Additional lactate dehydrogenase (LDH) isoenzymes in normal testis and spermatozoa of adult man

Summary Adult human testicular tissue contains up to six previously undescribed lactate dehydrogenase (LDH) isoenzymes in addition to the five LDH isoenzymes normally found and the sixth found in spermatogenic cells and spermatozoa, LDH-X. Additional LDH isoenzymes were also found in spermatozoa but not in seminal fluid or in serum. After electrophoresis one additional LDH isoenzyme of testicular tissue was localized between LDH-1 and LDH-2, two between LDH-2 and LDH-3, two between LDH-3 and LDH-4, and two between LDH-4 and LDH-5. These localizations indicate that the additional LDH isoenzymes are tetramers combining the A and B subunits of the five normal LDH isoenzymes and the C subunit of LDH-X. The additional LDH isoenzymes may be important in the metabolism of spermatogenic germ cells and spermatozoa.     

Changes in the lactate dehydrogenase isoenzyme spectrum during T-lymphocyte differentiation

The pattern of lactate dehydrogenase isoenzyme spectrum changes on different stages of T-lymphocyte differentiation was studied An enriched population of stem cells has LDH-5, 4 and 3 isoenzymes, and much less LDH-2 activity. The isoenzyme pattern of thymic cell precursors consists of LDH-5, 4, 3 and 2. All the five LDH isoenzymes were found in cortical thymocytes. Medullary thymocytes reveal LDH-5, 4 and 3 isoenzymes. T-lymphocytes of peripheral lymphoid organs contain mainly LDH-5 and in a lesser degree LDH-4 activity.     

Peripherin Is Tyrosine-Phosphorylated at Its Carboxyl-Terminal Tyrosine

Abstract: Peripherin is a type III intermediate filament present in peripheral and certain CNS neurons. We report here that peripherin contains a phosphotyrosine residue and, as such, is the only identified intermediate filament protein known to be modified in this manner. Antiserum specific for phosphotyrosine recognizes peripherin present in PC12 cells (with or without nerve growth factor treatment) and in rat sciatic nerve as well as that expressed in Sf-9 cells and SW-13 cl. 2 vim⁻ cells. The identity of peripherin as a tyrosine-phosphorylated protein in PC12 cells was confirmed by immunoprecipitation, two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and phosphoamino acid analysis. Unlike serine/threonine phosphorylation, tyrosine phosphorylation of peripherin is not regulated by depolarization or nerve growth factor treatment. To identify the site of tyrosine phosphorylation, rat peripherin was mutated at several tyrosine residues and expressed in SW-13 cl. 2 vim⁻ cells. Tyrosine phosphorylation was selectively lost only for peripherin mutants in which the carboxy-terminal tyrosine (Y474) was mutated. Indirect immunofluorescence staining indicated that both wild-type peripherin and peripherin Y474F form a filamentous network in SW-13 cl. 2 vim⁻ cells. This indicates that tyrosine phosphorylation of the peripherin C-terminal residue is not required for assembly and leaves open the possibility that this modification serves other functions.     

Selective inactivation of lactate dehydrogenase of rat tissues by sodium deoxycholate.

Soluble lactate dehydrogenase (EC 1.1.1.27) extracted from brain, skeletal and cardiac muscle and liver of rats, and purified isoenzymes LDH-1 and LDH-5, were incubated with sodium deoxycholate. Deoxycholate almost totally inactivated isoenzyme LDH-5 (A4), whereas it left isoenzyme LDH-1 (B4) unaffected. Tissue lactate dehydrogenase was inactivated to different degrees depending on the origin of the enzyme. Electrophoretic isoenzyme studies of tissue lactate dehydrogenase showed the loss of activity to be quantitatively related to the overall percentage of subunit A distributed among the homotetramer LDH-5 and the heterotetramers LDH-2, LDH-3 and LDH-4. It was concluded that subunit A of lactate dehydrogenase interacts selectively with deoxycholate, irrespective of its association with subunit B. Distinct changes in electrophoretic mobilities of deoxycholate-treated isoenzymes strongly indicated an indiscriminate binding of deoxycholate by all LDH isoenzymes, probably through hydrophobic interactions. The results suggest that the inactivation of the enzyme is non-competitive, but the basis of the selectivity of deoxycholate towards subunit A is not known at present.     

