A study on lactate dehydrogenase isozymes in rat ova

The presence of subunits A and B has been demonstrated in mature rat ova by several means, including the immunohistochemical method of Coons, use of antisera against LDH-1 (B₄) and LDH-5 (A₄) isozymes, and polyacrylamide gel electrophoresis.     

Structural relatedness of mouse lactate dehydrogenase isozymes,A4 (muscle), B4 (heart), and C4 (testis)

Three homotetrameric lactate dehydrogenase isozymes, LDH-M(A₄), LDH-H(B₄), and LDH-X(C₄), from DBA/2J mice have been purified by affinity chromatography. The amino acid compositions of the subunits A, B, and C, based on a molecular weight of 36,000, have been determined. The compositional relatedness of these isozymes indicates that subunits A (muscle) and B (heart) are more closely related to each other than to subunit C (testis). Tryptic peptide maps and amino acid compositions of some active site peptides appear to confirm the compositional relatedness among these isozymes. The sequence of the loop region of mouse C subunit seems to be markedly different from all known A and B sequences, and the structural and functional implications are discussed.     

山蝠和黑斑蛙乳酸脱氨酶同工酶及血糖浓度的季节变化

本工作采用聚丙烯酸胺凝胶电泳等方法,对山蝠（Ｎｙｃｔｕｓｎｏｃｔｕｌａ）和黑斑蛙（Ｒａｎａｎｉｇｒｏｍａｃｕｌａｔａ）在冬眠或蛰伏期与活动期的血清、脑、骨胳肌中的乳酸脱氢酶（ｌａｃｔａｔｅｄｅｈｙｄｒｏｇｅｎａｓｅ,ＬＤＨ）同工酶和血糖浓度进行了比较研究。ＬＤＨ同工酶及血糖浓度表现出明显的季节变化。与活动期的动物相比,在冬眠或蛰伏期中的动物的ＬＤＨ１缺乏、ＬＤＨ５明显增多（Ｐ＜０．００１）,血糖浓度明显升高（Ｐ＜０．００１）。此外,ＬＤＨ同工酶和血糖浓度的季节变化在冬眠动物和蛰伏动物之间还存在着明显的差异。这些结果提示,ＬＤＨ同工酶含量和血糖浓度的季节变化不仅与动物的能量代谢水平相适应,而且显示了冬眠和蛰伏两种生理过程中的可能差异。     

Isozyme Patterns of Lactate Dehydrogenase from Tissues of Mink and Arctic Fox during Postnatal Development

Isozymes of lactate dehydrogenase extracted from heart, kidney, and liver of mink (Mustela vison Briss.) and Arctic fox (Alopex lagopus L.) during postnatal development were separated by agarose gel electrophoresis. Tissue-specific isozyme pattern of lactate dehydrogenase can be revealed at the age of one month, while the definitive pattern is formed at the age of four months. The isozyme patterns of lactate dehydrogenase in the studied tissues of mink and Arctic fox share the properties specific for animal species of various ecogenesis.     

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.     

Electrophoretic variation of supernatant malate dehydrogenase in marsupials

Starch gel electrophoresis of supernatant malate dehydrogenase (MDH A₂) was performed on erythrocyte samples from 505 individual animals representative of 33 marsupial species. Most species exhibited electrophoretically identical forms of MDH A₂ activity with the exception of the grey kangaroos, Trichosurus possums, and bandicoots, thus confirming the phylogenetic relatedness of animals within each group and the conservative nature of this enzyme. Polymorphisms were observed in two of the six species analyzed whose mobilities were non-standard. Allelic isozyme patterns and those from interspecies F₁ hybrids between grey kangaroos and other macropods were consistent with a dimeric subunit structure and an autosomal locus (MDH-A) encoding the enzyme.Supported in part by grants from the Australian Research Grants Committee.     

Regulation of the expression of lactate dehydrogenase isozymes in human lymphocytes

Mitogen activation of human peripheral lymphocytes leads to a switch in the isozymes of LDH; resting cells contain low activities of only the B₄ and B₃A forms, whereas activated cells contain high activities of the A₄ and A₃B forms. B₄ LDH is not altered in activated cells. In this study we show that the appearance of the A subunits occurs concomitantly with a several fold increase in the steady state levels of LDH-A mRNA. Responses in LDH-A mRNA are observed within 12 hrs of activation, and are, thus, associated with the G0/G1 transition or with early G1 (Marjanovicet al. Exp. Cell Res. (1991) 193: 425–431). Maximal expression of LDH-A mRNA requires both phorbol ester and concanavalin A, implying a complex regulatory pathway involving cascade systems activated through both the antigen receptor (TR) and protein kinase C.     

