of striatal ACE were recognized by a polyclonal antibody to kidney ACE. No significant differences in substrate specificity or inhibitor sensitivity between kidney and striatal ACE could be detected. In particular, the amidated neuropeptide, substance P, was hydrolysed identically by both preparations and no significant hydrolysis of the related tachykinin peptides neurokinin A and neurokinin B could be detected. After chemical or enzymic deglycosylation, kidney and both forms of striatal ACE migrated identically on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with an apparent Mr of 150,000. We suggest that the two detectable forms of ACE in pig brain are not isoenzymes but are the result of differential glycosylation in different cell types in the brain. It appears that ACE, unlike endopeptidase-24.11, does not have the general capacity to hydrolyse and inactivate the tachykinin peptides at a significant rate in brain.

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TL;DR: It is shown that both thiourea and dimethylthiOUrea are scavengers of HOCl, a powerful oxidant produced by neutrophil myeloperoxidase, and hence the ability of dim methylthiou Andrea to protect against neutrophIL-mediated tissue damage cannot be used as evidence for a role of .OH in causing such damage.
Abstract: Thiourea and dimethylthiourea are powerful scavengers of hydroxyl radicals (OH), and dimethylthiourea has been used to test the involvement of OH in several animal models of human disease It is shown that both thiourea and dimethylthiourea are scavengers of HOCl, a powerful oxidant produced by neutrophil myeloperoxidase Hence the ability of dimethylthiourea to protect against neutrophil-mediated tissue damage cannot be used as evidence for a role of OH in causing such damage Dimethyl sulphoxide also reacts with HOCl, but at a rate that is probably too low to be biologically significant at dimethyl sulphoxide concentrations up to 10 mM Neither mannitol nor desferrioxamine, at the concentrations normally used in radical-generating systems, appears to react with HOCl

Journal ArticleDOI
TL;DR: The observed homologies demonstrate that the arom polypeptide is a mosaic of functional domains and are consistent with the hypothesis that the ARO1 gene evolved by the linking of ancestral E. coli-like genes.
Abstract: The nucleotide sequence of the Saccharomyces cerevisiae ARO1 gene which encodes the arom multifunctional enzyme has been determined. The protein sequence deduced for the pentafunctional arom polypeptide is 1588 amino acids in length and has a calculated Mr of 174555. Functional regions within the polypeptide chain have been identified by comparison with the sequences of the five monofunctional Escherichia coli enzymes whose activities correspond with those of the arom multifunctional enzyme. The observed homologies demonstrate that the arom polypeptide is a mosaic of functional domains and are consistent with the hypothesis that the ARO1 gene evolved by the linking of ancestral E. coli-like genes.