Energy enzymes of the flight muscle of the house sparrow, Passer domesticus--III. Comparative physical properties of heart and flight muscle lactate dehydrogenase

Purification of heart (LDH-4) and flight muscle (LDH-2 and LDH-3) lactate dehydrogenase isoenzymes from the house sparrow, Passer domesticus, has been accomplished. Although these isoenzymes electrophoretically migrate reversed to most other vertebrate LDH isoenzymes, comparison of the amino acid compositions of LDH-4 and LDH-2-LDH-3 fails to reveal the basis for their reversed electrophoretic migration. Amino acid compositions did reveal mol. wts between 141,000-142,000 as well as vp of 0.744 ml/g (LDH-4) and 0.745 ml/g (LDH-2-LDH-3). SDS-gel electrophoresis yielded single bands for each preparation with mol. wts of 35,000 suggesting that LDH in this species exists as a tetramer. LDH-4 has a lower Km for both pyruvate (0.005 mM) and NADH (0.002 mM) than does LDH-2-LDH-3 (0.062 mM for pyruvate, 0.013 mM for NADH).     

Cytoplasmic retention of protein tyrosine kinase 6 promotes growth of prostate tumor cells

Protein tyrosine kinase 6 (PTK6) is an intracellular tyrosine kinase that is nuclear in epithelial cells of the normal prostate, but cytoplasmic in prostate tumors and in the PC3 prostate tumor cell line. The impact of altered PTK6 intracellular localization in prostate tumor cells has not been extensively explored. Knockdown of endogenous cytoplasmic PTK6 resulted in decreased PC3 cell proliferation and colony formation, suggesting that cytoplasmic PTK6 stimulates oncogenic pathways. In contrast, reintroduction of PTK6 into nuclei of PC3 cells had a negative effect on growth. Enhanced tyrosine phosphorylation of the PTK6 substrate Sam68 was detected in cells expressing nuclear-targeted PTK6. We found that mechanisms regulating nuclear localization of PTK6 are intact in PC3 cells. Transiently over-expressed PTK6 readily enters the nucleus. Ectopic expression of ALT-PTK6, a catalytically inactive splice variant of PTK6, did not affect localization of endogenous PTK6 in PC3 cells. Using leptomycin B, we confirmed that cytoplasmic localization of endogenous PTK6 is not due to CRM-1/exportin-1 mediated nuclear export. In addition, over-expression of the PTK6 nuclear substrate Sam68 is not sufficient to bring PTK6 into the nucleus. While exogenous PTK6 was readily detected in the nucleus when transiently expressed at high levels, low-level expression of inducible wild type PTK6 in stable cell lines resulted in its cytoplasmic retention. Our results suggest that retention of PTK6 in the cytoplasm of prostate cancer cells disrupts its ability to regulate nuclear substrates and leads to aberrant growth. In prostate cancer, restoring PTK6 nuclear localization may have therapeutic advantages.     

大豆黄酮对衰老小鼠脑组织SOD、LDH同工酶的影响

利用SOD和LDH同工酶电泳分析,研究大豆黄酮对衰老小鼠的抗氧化作用。结果显示大豆黄酮没有改变SOD和LDH同工酶谱的特征,但对因衰老引起的小鼠脑组织LDH和SOD同工酶活性、各组分的相对活性和比活力的变化有不同程度的改善作用,即LDH同工酶中LDH-2、LDH-3的活性明显下降,LDH-1的活性下降最为明显,而LDH-4的活性有所下降,但不显著,LDH-5的活性几乎没有变化,SOD同工酶的SOD-1和SOD-2的活性有不同程度的升高。这表明大豆黄酮是通过抑制LDH同工酶H亚基的合成来降低LDH的活性,而对M亚基的合成没有影响,并且能够促进SOD同工酶SOD-1和SOD-2的合成,不影响其遗传稳定性。     

