Enzyme & protein最新文献

{"title":"Kinetics of irreversible inhibition of creatine kinase during modification by o-phthaldehyde.","authors":"Z F Wang,&nbsp;Y K Xu,&nbsp;H M Zhou","doi":"10.1159/000474963","DOIUrl":"https://doi.org/10.1159/000474963","url":null,"abstract":"<p><p>It has been previously reported that, with a fluorescence probe formed from o-phthaldehyde (OPTA) and the thiol and amino groups at or near the active site of creatine kinase, inactivation and exposure of the probe take place simultaneously and well before unfolding of the molecule as a whole. In this study, the inactivation and modification kinetics of purified rabbit muscle creatine kinase by OPTA have been compared, the former by following the substrate reaction in the presence of a previously described inactivator. The microscopic rate constants for the reaction of the inactivator with the free enzyme and with the enzyme-substrate complexes were determined. From the results obtained it appears that OPTA is noncompetitive with respect to both substrates. The inactivation kinetics is monophasic with OPTA, and neither ATP nor creatine alone affect the rate constant of inactivation of the enzyme, indicating that the irreversible inhibition of creatine kinase by OPTA is of the noncompetitive type.</p>","PeriodicalId":11854,"journal":{"name":"Enzyme & protein","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"1994-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000474963","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"18787475","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}

{"title":"Proteasome workshop at Colorado State University. Fort Collins, Colo., July 1-3, 1995. Report and abstracts.","authors":"D L Mykles,&nbsp;G N DeMartino,&nbsp;P M Kloetzel","doi":"10.1159/000475003","DOIUrl":"https://doi.org/10.1159/000475003","url":null,"abstract":"<p><p>An international workshop on the proteasome was held on the campus of Colorado State University, Fort Collins, July 1-3, 1995. The program consisted of five oral sessions and one poster session. The abstracts of the posters and many of the oral presentations appear in this issue of Enzyme and Protein. The oral sessions were organized around four themes: structure and catalytic properties; allosteric regulators and synthetic inhibitors; the 26S proteasome; and physiological functions, developmental expression, and endogenous substrates. Since entire issues of Enzyme and Protein [vol. 47, No. 4-6, 1993] and Molecular Biology Reports [vol. 21, No. 1, 1995] have been devoted to the proteasome, this summary emphasizes recent results and current research of many of the laboratories studying this fascinating complex.</p>","PeriodicalId":11854,"journal":{"name":"Enzyme & protein","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"1994-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000475003","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19765124","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}

{"title":"Bradykinin-induced airway microvascular leakage is potentiated by enalaprilat but not by phosphoramidon.","authors":"D Klitzman,&nbsp;P L Almenoff,&nbsp;C Cardozo,&nbsp;M Lesser","doi":"10.1159/000474988","DOIUrl":"https://doi.org/10.1159/000474988","url":null,"abstract":"<p><p>The objective of the study was to determine if bradykinin-induced airway microvascular leakage in rats was altered by pretreatment of animals with enalaprilat, an inhibitor of angiotensin-converting enzyme (ACE), or phosphoramidon, an inhibitor of endopeptidase 24.11 (EP 24.11). We found that the intravascular infusion of bradykinin induced microvascular leakage of Evans blue dye in tracheal tissue (0.088 +/- 0.035 micrograms/mg tissue) that was significantly amplified by pretreatment with 3.27 mM enalaprilat (0.458 +/- 0.226 micrograms/mg tissue), but not by pretreatment with 10 mM phosphoramidon (0.082 +/- 0.0453 micrograms/mg tissue). Leakage in carinal tissue was also amplified by pretreatment with 3.27 mM enalaprilat (0.205 +/- 0.050 vs. 0.036 +/- 0.006 micrograms/mg tissue for bradykinin alone), whereas no amplification was observed in parenchymal tissue by pretreatment with either inhibitor. These findings indicate that in the rat, ACE, but not EP 24.11, modulates bradykinin-induced airway microvascular leakage following intravascular infusion of these agents.</p>","PeriodicalId":11854,"journal":{"name":"Enzyme & protein","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"1994-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000474988","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19789837","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}

