Properties of the testicular lactate dehydrogenase isoenzyme.

1. Studies were carried out with pure lactate dehydrogenase isoenzymes C4 (LDH isoenzyme X), B4, (LDH isoenzyme 1) and A4 (LDH isoenzyme 5) isolated from mouse testis, heart and muscle tissue respectively; with LDH isoenzyme X purified from pigeon testes and with crude lysates of spermatozoa from man, bull and rabbit. 2. LDH isoenzyme X from all species showed greater ability than the other isoenzymes to catalyse the NAD+-linked interconversions of 2-oxobutanoate into 2-hydroxybutanoate and of 2-oxopentanoate into 2-hydroxypentanoate. 3. Mouse LDH isoenzyme X presented the broadest spectrum of substrate specificity. It exhibited very similar Km values for a variety of 2-oxo acids: 2-oxopropanoate (pyruvate), 2-oxobutanoate, 2-oxo-3-methylbutanoate, 2-oxopentanoate, 2-oxo-3-methylpentanoate, 2-oxo-4-methylpentanoate, 2-oxohexanoate and 2-oxo-3-phenylpropanoate (phenylpyruvate). The corresponding 2-hydroxy acids were also readily utilized in the reverse reaction. A strong inhibition by substrate and product was demonstrated for the direct reaction. 4. Intracellular distribution of LDH isoenzyme X was investigated in mouse testes. LDH isoenzyme X activity was located in the fraction of "heavy mitochondria" and in the soluble phase. 5. A possible functional role for LDH isoenzyme X is proposed: the redox couple-2-oxo acid-2-hydroxy acid could integrate a shuttle system transferring reducing equivalents from cytoplasm to mitochondria.     

Changes in cytosolic Ca2+ levels correspond to fluctuations of lactate levels in crosstalk of astrocyte-neuron cell lines

Neurons and astrocytes differentially express isoenzymes of lactate dehydrogenase (LDH). The metabolic consequences for the variations in mRNA expression of LDH isoenzyme subtypes in neurons and astrocytes control cerebral vasoregulation. Moreover, cellular signalling consequences for functional neurovascular control may also be dependent on LDH isoenzyme subtype profiles. Initial computer simulations revealed glutamate-induced calcium waves in connected astrocytes, and showed concomitant changes in the expression of nitric oxide synthase (NOS) and lactic acid metabolism. To validate these findings, the nature and extent of glutamate-dependent signalling crosstalk in murine cell lines were investigated through correlated lactate levels and calcium upregulation. Neuro2A and C8D1A cells were separately treated with timed supernatant extracts from each other and their LDH1 and LDH5 isoenzyme responses were recorded. Western blot analysis showed LDH1/LDH5 isoenzyme ratio in the astrocytes to be positively correlated with Neuro2A-derived lactate levels estimated by the amplitude of 1.33-ppm spectral peak in 1H-NMR, and LDH1/LDH5 isoenzyme ratio in neurons is negatively correlated with CSD1A-derived lactate levels. Significant modulations of the calcium-responsive protein pCamKII levels were also observed in both cell lines, particularly correlations between pCamKII and lactate in C8D1A cells, thus explaining the calcium dependence of the lactate response. Together, these observations indicate that lactate is a key indicator of the metabolic state of these cell types, and may be a determinant of release of vasoregulatory factors.     

LDH and LDH Isoenzymes of Synovial Fluid in the Horse

LDH is an intracellular enzyme, which when cells degenerate is released to the extracellular spaces and body fluids. Cells and organs in the mammalian body differ from each other with respect to their LDH isoenzyme patterns. These circumstances have led to the use of LDH isoenzyme determinations in laboratory diagnostic work. In the present investigation total LDH activity and LDH isoenzyme distribution in equine synovial fluid from healthy joints, joints with serous arthritis, osteochondrosis dissecans and arthrosis, were determined. The fluids from the diseased joints differed from normal synovial fluid with respect to total LDH activity, and the different joint diseases each seemed to give rise to a characteristic isoenzyme pattern. In order to examine possible sources of the increased LDH activity and altered isoenzyme patterns, blood plasma, red and white blood cells, synovial membrane and articular cartilage were also studied. It was found that LDH4 and LDH5 were present in high amounts in articular cartilage, and an increase in these isoenzymes was the most characteristic feature in synovial fluid from joints with arthrosis. The results were discussed in view of possible diagnostic value of isoenzyme determinations on synovial fluid.     

