Enzyme & protein最新文献

{"title":"Erythrocyte pyruvate kinase deficiency. The influence of physiologically important metabolites on the function of normal and defective enzymes.","authors":"M. Lakomek, H. Winkler, A. Pekrun, N. Krüger, M. Sander, P. Huppke, W. Schröter","doi":"10.1159/000474982","DOIUrl":"https://doi.org/10.1159/000474982","url":null,"abstract":"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.","PeriodicalId":11854,"journal":{"name":"Enzyme & protein","volume":"198 1","pages":"149-63"},"PeriodicalIF":0.0,"publicationDate":"1994-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"74223963","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":"Measurement of ATPases in red cells: setting up and validation of a highly reproducible method.","authors":"E Matteucci,&nbsp;F Cocci,&nbsp;L Pellegrini,&nbsp;G Gregori,&nbsp;O Giampietro","doi":"10.1159/000474976","DOIUrl":"https://doi.org/10.1159/000474976","url":null,"abstract":"<p><p>Our aim was to set up and validate a reproducible method to study ATPase family on erythrocyte membranes. We compared several methods for erythrocyte washing and hemolysis and succeeded in preparing completely hemoglobin-free membrane ghosts still bearing intact ATPases. We compared the conventional incubation procedure with the coupled enzyme assay to measure Na-K-Mg, Ca-Mg and Mg ATPase on the membranes. A significant difference was constantly observed between the results by these methods, the values by the incubation procedure being 28, 57 and 58% of the respective values obtained by the linked enzyme assay. By adopting this last one, we obtained uniform and reproducible results in 31 healthy subjects. The following activities of the measured pumps resulted: Na-K-Mg ATPase 0.026 +/- 0.007, mean +/- SD; Ca-Mg ATPase 0.030 +/- 0.010, and Mg ATPase 0.017 +/- 0.003 U/mg protein, respectively. Finally, we investigated the effect of membrane storage time and temperature on ATPase results.</p>","PeriodicalId":11854,"journal":{"name":"Enzyme & protein","volume":"48 2","pages":"105-19"},"PeriodicalIF":0.0,"publicationDate":"1994-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000474976","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"18586204","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":"Quantitation of endopeptidase 24.11 and endopeptidase 24.15 in human blood leukocytes.","authors":"L Casale,&nbsp;C Cardozo,&nbsp;T Kalb,&nbsp;M Lesser","doi":"10.1159/000474981","DOIUrl":"https://doi.org/10.1159/000474981","url":null,"abstract":"<p><p>Endopeptidase 24.11 (EP 24.11; also called neutral endopeptidase, enkephalinase, CALLA, or CD10) and endopeptidase 24.15 (EP 24.15) are widely distributed neutral metalloendopeptidases that degrade a number of bioactive peptides including substance P, bradykinin, neurotensin, and chemotactic peptides. In this study we used sensitive substrates and specific inhibitors to quantitate the levels of these enzymes in purified peripheral human blood leukocytes obtained from healthy blood donors. We found that neutrophils did not contain detectable amounts of EP 24.15. In contrast, T lymphocytes, B lymphocytes, and monocytes contained significant amounts of the enzyme (446 +/- 248,314 +/- 183, and 484 +/- 212 nmol/mg protein/h, respectively). Neutrophils contained significant amounts of EP 24.11 (266 +/- 130 nmol/mg protein/h). Significantly lower levels of the enzyme were found in T lymphocytes, B lymphocytes, and monocytes (94 +/- 31, 87 +/- 38, and 20 +/- 13 nmol/mg protein/h, respectively). These findings suggest that the effects of some bioactive peptides on peripheral blood leukocyte function may be modulated by these enzymes.</p>","PeriodicalId":11854,"journal":{"name":"Enzyme & protein","volume":"48 3","pages":"143-8"},"PeriodicalIF":0.0,"publicationDate":"1994-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000474981","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19569774","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-fluorenylmethoxycarbonyl glutathione and diesters: inhibition of mammalian glyoxalase II.","authors":"M K Chyan,&nbsp;A C Elia,&nbsp;G B Principato,&nbsp;E Giovannini,&nbsp;G Rosi,&nbsp;S J Norton","doi":"10.1159/000474983","DOIUrl":"https://doi.org/10.1159/000474983","url":null,"abstract":"<p><p>Inhibitors having high specificity toward mammalian glyoxalase II, but not glyoxalase I, were sought as part of a program to study glyoxalase enzyme function in mammalian cells. The compound, S-fluorenylmethoxycarbonyl glutathione (FMOC-G), was synthesized and found to be a competitive inhibitor of purified calf liver glyoxalase II (Ki = 2.1 mumol/l). Inhibition constants (Ki values) for the other glyoxalase enzyme, glyoxalase I, and the glutathione-requiring enzyme, glutathione S-transferase, from other sources, were found to be 17 and 25 mumol/l, respectively. FMOC-G is a very poor inhibitor of glutathione reductase and glutathione peroxidase. Diesters (dimethyl, diethyl, diisopropyl) of FMCO-G were also synthesized, as proinhibitors, to improve transport of FMOC-G into mammalian tumor cells (rat adrenal pheochromocytoma, PC-12) in culture. The diesters were inhibitory to cell growth and variability; the most effective of these, diisopropyl FMOC-G, exhibited an [I]0.5 value of approximately 275 mumol/l. Diesters of FMOC-G may be useful in studies of the glyoxalase enzyme system in cultured mammalian cells.