scispace - formally typeset
Search or ask a question

Showing papers in "Biochemical Journal in 1985"



Journal ArticleDOI
TL;DR: Evidence derived from its chromatographic behaviour, and from the structure of the products formed in partial dephosphorylation experiments, suggests that it is probably D-myo-inositol 1,3,4,5-tetrakisphosphate, which is likely to be a second messenger.
Abstract: Carbachol stimulation of muscarinic receptors in rat cortical slices prelabelled with myo-[2-3H]inositol caused the rapid formation of a novel inositol polyphosphate. Evidence derived from its chromatographic behaviour, and from the structure of the products formed in partial dephosphorylation experiments, suggests that it is probably D-myo-inositol 1,3,4,5-tetrakisphosphate. An enzyme in human red cell membranes specifically removes the 5-phosphate from it to form inositol 1,3,4-trisphosphate. It is suggested that inositol 1,3,4,5-tetrakisphosphate is likely to be a second messenger, and that it is the precursor of inositol 1,3,4-trisphosphate and possibly of inositol 1,4,5-trisphosphate.

546 citations


Journal ArticleDOI
TL;DR: Antigenicity of sialoprotein II was not affected by reduction and alkylation, was only partially lost upon trypsin digestion and was completely lost upon fragmentation of the core protein by alkaline-borohydride treatment, indicating that all antigenic sites were located in the protein portion.
Abstract: Two different sialoproteins were isolated from the mineralized matrix of bovine bone by using extraction with guanidinium chloride first without and then with EDTA. The sialoproteins were purified by chromatography on DEAE-cellulose eluted with a sodium acetate gradient in 7 M-urea, pH 6. Two sialoproteins (I and II) were then separated by chromatography on DEAE-cellulose eluted with a sodium chloride gradient in 7 M-urea, pH 4. The ratio between recovered sialoprotein I and II was 1:5. The chemical analysis of the two sialoproteins showed that they differed. Both, however, had very high contents of aspartic acid/asparagine and glutamic acid/glutamine though they differed markedly in contents of leucine and glycine. Both sialoproteins contained phosphate, sialoprotein I more than sialoprotein II. Content of sialic acid was substantially higher in the more prominent sialoprotein II (13.4% of dry weight) than in sialoprotein I (4.8% of dry weight). The peptide patterns produced by trypsin digests of [125I]iodinated sialoproteins I and II showed both structural similarities and structural differences. Sialoprotein II, being the major component, was characterized further. Its molecular mass was 57300 Da determined by sedimentation-equilibrium centrifugation in 6 M-guanidinium chloride, and its sedimentation coefficient (S0(20),w) was 2.53 S. Upon rotary shadowing, sialoprotein II appeared as an extended rod, having a core with an average length of 40 nm. Two types of oligosaccharides, N-glycosidically and O-glycosidically linked to the core protein, were isolated from sialoprotein II. Contents of mannose and sialic acid in the O-linked oligosaccharide were surprisingly high. Antibodies against sialoprotein II were raised in rabbits and an enzyme-linked immunosorbent assay was developed. Antigenicity of sialoprotein II was not affected by reduction and alkylation, was only partially lost upon trypsin digestion and was completely lost upon fragmentation of the core protein by alkaline-borohydride treatment, indicating that all antigenic sites were located in the protein portion. Sialoprotein I expectedly showed only partial immunological cross-reactivity with sialoprotein II. The quantity of sialoprotein II in bone extracts was found to be about 1.5 mg/g wet wt. of bone, but the protein was not detected in extracts of a number of other bovine tissues i.e. aorta, cartilage, dentine, kidney, liver, muscle, sclera, skin and tendon.

531 citations


Journal ArticleDOI
TL;DR: Data indicate that, provided that inositol 1,4,5-trisphosphate is studied independently of inositl 1,3,4-trishosphate, the former shows metabolic characteristics consistent with its proposed role as a second messenger for calcium mobilization.
Abstract: A complete separation of myo-inositol 1,4,5-[4,5-(32)P]trisphosphate prepared from human erythrocytes, and myo-[2-3H]inositol 1,3,4-trisphosphate prepared from carbachol-stimulated rat parotid glands [Irvine, Letcher, Lander & Downes (1984) Biochem. J. 223, 237-243], was achieved by anion-exchange high-performance liquid chromatography. This separation technique was then used to study the metabolism of these two isomers of inositol trisphosphate in carbachol-stimulated rat parotid glands. Fragments of glands were pre-labelled with myo-[2-3H]inositol, washed, and then stimulated with carbachol. At 5s after stimulation a clear increase in inositol 1,4,5-trisphosphate was detected, with no significant increase in inositol 1,3,4-trisphosphate. After this initial lag however, inositol 1,3,4-phosphate rose rapidly; by 15s it predominated over inositol 1,4,5-trisphosphate, and continued to rise so that after 15 min it was at 10-20 times the radiolabelling level of the 1,4,5-isomer. In contrast, after the initial rapid rise (maximal within 15s), inositol 1,4,5-trisphosphate levels declined to near control levels after 1 min and then rose again very gradually over the next 15 min. When a muscarinic blocker (atropine) was added after 15 min of carbachol stimulation, inositol 1,4,5-trisphosphate levels dropped to control levels within 2-3 min, whereas inositol 1,3,4-trisphosphate levels took at least 15 min to fall, consistent with the kinetics observed earlier for total parotid inositol trisphosphates [Downes & Wusteman (1983) Biochem. J. 216, 633-640]. Phosphatidylinositol bisphosphate (PtdInsP2) from stimulated and control cells were degraded chemically to inositol trisphosphate to seek evidence for 3H-labelled PtdIns(3,4)P2. No evidence could be obtained that a significant proportion of PtdInsP2 was this isomer; in control tissues it must be less than 5% of the total PtdInsP2 radiolabelled by myo-[2-3H]inositol. These data indicate that, provided that inositol 1,4,5-trisphosphate is studied independently of inositol 1,3,4-trisphosphate, the former shows metabolic characteristics consistent with its proposed role as a second messenger for calcium mobilization. The metabolic profile of inositol 1,3,4-trisphosphate is entirely different, and its function and source remain unclear.

