Glucose-induced degradation of the MDH2 isozyme of malate dehydrogenase in yeast.

MDH2, the nonmitochondrial isozyme of malate dehydrogenase in Saccharomyces cerevisiae, was determined to be a target of glucose-induced proteolytic degradation. Shifting a yeast culture growing with acetate to medium containing glucose as a carbon source resulted in a 25-fold increase in turnover of MDH2. A truncated form of MDH2 lacking amino acid residues 1-12 was constructed by mutagenesis of the MDH2 gene and expressed in a haploid yeast strain containing a deletion disruption of the corresponding chromosomal gene. Measurements of malate dehydrogenase specific activity and determination of growth rates with diagnostic carbon sources indicated that the truncated form of MDH2 was expressed at authentic MDH2 levels and was fully active. However, the truncated enzyme proved to be less susceptible to glucose-induced proteolysis, exhibiting a 3.75-fold reduction in turnover rate following a shift to glucose medium. Rates of loss of activity for other cellular enzymes known to be subject to glucose inactivation were similarly reduced. An extended lag in attaining wild type rates of growth on glucose measured for strains expressing the truncated MDH2 enzyme represents the first evidence of a selective advantage for the phenomenon of glucose-induced proteolysis in yeast.     

两种虫生真菌酯酶同工酶酶谱多样性研究

采用聚丙烯酰胺凝胶电泳技术对不同来源的22株球孢白僵菌和21株粉拟青霉酯酶同工酶的酶谱多样性进行了研究,由酯酶图谱可知同种不同菌株间具有丰富的遗传多样性,可作为菌株亲缘关系的可靠的遗传标记。遗传距离进行聚类分析,结果表明球孢白僵菌、粉拟青霉不同菌株间亲缘关系的远近与寄主范围无任何相关性,与菌株来源地在某些菌株间存在一定的相关性。     

Isolation and characterization of the yeast gene encoding the MDH3 isozyme of malate dehydrogenase.

The MDH3 isozyme of Saccharomyces cerevisiae was purified from a haploid strain containing disruptions in genomic loci encoding the mitochondrial MDH1 and nonmitochondrial MDH2 isozymes. Partial amino acid sequence analysis of the purified enzyme was conducted and used to plan polymerase chain reaction techniques to clone the MDH3 gene. The isolated gene was found to encode a 343-residue polypeptide with a molecular weight of 37,200. The deduced amino acid sequence was closely related to those of MDH1 (50% residue identity) and of MDH2 (43% residue identity). The MDH3 sequence was found to contain a carboxyl-terminal SKL tripeptide, characteristic of many peroxisomal enzymes, and immunochemical analysis was used to confirm organellar localization of the MDH3 isozyme. Levels of MDH3 were determined to be elevated in cells grown with acetate as a carbon source, and under these conditions, MDH3 contributed approximately 10% of the total cellular malate dehydrogenase activity. Disruption of the chromosomal MDH3 locus produced a reduction in cellular growth rates on acetate, consistent with the presumed function of this isozyme in the glyoxylate pathway of yeast. Combined disruption of MDH1, MDH2, and MDH3 loci in a haploid strain resulted in the absence of detectable cellular malate dehydrogenase activity.     

Differential alcohol dehydrogenase and malate dehydrogenase isozyme expression in long-term callus tissue cultures ofCereus peruvianus (Cactaceae)

Alcohol dehydrogenase (ADH) and mitochondrial malate dehydrogenase (mMDH) isozymes were tested as markers to study the effect of a high kinetin concentration on isozyme phenotypes and on the development ofCereus peruvianus callus tissue culture. Three-year-old callus tissues were used as samples. Callus tissue samples grown on 4.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and on 4.0 and 8.0 mg/LN-(2-furanylmethyl)-1H-purine-6 amine (kinetin) were cut and transferred to fresh medium containing 4.0 mg/L 2,4-D and 4.0, 8.0, 16.0, and 32 mg/L kinetin combinations. The pattern of changes observed in the ADH and mMDH isozymes as well as the growth of callus tissues was independent of the concentrations tested. The various ADH and mMDH isozymes seem to be products of differential association of subunits of the twoAdh and twomMdh genes. Both genes are active throughout callus tissue development; however, gene expression changed with various callus culture conditions. This study addresses how long-term callus culture conditions affect constitutive and differential gene expression of theAdh andmMdh genes inC. peruvianus.     

Genetic variants of soluble malate dehydrogenase in new guinea populations

Summary 10 cases of an S-MDH variant have been detected in New Guinea. 3 cases were found among 199 samples from the Fore linguistic group and 6 cases among 9 related members of a family from the Agarabi linguistic group, both groups being located in the Eastern Highlands. 1 case was found in 24 samples from the Sepik district. The new variant has been given the trivial name S-MDH New Guinea-1.     

Changes in the activity and isozyme patterns of malate dehydrogenase in root nodules of yellow lupine

The malate dehydrogenase present in the cytoplasmic fraction of plant origin and bacteroids from yellow lupine root nodules was investigated. The plant enzyme was 14 times more active in nodules than in roots and it contained 6 molecular forms in nodules compared with 3 forms detected in roots. The highest malate dehydrogenase activity in plant fraction and bacteroids was noted in 50-day old plants. Changes in the isoenzymatic patterns of malate dehydrogenase in plant fraction and bacteroids accompanying ageing of the lupine root nodules were observed. Possible physiological role of malate pathway in metabolism of lupine root nodules is discussed.     