{"title":"Erythrocyte pyruvate kinase deficiency. The influence of physiologically important metabolites on the function of normal and defective enzymes.","authors":"M Lakomek,&nbsp;H Winkler,&nbsp;A Pekrun,&nbsp;N Krüger,&nbsp;M Sander,&nbsp;P Huppke,&nbsp;W Schröter","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>The dependence of the erythrocyte pyruvate kinase (PK)-catalyzed reaction on the glycolytic intermediates glucose-6-phosphate (Gluc-6-P), 2,3-diphosphoglycerate (2,3-DPG) and the nucleotides ADP and ATP was studied in normal individuals and 14 patients with PK deficiency. The Gluc-6-P concentrations in the erythrocytes are markedly elevated (4- to 6-fold) in 9 patients with severe hemolytic anemia compared to those 5 exhibiting a mild clinical course (up to 2-fold increased). 2,3-DPG is elevated up to 2 times compared to the controls whereas the measured ADP and ATP only slightly deviate from the normal range. Control experiments showed that these elevations of Gluc-6-P and 2,3-DPG do not depend on the number of reticulocytes. In enzyme kinetic terms, Gluc-6-P shifts the Hill coefficient to smaller values, i.e. suppresses the positive cooperativity (sigmoidal reaction kinetics), found in normal and some of the mutant enzymes and shift the noncooperative enzymes of some patients to an enzyme exhibiting negative cooperativity. The negative cooperativity already present in the enzymes of some of the patients suffering from severe hemolytic anemia becomes more pronounced upon addition of Gluc-6-P. Apparently 2,3-DPG acts as an antagonist to Gluc-6-P in increasing the Hill coefficient, i.e. enhancing the positive cooperativity of the normal enzyme. It shifts the hyperbolic patients' enzymes to a sigmoidal reaction type and the enzymes of those patients with negative cooperativity to a hyperbolic type. ADP and ATP show a similar behavior as 2,3-DPG, but additionally inhibit the enzyme at higher concentrations. The influence of all four phosphates on the Michaelis constant varies depending on the type of cooperativity, in some cases increasing and in some cases decreasing K0.5 PEP. With 7 of the patients, all of them with severe clinical course, a genetic analysis of their R-type PK gene was performed and genetic defects have been identified in the coding sequence. The found changes in the amino acid sequence and their corresponding location in the tertiary structure of the PK subunit can satisfactorily explain the alterations of the regulatory properties of the mutant enzymes thus allowing to establish a good correlation between altered structural and functional properties of the deficient enzyme and the severeness of the course of the disease.</p>","PeriodicalId":11854,"journal":{"name":"Enzyme & protein","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"1994-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19569663","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}

{"title":"Carboxypeptidase A hydrolyses benzoylglycyl-histidyl-leucine but not furylacryloyl-phenylalanyl-glycyl-glycine, two usual substrates for angiotensin I-converting enzyme.","authors":"B Baudin,&nbsp;J Giboudeau","doi":"10.1159/000474973","DOIUrl":"https://doi.org/10.1159/000474973","url":null,"abstract":"<p><p>We compared angiotensin I-converting enzyme (ACE) and carboxypeptidase A (CPA), two zinc metallopeptidases, for the hydrolysis of the usual ACE synthetic substrate benzoylglycyl-histidyl-leucine (HHL) investigating the possible interference by CPA in the determination of ACE activity in biological fluids. Both purified enzymes hydrolyse HHL in a radiochemical assay with the same optimal pH, a characteristic divalent metal requirement, a close similar behavior against inhibitors of other metallopeptidases, such as enkephalinase and kininase I, and the involvement of arginine and lysine residues in their active site. Conversely, CPA does not show the other catalytic properties of ACe, i.e. chloride dependence, low Km for HHL, inhibition by specific synthetic ACE inhibitors and antibody, also hydrolysis of the other ACE substrate furylacryloylphenylalanyl-glycyl-glycine (FAPGG). We advise the use of ACE inhibitors to validate ACE measurement with HHL or, alternatively, FAPGG, which is a more specific substrate for ACE, must be preferred, although the poor sensitivity of the spectrophotometric assay with this substrate limits its use to blood samples.</p>","PeriodicalId":11854,"journal":{"name":"Enzyme & protein","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"1994-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000474973","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"18587466","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}