385 citations


Journal ArticleDOI
TL;DR: The stoichiometric evaluation of fatty acid loss and the production of malonaldehyde and other carbonyls suggests that the pathways of lipid peroxidation triggered by CCl4 and ADP-iron are different.
Abstract: Carbonyl products were separated and identified in suspensions of rat liver microsomal fractions and in isolated hepatocytes, after stimulation of lipid peroxidation by incubation with the pro-oxidants CCl4 and ADP-iron. The carbonyl products were allowed to react with 2,4-dinitrophenylhydrazine, and the derivatives were extracted and separated by t.l.c. into three zones of non-polar materials, and one fraction of polar derivatives that remained at the origin. Separation of the individual non-polar hydrazones in each zone by h.p.l.c. demonstrated that zone I prepared from microsomal fraction or hepatocytes incubated with CCl4 or ADP-iron contained mainly 4-hydroxyhex-2-enal, 4-hydroxynon-2-enal and 4-hydroxynona-2,5-dienal. Zone III consisted mainly of the alkanals propanal, pentanal and hexanal, the 2-alkenals propenal, pent-2-enal, hex-2-enal, hept-2-enal, oct-2-enal and non-2-enal, the ketones butanone, pentan-2-one and pentan-3-one, and deca-2,4-dienal. Incubation of a microsomal fraction with ADP-iron was much more effective in producing malonaldehyde and other carbonyl products than an incubation with CCl4. Despite such quantitative differences, there were no obvious qualitative differences in the h.p.l.c. spectra obtained from zones I and III. However, the stoichiometric evaluation of fatty acid loss and the production of malonaldehyde and other carbonyls suggests that the pathways of lipid peroxidation triggered by CCl4 and ADP-iron are different. The accumulation of carbonyl products of lipid peroxidation in isolated hepatocytes is strongly affected by their metabolism; in particular, 4-hydroxyalkenals were found to be metabolized very rapidly. Nonetheless, both CCl4 and ADP-iron produced stimulation in the production of malonaldehyde and non-polar carbonyl production. After incubation of rat hepatocytes with CCl4 or ADP-iron it was found that approx. 50% of the total amount of non-polar carbonyls produced during incubation escaped into the external medium. This was not leakage from dead cells, as 90-95% of the hepatocytes had retained their integrity at the end of the incubation. Release of carbonyl products from cells stimulated to undergo lipid peroxidation may be a mechanism for spreading an initial intracellular disturbance to affect critical targets outside the parent cell.

362 citations


Journal ArticleDOI
TL;DR: A quantitative method allowing determination of glucose metabolism in vivo in muscles and white adipose tissue of the anaesthetized rat is presented and results corresponded qualitatively and quantitatively to the known physiological characteristics of the tissues studied.
Abstract: A quantitative method allowing determination of glucose metabolism in vivo in muscles and white adipose tissue of the anaesthetized rat is presented. A tracer dose of 2-deoxy[3H]glucose was injected intravenously in an anaesthetized rat and the concentration of 2-deoxy[3H]glucose was monitored in arterial blood. After 30-80 min, three muscles, the soleus, the extensor digitorum longus and the epitrochlearis, periovarian white adipose tissue and brain were sampled and analysed for their content of 2-deoxy[3H]glucose 6-phosphate. This content could be related to glucose utilization during the same time period, since (1) the integral of the decrease of 2-deoxy[3H]glucose in arterial blood was known and (2) correction factors for the analogue effect of 2-deoxyglucose compared with glucose in the transport and phosphorylation steps were determined from experiments in vitro. Glucose utilization was then measured by this technique in the tissues of post-absorptive rats in the basal state (0.1 munit of insulin/ml of plasma) or during euglycaemic-hyperinsulinaemic glucose clamp (8 munits of insulin/ml of plasma) and of 48 h-starved rats. Results corresponded qualitatively and quantitatively to the known physiological characteristics of the tissues studied.

332 citations


Journal ArticleDOI
TL;DR: Myoblast fusion provides a naturally occurring process in which mechanistic proposals from the study of fusion in moder fusion systems can be tested and profitably applied to appropriate vesicular model systems.
Abstract: Skeletal muscle fibres are permanent multinucleated, non-mitotic cells. Consequently, there is considerable interest in how such a unique cell type is formed. Muscle fibres are derived from multinucleated myotubes which are themselves formed, during embryonic development, by the fusion of mononucleated myoblasts. Myoblast fusion provides a naturally occurring process in which mechanistic proposals from the study of fusion in moder fusion systems can be tested. Conversely, proposals derived from myoblast fusion studies can be profitably applied to appropriate vesicular model systems. In vivo, the normal development of muscle has been observed in many species. Although the transition from monoto predominantly multi-nucleated muscle cells can be observed in sections of embryonic muscle, this is asynchronous and poses almost insurmountable difficulties for the biochemical analysis of discrete stages. Tissue culture techniques dramatically simplify this analysis. Holtzer et al. (1958) were the first to show that the fusion ofmyoblasts occurred in primary culture, and since then fusion studies have emphasized the importance of this powerful model in vitro. Muscle cell fusion can be studied using both primary cultures and identified cell lines. Primary cells are prepared from embryonic tissues by mechanical or enzymic disaggregation (see Konigsberg, 1978) and cultured in medium supplemented with serum (horse

328 citations


Journal ArticleDOI
TL;DR: A simple spectrophotometric assay was developed for peroxisomal fatty acyl-CoA oxidase activity, based on the H2O2-dependent oxidation of leuco-dichlorofluorescein catalysed by exogenous peroxidase, which is more sensitive than methods previously described.
Abstract: A simple spectrophotometric assay was developed for peroxisomal fatty acyl-CoA oxidase activity. The assay, based on the H2O2-dependent oxidation of leuco-dichlorofluorescein catalysed by exogenous peroxidase, is more sensitive than methods previously described. By using mouse liver samples, cofactor requirements were assessed and a linear relationship was demonstrated between dye oxidation and enzyme concentration. By using this assay on subcellular fractions, palmitoyl-CoA oxidase activity was localized for the first time in microperoxisomes of rat intestine. The assay was also adapted to measure D-amino acid oxidase activity, demonstrating the versatility of this method for measuring activity of other H2O2-producing oxidases.

