Characterization of potato (Solanum tuberosum) lactate dehydrogenase isoenzymes by affinity chromatography and hybridization

The five isoenzymes of potato (Solanum tuberasum) lactate dehydrogenase have been resolved by affinity chromatography. Mixtures of isoenzymes LDH-1 and LDH-5 dissociate and reassociate during freezing and thawing to produce five isoenzymes. These results indicate that potato lactate dehydrogenase isoenzymes are primary isoenzymes of the vertebrate type, which are composed of two subunit types.     

Cytoplasmic enzyme patterns in isolated hamster pulmonary alveolar type II cells

Three cytoplasmic enzyme patterns were studied in pulmonary alveolar type II cells isolated from normal adult hamster lung: lactate dehydrogenase (total and isoenzymes), peroxidase, and beta-N-acetylglucosaminidase. Enzyme patterns of freshly-isolated type II cells were found to be different from those of freshly-isolated pulmonary hamster fibroblasts. After both types of cells had been cultured for seven days, no difference in cytoplasmic enzyme patterns remained. Lactate dehydrogenase isoenzyme patterns for type II cells were different from those obtained from polymorphonuclear leukocytes and alveolar macrophages. These data may be useful in detecting sources of lung injury by assessment of enzyme patterns in bronchoalveolar lavage fluid.     

Separation of rat lactate dehydrogenase isoenzyme C4 from other isoenzymes by affinity and ion-exchange chromatography.

Lactate dehydrogenase C, an isoenzyme composed of C polypeptide subunits and found only in mature testes and spermatozoa, differs kinetically, chemically and immunologically from the five common isoenzymes of lactate dehydrogenase, each of which is a tetramer of A and/or B subunits. In the rat lactate dehydrogenase C exists in two molecular forms, isoenzymes C4 and A1C3. In addition to these two forms of lactate dehydrogenase C, rat testicular homogenate contains all the five isoenzymes of A and B type. Purification of isoenzyme C4 requires its separation from the other six isoenzymes, of which isoenzymes A1C3 and A3B1 are the most difficult ones to separate. In the present study isoenzyme A3B1, along with other enzymes, was separated from isoenzyme C4 by AMP-Sepharose chromatography by using a gradient of increasing concentration of NAD+-pyruvate adduct. In the next step, isoenzyme A1C3 was separated from isoenzyme C4 by DEAD-cellulose chromatography, resulting in a pure lactate dehydrogenase isoenzyme C4 preparation.     

Developmental Changes in Lactate Dehydrogenase and Aldolase Activity of the A2G-adr Mouse with Abnormal Muscle Function: Further Comparison with the 129Re-dy Mutant

Abstract: Lactate dehydrogenase and aldolase activity were reduced in lateral gastrocnemius muscle from two mouse mutants, A2G- adr and 129Re- dy , with abnormal muscle function. The activities of both of these enzymes were significantly reduced in the lateral gastrocnemius muscle from the A2G- adr mice at ages varying from 2 weeks to 32 weeks, whereas the activities in the soleus, heart, liver, and brain were the same as in the control animals. The lactate dehydrogenase isoenzymes in the lateral gastrocnemius and soleus muscles from the A2G mice were quantified, and although those of the soleus were comparable in mutant and control muscle, the lateral gastrocnemius from the adr mutant had reduced activity of LDH 5 and increased activities of the other four isoenzymes. The findings suggest that the adr mutation is expressed in the white (Type II) muscle fibres and not in the red (Type I) fibres or in any of the organs studied. It is suggested that the initiation of differentiation into Type II fibres from the embryonic form is absent or delayed in the A2G mutant. The reduced activities of lactate dehydrogenase and aldolase in 129Re- dy muscle confirm the findings of other workers.     

