臭鼩血清蛋白和六种组织乳酸脱氢酶同工酶的研究

本实验对臭鼩的血清蛋白及心肌、骨骼肌、肾脏、脾脏、肝脏,睾丸6种组织器官的乳酸脱氢酶(LDH)同工酶进行了聚丙烯酰胺凝胶盘状电泳的分析研究。臭鼩血清蛋白存在15—17条带,各组织的LDH同工酶均由5条带构成,其中心肌LDH-1、LDH-2和肾脏LDH-1各出现1条亚带。     

Lactate dehydrogenase isozymes in type I,IIA and IIB fibres of rabbit skeletal muscles

Summary Lactate dehydrogenase (LDH) isozyme patterns were analysed by polyacrylamide (PAA) slab gel electrophoresis in extracts prepared from various rabbit skeletal muscles of defined fibre composition and by PAA microelectrophoresis of microdissected, histochemically typed single muscle fibres. The results obtained by electrophoresis of whole muscle extracts generally agreed with the data obtained from single fibre electrophoresis, i.e. the LDH isozyme pattern corresponded to that of the predominant fibre type. Type I Fibres from soleus and semitendinosus muscles were characterized by a unique pattern of all 5 LDH isozymes with a predominance of LDH-1, 2 and 3. The major fraction (80%) of the type II fibres from extensor digitorum longus and tibialis anterior muscles contained only LDH-5 (M₄). About 20% of the type II fibres contained in addition to LDH-5 small amounts of LDH-4 and LDH-3. The fraction of fibres containing LDH-5, LDH-4, and LDH-3 was similar (ca. 20%) in the histochemically defined IIA and IIB subpopulations In view of the fact that the major fractions of rabbit IIB fibres display low and of IIA fibres high aerobic oxidative capacities (Reichmann and Pette 1982), these data indicate that the expression of the H-subunit of LDH is not correlated with the aerobic-oxidative capacity of the fibre. It also appears not to be correlated with the presence of different myosin isoforms in IIA and IIB fibres.     

Significance of the variation in isozymes of liver lactate dehydrogenase with thermal acclimation in goldfish--II. Effect of pH.

1. Effect of pH on liver lactate dehydrogenase (LDH) and its isozymes was examined in the goldfish acclimated to different temperatures and some purification of the LDH was attempted. 2. The optimal pH and the Km value at 30 degrees C of the enzyme were independent of acclimation temperature. 3. the optimal pH of isozyme was more basic in the order of LDH-1, LDH-2, LDH-3, LDH-4 and LDH-5. Km values of isozymes at 30 degrees C were higher in the order of LDH-1, LDH-3 and LDH-5. 4. There was no change in the enzyme activity during thermal acclimation.     

Purification and some properties of isozymes 1 and 5 of lactate dehydrogenase from fox heart and skeletal muscles]

An improved method is described for the isolation of isozymes 1 and 5 of lactate dehydrogenase (LDH) from heart and skeletal muscles of foxes. The method includes salt fractionation with ammonium sulphate, chromatography on DEAE- and CM-celluloses and affinity chromatography on AMP-Sepharose. The preparations of LDH isozymes 1 and 5 turned out to be homogeneous both in 7,5% polyacrylamide gel electrophoresis and under immunodiffusion analysis. It is shown that the pH optimum for LDH-1 is 10.2-10.4 for LDH-5 it is 9.5-9.6 in the case of the direct reaction, and the pH optimum is 7.6-7.8 for LDH-1 and 7.3-7.4 for LDH-5 in the case of reverse reaction. The values of Mikhaelis constants were determined for substrates and coenzymes in direct and reverse reactions. It is found that the excess of lactate and pyruvate causes substrate inhibition of both LDH-1 and LDH-5. The activities of LDH-1 and LDH-5 showed an unexpected similar sensitivity to the inhibitory effect of high pyruvate concentrations.     