307 citations


Journal ArticleDOI
TL;DR: Fiction measurements showed that the human kneesynovial-fluid lubricin had equivalent lubricating ability in a test system in vitro to that observed for lubricin isolated from normal bovine synovial fluids.
Abstract: Lubricin was isolated from bovine ankle, metacarpophalangeal and knee and human knee synovial fluids. The lubricins isolated from the bovine joint fluids had the same amino acid and carbohydrate compositions, but differences were observed in the relative molecular masses. The Mr values of bovine metacarpophalangeal and ankle lubricin determined by light-scattering measurements were about 200 000, whereas values of 132 000 and 143 000 were obtained for the bovine knee lubricin. The human knee lubricin had a similar carbohydrate composition to bovine knee lubricin except for the higher glucosamine content, and the amino acid composition differed slightly. The human sample had a lower glutamic acid content and a leucine/isoleucine ratio of 2:1 compared with 1:1 in the bovine. The Mr value of the human knee lubricin (166 000) was also lower than that of the bovine metacarpophalangeal and ankle samples. The Mr value of the bovine knee lubricin determined by sedimentation-equilibrium measurements was 171 000. The length measurements determined by electron microscopy and also the sedimentation measurements showed considerable polydispersity and indicate that the degree of extension of lubricin molecules can vary. Friction measurements showed that the human knee synovial-fluid lubricin had equivalent lubricating ability in a test system in vitro to that observed for lubricin isolated from normal bovine synovial fluids. The lubricating ability of lubricin was concentration-dependent, and each lubricin sample was able to act as a lubricant in vitro in an equivalent manner to whole synovial fluid at concentrations that are thought to occur in vivo.

302 citations


Journal ArticleDOI
TL;DR: Xylose reductase from the xylose-fermenting yeast Pichia stipitis was purified to electrophoretic and spectral homogeneity via ion-exchange, affinity and high-performance gel chromatography and is an aldose reducase (EC 1.1.21).
Abstract: Xylose reductase from the xylose-fermenting yeast Pichia stipitis was purified to electrophoretic and spectral homogeneity via ion-exchange, affinity and high-performance gel chromatography. The enzyme was active with various aldose substrates, such as DL-glyceraldehyde, L-arabinose, D-xylose, D-ribose, D-galactose and D-glucose. Hence the xylose reductase of Pichia stipitis is an aldose reductase (EC 1.1.1.21). Unlike all aldose reductases characterized so far, the enzyme from this yeast was active with both NADPH and NADH as coenzyme. The activity with NADH was approx. 70% of that with NADPH for the various aldose substrates. NADP+ was a potent inhibitor of both the NADPH- and NADH-linked xylose reduction, whereas NAD+ showed strong inhibition only with the NADH-linked reaction. These results are discussed in the context of the possible use of Pichia stipitis and similar yeasts for the anaerobic conversion of xylose into ethanol.

293 citations


Journal ArticleDOI
TL;DR: Treatment of chick heart cells or 1321N1 human astrocytoma cells with pertussis toxin did not block muscarinic-receptor-mediated stimulation of phosphoinositide hydrolysis, as measured by [3H]inositol phosphate accumulation in the presence of Li+.
Abstract: Pertussis toxin was used to examine the role of the inhibitory guanine nucleotide regulatory protein, Ni, in muscarinic-receptor-mediated stimulation of phosphoinositide turnover and calcium mobilization. In cultured chick heart cells, pertussis-toxin treatment inhibited muscarinic-receptor-mediated attenuation of isoprenaline-stimulated cyclic AMP accumulation. This finding is consistent with the proposal that pertussis toxin blocks the capacity of Ni to couple muscarinic receptors to adenylate cyclase. In contrast, treatment of chick heart cells or 1321N1 human astrocytoma cells with pertussis toxin did not block muscarinic-receptor-mediated stimulation of phosphoinositide hydrolysis, as measured by [3H]inositol phosphate accumulation in the presence of Li+. Pertussis-toxin treatment also had little effect on basal and muscarinic-receptor-stimulated phosphatidylinositol synthesis, as measured by the incorporation of [3H]inositol into phosphatidylinositol. Activation of muscarinic receptors also enhances the rate of unidirectional 45Ca2+ efflux in 1321N1 cells; this response, like phosphoinositide hydrolysis, was not prevented by pertussis-toxin treatment. Our data suggest that muscarinic receptors are not coupled to phosphoinositide hydrolysis or calcium mobilization through Ni.

Journal ArticleDOI
TL;DR: It is concluded that acetylene is a suicide substrate for the mono-oxygenase, and the labelling experiment provides the first identification of a constituent polypeptide of ammonia mono-oxidizing enzyme in Nitrosomonas europaea.
Abstract: Acetylene brings about a progressive inactivation of ammonia mono-oxygenase, the ammonia-oxidizing enzyme in Nitrosomonas europaea. High NH4+ ion concentrations were protective. The inactivation followed first-order kinetics, with a rate constant of 1.5 min-1 at saturating concentrations of acetylene. If acetylene was added in the absence of O2, the cells remained active until O2 was re-introduced. A protective effect was also demonstrated with thiourea, a reversible non-competitive inhibitor of ammonia oxidation. Incubation of cells with [14C]acetylene was found to cause labelling of a single membrane polypeptide. This ran on dodecyl sulphate/polyacrylamide-gel electrophoresis with an Mr value of 28 000. It is concluded that acetylene is a suicide substrate for the mono-oxygenase. The labelling experiment provides the first identification of a constituent polypeptide of ammonia mono-oxygenase.

