bluuebunny/crossref_metadata_2025_split huggingface dataset:
Properties and Subcellular Localization of Acid Phosphatase Activity in Cultured Arterial Smooth Muscle Cells
Authors: LEAKE David S. , HEALD Benjamin , PETERS Timothy J. 
Published: July 2010

Smooth muscle cells were cultured from pig aortas and their acid phosphatase activity toward 4‐methylumbelliferyl phosphate was studied. Cell homogenates hydrolysed this substrate with a pH optimum of 5.0, with very little activity at alkaline pH. The apparent Km of the activity was approximately 20 μM at pH 5.0, and the activity was directly proportional to both the incubation time and to the concentration of homogenate. Acid phosphatase activity was inhibited by p‐chloromercuribenzoic acid, by fluoride ions, and by chloroquine.Postnuclear supernatants of the cells were subjected to analytical subcellular fractionation on sucrose density gradients. Acid phosphatase activity had a modal equilibrium density of 1.13 g/ml, which was similar to that of 5′‐nucleotidase and galactosyltransferase, marker enzymes for the plasma membrane and Golgi complex, respectively. The distribution of acid phosphatase activity also had a shoulder of equilibrium density about 1.16 g/ml, which may have corresponded to activity in the lysosomes. The activity of N‐acetyl‐β‐glucosaminidase, a lysosomal marker enzyme, had a very broad distribution with much of its activity equilibrating at low densities.When the cells were homogenized in sucrose medium containing digitonin, a selective membrane perturbant, the N‐acetyl‐β‐glucosaminidase activity remained almost entirely in the sample layer, showing that the lysosomes had been disrupted. The modal equilibrium density of 5′‐nucleotidase activity was increased to 1.19 g/ml, whereas the modal equilibrium density of galactosyltransferase activity was not significantly affected. Acid phosphatase activity showed a similar increase in modal equilibrium density to 5′‐nucleotidase, but had a shoulder on the least dense side of its peak, which may have corresponded to activity in the Golgi complex. To confirm the plasma membrane localization of a large proportion of the acid phosphatase activity, intact cells were treated with digitonin and were then washed and homogenized in sucrose medium without digitonin. The modal equilibrium densities of both 5′‐nucleotidase and acid phosphatase activity were increased to similar extents, whereas the equilibrium densities of the marker enzymes for the other organelles were not significantly affected.Thus, although acid phosphatase is a widely used marker enzyme for the lysosomes in subcellular fractionation studies, its activity towards 4‐methylumbelliferyl phosphate cannot be used as a marker for these organelles in pig arterial smooth muscle cells in culture.
Increase of Fidelity of Polypeptide Synthesis by Spermidine in Eukaryotic Cell‐Free Systems
Authors: IGARASHI Kazuei , HASHIMOTO Sanae , MIYAKE Atsuko , KASHIWAGI Keiko , HIROSE Seiyu 
Published: July 2010

The mechanism of spermidine‐induced increase of fidelity of polypeptide synthesis in a wheat germ cell‐free system has been studied. It was found that the increase of fidelity in the presence of spermidine occurred mainly at the level of binding of aminoacyl‐tRNA to ribosomes, that reduction of misreading was more marked at the 5′‐base than at the 3′‐base of the codon and that misreading caused by paromomycin and kanamycin C was not significantly decreased by spermidine. It was deduced from these results that spermidine inhibited low‐frequency misreading more strongly than high‐frequency misreading. In addition, spermidine was found to stimulate the rejection of non‐cognate aminoacyl‐tRNA mainly at an initial discrimination step during the binding of aminoacyl‐tRNA to ribosomes, and slightly at a subsequent GTP‐dependent discrimination step, the so‐called proofreading step.In yeast, rabbit reticulocyte, andArtemia salinacell‐free systems, spermidine was found to increase the fidelity of protein synthesis.
Specificities and Selectivities of Glycerol‐3‐Phosphate Acyltransferase and Monoacylglycerol‐3‐Phosphate Acyltransferase from Pea and Spinach Chloroplasts
Authors: FRENTZEN Margrit , HEINZ Ernst , McKEON Thomas A. , STUMPF Paul K. 
Published: July 2010