Changes in myoglobin and lactate dehydrogenase in muscle tissues of a diving bird, the Pigeon Guillemot, during maturation

1. The concentration of myoglobin (Mb) and the isozymic distribution and activity of lactate dehydrogenase (LDH) in heart and pectoralis muscle were investigated at three stages of maturation of the Pigeon Guillemot, Cepphus columba. 2. Mb is not detectable in chick pectoralis; it is present in fledgling pectoralis muscle and increases four-fold in adult pectoralis. Mb concentration in heart muscle is similar in chick and fledgling and doubles in the adult. 3. LDH activities in pectoralis muscle of fledgling and adult increase to about three times that of the chick. LDH activities in heart of chick, fledgling and adult are similar to one another. 4. All five isozymes of LDH are present in both heart and pectoralis muscle at all stages; the heart muscle shows predominantly LDH-1 isozyme, and the pectoralis, LDH-5. The relative amounts of the five isozymes in the heart extract were constant during maturation but pectoralis LDH isozymes changed during maturation towards a more even distribution of the five isozymes in the adult. 5. Changes in Mb and LDH in the Pigeon Guillemot correlate with the animal's maturation from a sedentary nest sitter to an active diver and flyer. The adult pectoralis muscle probably has both aerobic function for wing-propelled short dives and flying and anaerobic capacity for longer dives.     

Genetic regulation of the lactate dehydrogenase isoenzyme spectrum in mouse erythrocytes

The pattern of lactate dehydrogenase isozymes was investigated by means of electrophoresis in erythrocytes of CBA/Lac and DBA/2J mice homozygous for b and a alleles of the Ldr-1 locus. It is found that differences in the pattern of LDH isozymes, homozygous for the genes Ldr-1a and Ldr-1b, consist in increased activity of the isozyme LDH-4 in mice homozygous for the gene Ldr-1a (DBA/2J) within 12-14 days of postnatal development. Inhibition of the reaction between 125I-LDH-1 and the respective antibodies has demonstrated that increased LDH-4 activity during development is related to the higher content of B-subunits of LDH. It is suggested that the mechanism of the action of the gene Ldr-1 involves changes in the rate of the synthesis and degradation of B-subunit of LDH.     

Lactate dehydrogenase isoenzymatic spectrum in preimplantation murine embryos

The spectrum of LDH isozymes was studied using different concentration of X-100 triton for enzyme extraction from the eggs and embryos, different systems of electrophoresis and different methods of staining electrophoregrams. Similar results were obtained in all cases. The mouse eggs and embryos were shown to contain only LDH-1; the treatment of oviducal fluid with X-100 triton strengthened the activity of LDH-4 and LDH-5. The detection of the activity of LDH-5 and hybrid isozymes appears to be related to the presence of oviducal fluid in the sample under study.     

华南兔组织乳酸脱氢酶同工酶的研究

本文以聚丙烯酰胺凝胶电泳法分析华南兔(Lepus sinensis sinensis)11种组织乳酸脱氢酶同工酶的分布待征。分析结果:骨骼肌和肝组织5条同工酶带俱全;脑、肺、卵巢和盲肠等组织各含有4条谱带(LDH-1,-2,-3和-4);胃和肾组织含有3条谱带(LDH-1,-2,和-3);眼晶状体和睾丸组织也含有3条谱带,但前者是LDH-3,-4和-5,后者是LDH-1,-2和-5;谱带最少的是心肌组织,只有2条(LDH-1和-2)。此外,还对各组织中的亚基活性分布及电泳图谱特征进行了分析。     

镉对蟾蜍的4种器官乳酸脱氢酶同工酶的影响

以腹腔注射法对蟾蜍(Bufo bufo gargarizans)给镉,处理一周后,观察了4种镉中毒浓度(0．1、0．2、0．4、0．8mg／kg)条件下的蟾蜍心、肝、肾和睾丸中乳酸脱氢酶(LDH)同工酶的变化。结果表明：随着镉中毒浓度的升高,心脏LDH同工酶的活性明显升高,睾丸LDH同工酶的活性明显下降,肝中的LDH1、LDH2、LDH3、LDH5在0．4、0．8mg／kg浓度组酶活性明显增加,而LDH4则明显减弱,肾中LDH1的活性随镉浓度的升高而明显升高,其它各酶带活性出现先增强而后又逐渐减弱的现象。结果提示了镉对蟾蜍主要器官LDH同工酶的影响具有组织差异性。     

Use of the fish enzyme system in monitoring water quality: effects of mercury on tissue enzymes