Journal ArticleDOI
TL;DR: It is concluded that heparan sulphate and heparin are separate families of N-sulphated glycosaminoglycans.
Abstract: Heparan sulphate and heparin are chemically related alpha beta-linked glycosaminoglycans composed of alternating sequences of glucosamine and uronic acid. The amino sugars may be N-acetylated or N-sulphated, and the latter substituent is unique to these two polysaccharides. Although there is general agreement that heparan sulphate is usually less sulphated than heparin, reproducible differences in their molecular structure have been difficult to identify. We suggest that this is because most of the analytical data have been obtained with degraded materials that are not necessarily representative of complete polysaccharide chains. In the present study intact heparan sulphates, labelled biosynthetically with [3H]glucosamine and Na2(35)SO4, were isolated from the surface membranes of several types of cells in culture. The polysaccharide structure was analysed by complete HNO2 hydrolysis followed by fractionation of the products by gel filtration and high-voltage electrophoresis. Results showed that in all heparan sulphates there were approximately equal numbers of N-sulpho and N-acetyl substituents, arranged in a similar, predominantly segregated, manner along the polysaccharide chain. O-Sulphate groups were in close proximity to the N-sulphate groups but, unlike the latter, the number of O-sulphate groups could vary considerably in heparan sulphates of different cellular origins ranging from 20 to 75 O-sulphate groups per 100 disaccharide units. Inspection of the published data on heparin showed that the N-sulphate frequency was very high (greater than 80% of the glucosamine residues are N-sulphated) and the concentration of O-sulphate groups exceeded that of the N-sulphate groups. We conclude from these and other observations that heparan sulphate and heparin are separate families of N-sulphated glycosaminoglycans.

Journal ArticleDOI
TL;DR: The presence of indolylamine 2,3-dioxygenase was examined in human subjects by determining its activity with L-tryptophan as substrate.
Abstract: The presence of indolylamine 2,3-dioxygenase was examined in human subjects by determining its activity with L-tryptophan as substrate. Enzyme activity was detected in various tissues, and was relatively high in the lung, small intestine and placenta. Human indolylamine 2,3-dioxygenase, partially purified from the placenta, had an Mr of about 40 000 by gel filtration and exhibited a single pI of 6.9. The human enzyme required a reducing system, ascorbic acid and Methylene Blue, for maximal activity and was able to oxidize D-tryptophan, 5-hydroxy-L-tryptophan as well as L-tryptophan, but kinetic studies indicated that the best substrate of the enzyme was L-tryptophan.

Journal ArticleDOI
TL;DR: It is concluded that catabolin-treated cartilage synthesizes a smaller number of normal proteoglycan molecules, compared with controls, which had the same average hydrodynamic size and ability to aggregate.
Abstract: A homogeneous preparation of catabolin from pig leucocytes caused a reversible dose-dependent (0.01-1 nM) decrease in the synthesis of proteoglycan in slices of pig articular cartilage cultured in serum-free medium. The monomers that were synthesized and secreted in the presence of catabolin had the same average hydrodynamic size and ability to aggregate as the controls, and the core protein was substituted with the same number of glycosaminoglycan chains. The chains were the same average length and charge as normal and were sulphated to the same extent as the controls. Newly synthesized extracellular proteoglycan was not preferentially degraded. A 2-3-fold increase in glycosaminoglycan synthesis occurred in control and catabolin-treated cartilage in the presence of beta-D-xyloside (1 mM), more than 80% being secreted into the medium as free chains. Decreased incorporation of sulphate was not reversed in the presence of lysosomal-enzyme inhibitors, and there was no evidence in pulse-chase experiments of increased intracellular degradation of glycosaminoglycan chains before secretion. It is concluded that catabolin-treated cartilage synthesizes a smaller number of normal proteoglycan molecules.

Journal ArticleDOI
TL;DR: The effective conversion of the cytotoxic 4-hydroxynonenal and other reactive aldehydes to alcohols, which are probably less toxic, could play a role in the general defence system of the liver against toxic products arising from radical-induced lipid peroxidation.
Abstract: The metabolism of the lipid peroxidation product 4-hydroxynonenal and of several other related aldehydes by isolated hepatocytes and rat liver subcellular fractions has been investigated. Hepatocytes rapidly metabolize 4-hydroxynonenal in an oxygen-independent process with a maximum rate (depending on cell preparation) ranging from 130 to 230 nmol/min per 10(6) cells (average 193 +/- 50). The aldehyde is also rapidly utilized by whole rat liver homogenate and the cytosolic fraction (140 000 g supernatant) supplemented with NADH, whereas purified nuclei, mitochondria and microsomes supplemented with NADH show no noteworthy consumption of the aldehyde. In cytosol, the NADH-mediated metabolism of the aldehyde exhibits a 1:1 stoichiometry, i.e. 1 mol of NADH oxidized/mol of hydroxynonenal consumed, and the apparent Km value for the aldehyde is 0.1 mM. Addition of pyrazole (10 mM) or heat inactivation of the cytosol completely abolishes aldehyde metabolism. The various findings strongly suggest that hepatocytes and rat liver cytosol respectively convert 4-hydroxynonenal enzymically is the corresponding alcohol, non-2-ene-1,4-diol, according to the equation: CH3-[CH2]4-CH(OH)-CH = CH-CHO + NADH + H+→CH3-[CH2]4-CH(OH)-CH = CH-CH2OH + NAD+. The alcohol non-2-ene-1,4-diol has not yet been isolated from incubations with hepatocytes and liver cytosolic fractions, but was isolated in pure form from an incubation mixture containing 4-hydroxynonenal, isolated liver alcohol dehydrogenase and NADH and its chemical structure was confirmed by mass spectroscopy. Compared with liver, all other tissues possess only little ability to metabolize 4-hydroxynonenal, ranging from 0% (fat pads) to a maximal 10% (kidney) of the activity present in liver. The structure of the aldehyde has a strong influence on the rate and extent of its enzymic NADH-dependent reduction to the alcohol. The saturated analogue nonanal is a poor substrate and only a small proportion of it is converted to the alcohol. Similarly, nonenal is much less readily utilized as compared with 4-hydroxynonenal. The effective conversion of the cytotoxic 4-hydroxynonenal and other reactive aldehydes to alcohols, which are probably less toxic, could play a role in the general defence system of the liver against toxic products arising from radical-induced lipid peroxidation.

