Organ specificity and lactate-dehydrogenase activity. Differential inhibition by urea and related compounds

1. The effect of urea on the lactate-dehydrogenase activities of human-heart and -liver tissue extracts and on crystalline ox-heart and rabbit-muscle enzyme have been determined. Similar studies on electrophoretically separated isoenzyme fractions have shown an inverse relationship between sensitivity to urea inhibition and electrophoretic mobility. 2. With pyruvate as substrate a sharp change in the nature of the inhibition of tissue lactate dehydrogenase with increasing concentrations of urea occurs at 1 m or 4 m with the electrophoretically slow and fast isoenzymes respectively. 3. At concentrations of urea less than 1 m, inhibition of the purified enzymes is competitive with respect to pyruvate and 2-oxobutyrate. 4. Similar studies have been carried out with methylurea and hydantoic acid, both of which are more potent inhibitors than urea.     

Seasonal changes in the plasma isoenzymes of lactate: NADH oxidoreductase in Hereford cows under range conditions

Plasma levels of lactate: NADH oxidoreductase (LD) and the proportions of five major isoenzymes were estimated in samples taken in January, March, June, July and November from Hereford cows kept under range conditions. Seasonal changes were observed in both total LD and the proportions of the isoenzymes. Levels of total LD and all isoenzymes were low in winter and high in summer. Proportions of LD4 and LD5, the electrophoretically slow isoenzymes which predominate in muscle, increased in summer at the expense of LD1, the fast isoenzyme which predominates in liver and erythrocytes. LD1 was increased in November, at the time when other isoenzyme levels were decreasing from summer high levels. The seasonal changes observed in plasma LD were attributed to changes in diet and/or water supply and activity of the range cows.     

The preparation of crystalline human l-lactate–nicotinamide–adenine dinucleotide oxidoreductase isoenzyme 1 involving preparative polyacrylamide-gel electrophoresis

A method is presented for the preparation of human heart lactate dehydrogenase (l-lactate-NAD(+) oxidoreductase; EC 1.1.1.27) isoenzyme 1; this involves the use of polyacrylamide-gel electrophoresis as a preparative step. The yield was about 10% with a final specific activity of 220 units/mg of protein, one unit being defined as the amount of enzyme catalysing the oxidation of 1mumol of NADH/min at 25 degrees C, in the presence of 0.33mm-pyruvate. The crystalline preparation contained less than 2% of the other isoenzymes, was homogeneous in the ultracentrifuge and showed only a trace of protein contamination on polyacrylamide-gel electrophoresis. Some properties of the crystalline isoenzyme are reported; E(1%) (1cm)=13.2 at 280nm, s(0) (20,w)=7.43S, pI=4.6, and the apparent K(m) for pyruvate=1.02x10(-4)m. The human isoenzyme and the isoenzyme from pig heart differ with respect to amino acid composition, electrophoretic mobility and solubility. It is possible that these differences do not involve the active site, or sites, but are due to changes in amino acid residues elsewhere in the molecule. The importance of purified human LDH-1 isoenzyme with regard to enzyme radioimmunoassay is emphasized.     

实验细胞资源同功酶分析鉴定

琼脂糖凝胶电泳检测细胞同功酶的方法,可用于实验细胞系种间鉴别和交叉污染检测。该方法所需样品少,时间短,费用低,并且具有较高的灵敏度。本文对HeLa细胞进行了LD、MD、G6PD同功酶检测灵敏度分析,结果表明:HeLa细胞LD和MD同功酶在细胞为5×107个/L时可有效检测出来,而G6PD分析时细胞为109个/L。20种实验细胞的LD、MD同功酶检测证明,每种细胞种属来源清楚,且均未被其它细胞污染。     

Acanthamoeba healyi n. sp. and the isoenzyme and immunoblot profiles of Acanthamoeba spp., groups 1 and 3.

