ABSTRACT
We have developed a new automated method for the determination of malate dehydrogenase (MDH) isoenzymes in serum employing guanidine hydrochloride. Our proposed method showed good reproducibility; within-run precision coefficient of variations (CVs) were less than 2.5 (mean 13.6-42.9 U/L) for total MDH (T-MDH) and less than 6.7% (mean 6.3-23.5 U/L) for mitochondrial MDH (m-MDH) (n = 10). The upper detection limit of the proposed method exhibited good linearity up to 1,000 U/L for both T-MDH and m-MDH. In the proposed m-MDH reagent, the presence of up to 2,000 U/L of cytosolic MDH(c-MDH) activity had no effect on the outcome of m-MDH assay. Results of our proposed method (y) correlated well with those of the electrophoretic method (x) giving the regression equation: y = 1.46 x + 6.87 (N = 30); r = 0.99. Normal concentrations of various anticoagulants and bilirubin did not affect the assay results. Both ascorbic acid and glucose exhibited a slight positive interference with the proposed assay. Clinically, we found that m-MDH activity in serum had greater diagnostic predictive value than T-MDH activity for judging successful outcome of reperfusion therapy; the prognosis was poor when the m-MDH/T-MDH ratio was greater than 20%.
Subject(s)
Isoenzymes/analysis , Malate Dehydrogenase/analysis , Adult , Coenzymes/analysis , Coenzymes/blood , Electrophoresis , Female , Guanidine , Guanidines , Humans , Hydrogen-Ion Concentration , Isoenzymes/blood , L-Lactate Dehydrogenase/blood , L-Lactate Dehydrogenase/metabolism , Linear Models , Malate Dehydrogenase/blood , Male , Middle Aged , Myocardial Infarction/enzymology , Oxamic Acid/pharmacology , Prognosis , Protein Denaturation , Reference Values , Reproducibility of Results , Sensitivity and Specificity , Substrate Specificity , Time FactorsABSTRACT
A selective inactivating enzyme for the m-subunit of lactate dehydrogenase (LDH) was found in the culture filtrate of Penicillium citrinum KE-1, newly isolated from soil. The enzyme was purified from the culture filtrate by ammonium sulfate fractionation, column chromatography on CM-Sepharose CL-6B, and gel filtration on Sephadex G-100. The purification was 124-fold with an activity yield of 81%. The purified enzyme gave a single band, corresponding to a molecular weight of 32,000, on SDS polyacrylamide gel electrophoresis, and the isoelectric point was 9.5. The enzyme specifically inactivated the m-subunit of LDH but showed no activity on the h-subunit of LDH. The enzyme, named KE-1 proteinase, proved to be a serine-type proteinase. Limited proteolysis of native m-subunit of LDH was assumed to result in a loss of enzyme activity.
Subject(s)
L-Lactate Dehydrogenase/antagonists & inhibitors , Penicillium/enzymology , Serine Endopeptidases/isolation & purification , Serine Endopeptidases/metabolism , Amino Acid Sequence , Chromatography, Gel , Chromatography, High Pressure Liquid , Culture Media , Electrophoresis, Polyacrylamide Gel , Hydrolysis , Isoenzymes , Molecular Sequence Data , Molecular Weight , Oxidation-Reduction , Sequence Homology, Amino Acid , Serine Endopeptidases/chemistry , Serine Proteinase Inhibitors/pharmacology , Substrate SpecificityABSTRACT
We studied a new proteinase K assay method for human serum mitochondrial aspartate aminotransferase. We found that proteinase K showed no inactivation of human mitochondrial aspartate aminotransferase isoenzyme and complete inactivation of cytosolic aspartate aminotransferase. Previous studies have shown that selective proteolytic measurement for mitochondrial aspartate aminotransferase in serum using the protease 401 cleaved peptide bond at Leu 20 from the amino-terminal bond shows complete inactivation of cytosolic aspartate aminotransferase and slight inactivation of mitochondrial aspartate aminotransferase isoenzyme, depending on protease concentration. In this investigation, we found that the proteinase K method does not depend on protease concentration. The proteinase K enzyme inactivation of cytosolic aspartate aminotransferase is caused by the cleavage of the peptide bond at Ileu 21 from the aminoterminal bond. In studies with various animal cytosolic aspartate aminotransferase isoenzymes, proteinase K almost completely inactivated cytosolic aspartate aminotransferase. Precision and correlation using proteinase K for measurement of serum mitochondrial aspartate aminotransferase in human showed a good coefficient of variation (within-run < 4.45%) and a coefficient of correlation of r = 0.985 (N = 125).