Comparison of muscle fiber typing by quantitative enzyme assays and by myosin ATPase staining

Fibers in cross sections of human and rat muscle were typed by using histochemical ATPase stains, and the results were compared with those of quantitative enzyme assays of fragments of the same fibers dissected from serial freeze-dried sections. Two enzymes previously used to assess the metabolic type were measured in each case: lactate dehydrogenase and either adenylokinase (human fibers) or malate dehydrogenase (rat fibers). With human fibers there was essentially complete agreement between ATPase staining and the metabolic enzyme assays in distinguishing types I and II fibers. The agreement was less consistent with regard to type IIA and IIB fibers. A number of ATPase type IIC fibers were identified in one human muscle, and were found to fall between ATPase types I and IIA on the basis of metabolic enzyme assay results. Rat-fiber ATPase types I, IIA, and IIB from the plantaris muscle were rather well segregated on a two-dimensional lactate dehydrogenase-malate dehydrogenase grid. In the rat soleus muscle, ATPase types I and IIA fibers were shifted to lower lactate dehydrogenase levels, with IIC fibers interposed between them.     

Somatic and sperm-specific isoenzymes of glyceraldehyde-3-phosphate dehydrogenase: Comparative analysis of primary structures and functional features

The elucidation of the interdependence between structural features and functions of somatic and sperm-specific isoenzymes of glyceraldehyde-3-phosphate dehydrogenase (GAPD and GAPDS, respectively) was the goal of comparative analysis of their primary structures. GAPDS was shown to lack the sequence similar to the atypical nuclear export signal motif (NES) of the somatic isoenzyme GAPD. This finding is confirmed by experimental data on the absence of interaction between GAPDS and antibodies 6C5 recognizing the NES motif in the sequence of GAPD. The lack of NES correlates with functional peculiarities of the sperm-specific enzyme that is tightly bound to the fibrous sheath of the sperm flagellum. The sequences of the two isoenzymes were examined for the short motifs that might participate in apoptosis, endocytosis, and DNA repair. Sites of phosphorylation by different protein kinases have been revealed in both isoenzymes, and their characteristic features are discussed. These observations can serve as the basis for subsequent search for new ways of regulating the two isoenzymes.     

Demonstration of a heterogeneous distribution of glycolytic enzymes and of pyruvate kinase isoenzymes types M1 and M2 in unfertilized hen eggs

The intracellular distribution of the glycolytic enzymes hexokinase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase and the pyruvate kinase isoenzymes type M1 and type M2 within unfertilized hen eggs was studied. Most of glycolytic enzyme activities were found in the yolk fraction; 8-24% of total glycolytic enzyme activities were found in the vitelline membrane fraction. However, the specific activities of these enzymes in the vitelline membrane fraction are 19-72-fold higher (U/mg protein) and 45-178-fold more concentrated (U/g wet weight) than in the yolk fraction. The study of intracellular localization of pyruvate kinase isoenzymes shows that the blastodisc, latebra and vitelline membrane contain only pyruvate kinase type M2, whereas pyruvate kinase types M1 and M2 are found in the egg yolk. The exclusive occurrence of pyruvate kinase type M2 in the blastodisc is consistent with the concept that this isoenzyme is involved in the cell proliferation. The heterogeneous distribution of the glycolytic enzymes hexokinase, glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase, and the heterogeneous localization of the pyruvate kinase isoenzymes types M1 and M2 indicate that glycolysis is distributed heterogeneously within the unfertilized hen egg cell.     

Structure, function and tissue-specific gene expression of 3β-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase enzymes in classical and peripheral intracrine steroidogenic tissues