Journal ArticleDOI
TL;DR: Large and small proteoglycans were separately isolated from a number of connective tissues and compared to determine the extent of structural similarity, and two groups can be identified, although amino acid composition and protein core sizes are grossly similar.
Abstract: Large and small proteoglycans were separately isolated from a number of connective tissues and compared to determine the extent of structural similarity. This was studied by enzyme-linked immunosorbent assays and by the peptide patterns obtained when 125I-labelled proteoglycans were digested with trypsin. All the large proteoglycans, i.e. from tendon, sclera, cartilage and aorta, appear to contain the structure typical for the hyaluronic acid-binding region, both shown by enzyme-linked immunosorbent assay and by content of peptides unique for this region. These proteoglycans also share other structural features of the protein core, as indicated by immunological cross-reactivity and similar peptide patterns. The large proteoglycans from aorta in addition show the presence of unique structures both upon immunoassay and with regard to peptide pattern. Among the small proteoglycans two groups can be identified, although amino acid composition and protein core sizes are grossly similar. One group consists of the small proteoglycans from aorta and cartilage having similar peptide maps and showing immunological cross-reactivity in enzyme-linked immunosorbent assay. The other distinctly different group consists of the small proteoglycans from bone, cornea, sclera and tendon, which among them show identity in enzyme-linked immunosorbent assay and similar peptide patterns. Proteoglycans from the two groups, however, show partial immunological cross-reactivity.

Journal ArticleDOI
TL;DR: This review aims to summarize the current situation regarding these actions of the interferons and suggests areas in which the perception of such control of cell function may be expected to increase.
Abstract: The interferons are a widely studied group of proteins which were first identified by their ability to protect cells against virus infections (Stewart, 1979). They are synthesized and secreted by a variety of cell types in response to several classes of inducers (notably virus infections) and exert their effects in vivo as a result of interaction with other cells in distant parts of the body. In this sense the interferons are analogous to the polypeptide hormones. A functional similarity with hormones is further emphasized by the recent identification in target cells of specific membrane receptors for interferons and by the multiplicity of biological effects which result from the interactions of the interferons with these receptors. In recent years it has become clear that the interferons are capable of influencing cellular physiology and behaviour in a number of ways. Their effects include inhibition of cell growth and proliferation, regulation of the expression of specific genes, modulation of cell differentiation, and activation of certain cell types in the immune system (e.g. macrophages and natural killer cells). The mechanisms underlying the antiviral actions of the interferons have been widely studied and frequently reviewed (Baglioni, 1979; Hovanessian, 1979; Lengyel, 1982; Sen, 1982). In contrast, information concerning the effects of interferons on cell proliferation and differentiation has only been obtained during the last decade, as our understanding ofmechanisms ofgrowth regulation and gene expression in eukaryotes in general has increased. This review aims to summarize the current situation regarding these actions of the interferons and suggests areas in which our perception of such control of cell function may be expected to increase.

Journal ArticleDOI
TL;DR: It is reported that the presence of this label decreases the affinity of actin for myosin subfragment 1 by less than a factor of 2 and the rate ofActin binding is unaffected by the label and the dissociation rate is increased by up to a factors of 2.
Abstract: A pyrene label attached to Cys-374 of actin has been shown to be a useful probe for monitoring the interaction of actin with myosin subfragments [Kouyama & Mihashi (1981) Eur. J. Biochem. 114, 33-38]. We report that the presence of this label decreases the affinity of actin for myosin subfragment 1 by less than a factor of 2. The rate of actin binding is unaffected by the label and the dissociation rate is increased by up to a factor of 2. Both the rate of actin binding to, and the rate of actin dissociation from, heavy meromyosin show two phases when monitored by pyrene fluorescence. Thin filiments reconstituted from pyrene-labelled actin show a 5% increase in pyrene fluorescence on binding Ca2+.

Journal ArticleDOI
TL;DR: Data suggest that an increased loss of GSH from the liver constitutes an important mechanism for the decrease in GSH induced by ethanol, and an inhibition of G SH synthesis is observed.
Abstract: The effect of acute ethanol administration on rates of synthesis and utilization of hepatic glutathione (GSH) was studied in rats after a pulse of [35S]cysteine. A 35% decrease in hepatic GSH content 5h after administration of 4 g of ethanol/kg body wt. was accompanied by a 33% increase in the rate of GSH utilization. The decrease occurred without increases in hepatic oxidized glutathione (GSSG) or in the GSH/GSSG ratio. The rate of non-enzymic condensation of GSH with acetaldehyde could account for only 6% of the rate of hepatic GSH disappearance. The increased loss of [35S]GSH induced by ethanol was not accompanied by an increased turnover; rather, a 30% inhibition of GSH synthesis balanced the increased rate of loss, leaving the turnover rate unchanged. The rate of acetaldehyde condensation with cysteine in vitro occurred at about one-third of the rate of GSH loss in ethanol-treated animals. However, ethanol induced only a minor decrease in liver cysteine content, which did not precede, but followed, the decrease in GSH. The characteristics of 2-methylthiazolidine-4-carboxylic acid, the condensation product between acetaldehyde and cysteine, were studied and methodologies were developed to determine its presence in tissues. It was not found in the liver of ethanol-treated animals. Ethanol administration led to a marked increase (47%) in plasma GSH in the post-hepatic inferior vena cava, but not in its pre-hepatic segment. Data suggest that an increased loss of GSH from the liver constitutes an important mechanism for the decrease in GSH induced by ethanol. In addition, an inhibition of GSH synthesis is observed.