In addition to acyl‐CoA, purified glycerol‐3‐phosphate acyltransferases from pea and spinach chloroplasts can also use acyl‐(acyl‐carrier protein), acyl‐ACP, as a substrate for glycerol 3‐phosphate acylation. The enzyme fractions showed absolute specificity for glycerol 3‐phosphate as acyl acceptor. Dihydroxyacetone phosphate was ineffective. Glycerol 3‐phosphate was almost exclusively acylated at the C‐1 position. If mixtures of palmitoyl‐ACP, stearoyl‐ACP and oleoyl‐ACP were offered, the oleoyl group was preferred. These data fully agree with previous experiments on these enzymes carried out with various acyl‐CoA thioesters. Kinetic data determined with different acyl‐ACPs as substrates are consistent with the observed fatty acid selectivity for the oleoyl group. Double labelling experiments with mixtures of oleoyl‐ACP and oleoyl‐CoA demonstrated a preference for ACP‐thioesters.Monoacylglycerol‐3‐phosphate acyltransferase, localized in the envelope of chloroplasts, can also utilize acyl‐ACP as substrate. Envelope fractions of spinach as well as of pea showed a high specificity for the palmitoyl group when ACP‐thioesters or CoA‐thioesters were offered and directed this acyl group to the C‐2 position of the glycerol backbone. Results from competition experiments with [14C]palmitoyl‐ACP and [3H]palmitoyl‐CoA indicate that the membrane‐bound acyltransferase preferably uses ACP‐thioesters for the acylation of 1‐acylglycerol 3‐phosphate. According to fatty acid selectivities and specificities the main product of the recombined acyltransferase systems in chloroplasts of 16:3 plants as well as 18:3 plants is a phosphatidic acid with the oleoyl group at C‐1 and the palmitoyl group at C‐2 whereas molecules with the oleoyl group at both positions are not synthesized.Under appropriate conditions the phosphatidic acid formed by the soluble acyltransferase and spinach envelope was rapidly converted to monogalactosyl diacylglycerol in the presence of UDP‐galactose. In analogous assays with acyltransferase and envelope from pea only a low proportion of labelled phosphatidic acid was converted via diacylglycerol to monogalactosyl diacylglycerol. In both systems monogalactosyl diacylglycerol synthesized in the presence of C16:0‐thioesters and C18:1‐thioesters carried C18:1 at C‐1 and C16:0 at C‐2 in agreement with the fatty acid selectivity of the acyltransferase systems.
Is Cytochrome b Really the Antimycin‐Binding Component of the Cytochrome b—c1 Complex of Yeast Mitochondria
Authors: CHEVILLOTTE‐BRIVET Paule , MEUNIER‐LEMESLE Danielle , FORGET Nicole , PAJOT Patrick 
Published: July 2010

The antimycin binding sites were found to be independent of cytochrome(s) b synthesis in pseudo‐wild‐type revertants of cytochrome b mutants. These revertants, whose primary mutation is located in the introns of the cob‐box gene of mitochondrial DNA, have modified contents of cytochromes b‐562 and b‐565 and fully functional respiratory chain.
Missense mutations in three genetic loci allocated to the exons of the cytochrome b gene, abolish the strong affinity binding of antimycin.
It is proposed that the antimycin‐binding component is not cytochrome b itself, but interacts with it in such a way that an alteration of the cytochrome b structure affects the antimycin‐binding site.
Isoenzymes of cAMP‐Dependent Protein Kinase in Developing Rat Liver and in Malignant Hepatic Tissues
Authors: WITTMAACK Frank M. , WEBER Wolfgang , HILZ Helmuth 
Published: July 2010

Total R proteins and total cAMP‐dependent protein kinase activity during rat liver development reach highest values per unit DNA when the organ has attained full metabolic competence. The parallel changes indicate coordinate synthesis of R and C subunits during hepatic development.In contrast to total R2· C2, protein kinase activation and endogenous cAMP levels were highest around birth. Immunotitration with anti‐RI and anti‐RII in the presence of protein‐A–Sepharose of extracts obtained from various developmental stages and from hepatomas suggested a relation of both protein kinase I and II to the terminal differentiation of the organ rather than to cellular proliferation rates. The type‐II enzyme appears to be subject to additional regulations connected with neonatal adaptation phenomena.A non‐enzymic analysis of the protein kinase activation status is described. It is based on the determination of the ratio of amounts: R · cAMP/total R, which showed a linear correlation with the conventional protein kinase activity ratio (–cAMP/+cAMP).
Calmodulin Stimulation of Adenylate Cyclase of Intestinal Epithelium
Authors: AMIRANOFF Brigitte M. , LABURTHE Marc C. , ROUYER‐FESSARD Christiane M. , DEMAILLE Jacques G. , ROSSELIN Gabriel E. 
Published: July 2010