The membrane-bound enzyme 3β-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase (3β-HSD) catalyses an essential step in the transformation of all 5-pregnen-3β-ol and 5-androsten-3β-ol steroids into the corresponding 3-keto-4-ene-steroids, namely progesterone as well as all the precursors of androgens, estrogens, glucocorticoids and mineralocorticoids. We have recently characterized two types of human 3β-HSD cDNA clones and the corresponding genes which encode type I and II 3β-HSD isoenzymes of 372 and 371 amino acids, respectively, and share 93.5% homology. The human 3β-HSD genes containing 4 exons were assigned by in situ hybridization to the p11-p13 region of the short arm of chromosome 1. Human type I 3β-HSD is the almost exclusive mRNA species present in the placenta and skin while the human type II is the predominant mRNA species in the adrenals, ovaries and testes. The type I protein possesses higher 3β-HSD activity than type II. We elucidated the structures of three types of rat 3β-HSD cDNAs as well that of one type of 3β-HSD from bovine and macaque ovary λgt11 cDNA libraries, which all encode a 372 amino acid protein. The rat type I and II 3β-HSD proteins expressed in the adrenals, gonads and adipose tissue share 93.8% homology. Transient expression of human type I and II as well as rat type I and II 3β-HSD cDNAs in HeLa human cervical carcinoma cells reveals that 3β-ol dehydrogenase and 5-ene-4-ene isomerase activities reside within a single protein. These expressed 3β-HSD proteins convert 3β-hydroxy-5-ene-steroids into 3-keto-4-ene derivatives and catalyze the interconversion of 3β-hydroxy and 3-keto-5α-androstane steroids. By site-directed mutagenesis, we demonstrated that the lower activity of expressed rat type II compared to rat type I 3β-HSD is due to a change of four residues probably involved in a membrane-spanning domain. When homogenates from cells transfected with a plasmid vector containing rat type I 3β-HSD is incubated in the presence of dihydrotestosterone (DHT) using NAD? as co-factor, 5α-androstanedione was formed (A-dione), indicating an intrinsic androgenic 17β-hydroxysteroid dehydrogenase (17β-HSD) activity of this 3β-HSD. We cloned a third type of rat cDNA encoding a predicted type III 3β-HSD specifically expressed in the rat liver, which shares 80% similarity with the two other isoenzymes. Transient expression in human HeLa cells reveals that the type III isoenzyme does not display oxidative activity for the classical substrates of 3β-HSD. However, in common with the type I enzyme, it converts A-dione and DHT to the corresponding 3β-hydroxysteroids, thus showing an exclusive 3-ketosteroid reductase activity. When NADPH is used as co-factor, the affinity for DHT of the type III enzyme becomes 10-fold higher than that of the type I. Rat type III mRNA was below the detection limit in intact female liver. Following hypophysectomy, its concentration increased to 55% of the values measured in intact or hypophysectomized male rats, an increase which can be blocked by administration of ovine prolactin (oPRL). Treatment with oPRL for 10 days starting 15 days after hypophysectomy markedly decreased ovarian 3β-HSD mRNA accumulation accompanied by a similar decrease in 3β-HSD activity and protein levels. Treatment with the gonadotropin hCG reversed the potent inhibitory effect of oPRL on these parameters and stimulated 3β-HSD mRNA levels in ovarian interstitial cells. These data indicate that the presence of multiple 3β-HSD isoenzymes offers the possibility of tissue-specific expression and regulation of this enzymatic activity that plays an essential role in the biosynthesis of all hormonal steroids in classical as well as peripheral intracrine steroidogenic tissues.     

Ultrathin-layer microelectrophoretic determination of lactate dehydrogenase isoenzymes in corneal and conjunctival epithelium of the cow

In order to evaluate the impact of tissue oxygenation on the distribution pattern of lactate dehydrogenase isoenzymes, activities of the isoenzymes were measured in microdissected samples of bovine tissue. A highly sensitive ultrathin-layer electrophoretic technique was used to determine the distribution pattern of lactate dehydrogenase isoenzymes in basal, intermediate and superficial layers of the epithelium of central and peripheral cornea and in the epithelium of the bulbar conjunctiva. Measurements revealed almost homogeneous intraepithelial distribution patterns of lactate dehydrogenase isoenzymes in both tissues. In the cornea the lactate dehydrogenase isoenzymes 4 and 5, which are regarded to be specialized for anaerobic glucose metabolism, were found to predominate. In the well-oxygenated conjunctival epithelium most of the activity could be ascribed to the lactate dehydrogenase isoenzyme 3. In contrast to the isoenzymatic activities, total activity of lactate dehydrogenase was inhomogeneously distributed; maximum activities were found in the basal layer of corneal epithelium and in the intermediate layer of conjunctival epithelium. The results indicate that oxygen supply is relevant rather for the intraepithelial distribution of total enzyme activity than for the expression of lactate dehydrogenase isoenzymes.Parts of this study were presented as an inaugural dissertation to the Medical Faculty of the University of Basel by K. Krieger     