1. Rosy barb (Puntius conchonius) were exposed to 181 micrograms/l mercuric chloride for 48 h and the activity of acid and alkaline phosphatases (AcP and AIP), aspartate aminotransferase (AAT), alanine aminotransferase (AIAT), lactic dehydrogenase (LDH), and acetylcholinesterase (AchE) were measured in vivo in several organs. 2. The AcP activity was inhibited in the liver, gills, kidneys, and gut but stimulated in the gonads. With the exception of kidney, the AIP activity showed an increase in all the organs examined. The AAT and AIAT were generally inhibited in different organs. An increase in LDH activity occurred in the cardiac and skeletal muscles while the AchE activity was considerably lowered in the brain, gills, and liver. 3. In vitro exposure to mercury at concentrations ranging between 10(-10) and 10(-4) M, inhibited the AIP, AAT, AIAT, LDH, and AchE activities in the tissues examined. The AcP activity was also depressed in all the tissues except in the testes, in which a marginal increase was noted. 4. The in vivo and in vitro effects of Hg were not of similar quality implying sequestration of toxic cations in the intact animals.     

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.     

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 isoenzyme spectrum of fast and slow muscles with innervation disorders

A relative content of muscle fibers of various types and the spectrum of lactate dehydrogenase (LDH) isozymes were studied in fast-twitch (extensor digitorum longus) and slow-twitch (soleus) muscles of newborn rats, of those aged 2, 3 weeks and one month and of adult rats after neonatal sciatic denervation and application of 0.5 mM colchicine solution to the sciatic nerve. No muscle fibers of various types were found (from the level of succinate dehydrogenase activity) in one-month-old rats, whereas the control and fast-twitch muscles showed A, B and C types and the slow-twitch one B and C types. The denervation brought about an increase in the content of LDH4 and LDH5 in both the muscles, while colchicine application gave rise to an increase in LDH2 activity, diminution of LDH1 in the fast-twitch muscle and elevation of LDH4 in the slow-twitch one. The data obtained attest to the retardation of muscle differentiation under application of the colchicine-induced blockade of axoplasmic transport.     

UV-induced changes of structural and functional properties of blood lactate dehydrogenase isoenzymes in free state and in the presence of serotonin

Photoinduced changes of structural and functional properties of lactatedehydrogenase isoenzymes from human erythrocytes in free state and in the presence of serotonin have been studied by means of gel chromatography, electrophoresis, IR-spectrophotometry and by the method of definition of catalytic activity. UV-light influence induces photoinactivation of erythrocyte's LDH, while its inhibitory effect intensifies with the increase of irradiation dose. The complicated character of changes in electrophoretic mobility and percentage content of isoenzymes LDH-1, LDH-2, LDH-3 under the influence of UV-rays testifies that the decrease of total enzyme activity of these isoforms in connected with their different photosensitiveness and represents the result of many-staged process which is characterized both by the consistent and parallel proceeding of its individual photochemical reactions. A pronounced photoprotective effect of serotonin towards the molecules of erythrocytic LDH isoenzymes has been discovered. It seems to be caused by formation of enzyme--biogenous amine complex affecting the secondary protein structure.     

Lactate dehydrogenase in rat mitochondria

Small but persistent amounts of L-lactate dehydrogenase (LDH) activity were found in mitochondrial preparations isolated from rat heart, kidney, liver, and lymphocytes. Brain mitochondrial preparations were also isolated, but the results were inconclusive. A variety of cytosolic markers were used and it was found that essentially no cytosolic contamination was present except in brain preparations. A bacterial protease was used along with digitonin fractionation to determine localization of the mitochondrial LDH. Approximately 80% of the LDH activity associated with heart and kidney mitochondrial preparations was on the inside compared to about 40% for liver. Lymphocyte mitochondrial LDH activity was about 70% on the inside. Cytosolic LDH-5 preferentially adheres to outer mitochondrial membrane of liver, kidney, and heart. Agarose gel electrophoresis showed LDH isozymes in mitochondria qualitatively similar to that of the corresponding cytosol except in kidney mitochondrial preparations, where a specific electrophoretic band was found which did not correspond to any of the common LDH isozymes.     

Adaptations in metabolic capacity of rat soleus after paralysis.

