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PubMedDate not supplied

Isoenzyme specific inhibition of the reactivation of in vitro dissociated lactic dehydrogenase isoenzymes by two different peptides isolated from human liver.

The catalytic activity of the LDH-isoenzymes depends on their tetrameric structure. Low pH or other denaturants leads to dissociation into monomers and to the loss of the specific activity. After removal of the denaturing conditions reassociation and reactivation occur spontaneously. Neither NADH nor NAD+ shows a significant effect on the reactivation. We have isolated two different peptides which isoenzyme specifically inhibit the reactivation of dissociated LDH. Inhibition was abolished by treating with proteases. Additionally, NAD+ and NADH were found to be antagonists of the inhibitors. The heart-type enzyme-inhibitor system is especially susceptible for NADH whereas NAD+ affects the inhibition only slightly. The muscle-type system shows the opposite behavior, e.g., the completely inhibited system can be fully reactivated by NAD+ but not by NADH. These findings together with first kinetic studies suggest a possible specific regulatory function of these peptides.

Amino Acids↗
PubMed1979

Dual-detector-post-column reactor system for the detection of isoenzymes separated by high-performance liquid chromatography. II. Evaluation and application to lactate dehydrogenase isoenzymes.

We describe the separation of lactate dehydrogenase isoenzymes by high-performance liquid chromatography-anion-exchange columns and their quantitation by a computer-controlled, dual-detector post-column reaction system. The recoveries from the separation column were ca. 90%. The dynamic range of the system was linear over about three orders of magnitude from 3 to 1500 U/l. The coefficient of variation for isoenzyme peak areas was ca. 2%. The method is compared to the classical electrophoresis measurement and shows increased speed, resolution, precision and accuracy.

Chromatography, High Pressure Liquid↗
PubMed2004

Regulation of the insulin receptor by protein kinase C isoenzymes: preferential interaction with beta isoenzymes and interaction with the catalytic domain of betaII.

We analysed the effects of high glucose in rat1 cells overexpressing insulin receptor. High (25 mM) glucose inhibited insulin-stimulated tyrosine kinase activity completely at insulin concentrations of 1 and 5 ng/ml. Decapeptides modelled on insulin receptor sequences surrounding serines 1035 and 1270 were found to inhibit protein kinase C activity in vitro and after microinjection into cells blocked the inhibition of mitogenesis induced by glucose. Purification of receptor from 3T3L1 adipocytes revealed that only the isoenzymes beta1, betaII and delta were detected. The site of the interaction was mapped to the catalytic domain of betaII. These results demonstrate that the inhibition of insulin receptor tyrosine kinase activity can be ameliorated using insulin receptor peptide sequences and there is constitutive and differential interaction of individual PKC isoenzymes with the insulin receptor, and in the case of betaII, this interaction maps to the catalytic domain rather than the regulatory domain.

Animals↗
PubMed2000

Lactate dehydrogenase isoenzyme 1 is the most important LD isoenzyme in patients with testicular germ cell tumor.

We examined the clinical utility of serum lactate dehydrogenase (LD) isoenzyme catalytic concentrations in 58 patients with testicular germ cell tumors (TGCT) (13 with seminoma and 45 with non-seminomatous tumors). Twenty-one patients with no evidence of disease (NED) all had serum LD isoenzyme 1 catalytic concentrations (LD-1) and LD-1/LD fractions below the upper limit of the reference values (ULR). LD-1 and the LD-1/LD fraction discriminated significantly between evidence of disease (ED) and NED (p = 0.00009 and p = 0.028, respectively, Mann Whitney U-test). Twenty of the 37 patients with ED had raised values of LD-1. The 17 patients with an LD-1 < 1.0 x ULR had a better survival than the 10 patients with LD-1 between 1.0 and 2.9 x ULR, the 7 with LD-1 between 3.0 and 5.9 x ULR, and the 3 patients with LD-1 > 6.0 x ULR (p = 0.006, log-rank test, chi2 test for trend)). Twenty-three patients with an LD-1/LD fraction < or = 0.25 had a better survival than the 14 with an LD-1/LD fraction > 0.25 (p = 0.013). Nineteen patients with LD-5 < 105 U/L and the 15 with LD-5 > 105 U/L had a similar rate of survival (p = 0.85). Our findings add to the evidence showing LD-1 in preference to LD as a serum tumor marker of TGCT.