Affinity chromatography of lactate dehydrogenase on immobilized nucleotides

The interaction of two isoenzymes of lactate dehydrogenase from pig heart muscle (H(4)) and rabbit skeletal muscle (M(4)), with immobilized nucleotides was examined: the effects of pH and temperature on the binding of lactate dehydrogenase were studied with immobilized NAD(+) matrices. The influence of substrate, product and sulphite on the binding of heart muscle lactate dehydrogenase to immobilized NAD(+) was investigated. The interaction of both lactate dehydrogenase isoenzymes with immobilized pyridine and adenine nucleotides and their derivatives were measured. The effects of these parameters on the interaction of lactate dehydrogenase with immobilized nucleotides were correlated with the known kinetic and molecular properties of the enzymes in free solution.     

Parallel evolution of pairs of dehydrogenase isoenzymes

Summary Lactate dehydrogenase and glycerol 3-phosphate dehydrogenase are metabolically coupled by the anaerobic dismutation of glyceraldehyde 3-phosphate and by the NAD redox state. This causes the concentrations of lactate and glycerol 3-phosphate to accumulate proportionally during anaerobic muscle contraction; these concentrations are high relative to those in aerobic tissues such as liver. We show that the isoenzymes of lactate dehydrogenase and glycerol 3-phosphate dehydrogenase from chicken breast muscle haveKm values for lactate and glycerol 3-phosphate, respectively, that are 10-fold higher than theKm values measured for the lactate dehydrogenase and glycerol 3-phosphate dehydrogenase isoenzymes from chicken liver. The association of proportionally higherKm values with the potential for proportionally higher accumulation of substrates suggests that the isoenzymes of lactate dehydrogenase and glycerol 3-phosphate dehydrogenase from chicken muscle have evolved in parallel as a coupled metabolic unit distinct from the coupled isoenzymes in liver. The parallelism observed for the reduced substrates extends to the oxidized substrates, and to the coenzymes, NAD⁺ and NADH.     

Pyruvate shuttle in muscle cells: high-affinity pyruvate transport sites insensitive to trans-lactate efflux

The specificity of the transport mechanisms for pyruvate and lactate and their sensitivity to inhibitors were studied in L6 skeletal muscle cells. Trans- and cis-lactate effects on pyruvate transport kinetic parameters were examined. Pyruvate and lactate were transported by a multisite carrier system, i.e., by two families of sites, one with low affinity and high capacity (type I sites) and the other with high affinity and low capacity (type II). The multisite character of transport kinetics was not modified by either hydroxycinnamic acid (CIN) or p-chloromercuribenzylsulfonic acid (PCMBS), which exert different types of inhibition. The transport efficiency (TE) ratios of maximal velocity to the trans-activation dissociation constant (Kt) showed that lactate and pyruvate were preferentially transported by types I and II sites, respectively. The cis-lactate effect was observed with high Ki values for both sites. The trans-lactate effect on pyruvate transport occurred only on type I sites and exhibited an asymmetric interaction pattern (Kt of inward lactate > Kt of outward lactate). The inability of lactate to trans-stimulate type II sites suggests that intracellular lactate cannot recruit these sites. The high-affinity type II sites act as a specific pyruvate shuttle and constitute an essential relay for the intracellular lactate shuttle.     

Cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase activity in the somatic cells of the seminiferous tubules. I. Isoform analysis

In this work firstly are reported the chromatographic analysis of cAMP dependent protein kinases present in the cytosols obtained from rat Sertoli cells and peritubular cells. In both cell types two different isoenzymes have been detected, one eluting at 40-80 mM KC1 (type I) and a second one eluting at 150-200 mM KC1 (type II). Only the type I was strongly stimulated by cAMP whereas the type II was slightly cAMP dependent both in the Sertoli cells and in the peritubular cells.     

Physical and chemical properties of lactate dehydrogenase in Homarus americanus.