{"title":"Characterization of human wild-type and mutant argininosuccinate synthetase proteins expressed in bacterial cells.","authors":"N Shaheen,&nbsp;K Kobayashi,&nbsp;H Terazono,&nbsp;T Fukushige,&nbsp;M Horiuchi,&nbsp;T Saheki","doi":"10.1159/000474998","DOIUrl":"https://doi.org/10.1159/000474998","url":null,"abstract":"<p><p>Argininosuccinate synthetase (ASS) is a urea cycle enzyme with a tetrameric structure composed of identical subunits. Citrullinemia is an autosomal recessive disease caused by a deficiency of ASS. We have previously identified 20 mutations in ASS mRNA of human classical citrullinemia. However, it is difficult to evaluate the effects of each mutation on the enzyme structure and function, since most of the patients are compound heterozygotes. In the present study, wild-type ASS and 12 mutant ASSs were expressed with a bacterial expression system and analyzed enzymologically and immunochemically. The properties of the purified recombinant protein with wild-type human ASS showed good agreement with native enzyme purified from human liver. Mutant ASS proteins with an expected molecular mass, except for delta 7b/Ex16, were highly expressed in the bacterial cells. It was difficult to extract ASS proteins with some mutations (A118T, delta Ex7, R157H, R363W, R363L, G390R and ins37b/Ex15&16) from cells by freezing and thawing. Extractable mutant proteins were as follows: G280R mutant was extracted with an amount of ASS protein similar to wild-type but with no ASS activity, and A192V, R272C and R304W mutants detected various amounts of ASS protein (13, 110 and 33% of wild-type, respectively) with a low ASS activity and abnormal kinetics. Higher Km values for citrulline were obtained in mutant ASSs with A192V (15 mmol/1), R272C (4.2 mmol/l) and R304W. (190 mmol/l) than in wild-type ASS (0.056 mmol/l). The results confirm that these mutations are responsible for ASS deficiency and also indicate that these amino acid residues are important for the function and structure of ASS protein.</p>","PeriodicalId":11854,"journal":{"name":"Enzyme & protein","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"1994-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000474998","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19763965","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}

{"title":"Alkaline unfolding and salt-induced folding of aminoacylase at high pH.","authors":"M Q Huang,&nbsp;H M Zhou","doi":"10.1159/000474993","DOIUrl":"https://doi.org/10.1159/000474993","url":null,"abstract":"<p><p>The conformational changes of aminoacylase during unfolding at alkaline pH have been followed by fluorescence emission, circular dichroism (CD) and ultraviolet difference spectra. The results of comparison of inactivation and conformation show that much lower values of alkaline pH are required to bring about inactivation than significant conformational change of the enzyme molecule. At pH above 12, although the enzyme has been inactivated, the apparently fully unfolded enzyme retains some ordered secondary structure. At pH 12 by adding KCl, the relatively unfolded state of denatured enzyme changes into a compact conformational state by hydrophobic collapsing, but no new secondary structure is formed. On decreasing the pH from pH 12 to approximate neutrality, the unfolded enzyme also undertakes the similar conformational transition. It can be suggested that hydrophobic collapsed intermediate may be a general intermediate conformational state from alkaline unfolded state to native state.</p>","PeriodicalId":11854,"journal":{"name":"Enzyme & protein","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"1994-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000474993","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19789763","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}