Two strains of Acanthamoeba isolated from human brain tissue and a strain of Acanthamoeba isolated from a fish were compared with 10 species of Acanthamoeba belonging to groups 1, 2 and 3 based on their isoenzyme profiles and antigenic characteristics. A total of 12 enzymes were studied. The isoenzymes and antigens were electrophoretically separated on polyacrylamide gradient gels, and the patterns obtained were compared after appropriate staining for particular enzymes and reactivities with homologous and heterologous rabbit anti-Acanthamoeba antisera. One of the human strains (CDC:1283:V013) was identified as A. healyi n. sp. because of its unique isoenzyme profiles for 11 of the 12 enzymes tested. The other human isolate was reidentified as A. culbertsoni because its isoenzyme profiles for 10 of 12 enzymes resembled those of A. culbertsoni, Lilly A-1 strain. Since the isoenzyme profiles and the antigenic patterns of the fish isolate as well were remarkably similar to those of A. royreba, it was considered as a strain of A. royreba. Polyacrylamide gradient gel electrophoresis appears to be a powerful technique for the study of isoenzymes and antigens of Acanthamoeba.     

Acanthamoeba healyi N. Sp. and the Isoenzyme and Immunoblot Profiles of Acanthamoeba spp., Groups 1 and 3

ABSTRACT Two strains of Acanthamoeba isolated from human brain tissue and a strain of Acanthamoeba isolated from a fish were compared with 10 species of Acanthamoeba belonging to groups 1, 2 and 3 based on their isoenzyme profiles and antigenic characteristics. A total of 12 enzymes were studied. The isoenzymes and antigens were electrophoretically separated on polyacrylamide gradient gels, and the patterns obtained were compared after appropriate staining for particular enzymes and reactivities with homologous and heterologous rabbit anti- Acanthamoeba antisera. One of the human strains (CDC:1283:V013) was identified as A. healyi n. sp. because of its unique isoenzyme profiles for 11 of the 12 enzymes tested. The other human isolate was reidentified as A. culbertsoni because its isoenzyme profiles for 10 of 12 enzymes resembled those of A. culbertsoni , Lilly A-1 strain. Since the isoenzyme profiles and the antigenic patterns of the fish isolate as well were remarkably similar to those of A. royreba , it was considered as a strain of A. royreba . Polyacrylamide gradient gel electrophoresis appears to be a powerful technique for the study of isoenzymes and antigens of Acanthamoeba .     

Isoenzymes of p-coumarate: CoA ligase from cell suspension cultures of Glycine max.

Two isoenzymes of p-coumarate: CoA ligase were isolated from cell suspension cultures of soybean (Glycine max L., var. Mandarin). Separation and partial purification of the enzymes were achieved by precipitation with MnCl2 and (NH4)2SO4, and column chromatography on DEAE-cellulose, Sephadex G-100 and hydroxyapatite. The isoenzymes had approximately the same molecular weight, but differed significantly with respect to their substrate specificity, their inhibition constants for AMP, their dependence on pH and ionic strength for optimum activity, and their fractionation pattern during the purification procedure or upon analytical disc-gel electrophoresis. Both coumarate: CoA ligases were specific for the activation of various substituted cinnamic acids. Of the cinnamic acids tested, ferulic, sinapic, 5-hydroxyferulic, p-coumaric, and caffeic acids were the substrates with the lowest apparent Km values (on all the order of 1 to 4 x 10(-5) M) for isoenzyme 1. The lowest apparent Km values (from about 1 to 9 x 10(-5) M) for isoenzyme 2 were obtained for caffeic, p-coumaric, m-coumaric, and o-coumaric acids. Sinapic acid and several methoxycinnamic acids were efficient substrates of isoenzyme 1 but were not activated at all by isoenzyme 2. The possible roles of the two p-coumarate: CoA ligase isoenzymes in the phenylpropanoid metabolism of the cell cultures are discussed.     

Application of the Isoenzyme Profile in the Development of Cell‐Based Devices

To detect cell cross‐contamination and verify the origin of the cells of an artificial organ, the sensitive isoenzyme assay was chosen to monitor the quality test of cell‐based devices. Authoritative cell evaluation of artificial skin products has been established in this study. Human and porcine cell suspensions with total cell counts of between 1×10⁵ and 4×10⁶ were individually tested to determine the activity of isoenzymes. Human fibroblast, mixed with 1% to 100% of porcine fibroblast, could be significantly distinguished in the isoenzyme assay. Based on the glucose‐6‐phosphophate‐dehydrogenase analysis, the human fibroblast tested in this study belonged to the B type human cells. Lactate dehydrogenase (LD), malate dehydrogenase (MD) and mannose phosphate isomerase isoenzyme (MPI) activities obviously revealed that a different pattern corresponds to the percentage of human and porcine cell mixtures. The discriminatory limit of MPI, LD and MD activity can reach up to 1% of sensitivity of the isoenzyme analysis. This sensitive isoenzyme analysis method allows us to routinely test cellular biomaterials whether interspecies cell line cross‐contamination has occurred in the development of artificial organs.     