Aldehyde dehydrogenase isozyme deficiency and alcohol sensitivity in four different Chinese populations

Four different populations of China were studied regarding aldehyde dehydrogenase isozyme variation and incidence of alcohol sensitivity. While Korean and Mongolian minorities in the north showed an isozyme I deficiency with a frequency of about 25 and 30%, 45-50% of Zhuang and Han were deficient, respectively. Adverse reactions after alcohol drinking were mainly reported by those subjects who showed the lack of aldehyde dehydrogenase isozyme I.     

Characterization of a cytosolic malate dehydrogenase cDNA which encodes an isozyme toward oxaloacetate reduction in wheat

Malate dehydrogenase (MDH), which is ubiquitous in nature, catalyzes the interconversion of oxaloacetate and malate. Higher plants contain multiple forms of MDH that differ in co-enzyme specificity, subcellular localization and physiological function. Cytosolic NAD-dependent MDH (cyMDH) is one class of MDH that has not been extensively characterized in plants. Here we report the cloning of a cDNA from wheat by RT-PCR and cDNA library screening, which is designated as TaMDH. Sequence analysis indicated that TaMDH exhibits a highly similarity to other plant cyMDHs. Immunological analysis confirmed that TaMDH encoded a cytosolic NAD-dependent MDH. The secondary and three-dimensional structures of TaMDH were analyzed by molecular modeling. DNA gel-blot analyses demonstrated that TaMDH gene exists as two copies in the wheat genome. RNA and protein gel-blot hybridization indicated that both TaMDH mRNA and protein were constitutively expressed in vegetative tissues of wheat, with slightly lower levels in roots than in leaves and stems. In silico analysis indicated that TaMDH was also expressed in various reproductive tissues and tissues under many different stress conditions. Kinetic analysis of bacterially expressed and purified protein confirmed that TaMDH catalyzed a reaction driven towards malate synthesis, which is consistent with other cyMDHs. Evolutionary analysis showed that this class of genes evolved from a very ancestral gene. The cyMDH represents an ancestral form of MDH, which is highly conserved in plants, animals and bacteria. This implies that cyMDHs are housekeeping genes and may have very essential functions in plant metabolism.     

Adaptation of malate dehydrogenase to environmental temperature variability in two populations of Potentilla glandulosa Lindl.

Summary The responses of the kinetic properties of malate dehydrogenase to environmental temperature variability were compared for two populations of Potentilla glandulosa (Rosaceae). The two populations are native to regions of contrasting climates, with the inland population experiencing a high level of temperature variability during growth and the coastal populaton a low level of temperature variability. The substrate binding ability, as measured by apparent K _m of both populations was relatively insensitive to assay temperature (Q ₁₀<2.0) over the range of temperatures likely to be encountered during growth. The breadth of this thermal optimum was different for the two populations with the K _m of the inland plants exhibiting relative temperature insensitivity over a much wider range of temperatures than the K _m of the coastal plants. There was no difference between the two populations in the thermal stability of MDH activity.     

酯酶同工酶在鞘翅目昆虫分类中的应用简介

简要介绍了酯酶同工酶电泳技术在鞘翅目昆虫分类研究中的应用。包括叶甲科Chrysomelidae、天牛科Cerambycidae、小蠹科Scolytidae、瓢虫科Coccinelidae、豆象科Bruchidae、金龟子科Scarabaeidae、萤科Lampyridae和苔水龟虫科Hydraenidae等。     

Subunit dissociation of mitochondrial malate dehydrogenase.

Fluorescence polarization studies of porcine mitochondrial malate dehydrogenase labeled with fluorescein isothiocyanate or fluorescamine indicated a concentration-dependent dissociation of the dimeric molecule with a KD OF 2 X 10(7) N at pH 8.0. These results were confirmed by the concentration dependence of the stability of the enzyme at elevated temperatures and the creation of hybrid molecules with fluorescein and Rhodamine B labeled subunits, in which energy transfer was observed. The binding of NADH resulted in a small shift of the subunit dissociation curve toward monomer, demonstrating that monomer has twice the affinity for reduced coenzyme. NAD+ binding prevented dissociation of the dimer, even at concentrations below 10(-8) N. These results indicate that binding of reduced or oxidized coenzymes results in different conformation changes, which are transferred to the subunit interface.     

A Bacillus subtilis malate dehydrogenase gene.

A Bacillus subtilis gene for malate dehydrogenase (citH) was found downstream of genes for citrate synthase and isocitrate dehydrogenase. Disruption of citH caused partial auxotrophy for aspartate and a requirement for aspartate during sporulation. In the absence of aspartate, citH mutant cells were blocked at a late stage of spore formation.     

Human liver aldehyde dehydrogenase. Esterase activity.

Human liver aldehyde dehydrogenase has been found to be capable of hydrolyzing p-nitrophenyl esters. Esterase and dehydrogenase activities exhibited identical ion exchange and affinity properties, indicating that the same protein catalyzes both reactions. Competitive inhibition of esterase activity by glyceraldehyde and chloral hydrate furnished evidence that p-nitrophenyl acetate was hydrolyzed at the aldehyde binding site for dehydrogenase activity. Pyridine nucleotides modified esterase activity; NAD+ accelerated the rate of p-nitrophenyl acetate hydrolysis more that 5-fold, whereas NADH increased activity by a factor of 2. Activation constants of 117 muM for NAD+ and 3.5 muM for NADH were obtained from double reciprocal plots of initial rates as a function of modifier concentration at pH 7. The kinetics of activation of ester hydrolysis were consistent with random addition of pyridine nucleotide modifier and ester substrate to this enzyme.