{"title":"ADP-ribosylation of myelin basic protein and inhibition of phospholipid vesicle aggregation.","authors":"C Yamamori,&nbsp;M Terashima,&nbsp;H Ishino,&nbsp;M Shimoyama","doi":"10.1159/000474990","DOIUrl":"https://doi.org/10.1159/000474990","url":null,"abstract":"<p><p>Four isoforms of myelin basic protein (MBP) from chicken brain were ADP-ribosylated by chicken heterophil ADP-ribosyltransferase. The 21-kD isoform was the most preferential substrate of this transferase. With this isoform, the Km values were estimated to be 330 mumol/l for NAD and 30 mumol/l for MBP, and the optimal pH for ADP-ribosylation was 8.5. The stoichiometry of ADP-ribose incorporation into 21-kD MBP was 3.5 mol of ADP-ribose/mol MBP. We found the inhibition of ADP-ribosylation of MBP by hydroxylamine and L-arginine indicating that this modification was likely to be mediated by arginine residues. Proteolytic peptide maps of ADP-ribosylated MBP by chicken ADP-ribosyltransferase and cholera toxin showed partially different radio active bands. When 21-kD MBP was ADP-ribosylated by chicken transferase, the potential for phospholipid vesicle aggregation was reduced in proportion of the degree of ADP-ribosylation. The possibility that ADP-ribosylation of MBP may control stabilization of myelin through regulation of its affinity for phospholipid in vivo would need to be considered.</p>","PeriodicalId":11854,"journal":{"name":"Enzyme & protein","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"1994-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000474990","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19789839","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}

{"title":"Human colon sialidase: characterization and activity levels in normal mucosa and colonic adenocarcinoma.","authors":"V S Martínez-Zorzano,&nbsp;C Feijoo,&nbsp;M Páez de la Cadena,&nbsp;M Butrón,&nbsp;A Fernández-Briera,&nbsp;F J Rodríguez-Berrocal","doi":"10.1159/000475001","DOIUrl":"https://doi.org/10.1159/000475001","url":null,"abstract":"<p><p>Human colon sialidase has been characterized, and its activity levels in normal mucosa and colonic adenocarcinoma have been determined. Sialidase activity was maximal at pH 5.5, and was unstable with storage at 4 and -20 degrees C. The bulk of activity was pellet-associated, and could not be released with triton X-100 or 3-([3-cholamidopropyl]- dimethylammonio)-1-propanesulfonate. Using 2'-(4-methylumbelliferyl)alpha-D-N-acetylneuraminic acid as substrate, the Km and Vmax values were estimated to be 0.140 mmol/l and 63 mU/g, respectively. Furthermore, an inhibition by substrate concentrations above 1.5 mmol/l was detected. Neuraminic acid caused a competitive inhibition with a Ki of 3.5 mmol/l. A statistically significant increase (p < 0.001) in the sialidase specific activity was found in primary colonic adenocarcinoma (104.20 +/- 8.00 mU/g) compared to that of the normal mucosa (72.50 +/- 7.67 mU/g).</p>","PeriodicalId":11854,"journal":{"name":"Enzyme & protein","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"1994-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000475001","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19765123","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}

{"title":"S-acetyl- and S-phenylacetyl-glutathione as glutathione precursors in rat plasma and tissue preparations.","authors":"L Galzigna,&nbsp;V Rizzoli,&nbsp;P Schiappelli,&nbsp;C Moretto,&nbsp;A Bernareggi","doi":"10.1159/000474975","DOIUrl":"https://doi.org/10.1159/000474975","url":null,"abstract":"<p><p>S-acetyl- and S-phenylacetyl-glutathione derivatives were synthesized by using a new procedure. The derivatives were incubated with rat plasma and red blood cells, and also with cytosol from rat liver, kidney and heart, or tissue slices from rat heart, kidney and liver. A limited hydrolysis of the compounds occurs in plasma, whereas hydrolysis occurs to a larger extent in tissue cytosols. Both purified and crude gamma-glutamyl-transpeptidase from different sources recognized the S-acetyl- and S-phenylacetyl derivatives as substrates. Intracellular glutathione increases after incubating the derivatives with red blood cells. A potential role of S-acetyl- and S-phenylacetyl-glutathione in replenishing cells with exogenous glutathione is envisaged.</p>","PeriodicalId":11854,"journal":{"name":"Enzyme & protein","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"1994-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000474975","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"18587469","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}