Pub Date : 2005-03-03DOI: 10.1111/J.1432-1033.1980.TB06148.X
J. Hofsteenge, J. Vereijken, W. Weijer, J. Beintema, R. Wierenga, J. Drenth
p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens contains six methionine residues, one of which is N-terminal. After CNBr cleavage five peptides, ranging from 13 to 158 residues in length, and free homoserine were isolated and purified by repeated gel filtration. The alignment of the CNBr fragments was deduced from a 0.25-nm electron density map and sequence data. The isolated fragments account for the entire polypeptide chain. The amino acid sequence of the N-terminal quarter of the polypeptide chain was determined. The X-ray results together with the sequence data yielded details of the binding of FAD. The AMP moiety was bound to a beta alpha beta unit resembling that found in the dehydrogenases. Hydrogen bonds were present between the protein and the ribityl residue and the isoalloxazine ring. Furthermore, a homology was found between the N-terminal amino acid sequence of p-hydroxybenzoate hydroxylase and another enzyme containing FAD, viz. D-amino acid oxidase. This finding suggests the presence of a mononucleotide binding fold at the N terminus of the latter.
{"title":"Primary and tertiary structure studies of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. Isolation and alignment of the CNBr peptides; interactions of the protein with flavin adenine dinucleotide.","authors":"J. Hofsteenge, J. Vereijken, W. Weijer, J. Beintema, R. Wierenga, J. Drenth","doi":"10.1111/J.1432-1033.1980.TB06148.X","DOIUrl":"https://doi.org/10.1111/J.1432-1033.1980.TB06148.X","url":null,"abstract":"p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens contains six methionine residues, one of which is N-terminal. After CNBr cleavage five peptides, ranging from 13 to 158 residues in length, and free homoserine were isolated and purified by repeated gel filtration. The alignment of the CNBr fragments was deduced from a 0.25-nm electron density map and sequence data. The isolated fragments account for the entire polypeptide chain. The amino acid sequence of the N-terminal quarter of the polypeptide chain was determined. The X-ray results together with the sequence data yielded details of the binding of FAD. The AMP moiety was bound to a beta alpha beta unit resembling that found in the dehydrogenases. Hydrogen bonds were present between the protein and the ribityl residue and the isoalloxazine ring. Furthermore, a homology was found between the N-terminal amino acid sequence of p-hydroxybenzoate hydroxylase and another enzyme containing FAD, viz. D-amino acid oxidase. This finding suggests the presence of a mononucleotide binding fold at the N terminus of the latter.","PeriodicalId":11817,"journal":{"name":"European journal of biochemistry","volume":"8 1","pages":"141-50"},"PeriodicalIF":0.0,"publicationDate":"2005-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"87839239","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2005-03-03DOI: 10.1111/j.1432-1033.1982.tb06958.x
R. Morgenstern, C. Guthenberg, J. Depierre
Rat liver microsomal glutathione S-transferase was activated with N-ethylmaleimide, solubilized with Triton X-100, and purified by chromatography on hydroxyapatite and CM-Sepharose. A 36-fold purification resulted in a 36% yield, indicating that the glutathione S-transferase accounts for 2.5-3% of the original microsomal protein. The purified protein moved as a band with an apparent molecular weight of 14 000 on sodium dodecyl sulphate gel electrophoresis and appeared to be nearly homogeneous. The complex formed between the purified microsomal glutathione S-transferase and Triton X-100 has a sedimentation coefficient of 3.2 S, a partial specific volume of 0.844 cm3/g, and a Stokes radius of 5.5 nm. The complex has a molecular weight of 127 000 and contains three or four polypeptide chains and 112-134 detergent molecules. Antibodies directed against soluble glutathione S-transferases A, B and C do not react with the purified microsomal enzyme. This finding, together with differences in molecular weight and substrate specificity, demonstrate that the microsomal glutathione S-transferase is an enzyme distinct from the cytosolic glutathione S-transferases.