The occurrence of 4-methylthio-2-hydroxybutyrate in human urine

A method for determination of 4-methylthio-2-hydroxybutyrate and 4-methylthio-2-oxobutyrate in human urine has been devised, based on metoxime formation of the keto acid and a clean-up procedure using a strong anion-exchange resin AG 2 X 8 and ethyl acetate extraction. After alkylation, the compounds were quantified by GC, using a flame photometric sulfur-selective detector. A normal urinary excretion of 0.14 to 0.25 mmol/mol creatinine and 0.07 to 0.22 mmol/mol creatinine of the alpha-hydroxy and alpha-keto acid, respectively, was found, whereas a markedly elevated excretion of the hydroxy acid was noted in subjects with hypermethioninemia. The enzymatic reduction of 4-methylthio-2-oxobutyric acid by lactate dehydrogenase: NAD+ oxidoreductase (EC 1.1.1.17) was also studied. The Km and Kequil values for 4-methylthio-2-oxobutyrate were 1.41 mM and 0.92 X 10(8) M-1. The Vmax value of the enzyme at infinite concentrations of the two substrates was 7.2 mumol/s/mumol enzyme, which indicates low affinity and reduced catalytic activity compared to other known substrates of lactate dehydrogenase. The reaction product 4-methylthio-2-hydroxybutyrate was not inhibitory on the reaction. The M4 isoenzyme of lactate dehydrogenase (rabbit and pig muscle) possessed approximately 20% of the activity of the H4 isoenzyme (pig heart) for the substrate.     

Activity and isoenzymes of lactate dehydrogenase in tissues of amphibians (Xenopus laevis, Ambystoma mexicanum, Triturus alpestris and vulgaris) and the response in the fat body of Xenopus males to gonadotropin injection (author's transl)]

Lactate-dehydrogenase activity was determined by the optical test method using pyruvate as substrate and the isoenzymes were separated by vertical starch gel electrophoresis. Species-specific and organ-specific characteristics of the total activity and the isoenzyme patterns of the four amphibian species are compared with those of rat and mouse. Application of gonadotropin increases the amount of soluble protein and the lactate-dehydrogenase activity in the fat body of Xenopus and the isoenzyme pattern shows a shift of intensity towards the basic bands. Testis does not respond to this treatment.     

Evidence for the existence of two soluble NAD(+) kinase isoenzymes in Euglena gracilis Z

Two soluble NAD(+) kinase isoenzymes (isoenzymes 1 and 2) from Euglena gracilis were separated by preparative electrophoresis and characterized. They display several similar properties: both have an identical apparent molecular weight of 68 kDa and their activities are independent on calmodulin, insensitive to 2-mercaptoethanol but inhibited by p-chloromercurybenzoate, 5, 5'-dithiobis(2-nitrobenzoate) and, surprisingly, by low dithiothreitol concentrations, the inhibition by dithiothreitol being irreversible for isoenzyme 1 but reversible for isoenzyme 2. Nevertheless, the two isoenzymes mainly differ by their specificities towards triphosphate nucleotides and their catalytic mechanisms. Isoenzyme 1 is as active in the presence of ATP as of GTP and acts by a ping-pong mechanism with a k(M) for NAD(+) of 0.26 mM and a k(M) for low MgATP(2-)concentrations of 0.03 mM. Isoenzyme 2 is three-fold more active in the presence of GTP than of ATP and operates by a sequential mechanism with k(M)s for NAD(+) and MgGTP(2-) of 1.03 and 0.20 mM, respectively. This study shows the evidence for the existence of two structurally similar but catalytically different NAD(+) kinase isoenzymes in E. gracilis. One resembles the enzyme previously described in bacteria. The other displays a catalytic mechanism identical to that of NAD(+) kinase from other organisms but remains unique among all the NAD(+) kinases studied to-date regarding its specificity towards GTP.     