Adolescent↗
PubMed1990

Mass concentration of creatine kinase MB isoenzyme and lactate dehydrogenase isoenzyme 1 in diagnosis of perioperative myocardial infarction after coronary bypass surgery.

Recent advances in methodology allow the mass concentration of creatine kinase MB isoenzyme (CK-MB), and of lactate dehydrogenase isoenzyme 1 (LD1) to be determined quickly and easily as routine, emergency tests. We evaluated these tests as diagnostic criteria of perioperative myocardial infarction (PMI) after coronary bypass surgery. These tests were compared with the usual measurements of CK-MB activity by immunoinhibition and LD1 by electrophoresis and with other biological markers of myocardial infarction such as total CK, total LD, and aspartate aminotransferase. Sixty-one patients who underwent coronary bypass grafting were followed pre- and postoperatively by enzyme determinations and electrocardiography; a subgroup was monitored by myocardial scintigraphy. CK-MB mass appeared to be the best marker of PMI during the first 48 h, although LD1 was the marker of choice from days 2 to 4.

Adult↗
PubMed1982

Biosynthesis and topogenesis of aspartate aminotransferase isoenzymes in chicken embryo fibroblasts. The precursor of the mitochondrial isoenzyme is either imported into mitochondria or degraded in the cytosol.

In chicken embryo fibroblasts pulsed wih [35S]methionine, a precursor of mitochondrial aspartate aminotransferase with higher molecular weight (delta Mr approximately 3000) was detected by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide mapping of the precursor and the mature enzyme confirmed their precursor-product relationship. No precursor of the homologous cytosolic isoenzyme was found. The precursor of the mitochondrial isoenzyme is synthesized on membrane-free polysomes in the cytosol (Sonderegger, P., Jaussi, R., Christen, P., and Gehring, H. (1982) J. Biol. Chem. 257, 3339-3345); its half-life is 30 to 60 s. The pronounced susceptibility of the precursor toward exogenous proteases contrasts the stability of the mature enzyme and thus indicates that the conformation or the quarternary structure of the protein must change concomitantly with its import into mitochondria. Administration of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) to the cell cultures blocks the import of many matrix and inner membrane proteins into mitochondria. The precursor of mitochondrial aspartate aminotransferase is found to be accumulated in the cytosol. However, its steady state concentration in CCCP-treated cells exceeds the concentration in untreated cells by not more than 1 order of magnitude. During a chase, the radioactive precursor disappears with a half-life of approximately 5 with min without formation of mature enzyme. Thus, in CCCP-treated cells, a degradative process is limiting the accumulation of the precursor in the cytosol. When the chase is performed in the presence of cysteamine, an antagonist of CCCP, the precursor is processed to the mature enzyme. Newly synthesized cytosolic aspartate aminotransferase is not degraded.

Animals↗
PubMed1981

Lactate dehydrogenase isoenzyme 1 with the centrifugal analyzer after immunochemical removal of other isoenzymes.

We describe a centrifugal analyzer method for measuring the LD-1 isoenzyme of lactate dehydrogenase (EC 1.1.1.27) in serum, after immunochemical precipitation of the other four isoenzymes. Enzymic activity was measured kinetically at 30 degrees C with the pyruvate-to-lactate assay. The method for LD-1 was linear to 1000 U/L. The precision (CV) of the assay was 1.0--2.2% within-run and 3.1--4.5% day-to-day. The reference interval was 26--73 U/L (n = 51), corresponding to 21--35% of total LD activity.

Antigen-Antibody Complex↗
PubMed1986

Chorismate Mutase Isoenzymes from Selected Plants and Their Immunological Comparison with the Isoenzymes from Sorghum bicolor.

The isoenzyme pattern of chorismate mutase (EC 5.4.99.5) was examined by diethylaminoethyl-cellulose chromatography in a wide variety of plants. All plants contained a regulated form of chorismate mutase (CM-1), and most contained an additional, unregulated form (CM-2). The regulatory properties of CM-1 differed significantly between plants. Antisera prepared against CM-1 and CM-2 from Sorghum bicolor were used to test immunological cross reaction of chorismate mutases from other plants. There was a high degree of similarity between chorismate mutase isoenzymes from Sorghum bicolor and Zea mays and some with Hordeum vulgare, but all other species studied were antigenically distinct from sorghum. No homology between the structure of CM-1 and CM-2 was detected within any species.