{"title":"Microsomal glutathione S-transferase. Purification, initial characterization and demonstration that it is not identical to the cytosolic glutathione S-transferases A, B and C.","authors":"R. Morgenstern, C. Guthenberg, J. Depierre","doi":"10.1111/j.1432-1033.1982.tb06958.x","DOIUrl":"https://doi.org/10.1111/j.1432-1033.1982.tb06958.x","url":null,"abstract":"Rat liver microsomal glutathione S-transferase was activated with N-ethylmaleimide, solubilized with Triton X-100, and purified by chromatography on hydroxyapatite and CM-Sepharose. A 36-fold purification resulted in a 36% yield, indicating that the glutathione S-transferase accounts for 2.5-3% of the original microsomal protein. The purified protein moved as a band with an apparent molecular weight of 14 000 on sodium dodecyl sulphate gel electrophoresis and appeared to be nearly homogeneous. The complex formed between the purified microsomal glutathione S-transferase and Triton X-100 has a sedimentation coefficient of 3.2 S, a partial specific volume of 0.844 cm3/g, and a Stokes radius of 5.5 nm. The complex has a molecular weight of 127 000 and contains three or four polypeptide chains and 112-134 detergent molecules. Antibodies directed against soluble glutathione S-transferases A, B and C do not react with the purified microsomal enzyme. This finding, together with differences in molecular weight and substrate specificity, demonstrate that the microsomal glutathione S-transferase is an enzyme distinct from the cytosolic glutathione S-transferases.","PeriodicalId":11817,"journal":{"name":"European journal of biochemistry","volume":"88 1","pages":"243-8"},"PeriodicalIF":0.0,"publicationDate":"2005-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86677906","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2005-03-03DOI: 10.1111/J.1432-1033.1983.TB07127.X
Michael A. Lawton, Richard A. Dixon, Klaus Hahlbrock, Christopher J. Lamb
Changes in the activity levels of mRNAs encoding phenylalanine ammonia-lyase and chalcone synthase, two characteristic enzymes of phenylpropanoid biosynthesis, in elicitor-treated cells of dwarf French bean (Phaseolus vulgaris L.) have been investigated by immunoprecipitation of [35S]methionine-labelled enzyme subunits synthesised in vitro in an mRNA-dependent rabbit reticulocyte lysate translation system. Elicitor heat-released from cell walls of Colletotrichum lindemuthianum, the causal agent of anthracnose disease of bean, causes marked rapid increases in the polysomal activities of the mRNAs encoding the two enzymes concomitant with the phase of rapid increase in enzyme activity at the onset of phaseollin accumulation during the phytoalexin defence response. Increased polysomal mRNA activities encoding the two enzymes can be observed 30 min after elicitor treatment. The patterns of induction of the mRNA activities are broadly similar with respect to time and elicitor concentration although small but distinct differences between the enzymes were observed in the elicitor concentration giving maximum induction. There is a close correlation between the induction of polysomal mRNA activity and the induction of enzyme synthesis in vivo by elicitor treatment with respect to both the kinetics of induction and the dependence on elicitor concentration. The data indicate that elicitor stimulation of phenylalanine ammonia-lyase and chalcone synthase synthesis in vivo is largely a result of increased polysomal activity of the mRNAs encoding these enzymes. Similar patterns of induction of polysomal mRNA activity are observed with elicitor preparations from a variety of sources. The marked increases in polysomal mRNA activities encoding phenylalanine ammonia-lyase and chalcone synthase are increases as a proportion of total cellular mRNA activity, indicating that elicitor does not increase these polysomal mRNA activities by stimulation of selective recruitment from the total pool of cellular mRNA.