Two isoenzymes of lactate dehydrogenase have been purified from Homarus americanus: One is found predominantly in the tail muscles; the other, in the walking leg muscles. This is the first demonstration of multiple forms of l-specific lactate dehydrogenase in an invertebrate organism. These proteins contain four essential sulfhydryl groups titratable by p-hydroxymercuribenzoate and 5,5′-dithiobis(2-nitrobenzoic acid). The molecular weights of these isoenzymes are dependent upon ionic strength. The native tetramer (M_r 145,000) exists in low ionic strength solutions; the active dimer (M_r 75,000), in high ionic strength solutions; this is the only example of lactate dehydrogenase disaggregation without concomitant loss in enzymatic activity. Microcomplement fixation studies suggest that there may be less than 4% difference in the primary structures of these two proteins.     

Occurrence of two 3-hydroxyacyl-CoA dehydrogenases in rat liver.

3-Hydroxyacyl-CoA dehydrogenase was assayed for acetoacetyl pantetheine-reducing and acetoacetyl-CoA reducing activities in rat liver homegenates. Two isoenzymes of the enzyme, types I and II, were distinguished by the following procedures: trypsin treatment, heat treatment, CM-cellulose chromatography, antibody titration, and sucrose density gradient centrifugation of the light mitochondrial fraction. Type I enzyme was localized in mitochondria, and catalyzed the reduction of both acetoacetyl pantetheine and acetoacetyl-CoA. Type II enzyme was found mainly in peroxisomes, accompanied by a low activity in mitochondria or some other organelles, and was active with acetoacetyl-CoA but not with aceto acetylpantetheine. Both isozymes were induced by the administration to the rats of di-(2-ethylhexyl)phthalate, which enhances the peroxisomal beta-oxidation activity, but the extent of the induction of type II enzyme was much higher than that of type I enzyme. The activity of the former was found only in diethylhexylphthalate-treated rats.     

Limited proteolysis of bovine muscle and heart lactate dehydrogenase is inhibited by phospholipid liposome interaction

Limited proteolysis of phospholipid complexes of heart and muscle bovine lactate dehydrogenase by trypsin and chymotrypsin has been studied under nondenaturing condition at pH 7.5. Chymotrypsin cleaves the polypeptide chain of heart and muscle lactate dehydrogenase into two principal fragments and LDH subunits were protected by lipids towards the proteinase attack. Enzymatic activity of heart and muscle lactate dehydrogenase was abolished by limited proteolytic cleavage. In complexes, both isoenzymes were protected against proteinases attack by lipids.     

Comparative analysis of the malate dehydrogenases isolated from the cytosolic fraction of several tissues of guinea-pig Cavia porcellus

The specific activities of the malate dehydrogenase and lactate dehydrogenase present in the soluble fraction of several guinea-pig tissues are reported. The electrophoretic patterns showed always two forms (A and B) with malate dehydrogenase activity and the five isoenzymes of lactate dehydrogenase. Chromatography of the different soluble fractions through 5' AMP-Sepharose allowed both molecular forms of malate dehydrogenase to be separated and obtained free from lactate dehydrogenase. Comparative studies of the two forms of malate dehydrogenase evidenced that the A and B forms exhibited cytosolic and mitochondrial characteristics, respectively.     

Targeted disruption of the inosine 5'-monophosphate dehydrogenase type I gene in mice

Inosine 5'-monophosphate dehydrogenase (IMPDH) is the critical, rate-limiting enzyme in the de novo biosynthesis pathway for guanine nucleotides. Two separate isoenzymes, designated IMPDH types I and II, contribute to IMPDH activity. An additional pathway salvages guanine through the activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) to supply the cell with guanine nucleotides. In order to better understand the relative contributions of IMPDH types I and II and HPRT to normal biological function, a mouse deficient in IMPDH type I was generated by standard gene-targeting techniques and bred to mice deficient in HPRT or heterozygous for IMPDH type II. T-cell activation in response to anti-CD3 plus anti-CD28 antibodies was significantly impaired in both single- and double-knockout mice, whereas a more general inhibition of proliferation in response to other T- and B-cell mitogens was observed only in mice deficient in both enzymes. In addition, IMPDH type I(-/-) HPRT(-/0) splenocytes showed reduced interleukin-4 production and impaired cytolytic activity after antibody activation, indicating an important role for guanine salvage in supplementing the de novo synthesis of guanine nucleotides. We conclude that both IMPDH and HPRT activities contribute to normal T-lymphocyte activation and function.