Journal Article↗
PubMed1977

Studies on alkaline phosphatase isoenzymes. Relation to gamma-glutamyltransferase and lactate dehydrogenase isoenzymes.

Gamma-Glutamyltransferase (GT) and isoenzymes of alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) have been studied in 282 cases with increased S-ALP and in 18 chronic alcoholics with normal routine liver tests. There was a high degree of correlation between S-GT and the bile (alpha 1) and liver (alpha 2) fractions of S-ALP. Fractionation of alkaline phosphatases sometimes yielded clinical information, which could not be obtained by determinations of S-ALP and S-GT only. The presence of alpha 1-ALP and increased S-GT appeared to be more sensitive indicators of ethanol-induced liver involvement than other liver tests, including LDH-5/LDH-4 ratios.

Alcoholism↗
PubMed1988

Structural organization of the mouse aspartate aminotransferase isoenzyme genes. Introns antedate the divergence of cytosolic and mitochondrial isoenzyme genes.

We have cloned and characterized a mouse cytosolic aspartate aminotransferase (AspAT) (EC 2.6.1.1) gene, which is about 32,000 base-pairs long and is interrupted by eight introns. The 5' and 3'-flanking regions, and the exact sizes and boundaries of the exon blocks, including the transcription-initiation sites, were determined. The 5' end of the gene lacks the TATA and CAAT boxes characteristic of eukaryotic promoters, but contains G + C-rich sequences, three putative binding sites for a cellular transcription factor, Sp1, and multiple transcription-initiation sites. The sequences around the transcription-initiation sites are compatible with the formation of a number of potentially stable stem-loop structures. We compared the structural organization of the mouse cytosolic AspAT gene with that of the mouse mitochondrial AspAT gene, which has nine introns. We found that the promoter regions share a high level of homology and five of the introns are at identical places. This close matching leads to the tentative conclusion that the introns were in place before the divergence of cytosolic and mitochondrial isoenzyme genes.

Amino Acid Sequence↗
PubMed1984

The microdistribution of isoenzymic strains of Trypanosoma cruzi in southern Bolivia; new isoenzyme profiles and further arguments against Mendelian sexuality.

132 Trypanosoma cruzi stocks were collected in southern Bolivia (99 stocks in Tupiza, 33 in Tarija), and were characterized using five enzymes (six loci). From these 132 stocks, a sample of 21 was studied using 10 enzymes (12 loci) to establish the genetic distances between them. Only five different isoenzymic strains were registered among the 132 stocks: the taxonomic status of these strains is discussed. The distribution of the strains indicated that a Founder effect was not a constant fact at the level of the house and of the suburb, but that a Founder effect was more apparent for greater geographical distances. All strains were transmitted sympatrically by the same vector Triatoma infestans. Genotype frequencies demonstrated the lack of Mendelian sexuality among stocks of T. cruzi from southern Bolivia, confirming our previous results.

Alcohol Oxidoreductases↗
PubMed1990

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (NADP): amino acid sequence of the subunits from isoenzyme I and structural relationship with isoenzyme II.

The structural relationship between isoenzymes I and II of chloroplast glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NADP+ oxidoreductase (phosphorylating) EC 1.2.1.13) has been established at the protein level. The complete primary structure of subunits A and B of glyceraldehyde-3-phosphate dehydrogenase I from Spinacia oleracea has been determined by sequence analysis of the corresponding tryptic peptides, aligned by fragments derived from cyanogen bromide and Staphylococcus proteinase V8 digestions and by partially sequencing each intact subunit. Subunit A has an Mr of 36,225 and consists of 337 amino acid residues, whilst subunit B (Mr 39,355) consists of 368 residues. The amino acid sequence of subunit B, as determined through direct analysis of the protein, is identical to that recently deduced at cDNA level (Brinkmann et al. (1989) Plant Mol. Biol. 13, 81-94). The two subunits share a common portion of amino acid sequence which differs by 66 amino acid residues. Subunit B has an extra C-terminal sequence of 31 amino acid residues. Chloroplast glyceraldehyde-3-phosphate dehydrogenase II was partially characterized by sequencing the N-terminal portion of the intact protein and some of its tryptic peptides. The sequences of all the examined fragments fit precisely that of the corresponding regions of subunit A from glyceraldehyde-3-phosphate dehydrogenase I.