{"title":"Elicitor induction of mRNA activity. Rapid effects of elicitor on phenylalanine ammonia-lyase and chalcone synthase mRNA activities in bean cells.","authors":"Michael A. Lawton, Richard A. Dixon, Klaus Hahlbrock, Christopher J. Lamb","doi":"10.1111/J.1432-1033.1983.TB07127.X","DOIUrl":"https://doi.org/10.1111/J.1432-1033.1983.TB07127.X","url":null,"abstract":"Changes in the activity levels of mRNAs encoding phenylalanine ammonia-lyase and chalcone synthase, two characteristic enzymes of phenylpropanoid biosynthesis, in elicitor-treated cells of dwarf French bean (Phaseolus vulgaris L.) have been investigated by immunoprecipitation of [35S]methionine-labelled enzyme subunits synthesised in vitro in an mRNA-dependent rabbit reticulocyte lysate translation system. Elicitor heat-released from cell walls of Colletotrichum lindemuthianum, the causal agent of anthracnose disease of bean, causes marked rapid increases in the polysomal activities of the mRNAs encoding the two enzymes concomitant with the phase of rapid increase in enzyme activity at the onset of phaseollin accumulation during the phytoalexin defence response. Increased polysomal mRNA activities encoding the two enzymes can be observed 30 min after elicitor treatment. The patterns of induction of the mRNA activities are broadly similar with respect to time and elicitor concentration although small but distinct differences between the enzymes were observed in the elicitor concentration giving maximum induction. There is a close correlation between the induction of polysomal mRNA activity and the induction of enzyme synthesis in vivo by elicitor treatment with respect to both the kinetics of induction and the dependence on elicitor concentration. The data indicate that elicitor stimulation of phenylalanine ammonia-lyase and chalcone synthase synthesis in vivo is largely a result of increased polysomal activity of the mRNAs encoding these enzymes. Similar patterns of induction of polysomal mRNA activity are observed with elicitor preparations from a variety of sources. The marked increases in polysomal mRNA activities encoding phenylalanine ammonia-lyase and chalcone synthase are increases as a proportion of total cellular mRNA activity, indicating that elicitor does not increase these polysomal mRNA activities by stimulation of selective recruitment from the total pool of cellular mRNA.","PeriodicalId":11817,"journal":{"name":"European journal of biochemistry","volume":"23 1","pages":"131-9"},"PeriodicalIF":0.0,"publicationDate":"2005-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"87050098","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2005-03-03DOI: 10.1111/J.1432-1033.1983.TB07105.X
A. Abdelal, Lee Bussey, Leland P. Vickers
Carbamoylphosphate synthetase from Pseudomonas aeruginosa is subject to repression by pyrimidines and significant derepression by limitation of arginine or pyrimidines. Carbamoylphosphate synthetase was purified to homogeneity from a derepressed strain of P. aeruginosa. The molecular weight of the enzyme was estimated to be 168 000 by sucrose gradient ultracentrifugation. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the enzyme is composed of two non-identical subunits with molecular weights of 122 000 and 44 000. Cross-linking the enzyme prior to electrophoresis yielded an additional band corresponding to a molecular weight of 165 000, showing that the enzyme is composed of one of each subunit. The enzyme utilized either glutamine (Km 0.15 mM) or NH3 (Km 17 mM) and requires free Mg2+ for maximal activity with the optimal level between 4 mM and 10 mM. Hill plots of MgATP saturation data yielded coefficients of 1.2 and 1.4 at pH 8.0 and 8.5, respectively. A Hill equation was derived on the assumptions that MgATP binds at the same time to two distinct sub-sites as was shown to be the case for carbamoylphosphate synthetase from Escherichia coli and that these sub-sites are strictly non-interacting. The resulting theoretical Hill coefficients correspond very closely to the experimental coefficients. Carbamoylphosphate synthetase activity was subject to activation by ornithine and N-acetylornithine and feedback inhibition by UMP. Carbamoylphosphate synthetase from P. aeruginosa does not associate under all conditions examined, establishing that self-association does not play a role in regulation of enzyme activity as suggested by other workers for the enzyme from E. coli.