The present work analyzes the calmodulin stimulation of rat intestinal epithelial adenylate cyclase. It is shown that: (1) activation of intestinal adenylate cyclase by calmodulin occurs in the presence of 1 – 30 μM free Ca2+; (2) in the 0.5 – 10 mM MgCl2concentration range, the pattern of calmodulin action is not modified by Mg2+; calmodulin acts by increasing the maximal velocity of the intestinal adenylate cyclase without altering its affinity for ATP and Mg2+; (3) the calmodulin effect does not require the presence of guanine nucleotide and is even additive to that induced by GTP or its synthetic non‐hydrolyzable analog guanosine 5′‐[β,γ‐imido]triphosphate; (4) calmodulin and sodium fluoride exert an additive effect on the enzyme activity; (5) calmodulin and prostaglandins E1or E2display additive stimulatory actions, whatever the dose of prostaglandins tested; calmodulin, therefore, does not modify the affinity of intestinal adenylate cyclase for its hormonal effectors; (6) trifluopeazine in the 25–200 μM range of concentrations (K0.5= 85 μM) suppresses the adenylate cyclase activity which has been stimulated by 5 μM calmodulin; (7) calmodulin is active in the 0.1 – 10 μM concentration range, half‐maximal stimulation is induced by 0.9 μM protein, i.e. a dose of calmodulin that is much below the calmodulin content of the intestinal epithelial cell; (8) at low (0.5 mM) and high (5 mM) Mg2‐concentration, a sixfold and threefold maximal increase of basal activities are observed, respectively.Our data indicate that calmodulin stimulates intestinal adenylate cyclase by a mechanism that is different from the hormonal activation and suggest the presence of a specific regulatory site for calmodulin on the enzyme.
Latent and Active Human Polymorphonuclear Leukocyte Collagenases
Authors: MACARTNEY Henry W. , TSCHESCHE Harald 
Published: July 2010

Latent and active human polymorphonuclear leukocyte collagenase could be purified to apparent homogenei by a rapid and reproducible method involving affinity chromatography on concanavalin‐A–Sepharose, collage Sepharose and activated thiol‐Sepharose 4‐B, gel filtration on Sephacryl S‐300 and ion‐exchange chromatograpl on DEAE‐Sephacel.The latent and active collagenase were shown to be single entities with apparent Mr 91000 and 6400 respectively, as determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis without mercapto ethanol. Dodecyl sulfate gel electrophoresis of the latent enzyme in the presence of mercaptoethanol showed two bands with apparent Mr of 64000 and 24000 indicating the cleavage of a disulfide bond.Activation of the native latent collagenase could be achieved by_ disulfide‐containing compounds. A concomitant decrease in molecular weight of approximately 25000 was observed on activation of the latent enzyme. The activators proved ineffective after alkylation of the latent enzyme with iodoacetamide. This lends support to the proposed thiol/disulfide interchange mechanism in the activation reaction [Macartney, H. W. and Tschesche, H (1980) FEBS Lett. 119, 327–332]. The active collagenase could be completely inhibited by an inhibitor dissociated from the latent enzyme, i.e. a complex of enzyme and inhibitor. Regulation of the collagenase activity could be achieved by the redox potential of the glutathione cycle [Tschesche, H. and Macartney, H. W. (1981) Eur. J. Biochem. 120, 183–190].The latent and active enzymes were shown to be acidic proteins with isoelectric points at pH 5.5 and 6.4 respectively. Amino acid analyses of both enzymes have been obtained and indicate a difference of about 222–223 amino acid residues between the latent and active collagenases. This corresponds to a difference of approximately Mr25000 between the active and the latent enzyme, the latter being a mixed disulfide of the enzyme and its inhibitor.The optimum pH for collagenolytic activity was observed near neutrality. The enzyme specificity was revealed to be that of a true mammalian collagenase splitting the native type I tropocollagen molecule into three‐quarter and one‐quarter fragments according to dodecyl sulfate/polyacrylamide gel electrophoresis. No other contaminating proteolytic activity was observed in the purified latent and active collagenase preparations. The active enzyme was found to be stable for over one year when stored at ‐ 70°C. The collagenase was capable of reducing the specific viscosity of a native type I collagen solution by approximately 40%.The amount of latent collagenase present in the leukocytes was estimated to 0.65 mg/1010 cells. The overall yield obtained after the purification to homogeneity was 75–80% of the theoretical value.
The Collagenase Inhibitor from Human Polymorphonuclear Leukocytes
Authors: MACARTNEY Henry W. , TSCHESCHE Harald 
Published: July 2010