Use of lactate dehydrogenase release to assess changes in culture viability

This study reports the use of lactate dehydrogenase release to monitor changes in culture viability in flask culture and fixed bed, porosphere bioreactor systems. Lactate dehydrogenase release shows good agreement with increase in non-viable cell numbers and decline in glucose utilisation in flask cultures. Studies with the immobilised system show that lactate dehydrogenase release can detect loss of viability which is not always indicated by a decrease in glucose utilisation. The data show that culture viability in a repeated-feed-and-harvest system is influenced markedly by both a) the medium change regime itself and b) the use of an immobilised bioreactor compared to a flask system for the same medium change regime.     

细菌乳酸脱氢酶的纯化及其性质研究

从乳酸杆菌发酵液经过两次柱层析,可以得到纯度较高的乳酸脱氢酶,酶的比活力高达６７８．９ｕ／ｍｇ,纯度提高８５．７倍。酶的热稳定性好,ｐＨ稳定范围较宽,在临床上可用于雨氨酸氨基较移酶活力的测定。     

Genetic polymorphism and activities of human lung alcohol and aldehyde dehydrogenases: Implications for ethanol metabolism and cytotoxicity

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) exhibit genetic polymorphism and tissue specificity. ADH and ALDH isozyme phenotypes from 39 surgical Chinese lung specimens were identified by agarose isoelectric focusing. The identity of the lung β-ADHs was further demonstrated by their characteristic pH-activity profiles for ethanol oxidation,K _m values for NAD and ethanol, and inhibition by 4-methylpyrazole or 1,10-phenanthroline. The β₂ allele, coding for β₂ polypeptide, was found to be predominant in the lung specimens studied. The ADH activities in the lungs with the homozygous phenotype ADH₂ 2-2 (exhibiting β₂β₂) and ADH₂ 1-1 (exhibiting β₁β₁) and the heterozygous phenotype ADH₂ 2-1 (exhibiting β₂β₂, β₂β₁, and β₁β₁) were determined to be 999±77, 48±17, and 494±61 nmol/min/g tissue, respectively. Fifty-one percent of the specimens studied lacked the ALDH2 activity band on the isoelectric focusing gels. The activities in the lung tissues with the ALDH2-active phenotype and the inactive phenotype were determined to be 30±3 and 17±1 nmol/min/g tissue, respectively. These findings indicate that human pulmonary ethanol-metabolizing activities differ significantly with respect to genetic polymorphism at both theADH ₂ and theALDH ₂ loci. The results suggest that individuals with highV _max β₂-ADH and deficient in low-K _m mitochondrial ALDH2, accounting for approximately 45% of the Chinese population, may end up with acetaldehyde accumulation during alcohol consumption, rendering them vulnerable to tissue injury caused by this highly reactive and toxic metabolite. This work was supported by Grants NSC 77-0412-B016-58 and NSC 80-0412-B016-21 from the National Science Council, Republic of China.     

Aldehyde dehydrogenase (ALDH) isozymes in the gray short-tailed opossum (Monodelphis domestica): Tissue and subcellular distribution and biochemical genetics of ALDH3

Polyacrylamide gel isoelectric focusing (PAGE-IEF), cellulose acetate electrophoresis, and histochemical techniques were used to examine the tissue and subcellular distribution, genetics and biochemical properties of aldehyde dehydrogenase (ALDH) isozymes in a didelphid marsupial, the gray short-tail opossum (Monodelphis domestica). At least 14 zones of activity were resolved by PAGE-IEF and divided into five isozyme groups and three ALDH classes, based upon comparisons with properties previously reported for human, baboon, rat, and mouse ALDHs. Opossum liver ALDHs were distributed among cytosol (ALDHs 1 and 5) and large granular (mitochondrial) fractions (ALDHs 2 and 5). Similarly, kidney ALDHs were distributed between the cytosol (ALDH5) and the mitochondrial fractions (ALDHs 2, 4, and 5), whereas a major isozyme (ALDH3), found in high activity in cornea, esophagus, ear pinna, tail, and stomach extracts, was localized predominantly in the cytosol fraction. Phenotypic variants of the latter enzyme were shown to be inherited in a normal Mendelian fashion, with two alleles at a single locus (ALDH3) showing codominant expression. The data provided evidence for genetic identity of corneal, ear pinna, tail, and stomach ALDH3 and supported biochemical evidence from other mammalian species that this enzyme has a dimeric subunit structure.     