Kinetic properties of human liver alcohol dehydrogenase: oxidation of alcohols by class I isoenzymes

Class I isoenzymes of alcohol dehydrogenase (ADH) were isolated by chromatography of human liver homogenates on DEAE-cellulose, 4-[3-[N-(6-aminocaproyl)-amino]propyl]pyrazole--Sepharose and CM-cellulose. Eight isoenzymes of different subunit composition (alpha gamma 2, gamma 2 gamma 2, alpha gamma 1, alpha beta 1, beta 1 gamma 2, gamma 1 gamma 1, beta 1 gamma 1, and beta 1 beta 1) were purified, and their activities were measured at pH 10.0 by using ethanol, ethylene glycol, methanol, benzyl alcohol, octanol, cyclohexanol, and 16-hydroxyhexadecanoic acid as substrates. Values of Km and kcat for all the isoenzymes, except beta 1 beta 1-ADH, were similar for the oxidation of ethanol but varied markedly for other alcohols. The kcat values for beta 1 beta 1-ADH were invariant (approximately 10 min-1) and much lower (5-15-fold) than those for any other class I isoenzyme studied. Km values for methanol and ethylene glycol were from 5- to 100-fold greater than those for ethanol, depending on the isoenzyme, while those for benzyl alcohol, octanol, and 16-hydroxyhexadecanoic acid were usually 100-1000-fold lower than those for ethanol. The homodimer beta 1 beta 1 had the lowest kcat/Km value for all alcohols studied except methanol and ethylene glycol; kcat values were relatively constant for all isoenzymes acting on all alcohols, and, hence, specificity was manifested principally in the value of Km. Values of Km and kcat/Km revealed for all enzymes examined that the short chain alcohols are the poorest while alcohols with bulky substituents are much better substrates. The experimental values of the kinetic parameters for heterodimers deviate from the calculated average of those of their parent homodimers and, hence, cannot be predicted from the behavior of the latter. Thus, the specificities of both the hetero- and homodimeric isoenzymes of ADH toward a given substrate are characteristics of each. Ethanol proved to be one of the "poorest" substrates examined for all class I isoenzymes which are the predominant forms of the human enzyme. On the basis of kinetic criteria, none of the isoenzymes of class I studied oxidized ethanol in a manner that would indicate an enzymatic preference for that alcohol.     

Comparison of human alkaline phosphatase isoenzymes. Structural evidence for three protein classes.

The structural relationships among human alkaline phosphatase isoenzymes from placenta, bone, kidney, liver and intestine were investigated by using three criteria. 1. Immunochemical characterization by using monospecific antisera prepared against either the placental isoenzyme or the liver isoenzyme distinguishes two antigenic groups: bone, kidney and liver isoenzymes cross-react with anti-(liver isoenzyme) serum, and the intestinal and placental isoenzymes cross-react with the anti-(placental isoenzyme) antiserum. 2. High-resolution two-dimensional electrophoresis of the 32P-labelled denatured subunits of each enzyme distinguishes three groups of alkaline phosphatase: (a) the liver, bone and kidney isoenzymes, each with a unique isoelectric point in the native form, can be converted into a single form by treatment with neuraminidase; (b) the placental isoenzyme, whose position also shifts after removal of sialic acid; and (c) the intestinal isoenzyme, which is distinct from all other phosphatases and is unaffected by neuraminidase digestion. 3. Finally, we compare the primary structure of each enzyme by partial proteolytic-peptide 'mapping' in dodecyl sulphate/polyacrylamide gels. These results confirm the primary structural identity of liver and kidney isoenzymes and the non-identity of the placental and intestinal forms. These data provide direct experimental support for the existence of at least three alkaline phosphatase genes.     

The amino acid sequence of cytosolic aspartate aminotransferase from human liver.