{"title":"Carbamoylphosphate synthetase from Pseudomonas aeruginosa. Subunit composition, kinetic analysis and regulation.","authors":"A. Abdelal, Lee Bussey, Leland P. Vickers","doi":"10.1111/J.1432-1033.1983.TB07105.X","DOIUrl":"https://doi.org/10.1111/J.1432-1033.1983.TB07105.X","url":null,"abstract":"Carbamoylphosphate synthetase from Pseudomonas aeruginosa is subject to repression by pyrimidines and significant derepression by limitation of arginine or pyrimidines. Carbamoylphosphate synthetase was purified to homogeneity from a derepressed strain of P. aeruginosa. The molecular weight of the enzyme was estimated to be 168 000 by sucrose gradient ultracentrifugation. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the enzyme is composed of two non-identical subunits with molecular weights of 122 000 and 44 000. Cross-linking the enzyme prior to electrophoresis yielded an additional band corresponding to a molecular weight of 165 000, showing that the enzyme is composed of one of each subunit. The enzyme utilized either glutamine (Km 0.15 mM) or NH3 (Km 17 mM) and requires free Mg2+ for maximal activity with the optimal level between 4 mM and 10 mM. Hill plots of MgATP saturation data yielded coefficients of 1.2 and 1.4 at pH 8.0 and 8.5, respectively. A Hill equation was derived on the assumptions that MgATP binds at the same time to two distinct sub-sites as was shown to be the case for carbamoylphosphate synthetase from Escherichia coli and that these sub-sites are strictly non-interacting. The resulting theoretical Hill coefficients correspond very closely to the experimental coefficients. Carbamoylphosphate synthetase activity was subject to activation by ornithine and N-acetylornithine and feedback inhibition by UMP. Carbamoylphosphate synthetase from P. aeruginosa does not associate under all conditions examined, establishing that self-association does not play a role in regulation of enzyme activity as suggested by other workers for the enzyme from E. coli.","PeriodicalId":11817,"journal":{"name":"European journal of biochemistry","volume":"247 1","pages":"697-702"},"PeriodicalIF":0.0,"publicationDate":"2005-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73266039","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2005-03-03DOI: 10.1111/J.1432-1033.1981.TB06243.X
M. Negishi, N. M. Jensen, G. S. Garcia, D. Nebert
Antibodies against mouse-liver microsomal cytochromes P1-450 and P-448, two polycyclic aromatic inducible cytochromes, were previously developed [Negishi, M. and Nebert, D.W. (1979) J. Biol. Chem. 254, 11015-11023]. Liver microsomes from 3-methylcholanthrene-treated and phenobarbital-treated and control adult mice and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated adult and fetal mice were examined. Immunoprecipitable radioactivity was measured, following labeling with pyridoxal phosphate/NaB[3H]4 or with 125I-labeled p-aminosulfobenzoic acid/NaNO2 in vitro or with [3H]leucine, [14C]glucosamine, or [32P]O4 in vivo. (a) Induction of cytochrome P1-450 occurs developmentally earlier in gestation than induction of cytochrome P-448 when the mother is treated with polycyclic aromatic compounds. (b) There appears to be a basal form of cytochrome P-448 but no cytochrome P1-450 in control liver microsomes; inducibility of cytochrome P-448 thus ranges between 5--12-fold, whereas that of P1-450 is infinite. (c) Phenobarbital pretreatment induces no detectable P1-450 or P-448. (d) P-448 appears to be either greater in concentration than P1-450 in the membrane or more exposed than P1-450 on the microsomal membrane surface. (e) By the radioimmunoassay methods used, 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced P1-450 and P-448 in Ah-nonresponsive mice are indistinguishable from those in Ah-responsive mice; this is true in both the fetus and the adult. (f) Compared with P-448 expression, the expression of P1-450 is more closely associated with 3-methylcholanthrene-induced aryl hydrocarbon hydroxylase activity, and these two structural gene products are apparently regulated independently. (g) P-448 but not P1-450 appears to be a glycoprotein. These data illustrate further differences between two forms of polycyclic aromatic-inducible P-450 in mouse liver. Neither P1-450 nor P-448 appears to be a phosphoprotein. Neither anti-(P1-450) nor anti-(P-448) precipitates any forms of liver microsomal P-450 from beta-naphthoflavone-treated adult rabbits and, conversely, anti-LM4 (the antibody to rabbit liver microsomal P-450 form 4) does not precipitate any forms of liver microsomal P-450 from 3-methylcholanthrene-treated C57BL/6N mice.