牛蛙乳酸脱氢酶同工酶的分离纯化及其性质研究

牛蛙心脏中乳酸脱氢酶在聚丙烯酰胺凝胶电泳上显示3种同工酶区带,分别命名为LDH1、LDH2、LDH3,其中LDH1的活力占绝对优势.采用HiTrap^TM Blue HP 亲和层析和DEAE-Sephadex A离子交换层析对牛蛙骨骼肌中的LDH3进行了分离纯化.纯化的LDH3比活力为295 U/mg,Km NADH=0.028,Km丙酮酸=1.242,在SDS-PAGE上显示两条带,提示该同工酶是由两种亚基组成的,亚基的分子量分别为35.3 kD和37.6 kD.     

Lactate Dehydrogenase Activity and Insulin and Lactate Levels at an Altitude Below Sea Level (–350 m) Compared to Those at an Altitude Above Sea Level (620 m) After Exercise

This study was designed to investigate the effect of exercise at 350 m below sea level altitude (–350 m) on the serum levels of lactate dehydrogenase (LDH), insulin, and lactate. The study was carried out on ten trained adult males with mean age of 23.3 ± 3.4 years following a 21-km noncompetitive run. Venous blood was withdrawn from the subjects before exercise and 5 min post exercise. For comparison purposes, a similar study was performed with the same subjects but at 620 m above sea level (+620 m). The results show a significant increase in LDH and lactate levels after exercise only at low altitude (–350 m). Serum insulin levels decreased significantly after exercise at both altitudes. These changes in serum levels of LDH, insulin, and lactate at different altitudes suggest that a type of metabolic adjustment is present that meets energy requirements during exercise.     

基于酶电极的乳酸检测生物传感器

乳酸(C₃H₆O₃),又名2-羟基丙酸、丙醇酸,属于羟基酸的一种。乳酸在食品工业、临床医学、生物技术等行业具有极其重要的意义,因此如何高通量检测不同样品中的乳酸成为目前业界研究的重点。传统乳酸检测方法操作繁琐、费时费力或需要昂贵的检测设备,乳酸生物传感器可以克服这些限制,不需要样品制备,能够快速、简便、可靠地定量测定食品或血浆中的乳酸,具有广阔的应用前景。乳酸酶电极生物传感器主要有两种类型——基于L-乳酸氧化酶(L-LOD)和L-乳酸脱氢酶(L-LDH)的乳酸生物传感器。本文综述了L-LOD和L-LDH结构特征、来源及催化机理,讨论了改善基于酶电极的乳酸传感器性能的3种策略(电极材料改造策略、酶固定化策略、酶分子工程改造策略),还根据用于制造乳酸生物传感器的不同载体包括膜、透明凝胶基质、水凝胶载体、纳米颗粒等对乳酸生物传感器进行了归类分析,最后本文将目前商品化应用的酶电极乳酸生物传感器特点进行了对比总结讨论,阐述了乳酸生物传感器的未来应用方向,并对未来发展前景进行了展望。     

Three alcohol dehydrogenase genes in wild and cultivated barley: Characterization of the products of variant alleles

Barley (Hordeum vulgare) and its wild progenitor (H. spontaneum) have three loci for alcohol dehydrogenase (EC 1.1.1.1; ADH). The Adh1 locus is constitutively expressed in seed tissues, whereas expression of the loci Adh2 and Adh3 requires anaerobic induction. The Adh3 gene is well expressed in aleurone and embryo tissues kept under N₂ for 2–3 days. Using N₂-treated embryos, a diverse collection of H. spontaneum was screened in starch gels for electrophoretic variants at the Adh3 locus. Four variants were found: two were conventional mobility variants (Adh3 S, Adh3 V); one was a null variant (Adh3 n); and the fourth (Adh3 I) variant lacked active homodimers and showed reduced heterodimer activity. The ³⁵S-labeled monomers induced under N₂ in the lines homozygous for Adh1, Adh2, or Adh3 variants were immunoprecipitated with antiserum raised against maize ADH. Fluorography after separation by SDS-PAGE and by urea-isoelectric focusing indicated that the Adh3 n allele was CRM- and that the Adh3 I gene product was smaller than normal. The Adh1 and Adh3 variants showed independent segregation.