Journal ArticleDOI
TL;DR: Inositol 1,4,5-trisphosphate was released rapidly, with no measurable latency on hormone stimulation, and, consistent with its proposed role as an intracellular messenger for Ca2+ mobilization, there was good temporal correlation between its formation and Ca2-mediated events in these tissues.
Abstract: The inositol trisphosphate liberated on stimulation of guinea-pig hepatocytes, pancreatic acinar cells and dimethyl sulphoxide-differentiated human myelomonocytic HL-60 leukaemia cells is composed of two isomers, the 1,4,5-trisphosphate and the 1,3,4-trisphosphate. Inositol 1,4,5-trisphosphate was released rapidly, with no measurable latency on hormone stimulation, and, consistent with its proposed role as an intracellular messenger for Ca2+ mobilization, there was good temporal correlation between its formation and Ca2+-mediated events in these tissues. There was a definite latency before an increase in the formation of inositol 1,3,4-trisphosphate could be detected. In all of these tissues, however, it formed a substantial proportion of the total inositol trisphosphate by 1 min of stimulation. In guinea-pig hepatocytes, where inositol trisphosphate increases for at least 30 min after hormone application, inositol 1,3,4-trisphosphate made up about 90% of the total inositol trisphosphate by 5-10 min. In pancreatic acinar cells, pretreatment with 20 mM-Li+ caused an increase in hormone-induced inositol trisphosphate accumulation. This increase was accounted for by a rise in inositol 1,3,4-trisphosphate; inositol 1,4,5-trisphosphate was unaffected. This finding is consistent with the observation that Li+ has no effect on Ca2+-mediated responses in these cells. The role, if any, of inositol 1,3,4-trisphosphate in cellular function is unknown.

Journal ArticleDOI
TL;DR: Significantly higher alanine aminotransferase, gamma-glutamyltransferase and lactate dehydrogenase activities and lower glutamate dehydrogenases and pyruvate kinase activities in periportal than in perivenous cells demonstrate marked separation.
Abstract: Intact rat liver cells from the perivenous region were isolated by collagenase perfusion after first destroying the periportal region by a brief portal infusion of digitonin. Periportal cells were isolated after retrograde digitonin infusion. Significantly higher alanine aminotransferase, gamma-glutamyltransferase and lactate dehydrogenase activities and lower glutamate dehydrogenase and pyruvate kinase activities in periportal than in perivenous cells demonstrate marked separation. The high yield allows further characterization in vitro of the cell populations.

Journal ArticleDOI
TL;DR: A proteolytic enzyme was purified from the post-myofibrillar fraction of rat skeletal muscle and appeared to be homogeneous as judged by disc electrophoresis in polyacrylamide gels and by immunoelectrophoresIS.
Abstract: A proteolytic enzyme was purified from the post-myofibrillar fraction of rat skeletal muscle. The purification procedure consisted of fractionation of the muscle extract by (NH4)2SO4, chromatography on DEAE-Sephacel, fast protein liquid chromatography on Mono Q and gel filtration on Sepharose 6B. The enzyme preparation appeared to be homogeneous as judged by disc electrophoresis in polyacrylamide gels and by immunoelectrophoresis. The isoelectric point of the proteinase is at 5.1-5.2. The enzyme has an Mr of about 650 000 and dissociates into eight subunits of Mr 25 000-32 000 when subjected to electrophoresis in sodium dodecyl sulphate/polyacrylamide gels. The proteinase contains hydrolytic activity against N-blocked tripeptide 4-methyl-7-coumarylamide substrates with an arginine or phenylalanine residue adjacent to the leaving group. Maximum activity with the first group of substrates was at pH 10.5, and this activity was inhibited by leupeptin, chymostatin and Ca2+. Maximum activity with the latter group of substrates was at pH 7.5, and was also inhibited by the two microbial inhibitors, but was activated by Ca2+ ions. By using [14C]methylcasein as a substrate, maximum activity was observed at pH9.0, and this proteolytic activity was not affected by leupeptin, was enhanced by chymostatin and inhibited by Ca2+. Similar effects were observed when benzyloxycarbonyl-Leu-Leu-Glu 2-naphthylamide was used as a substrate. These enzymic activities were abolished by p-hydroxymercuribenzenesulphonic acid or mersalyl acid, whereas a small activation was observed with cysteine or dithiothreitol.

Journal ArticleDOI
TL;DR: In incubated colonocytes isolated from rat colons, the rates of utilization O2, glucose or glutamine were linear with respect to time for over 30 min, and the concentrations of adenine nucleotides plus the ATP/ADP or ATP/AMP concentration ratios remained approximately constant for 30 min.
Abstract: In incubated colonocytes isolated from rat colons, the rates of utilization O2, glucose or glutamine were linear with respect to time for over 30 min, and the concentrations of adenine nucleotides plus the ATP/ADP or ATP/AMP concentration ratios remained approximately constant for 30 min. Glutamine, n-butyrate or ketone bodies were the only substrates that caused increases in O2 consumption by isolated incubated colonocytes. The maximum activity of hexokinase in colonic mucosa is similar to that of 6-phosphofructokinase. Starvation of the donor animal decreased the activities of hexokinase and 6-phosphofructokinase, whereas it increased those of glucose-6-phosphatase and fructose-bisphosphatase. Isolated incubated colonocytes utilized glucose at about 6.8 mumol/min per g dry wt., with lactate accounting for 83% of glucose removed. These rates were not affected by the addition of glutamine, acetoacetate or n-butyrate, and starvation of the donor animal. Isolated incubated colonocytes utilized glutamine at about 5.5 mumol/min per g dry wt., which is about 21% of the maximum activity of glutaminase. The major end-products of glutamine metabolism were glutamate, aspartate, alanine and ammonia. Starvation of the donor animal decreased the rate of glutamine utilization by colonocytes, which is accompanied by a decrease in glutamate formation and in the maximum activity of glutaminase. Isolated incubated colonocytes utilized acetoacetate at about 3.5 mumol/min per g dry wt. This rate was not markedly affected by addition of glucose or by starvation of the donor animal. When colonocytes were incubated with n-butyrate, both acetoacetate and 3-hydroxybutyrate were formed, with the latter accounting for only about 19% of total ketones produced.