针对小鼠肝微粒体细胞色素P1-450和P-448(两种多环芳香族诱导细胞色素)的抗体先前已被开发[Negishi, M. and Nebert, D.W. (1979) J. Biol.]。化学学报，2004,23(2):444 - 444。对3-甲基胆蒽处理和苯巴比妥处理的成年小鼠和2,3,7,8-四氯二苯并对二恶英处理的成年小鼠和胎儿小鼠的肝微粒体进行了检测。在体外用吡哆醛磷酸/NaB[3H]4或125i标记的对氨基磺酸/NaNO2或体内用[3H]亮氨酸、[14C]氨基葡萄糖或[32P]O4标记后，测量免疫可沉淀放射性。(a)细胞色素P1-450的诱导在妊娠期比细胞色素P-448的诱导发生得早。(b)在对照肝微粒体中似乎有细胞色素P-448的基础形式，但没有细胞色素P1-450;因此，细胞色素P-448的诱导率在5- 12倍之间，而P1-450的诱导率是无限的。(c)苯巴比妥预处理未诱导检测到P1-450或P-448。(d) P-448在膜上的浓度似乎高于P1-450，或在微粒体膜表面的暴露程度高于P1-450。(e)采用放射免疫测定方法，ah无应答小鼠体内2,3,7,8-四氯二苯并-对二恶英诱导的P1-450和P-448与ah应答小鼠体内的P1-450和P-448无法区分;胎儿和成人都是如此。(f)与P-448的表达相比，P1-450的表达与3-甲基胆蒽诱导的芳烃羟化酶活性的关系更为密切，这两种结构基因产物明显是独立调控的。(g) P-448是糖蛋白，P1-450不是。这些数据进一步说明了小鼠肝脏中两种形式的多环芳烃诱导P-450之间的差异。P1-450和P-448似乎都不是磷蛋白。抗(P1-450)和抗(P-448)都不能从β -萘黄酮处理的成年家兔中沉淀任何形式的肝微粒体P-450，相反，抗lm4(兔肝微粒体P-450形式4的抗体)也不能从3-甲基胆碱处理的C57BL/6N小鼠中沉淀任何形式的肝微粒体P-450。
{"title":"Structural gene products of the murine Ah complex. Differences in ontogenesis and glucosamine incorporation between liver microsomal cytochromes P1-450 and P-448 induced by polycyclic aromatic compounds.","authors":"M. Negishi, N. M. Jensen, G. S. Garcia, D. Nebert","doi":"10.1111/J.1432-1033.1981.TB06243.X","DOIUrl":"https://doi.org/10.1111/J.1432-1033.1981.TB06243.X","url":null,"abstract":"Antibodies against mouse-liver microsomal cytochromes P1-450 and P-448, two polycyclic aromatic inducible cytochromes, were previously developed [Negishi, M. and Nebert, D.W. (1979) J. Biol. Chem. 254, 11015-11023]. Liver microsomes from 3-methylcholanthrene-treated and phenobarbital-treated and control adult mice and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated adult and fetal mice were examined. Immunoprecipitable radioactivity was measured, following labeling with pyridoxal phosphate/NaB[3H]4 or with 125I-labeled p-aminosulfobenzoic acid/NaNO2 in vitro or with [3H]leucine, [14C]glucosamine, or [32P]O4 in vivo. (a) Induction of cytochrome P1-450 occurs developmentally earlier in gestation than induction of cytochrome P-448 when the mother is treated with polycyclic aromatic compounds. (b) There appears to be a basal form of cytochrome P-448 but no cytochrome P1-450 in control liver microsomes; inducibility of cytochrome P-448 thus ranges between 5--12-fold, whereas that of P1-450 is infinite. (c) Phenobarbital pretreatment induces no detectable P1-450 or P-448. (d) P-448 appears to be either greater in concentration than P1-450 in the membrane or more exposed than P1-450 on the microsomal membrane surface. (e) By the radioimmunoassay methods used, 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced P1-450 and P-448 in Ah-nonresponsive mice are indistinguishable from those in Ah-responsive mice; this is true in both the fetus and the adult. (f) Compared with P-448 expression, the expression of P1-450 is more closely associated with 3-methylcholanthrene-induced aryl hydrocarbon hydroxylase activity, and these two structural gene products are apparently regulated independently. (g) P-448 but not P1-450 appears to be a glycoprotein. These data illustrate further differences between two forms of polycyclic aromatic-inducible P-450 in mouse liver. Neither P1-450 nor P-448 appears to be a phosphoprotein. Neither anti-(P1-450) nor anti-(P-448) precipitates any forms of liver microsomal P-450 from beta-naphthoflavone-treated adult rabbits and, conversely, anti-LM4 (the antibody to rabbit liver microsomal P-450 form 4) does not precipitate any forms of liver microsomal P-450 from 3-methylcholanthrene-treated C57BL/6N mice.","PeriodicalId":11817,"journal":{"name":"European journal of biochemistry","volume":"65 1","pages":"585-94"},"PeriodicalIF":0.0,"publicationDate":"2005-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83421964","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2005-03-03DOI: 10.1111/J.1432-1033.1981.TB06167.X