A new collagenase inhibitor of latent human polymorphonuclear collagenase, initially reported by Macartney and Tschesche [Hoppe‐Seyler's Z. Physiol. Chem. 361, 298–299 (1980) and FEBS Lett. 119, 327–332 (1980)], has been purified to apparent homogeneity. We described several methods of purification of this inhibitor, but the fastest and most reproducible method is by passage of purified latent human polymorphonuclear leukocyte collagenase on activated thiol‐Sepharose 4‐B (preceding paper in this journal). Active collagenase is eluted from the column and the collagenase inhibitor is retarded. Elution of the inhibitor is achieved by eluting the column with thiol compounds in the equilibration buffer. Further purification of the inhibitor is obtained by gel filtration on Sephacryl S‐300 and ion‐exchange chromatography on DEAE‐Sephacel.The inhibitor exhibits a strict specificity for mammalian collagenases. It inhibits collagenases isolated from human synovial fluid, human normal and diabetic skin fibroblasts and human embryonic skin fibroblasts. The inactive complexes are all subject to activation by several disulfide compounds, such as oxidised glutathione by the thiol/disulfide interchange reaction already described.The purified inhibitor was shown by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (10% gel) to be homogeneous and displayed an apparent molecular weight of 24500. Titration of the inhibitor with Ellmans reagent revealed that it contains one free sulfhydryl group, a prerequisite for its inhibitory activity. The inhibitor inhibits human polymorphonuclear leukocyte collagenase in a strict 1:1 stoichiometric reaction. The amino acid composition of the complex formed from the inhibitor and active enzyme reproduces that of the latent collagenase thus confirming that the latent enzyme is a 1:1 complex.The inhibitor is an acidic protein having an isoelectric point at pH 5.5, which would be expected from the amino acid analysis. The composition reveals a glycoprotein of about 240 residues containing a carbohydrate moiety composed of N‐acetylglucosamine, mannose, galactose and glucose. The amino acid composition of the inhibitor is not identical to that of β1‐anticollagenase from human plasma.
Characterisation of β1‐Anticollagenase from Human Plasma and Its Reaction with Polymorphonuclear Leukocyte Collagenase by Disulfide/Thiol Interchange
Authors: MACARTNEY Henry W. , TSCHESCHE Harald 
Published: July 2010

A β1‐serum component, β1‐anticollagenase, capable of inhibiting various mammalian tissue collagenases, was isolated from human plasma by gel filtration, affinity chromatography and ion‐exchange chromatography. The inhibitor contains 1–2 free sulfhydryl groups, which are a preequiste for inhibitory activity and for binding to the thiol‐Sepharose affinity support. Alkylation of β1‐anticollagenase by iodoacetamide blocks inhibitory activity. The inhibitor was purified to apparent homogeneity and exhibited a Mr= 30500 determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The amino acid and carbohydrate composition was determined. According to its composition and the isoelectric focussing β1‐anticollagenase is an acidic protein with an isoelectric point of 5.6.Inhibition of human leukocyte collagenase proceeds in a strong 1:1 stoichiometric reaction. The mechanism of this association takes place by a disulfide/thiol interchange reaction as has been previously indicated for human leukocyte collagenases in forming the latent enzyme [Macartney, H. W. and Tschesche, H. (1980) FEBS Lett. 119, 327–332]. The β1‐anticollagenase‐leukocyte‐collagenase complex (latent enzyme) is activatable by disulfide‐containing compounds such as cystine, oxidised glutathione, insulin, relaxin, trypsinogen and others, but not by 179,203‐di(S‐carboxymethyl)trypsinogen, or its trypsin derivative. Compounds containing inaccessible disulfide bonds, e.g. chymotrypsin, or sulfhydryl groups, e.g. d‐penicillamine, do not activate the complex. Activation is, however, easily obtained with the oxidised‐glutathione‐generating system myeloperoxidase/H2O2/glutathione as was previously demonstrated for the human leukocyte latent collagenase activatable in a phagocytosis‐simulated respiratory burst [Tschesche, H. and Macartney, H. W. (1981) Eur. J. Biochem. 120, 183–190.]
Kinetics of Reduction of Putidamonooxin by NADH–Putidamonooxin Oxidoreductase, Sodium Dithionite and Superoxide Radicals
Authors: BERNHARDT Frithjof‐Hans , KUTHAN Hartmut 
Published: July 2010