Journal ArticleDOI
TL;DR: Energy metabolism in proliferating cultured rat thymocytes was compared with that of freshly prepared non-proliferating resting cells and formation of acetyl-CoA in the presence of pyruvate might explain the relatively enhanced oxidation of glutamate to CO2 by proliferatingThymocytes.
Abstract: Energy metabolism in proliferating cultured rat thymocytes was compared with that of freshly prepared non-proliferating resting cells. Cultured rat thymocytes enter a proliferative cycle after stimulation by concanavalin A and Lymphocult T (interleukin-2), with maximal rates of DNA synthesis at 60 h. Compared with incubated resting thymocytes, glucose metabolism by incubated proliferating thymocytes was 53-fold increased; 90% of the amount of glucose utilized was converted into lactate, whereas resting cells metabolized only 56% to lactate. However, the latter oxidized 27% of glucose to CO2, as opposed to 1.1% by the proliferating cells. Activities of hexokinase, 6-phosphofructokinase, pyruvate kinase and aldolase in proliferating thymocytes were increased 12-, 17-, 30- and 24-fold respectively, whereas the rate of pyruvate oxidation was enhanced only 3-fold. The relatively low capacity of pyruvate degradation in proliferating thymocytes might be the reason for almost complete conversion of glucose into lactate by these cells. Glutamine utilization by rat thymocytes was 8-fold increased during proliferation. The major end products of glutamine metabolism are glutamate, aspartate, CO2 and ammonia. A complete recovery of glutamine carbon and nitrogen in the products was obtained. The amount of glutamate formed by phosphate-dependent glutaminase which entered the citric acid cycle was enhanced 5-fold in the proliferating cells: 76% was converted into 2-oxoglutarate by aspartate aminotransferase, present in high activity, and the remaining 24% by glutamate dehydrogenase. With resting cells the same percentages were obtained (75 and 25). Maximal activities of glutaminase, glutamate dehydrogenase and aspartate aminotransferase were increased 3-, 12- and 6-fold respectively in proliferating cells; 32% of the glutamate metabolized in the citric acid cycle was recovered in CO2 and 61% in aspartate. In resting cells this proportion was 41% and 59% and in mitogen-stimulated cells 39% and 65% respectively. Addition of glucose (4 mM) or malate (2 mM) strongly decreased the rates of glutamine utilization and glutamate conversion into 2-oxoglutarate by proliferating thymocytes and also affected the pathways of further glutamate metabolism. Addition of 2 mM-pyruvate did not alter the rate of glutamine utilization by proliferating thymocytes, but decreased the rate of metabolism beyond the stage of glutamate significantly. Formation of acetyl-CoA in the presence of pyruvate might explain the relatively enhanced oxidation of glutamate to CO2 (56%) by proliferating thymocytes.

Journal ArticleDOI
TL;DR: Human cathepsin L proved to be similar to the rat and rabbit enzymes in regard to kinetic constants for the substrate benzyloxycarbonylphenylalanylarginine 7-(4-methyl)coumarylamide and rates of inactivation by the active-site-directed reagents.
Abstract: Cathepsin L was purified to apparent homogeneity from human liver obtained post mortem. It was necessary to treat the homogenate at pH 4.2 and 37 degrees C to release active enzyme. The purification procedure involved ion-exchange chromatography on carboxymethyl-Sephadex and the Mono S column of a Pharmacia fast-protein-liquid-chromatography system. The enzyme was found to consist of two polypeptide chains of Mr 25 000 and 5000. The larger chain was shown to contain the active-site cysteine residue. Human cathepsin L proved to be similar to the rat and rabbit enzymes in regard to kinetic constants for the substrate benzyloxycarbonylphenylalanylarginine 7-(4-methyl)coumarylamide and rates of inactivation by the active-site-directed reagents benzyloxycarbonylphenylalanylphenylalanyldiazomethane and benzyloxycarbonylphenylalanylalanyldiazomethane. Thus clear characteristics of cathepsin L are now emerging, and these should simplify the identification of the enzyme in other tissues and species.

Journal ArticleDOI
TL;DR: Rabbit forelimb tendons incubated for 15 or 21 days at 35 degrees C in the presence of 8 or 24 mg of glucose/ml were shown to change their chemical, biochemical and mechanical characteristics, indicating the formation of new covalent stabilizing bonds.
Abstract: Rabbit forelimb tendons incubated for 15 or 21 days at 35 degrees C in the presence of 8 or 24 mg of glucose/ml were shown to change their chemical, biochemical and mechanical characteristics. The tendons treated with glucose contained up to three times as much hexosyl-lysine and hexosylhydroxylysine as did control tendons as judged by assay of NaB3H4-reduced samples. Measurement of the force generated on thermal contraction showed significant increases in glycosylated tendons compared with controls, indicating the formation of new covalent stabilizing bonds. This conclusion was supported by the decreased solubility of intact tendons and re-formed fibres glycosylated in vitro, and by the evidence from peptide maps of CNBr-digested glucose-incubated tendons. The latter, when compared with peptide maps of control tendons, revealed the presence of additional high-Mr peptide material. These peptides appear to be cross-linked by a new type of covalent bond stable to mild thermal and chemical treatment. This system in vitro provides a readily controlled model for the study of the chemistry of changes brought about in collagen by non-enzymic glycosylation in diabetes.