Subject(s)
Aspartate Aminotransferases/blood , Isoenzymes/blood , Mitochondria/enzymology , Serine Endopeptidases/metabolism , Adult , Aged , Amino Acid Sequence , Aspartate Aminotransferases/metabolism , Cytosol/enzymology , Endopeptidase K , Humans , Hydrolysis , Isoenzymes/metabolism , Middle Aged , Molecular Sequence Data , Reference Values , Reproducibility of ResultsABSTRACT
We devised a method for assaying serum lactate dehydrogenase isoenzyme 1 (LD-1) activity specifically by preincubation with alpha-chymotrypsin and guanidine. Cleavage of phenylalanine bonds in the loop of A and B subunits of LD-3, LD-4, and LD-5 isoenzymes (residues 117-119) by incubation with alpha-chymotrypsin for a short time completely inactivated these isoenzymes and partially inactivated LD-2. Addition of guanidine (0.50 mol/L, pH 7.8) to the incubation mixture containing the chymotrypsin completed the inactivation of LD-2. As much as 4000 U/L of LD-2, LD-3, LD-4, and LD-5 were inactivated, whereas LD-1 was affected only slightly. Results by this method (y) correlated well with those by the Roche Isomune immunochemical LD-1 method (x): y = 0.98 x -0.11, r = 0.99 (n = 60). Within-run CVs were 0.5-2.5%. Several common interferents had no effect. In 500 healthy people, serum LD-1 ranged between 66 and 130 U/L, with a mean +/- SD of 88 +/- 15 U/L.
Subject(s)
Chymotrypsin/metabolism , L-Lactate Dehydrogenase/blood , Electrophoresis, Polyacrylamide Gel , Guanidine , Guanidines/metabolism , Humans , Immunoassay/methods , Immunoassay/statistics & numerical data , Isoenzymes , L-Lactate Dehydrogenase/antagonists & inhibitors , Phenylalanine/metabolism , Protein Denaturation , Reference ValuesABSTRACT
A new proteolytic measurement of serum mitochondrial aspartate aminotransferase was evaluated using cytosolic aspartate aminotransferase inactivating protease. Some of the proteases, such as, alpha-chymotrypsin, subtilisin and cytosolic aspartate aminotransferase inactivating protease 401 from Streptomyces species, also specifically inactivated cytosolic aspartate aminotransferase, but not mitochondrial, aspartate aminotransferase. The protease 401 was the most heat stable for storage and showed a higher inactivation rate for cytosolic aspartate aminotransferase--up to 7000 IU/L--more than 200-fold the upper limit. The coefficient of variation of the proteolytic method was less than 10%. Results by the present method correlated with those by the immunochemical method (r = 0.970) and the regression curve was Y = 0.95X + 1.60 (Y: immunochemical method; X: proteolytic method). In the present assay system, reference values for mitochondrial aspartate aminotransferase activity in 500 healthy people ranged from 2.0-7.2 U/L (mean 3.8 U/L).
Subject(s)
Aspartate Aminotransferases/blood , Mitochondria/enzymology , Aspartate Aminotransferases/antagonists & inhibitors , Humans , Peptide Hydrolases/metabolism , Reference ValuesABSTRACT
The optimal conditions for selective proteolytic inactivation of cytosolic aspartate aminotransferase (c-AST) to determine mitochondrial aspartate aminotransferase (m-AST) in serum were studied. Protease 401 was found to be effective over a pH range of 6.0-10.0. A pH of 9.5 with 0.5% albumin in the reagent mixture was determined to be optimal for inactivation of c-AST and preservation of m-AST, lactic dehydrogenase (LDH), and malic dehydrogenase (MDH) in the assay procedure. The presence of serum endogenous protein inhibitors such as alpha 1-antitrypsin and alpha 2-macroglobin did not inhibit protease 401.