1. The cytosolic aspartate aminotransferase was purified from human liver. 2. The isoenzyme contains four cysteine residues, only one of which reacts with 5,5'-dithiobis-(2-nitrobenzoic acid) in the absence of denaturing agents. 3. The amino acid sequence of the isoenzyme is reported, as determined from peptides produced by digestion with trypsin and with CNBr, and from sub-digestion of some of these peptides with Staphylococcus aureus V8 proteinase. 4. The isoenzyme shares 48% identity of amino acid sequence with the mitochondrial form from human heart. 5. Comparisons of the amino acid sequences of all known mammalian cytosolic aspartate aminotransferases and of the same set of mitochondrial isoenzymes are reported. The results indicate that the cytosolic isoenzymes have evolved at about 1.3 times the rate of the mitochondrial forms. 6. The time elapsed since the cytosolic and mitochondrial isoenzymes diverged from a common ancestral protein is estimated to be 860 x 10(6) years. 7. Experimental details and confirmatory data for the results presented here are given in a supplementary paper that has been deposited as a Supplementary Publication SUP 50158 (25 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1990) 265, 5.     

Expression of human glutathione S-transferase 2 in Escherichia coli. Immunological comparison with the basic glutathione S-transferases isoenzymes from human liver.

A plasmid, termed pTacGST2, which contains the complete coding sequence of a GST2 (glutathione S-transferase 2) subunit and permits the expression of the protein in Escherichia coli was constructed. The expressed protein had the same subunit Mr as the enzyme from normal human liver and retained its catalytic function with both GST and glutathione peroxidase activity. Antiserum raised against the bacterially synthesized protein cross-reacted with all the basic GST isoenzymes in human liver. The electrophoretic mobility in agarose of the bacterially expressed isoenzyme suggested that its pI is identical with that of the cationic isoenzyme from human liver previously termed GST2 type 1. The available evidence suggests that the three common cationic isoenzymes found in human liver are the products of two very similar gene loci.     

Kinetics of bromodichloromethane metabolism by cytochrome P450 isoenzymes in human liver microsomes

The kinetic constants for the metabolism of bromodichloromethane (BDCM) by three cytochrome P450 (CYP) isoenzymes have been measured in human liver microsomes. The three CYP isoenzymes, CYP2E1, CYP1A2 and CYP3A4, have been identified previously as important in the metabolism of this compound. To measure the constants for each isoenzyme, enzyme-specific inhibitory antibodies were used to block the activities for two of the three isoenzymes. CYP2E1 was found to have the lowest K(m), 2.9 microM, and the highest catalytic activity, k(cat). The K(m) for the other isoenzymes, CYP1A2 and CYP3A4, were about 60 microM with lower values of k(cat). Apparent kinetic constants obtained from two microsomal samples that were not inhibited were consistent with these results. In addition, 11 human microsome samples characterized for 10 CYP activities were correlated with the metabolism of 9.7 microM BDCM by each sample; statistical analysis showed a correlation with CYP2E1 activity only. This result is consistent with the finding that CYP2E1 is the only isoenzyme with a K(m) lower than the BDCM concentration used. The kinetic constants obtained from the inhibited microsomes were compared to similar results from recombinant human isoenzyme preparations containing only one CYP isoenzyme. The results for CYP2E1 were very similar, while the results for CYP1A2 were somewhat less similar and there was a substantial divergence for CYP3A4 in the two systems. Possible reasons for these differences are differing levels of CYP reductase and/or differing makeup of the membrane lipid environment for the CYPs. Because of the low levels of BDCM exposure from drinking water, it appears likely that CYP2E1 will dominate hepatic CYP-mediated BDCM metabolism in humans.     

Kinetic Properties of the ATP-Dependent Phosphofructokinase Isoenzymes from Cucumber Seeds