LDH calcutta-1: A mutation of the B subunit of human lactate dehydrogenase

The electrophoretic variant of human LDH, Calcutta-1, occurs at phenotypic frequencies of 0–4% throughout India. The variant was examined by various electrophoretic techniques and by heat stability studies. The LD₁ (B₄) isoenzyme was purified from normal and variant bloods by affinity chromatography and ion-exchange chromatography. A minimum of five Calcutta-1 LD₁ bands was demonstrated by isoelectric focusing. Electrophoresis of variant LD₁ in high-molar urea-acrylamide denaturing gels resulted in two Calcutta-1 B subunit bands, while normal gels yielded only a single band. Homozygote Calcutta-1 LDH from red cells demonstrated a decreased heat stability, while heterozygote variant LDH showed a normal heat stability. This effect was confirmed when purified LD₁'s were compared. Evidence is presented suggesting a B-subunit variant showing thermolability in the homozygous form.The author was supported by an Australian National University Scholarship.     

幼年和成年小白鼠LDH与SOD动态变化的电泳分析

采用聚丙烯酰胺凝胶圆盘电泳分析方法,研究了幼年鼠和成年鼠睾丸乳酸脱氢酶（ＬＤＨ）同工酶与超氧化物歧化酶（ＳＯＤ）的酶带变化。观察结果表明,成年鼠睾丸的凝胶电泳胶条,多数显现六种ＬＤＨ同功酶带,其中ＬＤＨ５和ＬＤＨ４占优势,ＬＤＨ１、ＬＤＨ２、ＬＤＨ３显带较弱,在正极端ＬＤＨ的下面是第６条酶带,相当于Ｃ亚单位（ＬＤＨｃ）的Ｃ４。幼年鼠睾丸的凝胶电泳胶条,只显现ＬＤＨ１和ＬＤＨ２两条同功酶带,以ＬＤＨ     

Lactate dehydrogenase: inhibition of subunit A by the sulfhydryl reagent AgNO3.

Lactate dehydrogenase isoenzymes including the subunit A--LDH 5 (A4), LDH 4 (A3B), LDH 3 (A2B2), and LDH 2 (AB3)--are inhibitable by the sulfhydryl reagent AgNO3, while the isoenzyme LDH 1 (B4) is not.     

Comparative binding of lactate dehydrogenase to mitochondrial fractions

Beef liver mitochondrial fraction showed LDH activity (1.76 +/- 0.25 U/g pellet). Sixty seven% of the initial mitochondrial pellet LDH activity (almost M4 isoenzyme) was released when suspended in NaCl 0.15 M. When the washed particles were sonicated in a 0.15 M NaCl medium, the solubilized LDH activity (all five isoenzymes as cytosoluble fraction) was 5-fold higher than the initial pellet activity. The different isoenzymatic composition of intramitochondrial and externally bound forms of the enzyme should be taken into account when investigating the physiological role of intramitochondrial LDH. Beef liver cytosoluble LDH (very little content of M4 isoenzyme) showed no affinity for the beef liver mitochondrial fraction but purified M4-LDH isoenzyme was able to bind to the particulate fraction from the same source. This suggests an isoenzyme specificity for the interaction. The maximum amount of cytosoluble LDH bound to the mitochondrial fraction depends on the enzyme and the particulate fraction source. Therefore, binding capacity to the mitochondrial fraction depends not only on the net charge of LDH isoenzymes, which play a predominant role in the binding, but also on individual characteristics of the LDH isoenzymes and mitochondrial fractions from different sources. This suggests that electrostatic forces are not the only ones involved in the binding process.     

Lactate dehydrogenase activity and its isoenzyme spectrum in the human pancreas in the prenatal period

Lactate dehydrogenase (LDH) activity and the character of its isoenzyme distribution in pancreas of the human embryos and feti of the 5th-13th-week development were studied. It is shown that LDH activity was rather high already in early periods of the embryonic development, peaks of the enzymic activity were observed after 7-8 and 12-13 weeks. The isoenzymic LDH spectrum was characterized by the presence of four isoenzymes: LDH1, LDH2, LDH3, LDH5. Isoenzyme LDH4 was absent in the human pancreas in all the studied periods of embryonic development. The data obtained evidence for intensity of the glycolysis processes at LDH reaction level in the prenatal ontogenesis period and they reflect most probably the processes of development and differentiation ox cellular populations in the given organ.     