</p>","PeriodicalId":11854,"journal":{"name":"Enzyme & protein","volume":"48 3","pages":"164-73"},"PeriodicalIF":0.0,"publicationDate":"1994-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000474983","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19569782","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":"Clinical evaluation of serum ornithine carbamoyltransferase by enzyme-linked immunosorbent assay in patients with liver diseases.","authors":"Y Watanabe,&nbsp;S Mori,&nbsp;S Fujiyama,&nbsp;T Sato,&nbsp;M Mori","doi":"10.1159/000474965","DOIUrl":"https://doi.org/10.1159/000474965","url":null,"abstract":"<p><p>We developed a sensitive enzyme-linked immunosorbent assay (ELISA) for serum ornithine carbamoyltransferase (OCT) protein, and examined serum OCT concentrations in patients with various liver diseases. OCT concentrations were markedly elevated in cases of hepatic encephalopathy, 'acute on chronic', and those with the acute phase of acute hepatitis, moderately in chronic hepatitis, liver cirrhosis, hepatocellular carcinoma, primary biliary cirrhosis, and slightly in those with a fatty liver. High percentages (92-98%) of patients with chronic hepatitis, liver cirrhosis and hepatocellular carcinoma had higher than normal concentrations of serum OCT protein. There was a close correlation with aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities and moderate correlations with those of mitochondrial AST, glutamate dehydrogenase and gamma-glutamyltranspeptidase. The OCT/ALT ratio was higher in patients with liver cirrhosis than in those with chronic hepatitis (p < 0.001), and was still higher in cases of hepatocellular carcinoma (p < 0.05). In 2 patients with 'acute on chronic' disease, OCT concentrations decreased similarly with or more rapidly than AST or ALT activities after admission. In 2 patients with hepatic encephalopathy, the OCT concentrations changed similarly with AST and ALT activities. This OCT ELISA system will aid in diagnosing various liver diseases and in the follow-up of the patients, and the OCT/ALT ratio may serve for a differential diagnosis of liver diseases.</p>","PeriodicalId":11854,"journal":{"name":"Enzyme & protein","volume":"48 1","pages":"18-26"},"PeriodicalIF":0.0,"publicationDate":"1994-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000474965","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"18787421","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":"Structural characterization of cytosolic NADP(+)-dependent isocitrate dehydrogenase from rat ovary.","authors":"S Sechi,&nbsp;D Parmelee,&nbsp;P P Roller,&nbsp;G T Jennings","doi":"10.1159/000474966","DOIUrl":"https://doi.org/10.1159/000474966","url":null,"abstract":"<p><p>Cytosolic NADP(+)-dependent isocitrate dehydrogenase was purified to homogeneity from superovulated rat ovaries. Amino acid sequence information was obtained by analyzing peptides generated by digestion with either cyanogen bromide or trypsin. Eleven peptides were sequenced and a total of 146 amino acids were identified. Nine of these peptides were found to be 60-100% identical with sequences from mitochondrial NADP(+)-dependent isocitrate dehydrogenase. Conservation of amino acids was observed for residues that were previously identified as potentially binding isocitrate-Mg2+. Circular dichroism measurements showed that the structure is composed of approximately 35% alpha-helix and 21% beta-sheet segments. Temperature denaturation studies indicated that the enzyme is more stable in the presence of isocitrate.</p>","PeriodicalId":11854,"journal":{"name":"Enzyme & protein","volume":"48 1","pages":"27-36"},"PeriodicalIF":0.0,"publicationDate":"1994-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000474966","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"18787423","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":"Purification of human and rat kidney aldose reductase.","authors":"G Moeckel,&nbsp;J Hallbach,&nbsp;W G Guder","doi":"10.1159/000474968","DOIUrl":"https://doi.org/10.1159/000474968","url":null,"abstract":"<p><p>In the present study we report on a rapid two-step affinity chromatographic procedure to purify aldose reductase from human and rat kidney papilla and inner medulla. This enzyme, which is responsible for sorbitol formation in the kidney, was purified 145-fold from rat and 76-fold from human kidneys by consecutive Blue Sepharose and Matrex Orange chromatography. SDS-PAGE showed a single band of 38 kD for the human enzyme and a doublet of similar molecular weight for the rat kidney aldose reductase. The enzyme was characterized by substrate specificity and kinetic constants found identical to that of other organs purified previously.