Amino Acid Sequence↗
PubMed1988

Phenotyping of phosphoglucomutase (PGM1) isoenzymes--a new method for the follow-up of chimerism after bone marrow transplantation. Follow-up of chimerism after bone marrow transplantation by phenotyping of phosphoglucomutase (PGM1) isoenzymes.

In this study, phenotyping of phosphoglucomutase isoenzymes (PGM1, EC 2.7.5.1) in erythrocytes of bone marrow donors and hosts was shown to present a simple and exact method for the follow-up of chimerism after bone marrow transplantation, aimed at a better assessment of the transplant state and function.

Bone Marrow Transplantation↗
PubMed1990

Time-related changes in the diagnostic utility of total lactate dehydrogenase, lactate dehydrogenase isoenzyme-1, and two lactate dehydrogenase isoenzyme-1 ratios in serum after myocardial infarction.

Using receiver-operating characteristic (ROC) curve and likelihood ratio analysis, we examined the diagnostic utility of total lactate dehydrogenase (LD; EC 1.1.1.27) activity (I). LD isoenzyme-1 activity (II), and the LD-1 percentage of total LD activity (III), LD-1 LD-2 (IV), and LD-1/LD-4 (V) in 347 persons admitted to the Cardiac Care Unit (of whom 173 were subsequently proven to have had myocardial infarction). Blood was sampled from these subjects at about 6-h intervals for up to 96 h from the onset of chest pain. Defining an "effective" test as one having an area under the ROC curve of greater than or equal to 0.9, we determined the ranked utility (greatest to least) of these tests as V = IV greater than III greater than II greater than I. Tests III, IV, and V had by this criterion, diagnostic effectiveness equivalent to measurements of creatine kinase-2 in serum but in samples obtained at later time intervals. The decision thresholds for both high (constant) test sensitivity and specificity varied with time, to differing extents, over the entire 96-h period, a finding with important diagnostic implications. We document positive and negative likelihood ratio values for each of these tests throughout the entire period of study.

Adult↗
PubMedDate not supplied

L.D.H. isoenzymes sub-unit ratio in leprosy and the effect of clofazimine treatment on L.D.H. isoenzymes sub-unit.

The clinical material for our studies of serum total LDH activity and LDH isoenzymes in leprosy included 255 patients consisting of Tuberculoid (74), Lepromatous leprosy (116), and Lepromatous leprosy with lepra reaction (65). 20 patients with suspected DDS resistance and repeated attacks of lepra reactions were selected for Clofazimine studies. All leprosy patients exhibited higher total LDH activity as compared to normals. M/H ratio was significantly increased in patients of Lepromatous leprosy and correlated closely with the clinical severity and advancement of disease. Tuberculoid leprosy patients showed values close to normals. Hence M/H ratio could demarcate two polar types of leprosy i.e. Tuberculoid and Lepromatous leprosy. Clofazimine treatment over a period of one year in patients with suspected DDS resistance and repeated attacks of lepra reaction decreased total LDH activity and M/H ratio considerably. Fall in M/H ratio during Clofazimine treatment could be attributed to the clearance of 'M' subunits by the drug due to removal of blockade of R.E.S. system produced by lepra bacilli.

Clofazimine↗
PubMed1983

[Taxonomic and medical status of isoenzyme strains of Trypanosoma cruzi. Considerations of the systematic and immunologic values of various isoenzymes].

The genetic distance calculations for 45 Bolivian stocks of Trypanosoma cruzi show the lack of continuum among them and allow to distinguish two main groups, which origin is perhaps a passed speciation. The presence of some heterogeneity within each group leads us to propose a temporary terminology: each isozymic variant is called "zymostrain" without prejudging its medical or taxonomic importance. The relationships between biochemical Taxonomy and medical data are discussed. The taxonomic value of the different isoenzymes is examined. The possible immunogenic properties of some of them (particularly Glutamate deshydrogenase) is discussed.

Alleles↗