T. Kouyama, K. Mihashi
A fluorescent reagent, N-(1-pyrenyl)iodoacetamide, was conjugated to rabbit skeletal muscle actin at the site of the most reactive sulfhydryl group, and fluorescence characteristics (excitation and emission spectra, quantum yields, lifetimes) of the conjugate were investigated. Associated with polymerization of labelled G-actin, the fluorescence intensity at 407 nm, after excitation at 365 nm, was enhanced by a factor of about 25. It was reduced to about 25% on the binding of heavy meromyosin (or subfragment 1). The results suggest that binding of heavy meromyosin to the protomer of F-actin alters the local structure of the protomer towards a G-actin-like one.
{"title":"Fluorimetry study of N-(1-pyrenyl)iodoacetamide-labelled F-actin. Local structural change of actin protomer both on polymerization and on binding of heavy meromyosin.","authors":"T. Kouyama, K. Mihashi","doi":"10.1111/J.1432-1033.1981.TB06167.X","DOIUrl":"https://doi.org/10.1111/J.1432-1033.1981.TB06167.X","url":null,"abstract":"A fluorescent reagent, N-(1-pyrenyl)iodoacetamide, was conjugated to rabbit skeletal muscle actin at the site of the most reactive sulfhydryl group, and fluorescence characteristics (excitation and emission spectra, quantum yields, lifetimes) of the conjugate were investigated. Associated with polymerization of labelled G-actin, the fluorescence intensity at 407 nm, after excitation at 365 nm, was enhanced by a factor of about 25. It was reduced to about 25% on the binding of heavy meromyosin (or subfragment 1). The results suggest that binding of heavy meromyosin to the protomer of F-actin alters the local structure of the protomer towards a G-actin-like one.","PeriodicalId":11817,"journal":{"name":"European journal of biochemistry","volume":"194 1","pages":"33-8"},"PeriodicalIF":0.0,"publicationDate":"2005-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79748024","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
1. The enzyme trehalase was prepared from whole flesh flies, Sarcophaga barbata. 2. The rate of mutorotation of glucose released by the enzyme was compared with that of alpha-D-glucose, beta-D-glucose and an equimolar mixture of the two. 3. Derivatives of the glucose released by the enzyme were prepared and analysed by gas chromatography for anomeric composition. 4. The enzyme was incubated with trehalose in a medium enriched with H218O and the glucose was analysed by gas chromatography/mass spectrometry. 5. It was found that an equimolar mixture of alpha-D-glucose and beta-D-glucose is formed on hydrolysis of trehalose by trehalase and 18O is incorporated into beta-D-glucose. 6. These results indicate that there is an inversion of configuration at C-1 of trehalose on hydrolysis and that the hydrolysis proceeds by a bimolecular nucleophilic substitution mechanism.