</p>","PeriodicalId":11854,"journal":{"name":"Enzyme & protein","volume":"48 1","pages":"45-50"},"PeriodicalIF":0.0,"publicationDate":"1994-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000474968","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"18787426","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":"Effects of pertussis toxin on signal transductions via P2-purinergic receptors in A-431 human epidermoidal carcinoma cells.","authors":"K Sugita,&nbsp;K Kurihara,&nbsp;K Hosoi,&nbsp;T Atsumi,&nbsp;T Takahashi,&nbsp;M Kohno,&nbsp;T Ueha","doi":"10.1159/000474992","DOIUrl":"https://doi.org/10.1159/000474992","url":null,"abstract":"<p><p>In A-431 cells, stimulation of P2-purinergic receptors with extracellular ATP caused production of inositol 1,4,5-trisphosphate (InsP3), followed by mobilization of Ca2+ from intracellular stores; Ca2+ influx from the extracellular fluid and breakdown of phosphatidylcholine (PtdCho) also accompanied this InsP3/Ca2+ signalling. When A-431 cells were pretreated with pertussis toxin (PTX), production of InsP3 and elevation of cytosolic Ca2+ were strongly inhibited. PTX also inhibited the Ca2+ influx, but the effect was much weaker than that for InsP3/Ca2+ elevation. No inhibitory effect was observed in ATP-stimulated PtdCho breakdown. These results suggest that there is a system(s) which mediates the functions of P2-purinergic receptors in addition to PTX-sensitive G-proteins.</p>","PeriodicalId":11854,"journal":{"name":"Enzyme & protein","volume":"48 4","pages":"222-8"},"PeriodicalIF":0.0,"publicationDate":"1994-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000474992","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19789761","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":"Enzyme-linked immunosorbent assay of carbamoylphosphate synthetase I: plasma enzyme in rat experimental hepatitis and its clearance.","authors":"M Ozaki,&nbsp;K Terada,&nbsp;M Kanazawa,&nbsp;S Fujiyama,&nbsp;K Tomita,&nbsp;M Mori","doi":"10.1159/000474991","DOIUrl":"https://doi.org/10.1159/000474991","url":null,"abstract":"<p><p>Carbamoylphosphate synthetase I (CPS I), a urea cycle enzyme, is located almost exclusively in the mitochondria of hepatocytes. The enzyme is unique in that it constitutes about 2-6% of total liver protein and is composed of a large subunit of 160 kD. We developed a sensitive enzyme-linked immunosorbent assay (ELISA) for measurement of the enzyme in plasma using an antibody against the rat enzyme. In galactosamine-induced rat acute hepatitis, plasma concentration of CPS I that was 1-2 micrograms/ml blood before the treatment, increased up to 125 micrograms/ml blood in 24 h after the treatment and decreased to a near control level in 72 h. Plasma concentration of ornithine carbamoyl-transferase (OCT), another urea cycle enzyme, reached a maximum in 24 h and then decreased a little more rapidly than that of CPS I. On the other hand, alanine aminotransferase activity reached a maximum in 36 h and decreased to a normal level in 96 h. In immunoblot analysis, the native CPS I polypeptide of 160 kD and its fragments of 140 and 125 kD were detected 24-48 h after the treatment. When purified rat CPS I and bovine OCT were injected intravenously into rats, the enzymes disappeared from blood roughly exponentially with apparent half-lives of about 67 and 18 min, respectively. Development of an ELISA for human CPS I and determination of the serum enzyme in various liver diseases remain to be performed.</p>","PeriodicalId":11854,"journal":{"name":"Enzyme & protein","volume":"48 4","pages":"213-21"},"PeriodicalIF":0.0,"publicationDate":"1994-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000474991","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19789760","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":"Utility of 3-O-methyl-N-acetyl-D-glucosamine, an N-acetylglucosamine kinase inhibitor, for accurate assay of glucokinase in pancreatic islets and liver.","authors":"I Miwa,&nbsp;Y Mita,&nbsp;T Murata,&nbsp;J Okuda,&nbsp;M Sugiura,&nbsp;Y Hamada,&nbsp;T Chiba","doi":"10.1159/000474980","DOIUrl":"https://doi.org/10.1159/000474980","url":null,"abstract":"<p><p>Glucokinase, an enzyme that catalyzes the phosphorylation of glucose, constitutes the key regulatory step in glucose metabolism in pancreatic islets and liver. We found that 3-O-methyl-N-acetyl-D-glucosamine (3-O-methyl-GlcNAc) potently inhibits glucose phosphorylation by N-acetylglucosamine kinase whereas glucokinase is not at all affected by this hexosamine. The addition of 3-O-methyl-GlcNAc to the assay system for glucokinase in rat liver extracts, which contain a high activity of glucokinase (glucose as substrate) relative to N-acetylglucosamine kinase (N-acetyl-D-glucosamine as substrate), affected neither Km nor Vmax values of glucokinase. On the other hand, both Km and Vmax values of glucokinase in rat pancreatic islet extracts, in which N-acetylglucosamine kinase activity is higher than glucokinase activity, were significantly lowered by the use of 3-O-methyl-GlcNAc as an inhibitor of N-acetylglucosamine kinase.</p>","PeriodicalId":11854,"journal":{"name":"Enzyme & protein","volume":"48 3","pages":"135-42"},"PeriodicalIF":0.0,"publicationDate":"1994-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000474980","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19569779","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}