Detection of age-related fluorescent substances in rat tissues

Studies are reported on the detection of fluorescent substances and their relationship to the accumulation of the aging pigment and lactate dehydrogenase (LDH) isoenzyme composition in tissues of the rat. Comparison of fluorescent substances in nine tissues in 6- and 100-week-old rats by thin-layer chromatography showed that one substance in particular accumulated with age. This substance exhibited the typical excitation-fluorescence spectrum of lipofuscin pigments and its detection in tissues correlated closely with histologic analysis of the aging pigment. Accordingly, it was designated as an age-related fluorescent substance (ARFS). Because of the high intensity of its fluorescence and its relatively small mass, it appeared to be a constituent of aging pigment that was dissociated and extracted upon treatment of the tissue with chloroform-methanol. Histochemical analysis revealed the presence of both hemosiderin deposits and a golden brown pigment which was devoid of iron. These substances as well as the ARFS appeared to accumulate more in tissues which were classified generally as aerobic based on their LDH isoenzyme composition.     

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.     

Lactate dehydrogenase isoenzyme patterns as a method of pregnancy detection in cattle

The objective of this study was to evaluate serum lactate dehydrogenase isoenzyme patterns in open and pregnant Holstein and Hereford cows as a method of detecting pregnancy. Serum samples were collected from 26 Holstein and 13 Hereford cows and lactate dehydrogenase isoenzyme patterns were examined by electrophoresis and quantitated by scanning densitometry. Lactate dehydrogenase isoenzyme(4) and LDH(5) were found in higher concentration (P 相似文献   

Changes in lactate dehydrogenase isoenzyme patterns of human lymphocytes before and after blast formation

The lactate dehydrogenase isoenzyme pattern of human lymphocitic cells has been determined in several people before and after stimulation by mitogenic lectins at different times after the start of the culture. A very significant change take place in the LDH 5 which can reach a greater concentration towards the other isoenzymes at the 72 h from the mitogenic stimulus, even if it starts from a smaller concentration.     

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.     

Production of monoclonal antibodies to lactate dehydrogenase (LDH) isoenzymes for immunohistochemical study on fixed tissue section

Summary Purified lactate dehydrogenase (LDH) isoenzyme 1 (H or B subunits) and isoenzyme 5 (M or A subunits) were used to prepare monoclonal antibodies (MAb) suitable for immunohistochemical detection on formalin fixed paraffin-embedded tissue sections. In the initial fusions, screening of the antibodies was based on enzyme linked immunosorbent assay (ELISA) against the immunogens. None of the antibodies obtained was satisfactory. There were various problems related to specificity, crossreactivity, affinity and also the properties of the monoclonal antibody itself. Using a combined system involving more than one method for screening, two suitable monoclonal antibodies, MAb65 (to H-type LDH) and MAb25 (to M-type LDH) were selected. Both antibodies reacted specifically with corresponding LDH isoenzymes as shown in a series of tests. Their reactivity in sections of formalin fixed paraffin-embedded tissue indicated that both antibodies are suitable reagents for immunohistochemical studies.     

Serum Enzyme Activity of Newborn Calves,Pigs, and Lambs

Serum activities of aspartate aminotransferase (AspAT = GOT), alanine aminotransferase (AlAT = GPT), and total lactate dehydrogenase (LDH) have been investigated in newborn calves, pigs, and lambs. In the two latter species the LDH isoenzyme distribution in serum was also studied. Blood samples were taken at frequent intervals from birth to 48–72 hrs. post partum. Calves and pigs were born with very low serum enzyme values, whereas lambs showed a picture more similar to what has been reported in human infants. In all species a marked temporary enzyme increase occurred during the first 24–48 hrs. This elevation was found not to be due to colostrum feeding, since a parallel increase was found in starved animals. Possible regulating mechanisms are discussed. The LDH isoenzyme pattern proved to be more stable than total LDH in the early post-natal period. The percentage isoenzyme distribution, however, showed characteristic differences from that found in adult animals of the same species.     

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.     