1. 以全蝇(Sarcophaga barbata)为原料制备海藻酶。2. 将该酶释放的葡萄糖的变形速率与α - d -葡萄糖、β - d -葡萄糖以及两者的等摩尔混合物的变形速率进行了比较。3.制备了该酶释放的葡萄糖衍生物，并用气相色谱法对其进行了分析。4. 将酶与海藻糖在富含H218O的培养基中孵育，用气相色谱/质谱法分析葡萄糖。5. 发现海藻糖酶水解海藻糖形成α - d -葡萄糖和β - d -葡萄糖的等摩尔混合物，18O被纳入β - d -葡萄糖中。6. 这些结果表明海藻糖在水解过程中存在C-1位构型的反转，水解过程是通过双分子亲核取代机制进行的。
{"title":"Stereochemistry of the hydrolysis of trehalose by the enzyme trehalase prepared from the flesh fly Sarcophaga barbata.","authors":"K. Clifford","doi":"10.1039/C39790000136","DOIUrl":"https://doi.org/10.1039/C39790000136","url":null,"abstract":"1. The enzyme trehalase was prepared from whole flesh flies, Sarcophaga barbata. 2. The rate of mutorotation of glucose released by the enzyme was compared with that of alpha-D-glucose, beta-D-glucose and an equimolar mixture of the two. 3. Derivatives of the glucose released by the enzyme were prepared and analysed by gas chromatography for anomeric composition. 4. The enzyme was incubated with trehalose in a medium enriched with H218O and the glucose was analysed by gas chromatography/mass spectrometry. 5. It was found that an equimolar mixture of alpha-D-glucose and beta-D-glucose is formed on hydrolysis of trehalose by trehalase and 18O is incorporated into beta-D-glucose. 6. These results indicate that there is an inversion of configuration at C-1 of trehalose on hydrolysis and that the hydrolysis proceeds by a bimolecular nucleophilic substitution mechanism.","PeriodicalId":11817,"journal":{"name":"European journal of biochemistry","volume":"20 1","pages":"337-40"},"PeriodicalIF":0.0,"publicationDate":"2005-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"75410283","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2005-03-03DOI: 10.1111/J.1432-1033.1982.TB06792.X
L. Ferrara, R. Napolitano, L. Paolillo, S. Wurzburger, S. Andini, C. Toniolo, G. Bonora
The three main fractions of clupeine, the protamine extracted from herring sperm, have been investigated by 13C nuclear magnetic resonance techniques. The dynamic behaviour, examined through the evaluation of the spin lattice relaxation times (T1) of individual carbon resonances assigned to both backbone and side chains, reveals interesting features. The relaxation times of backbone alpha-carbons, interpreted on the basis of an axially symmetric ellipsoid, point to the clupeine fractions as being essentially extended in aqueous solution. These times remain constant along the polypeptide chain and are of the order of 0.16 +/- 0.02 s. Conversely, the side chains show different flexibilities in the presence of monophosphate counterions, thus demonstrating a diverging behaviour which may be biologically relevant. In particular, the side-chain flexibilities of fraction YI decrease, while those of fractions Z and YII are either constant or increase. Comparison of these data with the viscosity measurements helps in explaining the viscosity changes observed in the presence of phosphate.