Putidamonooxin, a conjugated iron‐sulfur protein, functions as the oxygen‐activating component within the 4‐methoxybenzoate monooxygenase from Pseudomonas putida. A[2Fe–2S] cluster and a mononuclear non‐heme iron together form the oxygen‐activating units within the active sites of putidamonooxin. In the present paper the reduction kinetics of oxidized putidamonooxin were studied in the absence and in the presence of a substrate and/or the mononuclear non‐heme iron. The reducing agenst used were (a) NADH plus the NADH–putidamonooxin oxidoreductase and (b) artificial reductants, i.e. O2 radicals and sodium dithionite in the presence or in the absence of the NADH‐putidamonooxin oxidoreductase. The reduction kinetics of cofactor‐iron‐depleted putidamonooxin revealed that the addition of a substrate strongly enhanced the rate of reduction of the [2Fe–2S] chromophores by the native reductase but significantly decreased the reduction by artificial reductants. These effects were even more pronounced when the mononuclear non‐heme iron was bound to putidamonooxin in addition to the substrate. The presence of the native reductase markedly enhanced the rate of reduction of native putidamonooxin by sodium dithionite, though not to the optimal rate as measured in the presence of both NADH and the NADH–putidamonooxin oxidoreductase.The experimental findings are taken to show that the reduction of oxidized putidamonooxin in the absence and in the presence of its mononuclear non‐heme iron, when NADH–putidamonooxin oxidoreductase and artificial reductants are used as electron donors, may occur at two different sites, both of which are influenced by substrate binding. The most powerful reductant of putidamonooxin is the NADH–putidamonooxin oxidoreductase in its partially reduced state (which is characterized by its broad absorption band centered near 600 nm and its typical isotropic electron‐paramagnetic‐resonance signal with g= 2.0033), rather than in its fully reduced state.
The Role of Pyrrolo‐quinoline Semiquinone Forms in the Mechanism of Action of Methanol Dehydrogenase
Authors: DE BEER Ron , DUINE Johannis A. , FRANK Jzn , WESTERLING Jaap 
Published: July 2010

On storage of methanol dehydrogenase of Hyphomicrobium X as a solution, the electron spin resonance (ESR) spectrum becomes more complicated. Ka‐band (35 GHz) spectroscopy reveals that a paramagnetic species with a lower g‐vale is formed from pyrrolo‐quinoline semiquinone (PQQH) during the ageing process. From the way in which this second paramagnetic species is formed and the absence of an effect of substrate, activator or both on fresh or aged enzyme, it follows that the complex ESR spectrum cannot be used as an argument to support the view that a three‐electron reduced form of PQQ exists in methanol dehydrogenase.X‐band ESR spectroscopy of an enzyme solution reveals additional peaks at g= 2.03 and g= 2.01 only when the preparation contains O2 and is measured at liquid nitrogen temperature. The results point to the formation of a complex from PQQH and O2, most probably a peroxyl radical which only exists at low temperature.Direct reduction of methanol dehydrogenase, as it is isolated, is possible. The enzyme form thus produced has the same properties as that obtained from reduction of oxidized methanol dehydrogenase with substrate, containing only a low amount of PQQH‐but not other free radical species. This substantiates the earlier conclusion that the isolated enzyme is in a half‐oxidized form.A view on the mechanism of action of the enzyme is proposed which is quite opposite to the one that was recently advocated [Mincey et al. (1981) Biochemistry, 20, 7502–7509] since former and present results lead to the following conclusions: (a) there is no evidence for the involvement of a three‐electron reduced form of PQQ in the catalytic cycle of the enzyme; (b) enzyme molecules containing free radical species are not reduced by substrate; (c) the product is released before the enzyme is oxidized by electron acceptor.
UDPglucosyltransferase and Its Kinetic Fluorimetric Assay
Authors: VÄISÄNEN Matti V. T. , MACKENZIE Peter I. , HÄNNINEN Osmo O. P. 
Published: July 2010

A rapid, Kinetic assay for UDPglucosyltransferase has been developed using 1‐naphthol as substrate. It is based on the continuous fluorimetric monitoring of 1‐naphthyl glucoside formation during the reaction at physiological pH. The conjugate is easily distinguished from aglycone, since their fluorimetric properties differ.Glucoside biosynthesis in vitro by microsomal preparations isolated from the gut and fat body of cockroaches Periplaneta americana and Leucophaea maderae, and from the green gland and hepatopancreas of the crayfish Astacus astacus, has been demonstrated. The effects of buffer, pH, MgCl2, UDP‐glucuronic acid, UDP‐N‐acetylglucosamine, sodium cholate and sonication on the enzyme activity have been assessed. The kinetic parameters of 1‐naphthol and UDP‐glucose have also been determined.