Heat Stability of Serum Lactate Dehydrogenase and its Isoenzymes in Young and Adult Cattle and Sheep

The heat stability of lactate dehydrogenase (LDH) has been investigated in serum from young and adult cattle and sheep. The thermoresistance of the isoenzymes was determined by electrophoresis of serum samples preincubated at different temperatures. Marked differences were found in the percentage distribution of isoenzymes in serum from the two species as well as in the heat stability. LDH in serum from sheep was inactivated at a lower temperature than that in serum from cattle, and inactivation occurred at a lower temperature in young than in adult animals. The enzyme was in both species less tolerant to elevated temperatures than what is reported for human serum. Procedures worked out for a so-called relative heat stability test of LDH in human clinical diagnosis may therefore give misleading results if they were applied uncritically to sera from these animals. The LDH isoenzyme pattern of some main organs in calves and sheep indicates that a serum heat stability test may be useful in the diagnosis of skeletal muscle injuries in sheep. In cattle the tissue isoenzyme distribution is assumed to be too uniform to give information about specific organ lesions either by serum electrophoresis or by a heating technique. In contrast to what has been reported in man, serum levels of α-hydroxybutyrate dehydrogenase (HBD) in cattle and sheep, as earlier reported in swine, are found to be far better correlated to total LDH than to the most thermostable isoenzyme, LDH1.     

Lactate dehydrogenase isoenzymes in diabetic patients

Lactate dehydrogenase (LDH) isoenzyme profiles in human platelets and the sera of patients with type I and II diabetes mellitus and vascular complications, as well as normal subjects were measured utilizing a recently established, modified micromethod. LDH-3 was the predominating species in platelets (37.5 +/- 3.0%), with LDH-2, 1, 4 and 5 following in decreasing order of concentration. The LDH-3/LDH-4 ratio in platelets varied from 6.2 to 1.38. Type I and type II diabetic patients with vascular complications showed a significantly higher ratio for LDH-3/LDH-4 (3.99 +/- 1.20 for DM I, 2.16 +/- 0.25 for DM II patients) than the mean ratio for normal subjects (1.14 +/- 0.08). This platelet-specific LDH isoenzyme pattern may be the result of frequent in vivo platelet-vessel wall interactions in the diabetic patients whose platelets are known to be hyperaggregable in in vitro test systems. Since non-diabetic patients patients with vascular complications also displayed a similarly elevated LDH-3/LDH-4 ratio, a wider classification is preferable, although the measurement of the LDH isoenzyme pattern will be helpful in assessing diabetic vascular complications.     

Electrophoretic distribution and dissociation into subunits of lactate dehydrogenase derived from human myeloid leukemia cells before and after induction of differentiation

The electrophoretic distribution of lactate dehydrogenase (LDH) isoenzymes, Michaelis constant, reaction with substrate, and dissociation into subunits with guanidine hydrochloride was examined in undifferentiated and differentiated human myeloid leukemia cells. Differentiation was induced with 1/microgram/ml tunicamycin. Undifferentiated cells did not display phagocytic ability, and less than 5% of these cells had Fc receptors. After exposure to tunicamycin for 40 hr, 40% of these differentiated cells had Fc receptors, and 35% showed phagocytic activity after 160 hr. The majority of the LDH activity in the undifferentiated cells was found in fraction 3, and following differentiation almost a 50% reduction in LDH activity was observed in this fraction. In addition, LDH 3 isoenzyme levels were found to be greater in patients containing a high percentage of undifferentiated cells than in patients containing a high percentage of differentiated cells. Differentiated cells displayed LDH isoenzyme fraction pattern, Michaelis constant, and reaction with substrate similar to those found in the normal granulocytes. Differences in the dissociation of LDH into subunits with guanidine hydrochloride were found between undifferentiated and differentiated acute myeloid leukemia (AML) cells. Treatment with 0.75 M guanidine hydrochloride caused complete inactivation of LDH derived from normal differentiated cells, whereas similar treatment caused complete inactivation of LDH derived from AML or normal granulocytes. LDH isoenzymes derived from normal granulocytes and differentiated AML cells were also more sensitive to guanidine hydrochloride depression of fluorescence intensity. The sedimentation constant for single peak LDH at 5.5 M guanidine hydrochloride was calculated as 1.65 sec for differentiated and 1.70 sec for undifferentiated cells. The molecular weight of the polypeptide subunits for undifferentiated cells was 30,000 and for differentiated cells was 39,000. The apparent parallel between leukemic cells after induction of differentiation and normal granulocytes indicates that the leukemic cells retain their maturation potential when exposed to an inducer of differentiation.