{"title":"Nuclear magnetic resonance of protamines. A 13C relaxation study of the three main fractions of clupeine.","authors":"L. Ferrara, R. Napolitano, L. Paolillo, S. Wurzburger, S. Andini, C. Toniolo, G. Bonora","doi":"10.1111/J.1432-1033.1982.TB06792.X","DOIUrl":"https://doi.org/10.1111/J.1432-1033.1982.TB06792.X","url":null,"abstract":"The three main fractions of clupeine, the protamine extracted from herring sperm, have been investigated by 13C nuclear magnetic resonance techniques. The dynamic behaviour, examined through the evaluation of the spin lattice relaxation times (T1) of individual carbon resonances assigned to both backbone and side chains, reveals interesting features. The relaxation times of backbone alpha-carbons, interpreted on the basis of an axially symmetric ellipsoid, point to the clupeine fractions as being essentially extended in aqueous solution. These times remain constant along the polypeptide chain and are of the order of 0.16 +/- 0.02 s. Conversely, the side chains show different flexibilities in the presence of monophosphate counterions, thus demonstrating a diverging behaviour which may be biologically relevant. In particular, the side-chain flexibilities of fraction YI decrease, while those of fractions Z and YII are either constant or increase. Comparison of these data with the viscosity measurements helps in explaining the viscosity changes observed in the presence of phosphate.","PeriodicalId":11817,"journal":{"name":"European journal of biochemistry","volume":"17 1","pages":"389-94"},"PeriodicalIF":0.0,"publicationDate":"2005-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"75304138","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2005-03-03DOI: 10.1111/J.1432-1033.1980.TB06020.X
P. Moonen, R. Akeroyd, J. Westerman, W. Puijk, P. Smits, K. Wirtz
The phosphatidylcholine exchange protein from bovine liver consists of a single polypeptide chain and has a blocked N terminus. The protein contains an estimated 244 amino acid residues in accordance with a determined molecular weight of 28000. The protease from mouse submaxillaris gland cleaved the citraconylated and S-carboxymethylated derivative of the exchange protein at one specific site (Arg14-Glu15) close to the N terminus. Analysis of the two resulting peptides showed that N-acetyl-methionine was the N-terminal residue and gave the sequence of the first 41 residues. The modified protein was also fragmented with the protease from Staphylococcus aureus. The peptides isolated represented 88% of the protein; their sequences were determined by manual and automated Edman degradation. Alignment of a number of these peptides gave the complex sequence of the N-terminal half up to position 122.
{"title":"The primary structure of the phosphatidylcholine-exchange protein from bovine liver. Isolation and characterization of the staphylococcal protease peptides and the amino-acid sequence of the N-terminal half (residues 1--122).","authors":"P. Moonen, R. Akeroyd, J. Westerman, W. Puijk, P. Smits, K. Wirtz","doi":"10.1111/J.1432-1033.1980.TB06020.X","DOIUrl":"https://doi.org/10.1111/J.1432-1033.1980.TB06020.X","url":null,"abstract":"The phosphatidylcholine exchange protein from bovine liver consists of a single polypeptide chain and has a blocked N terminus. The protein contains an estimated 244 amino acid residues in accordance with a determined molecular weight of 28000. The protease from mouse submaxillaris gland cleaved the citraconylated and S-carboxymethylated derivative of the exchange protein at one specific site (Arg14-Glu15) close to the N terminus. Analysis of the two resulting peptides showed that N-acetyl-methionine was the N-terminal residue and gave the sequence of the first 41 residues. The modified protein was also fragmented with the protease from Staphylococcus aureus. The peptides isolated represented 88% of the protein; their sequences were determined by manual and automated Edman degradation. Alignment of a number of these peptides gave the complex sequence of the N-terminal half up to position 122.","PeriodicalId":11817,"journal":{"name":"European journal of biochemistry","volume":"24 1","pages":"279-90"},"PeriodicalIF":0.0,"publicationDate":"2005-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"80420484","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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