Two isoenzymes of ATP:D-fructose-6-phosphate 1-phosphotransferase(phosphofructokinase) are present in germinating cucumber seeds,one in the plastids and the other in the cytosol. Both isoenzymeswere purified and some of their kinetic properties studied.These two isoenzymes differ kinetically, the pH optimum of thecytosolic isoenzyme being 7.2 and that of the plastid isoenzymebeing 8.0. Both isoenzymes are activated by phosphate althoughthe concentration required for activation is much lower forthe plastid isoenzyme than cytosolic isoenzyme. Phosphate increasesthe affinity of the isoenzymes for fructose-6-phosphate andalso changes the sigmoidal kinetics of the plastid isoenzymefor this substrate to hyperbolic kinetics at pH 7.2. The fructose-6-phosphatesaturation kinetics of the cytosolic isoenzyme becomes moresigmoidal with an increase in pH while the opposite is truefor the plastid isoenzyme. The cytosolic isoenzyme has a higheraffinity for fructose-6-phosphate at pH 7.2 than pH 8.0 whilethe affinity of the plastid isoenzyme for fructose-6-phosphateis highest at pH 8.0. Both isoenzymes are inhibited by ATP andthe extent of inhibition is pH dependent. The cytosolic isoenzymeis more sensitive to ATP inhibition at pH 8.0 than pH 7.2 whilethe opposite holds for the plastid isoenzyme. Magnesium alleviatesthe ATP inhibition of the plastid isoenzyme suggesting thatfree ATP is the inhibitory form. In contrast the ATP inhibitionof the cytosolic isoenzyme apparently appears to be caused bythe magnesium-ATP complex. (Received May 19, 1987; Accepted January 18, 1988)     

Isolation of human lactate dehydrogenase isoenzyme X by affinity chromatography.

Human isoenzyme LDH-X (lactate dehydrogenase isoenzyme X) was isolated from seminal fluid of frozen semen samples by affinity chromatography by using oxamate-Sepharose and AMP-Sepharose. In the presence of 1.6 mM-NAD+, isoenzyme LDH-X does not bind to AMP-Sepharose, whereas the other lactate dehydrogenase isoenzymes do. This is the crucial point in the isolation of isoenzyme LDH-X from the other isoenzymes. The purified human isoenzyme LDH-X had a specific activity of 146 units/mg of protein.     

The analysis of soluble galactosyltransferase isoenzyme patterns using high resolution agarose isoelectricfocusing

A high resolution method has been developed to separate the isoenzymes of galactosyltransferase by combining isoelectricfocusing (IEF) in 245 mm long agarose gels with a highly sensitive enzyme activity assay. The resolution and sensitivity is such that the isoenzyme pattern of 10 microliters human serum can be resolved. Using this method normal human serum was shown to contain at least 12 isoenzyme forms of galactosyltransferase, the major forms having isoelectric points of 4.33, 4.43, 4.51, 4.61, 4.74, 4.87, 4.96, 5.16 and 5.23. Part of the isoenzyme pattern complexity is due to sialylation of some isoenzymes. Alpha-lactalbumin-affinity chromatography, a method widely used in the purification of galactosyltransferase, causes a preferential purification of some of the isoenzyme forms.     

The activities of fructose 1,6-diphosphatase, phosphofructokinase and phosphoenolpyruvate carboxykinase in white muscle and red muscle

1. The activities of fructose 1,6-diphosphatase were measured in extracts of muscles of various physiological function, and compared with the activities of other enzymes including phosphofructokinase, phosphoenolpyruvate carboxykinase and the lactate-dehydrogenase isoenzymes. 2. The activity of phosphofructokinase greatly exceeded that of fructose diphosphatase in all muscles tested, and it is concluded that fructose diphosphatase could not play any significant role in the regulation of fructose 6-phosphate phosphorylation in muscle. 3. Fructose-diphosphatase activity was highest in white muscle and low in red muscle. No activity was detected in heart or a deep-red skeletal muscle, rabbit semitendinosus. 4. The lactate-dehydrogenase isoenzyme ratio (activities at high and low substrate concentration) was measured in various muscles because a low ratio is characteristic of muscles that are more dependent on glycolysis for their energy production. As the ratio decreased the activity of fructose diphosphatase increased, which suggests that highest fructose-diphosphatase activity is found in muscles that depend most on glycolysis. 5. There was a good correlation between the activities of fructose diphosphatase and phosphoenolpyruvate carboxykinase in white muscle, where the activities of these enzymes were similar to those of liver and kidney cortex. However, the activities of pyruvate carboxylase and glucose 6-phosphatase were very low in white muscle, thereby excluding the possibility of gluconeogenesis from pyruvate and lactate. 6. It is suggested that the presence of fructose diphosphatase and phosphoenolpyruvate carboxykinase in white muscle may be related to operation of the alpha-glycerophosphate-dihydroxyacetone phosphate and malate-oxaloacetate cycles in this tissue.