Journal ArticleDOI
TL;DR: In contrast with the non-activated proteinase, the activated enzyme considerably degrades muscle cytoplasmic proteins in vitro, so it is not unlikely that, in vivo, potential activators such as fatty acids can induce the multicatalytic proteinase to participate in muscle protein breakdown.
Abstract: A multicatalytic proteinase from rat skeletal muscle contains active site(s) catalysing the degradation of benzoyl-Val-Gly-Arg 4-methyl-7-coumarylamide, succinyl-Ala-Ala-Phe 4-methylcoumarylamide and [14C]methylcasein as well as benzyloxy-carbonyl-Leu-Leu-Glu 2-naphthylamide. These activities are 7-14-fold activated by 1 mM-sodium dodecyl sulphate. The activation leads to a higher susceptibility to the proteinase inhibitor chymostatin and to a lower ability to be inhibited and precipitated by antibodies raised against the non-activated enzyme. Since no changes in Mr or subunit composition were observed in the SDS-activated form, some conformational changes seem to occur during the activation step. More pronounced activation was observed in the presence of physiological concentrations of fatty acids; oleic acid at 100 microM concentrations stimulated the proteinase about 50-fold. In contrast with the non-activated proteinase, the activated enzyme considerably degrades muscle cytoplasmic proteins in vitro. Thus it is not unlikely that, in vivo, potential activators such as fatty acids can induce the multicatalytic proteinase to participate in muscle protein breakdown.

Journal ArticleDOI
TL;DR: The purified protein interfered with the fast inactivation of thrombin clotting as well as amidolytic activity by antithrombin III-heparin complex, indicating the ability of S-protein to neutralize heparin activity.
Abstract: S-protein, the main inhibitor of the assembly of the membrane attack complex of complement, was isolated from human plasma by a simple purification procedure, which includes barium citrate adsorption, ammonium sulphate precipitation, chromatography on DEAE-Sephacel and Blue Sepharose and gel filtration on Sephacryl S-200. The homogeneous protein (sedimentation coefficient 4.6 S) was obtained in approx. 5% yield relative to its concentration in plasma, which was found to be 0.3-0.5 mg/ml. The final product did not cross-react with antisera against complement proteins or other proteinase inhibitors of human plasma. On polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, S-protein migrated as a single-chain band with an apparent Mr of 74000 under non-reducing conditions and as a doublet of Mr 78000 and 65000 upon reduction. In plasma or serum S-protein also existed in two forms of corresponding Mr values, as was evidenced by an immunoblot enzyme-linked immunosorbent assay technique. S-protein was found to be an acidic glycoprotein with 10% (W/W) carbohydrate content and several isoelectric points in the range pH 4.75-5.25, and it contained one free thiol group per molecule of protein. The functional properties of S-protein in the complement system were demonstrated by its ability to inhibit complement-dependent cell lysis in a concentration-dependent manner (Ki 0.6 microM) and by its incorporation into the nascent SC5b-7 complex. A new function for S-protein could be revealed in the blood coagulation system. The slow progressive inhibition of thrombin by antithrombin III was not affected by S-protein, whereas the purified protein interfered with the fast inactivation of thrombin clotting as well as amidolytic activity by antithrombin III-heparin complex. The acceleration of this inhibition reaction by heparin was counteracted by S-protein, indicating the ability of S-protein to neutralize heparin activity.

Journal ArticleDOI
TL;DR: Recombinant DNA technology has made it possible to isolate the mRNA and gene sequences of families such as that of the myosin alkali light chains, which can be used to address questions about the regulation of the corresponding genes.
Abstract: In all eukaryotes, actin and myosin play a role in the maintenance of cell shape and in cellular movement (e.g. reviewed by Korn, 1978). In the muscles of multicellular organisms actomyosin is the principal structural component of the sarcomere, the unit of the muscle fibre. Movement is effected by the sliding of actin and myosin filaments over each other, with accompanying hydrolysis of ATP providing energy for this process, which is regulated by calcium-sensitive myofibrillar proteins (see Bagshaw, 1982, for review). The myosin molecule consists oftwo heavy chains (MHC, ofMr about 220000) each with a globular head region where the ATPase activity is situated, and a filamentous, rod-like tail, and four associated light chains (of Mr 15000-30000),two so-called regulatory or DTNB light chains (MLC2) and two alkali light chains (MLC1, MLC3). It is the alkali myosin light chain proteins and their genes which are the subject of this review. The function of the alkali light chains is not clear. It has been shown that, although these light chains are associated with the globular region of the myosin molecule, probably near the head/tail junction (Flicker et al., 1983; Sellers & Harvey, 1984), they do not modify the ATPase activity of isolated myosin heavy chains (e.g. Wagner & Giniger, 1981; Sivaramakrishnan & Burke, 1982). Other experiments (e.g. Wagner & Weeds, 1977; Winstanley et al., 1979; Trayer & Trayer, 1985) would suggest that they play a role in the interaction of the head region of myosin with actin. It has become clear in the last few years, however, that distinct isoforms of the myosin alkali light chains are present in different muscles and in non-muscle cells, suggesting that these isoforms are associated with the different contractile properties of these tissues. This is also the case for the actins and myosin heavy chains. The presence of different isoforms of the contractile proteins in different adult and developing muscles (for review see Buckingham & Minty, 1983) raises interesting questions, not only about their function, but also about the regulation ofthe corresponding genes. Recombinant DNA technology has made it possible to isolate the mRNA and gene sequences of families such as that of the myosin alkali light chains, which can be used to address such questions. The contribution of this type of approach to research on muscle, and the interest of muscle as a model system for the molecular biologist in which to ask fundamental questions about gene regulations, are complementary aspects discussed in this review. The myosin alkali light chain isoforms and their genes