To determine whether long-term reductions in neuromuscular activity result in alterations in metabolic capacity, the activities of oxidative, i.e., succinate dehydrogenase (SDH) and citrate synthase (CS), and glycolytic, i.e., alpha-glycerophosphate dehydrogenase (GPD), enzyme markers were quantified in rat soleus muscles 1, 3, and 6 mo after a complete spinal cord transection (ST). In addition, the proportional content of lactate dehydrogenase (LDH) isozymes was used as a marker for oxidative and glycolytic capacities. The myosin heavy chain (MHC) isoform content of a fiber served as a marker of phenotype. In general, MHC isoforms shifted from MHC1 toward MHC2, particularly MHC2x, after ST. Mean SDH and CS activities were higher in ST than control at all time points. The elevated SDH and CS activities were indicative of an enhanced oxidative capacity. GPD activities were higher in ST than control rats at all time points. The increase in activity of SDH was larger than GPD. Thus the GPD-to-SDH (glycolytic-to-oxidative) ratio was decreased after ST. Compared with controls, total LDH activity increased transiently, and the LDH isozyme profile shifted from LDH-1 toward LDH-5, indicative of an enhanced glycolytic capacity. Combined, these results indicate that 1) the metabolic capacities of soleus fibers were not compromised, but the interrelationships among oxidative and glycolytic capacity and MHC content were apparently dissociated after ST; 2) enhancements in oxidative and glycolytic enzyme activities are not mutually exclusive; and 3) chronic reductions in skeletal muscle activity do not necessarily result in a reduced oxidative capacity.     

Kinetic studies on the lactate dehydrogenase isozymes of the brown trout, Salmo trutta L

Informative crosses have verified the genetic basis of a polymorphism at the Ldh-1 locus in brown trout and enzyme activity measurements indicate that the previously described polymorphism at this locus is best explained by a null allele. The LDH-1, LDH-2, LDH-3 and LDH-4 homotetrameric isozymes were purified and subjected to enzyme kinetic analysis. While LDH-1 and LDH-2 displayed catalytic equivalence, important kinetic differences were found between the LDH-3 and LDH-4 isozymes.     

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.     

社鼠组织器官同工酶的研究

用聚丙烯酰胺凝胶等电聚焦电泳方法,分析了社鼠的肝、肾、心肌、骨骼肌、肺、脾和脑等多种组织器官的ＬＤＨ、ＡＤＨ、ＥＳＴ、和ＳＯＤ４种同工酶,对各组织器官的酶带数目和分布,以及酶活性进行了比较研究。结果表明,社鼠的ＬＤＨ同工酶、ＡＤＨ同工酶和ＥＳＴ同工酶具有比较明显的组织特异性,而ＳＯＤ同工酶的组织特异性较低。肺和脾除ＥＳＴ同工酶活性较高外,脑除ＬＤＨ同工酶活性较高外,其它３种同工酶的活性均较低;而肝和肾中４种同工酶的活性普遍很高。心肌和骨骼肌因氧张力不同而使ＬＤＨ同工酶酶谱存在明显差异,但其它３种同工酶酶谱却非常相似。同工酶的组织特异性与各组织器官所执行的生理功能是相一致的     

Expression of lactate dehydrogenase isozyme 5 (LDH-5) in cultured mouse blastocysts in the absence of implantation and outgrowth

Extensive extraction studies with Triton X-100 revealed only LDH-1 (B₄) but no trace of LDH-5 (A₄) in one-cell and two-cell mouse and rat embryos. The LDH isozyme pattern of preimplantation mouse embryos changes from the maternally inherited B subunit isozyme (LDH-1) to a pattern dominated by LDH-5 when mouse blastocysts are cultured under conditions that prevent hatching but allow trophoblast giant cell transformation. During differentiation of mouse blastocysts in vitro, implantation is therefore not essential for the appearance of the A subunit form of LDH (LDH-5) coded for by the embryonic genome. Mechanisms controlling the expression of LDH-5 in mouse blastocysts during in vivo development are discussed.This work was supported by grants of the Deutsche Forschungsgemeinschaft awarded to the Sonderforschungsbereich 29—Embryonale Entwicklung and Differenzierung.