Lactate dehydrogenase isoenzymes in the larvae of Barathra brassicae and Galleria mellonella during microsporidan infection

Changes in the activities of lactate dehydrogenase isoenzymes in the gut and fat body of Galleria mellonella and Barathra brassicae larvac infected by the microsporidans Nosema plodiae and Pleistophora schubergi were studied by means of dise electrophoresis. In the normal last instar G. mellonella gut and fat body three isoenzymes, LDH-1, LDH-2-3, and LDH-4, and in B. brassicae two isoenzymes, LDH-1 and LDH-2-3, were present. In the fat body of both the animals infected by N. plodiae, the isoenzyme LDH-2-3 increased in activity substantially by the fifth day of infection. The gut LDH isoenzymes were not affected by the microsporidan. The same LDH-2-3 effect could be provoked by some enzymes toxic for G. mellonella larvae such as phospholipase-C and protease preparations.     

Cytoplasmic retention of protein tyrosine kinase 6 promotes growth of prostate tumor cells

Protein tyrosine kinase 6 (PTK6) is an intracellular tyrosine kinase that is nuclear in epithelial cells of the normal prostate, but cytoplasmic in prostate tumors and in the PC3 prostate tumor cell line. The impact of altered PTK6 intracellular localization in prostate tumor cells has not been extensively explored. Knockdown of endogenous cytoplasmic PTK6 resulted in decreased PC3 cell proliferation and colony formation, suggesting that cytoplasmic PTK6 stimulates oncogenic pathways. In contrast, reintroduction of PTK6 into nuclei of PC3 cells had a negative effect on growth. Enhanced tyrosine phosphorylation of the PTK6 substrate Sam68 was detected in cells expressing nuclear-targeted PTK6. We found that mechanisms regulating nuclear localization of PTK6 are intact in PC3 cells. Transiently overexpressed PTK6 readily enters the nucleus. Ectopic expression of ALT-PTK6, a catalytically inactive splice variant of PTK6, did not affect localization of endogenous PTK6 in PC3 cells. Using leptomycin B, we confirmed that cytoplasmic localization of endogenous PTK6 is not due to Crm-1/exportin-1 mediated nuclear export. In addition, overexpression of the PTK6 nuclear substrate Sam68 is not sufficient to bring PTK6 into the nucleus. While exogenous PTK6 was readily detected in the nucleus when transiently expressed at high levels, low-level expression of inducible wild type PTK6 in stable cell lines resulted in its cytoplasmic retention. Our results suggest that retention of PTK6 in the cytoplasm of prostate cancer cells disrupts its ability to regulate nuclear substrates and leads to aberrant growth. In prostate cancer, restoring PTK6 nuclear localization may have therapeutic advantages.Key words: PTK6, BRK, Sik, SAM68, prostate cancer, nuclear localization, tyrosine kinase     

Glycolytic enzymes in polyamine-treated bovine retina

The retina is characterized by glycolysis under aerobic conditions, mediated by lactate dehydrogenase isoenzyme-5 (LDH-5) as well as by the soluble isoenzyme of malate dehydrogenase. Bovine retina LDH and MDH isoenzymes and their activities were studied after polyamine treatment. Our results showed that LDH-5 isoenzyme presented the highest activity in untreated as well as in putrescine-treated retina. Decreased activity was present when the retina was treated with spermidine or spermine. It was demonstrated that retinic LDH-5 had a high affinity for lactate which enabled the isoenzyme to be more effective than the other LDH isoenzymes in the conversion of NADH to NAD. Therefore, the putrescine enhancing LDH-5 activity appeared to be capable of stimulating NAD-mediated rhodopsin regeneration. Putrescine induced a marked increase of both MDH isoenzymes--soluble (s-MDH) and mitochondrial (m-MDH), while spermine and spermidine mostly affected the soluble form of the enzyme. Putrescine induced a three-fold increase in s-MDH and m-MDH activities, while spermine and spermidine induced a four to five-fold increase in s-MDH. These results document the differential effects of polyamine treatment on LDH and MDH isoenzyme activities.     

L.D.H. and L.D.H. isoenzymes of the intra-uterine secretions of the cow during the estrous cycle

Uterine secretions were collected from 20 mature cows during estrus (day 0), metestrus (day 5), diestrus (day 10) and proestrus (day-1). Lactate dehydrogenase (LDH) and LDH isoenzymes activity were evaluated. No significant cyclic variations of LDH activity was found in the uterine secretions while the mean of the enzyme activity was higher during the estrogenic period of the cycle. The relative activity of LDH-1, LDH-2 and LDH-3 isoenzymes were higher during proestrus and estrus whereas LDH-5 activity was more important during metestrus. The LDH-3 seems to have the higher relative activity in uterine secretions of the cow.     

Tyrosine phosphorylation translocates beta-catenin from cell-->cell interface to the cytoplasm, but does not significantly enhance the LEF-1-dependent transactivating function

beta-catenin plays an essential role in cells, not only as a cadherin-associated complex, but also as a signaling molecule in the nucleus. Tyrosine phosphorylation of beta-catenin has been shown to correlate with tumorigenesis, cell migration, and developmental processes. However, its exact effects on downstream targets in the nucleus are not yet clear. In this study, we used HCT-15 colon carcinoma and NIH 3T3 fibroblasts as models to investigate the effects of a phosphotyrosine phosphatase (PTPase) inhibitor on the localization of beta-catenin, the binding affinity to LEF-1 (Lymphoid Enhancer Factor), and on LEF-1-dependent transactivation function. Treatment with a PTPase inhibitor, pervanadate, increased the tyrosine phosphorylation of beta-catenin in a time-dependent manner and led to its relocation from cell-cell interfaces to the cytoplasm. This phosphorylation/dephosphorylation of beta-catenin does not require its presence at cell-cell interfaces. However, tyrosine phosphorylation of beta-catenin does not change its binding affinity to LEF-1 nor enhance cyclin D1 transactivation, a nuclear target of beta-catenin/LEF-1. This result suggests that tyrosine phosphorylation of beta-catenin has effects on the binding to cadherins in the cytoplasm but not on its LEF-1-dependent transactivating function in the nucleus.     

Correlation among clinicopathological parameters of myocardial damage in rats treated with isoproterenol.

The correlation among clinicopathological parameters of myocardial damage was investigated in rats administered a single subcutaneous dose of isoproterenol at 0 (saline), 0.04, 0.4 and 4 mg/kg. Total lactate dehydrogenase (LDH), creatine kinase (CK), and their isoenzymes (LDH-1, LDH-2 and CK-MB), as well as troponin I and tropinin T, were measured 4 h after the administration of the drug. Troponin I was determined by a chemiluminescence method using Bayer Centaur and DPC Immulyze, as well as by ELISA. Troponin T was assayed semi-quantitatively using Trop T Sensitive. A high correlation was found among LDH isoenzymes, troponin I (Centaur) and troponin T. The present result provides a baseline for interpreting changes in the different parameters of myocardial damage assayed by different methods in toxicity studies.     

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.     

Adapter protein SH2-B beta undergoes nucleocytoplasmic shuttling: implications for nerve growth factor induction of neuronal differentiation

The adapter protein SH2-B has been shown to bind to activated nerve growth factor (NGF) receptor TrkA and has been implicated in NGF-induced neuronal differentiation and the survival of sympathetic neurons. However, the mechanism by which SH2-B enhances and maintains neurite outgrowth is unclear. We examined the ability of truncation mutants to regulate neuronal differentiation and observed that certain truncation mutants localized in the nucleus rather than in the cytoplasm or at the plasma membrane as reported for wild-type SH2-B beta. Addition of the nuclear export inhibitor leptomycin B caused both overexpressed wild-type and endogenous SH2-B beta to accumulate in the nucleus of both PC12 cells and COS-7 cells as did deletion of a putative nuclear export sequence (amino acids 224 to 233) or mutation of two critical lysines in that sequence. Deleting or mutating the nuclear export signal caused SH2-B beta to lose its ability to enhance NGF-induced differentiation of PC12 cells. Neither the NGF-induced phosphorylation of ERKs 1 and 2 nor their subcellular distribution was altered in PC12 cells stably expressing the nuclear export-defective SH2-B beta(L231A, L233A). These data provide strong evidence that SH2-B beta shuttles constitutively between the nucleus and cytoplasm. However, SH2-B beta needs continuous access to the cytoplasm and/or plasma membrane to participate in NGF-induced neurite outgrowth. These data also suggest that the stimulatory effect of SH2-B beta on NGF-induced neurite outgrowth of PC12 cells is either downstream of ERKs or via some other pathway yet to be identified.     

Lactate dehydrogenase isoenzyme study of Trichinella larvae of different ages

LDH isoenzymes of Trichinella larvae isolated from experimentally infected guinea pigs were investigated between days 30 and 400 inclusive after infection. It was found that on the 30 th day after infection the larvae have two isoenzymes while from the 40th to the 400 th day--three isoenzymes. The first of the latter is of fast responce and is located in the anod area near the indicator while the two others are of slow response and are located between the isoenzymes LDH-3 and LDH-5 of the muscular tissue of guinea pigs. This indicates that energetic metabolism of Trichinella larvae proceeds in muscular tissues by aerobic and anaerobic way. Since electrophoretic specificity of LDH-isoenzymes of Trichinella larvae differ from that of LDH-isoenzymes of muscular tissue the former are apparently characteristic of Trichinella larvae.     

Lactate dehydrogenase isoenzymatic analysis of murine lymphocyte populations: a parameter of cellular differentiation.

The lactate dehydrogenase isoenzyme pattern has been determined in different murine lymphocytic cell populations. In each cell population, the LDH activity was predominantly found in the LDH-4 and LDH-5 fractions. The percentage LDH-5 activity was significantly higher in B cells than in T cells. The same is true for lymphocytes from the spleen versus lymph node lymphocytes. The percentage LDH-5 activity is significantly higher in peripheral T lymphocytes than in thymocytes. Enrichment of the more mature thymocytes of the thymocyte cell pool by either cortisone treatment in vivo or gradient centrifugation on bovine serum albumin (BSA) results in a decrease of LDH-1 and LDH-2 fractions. In the cortisone-treated group, the shift in the LDH pattern is accompanied by a significant increase of LDH-5 and LDH-4 fractions, whereas in the BSA group only the LDH-4 fraction increases.     

Molecular hybridization of native and modified subunits of lactate dehydrogenase isoenzymes and aldolase A in rats]

Experimental conditions for the molecular hybridization in vitro between iodine and native subunits of isoenzymes 1 and 5 of lactate dehydrogenase (LDH) are described. It is also shown that the covalently fixed on the polyacrylamide beads rat J125 labelled LDH-5 and J125 labelled aldolase A, under conditions of complete dissociation of the quaternary structure of these enzymes, only one of the four subunits remain bound with the beads. Subunit of LDH-5, which is covalently bound with the polyacrylamide beads, is capable to hybridize (reassociated) with 3 native subunits. In addition, the immobilized LDH-5 subunits and aldolase A are capable to hybridize with J125 labelled subunits of these enzymes. Thus, when thyrosine, lysine and N-terminal amino acids are modified, subunits of LDH-5 and aldolase A retain their capacity to restore their quaternary structures.