Steffan J S, McAlister-Henn L
Department of Biological Chemistry, College of Medicine, University of California, Irvine 92717.
J Biol Chem. 1992 Dec 5;267(34):24708-15.
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.
酿酒酵母的MDH3同工酶是从一个单倍体菌株中纯化得到的，该菌株在编码线粒体MDH1和非线粒体MDH2同工酶的基因组位点上存在破坏。对纯化后的酶进行了部分氨基酸序列分析，并用于规划聚合酶链反应技术以克隆MDH3基因。发现分离出的基因编码一个由343个氨基酸残基组成的多肽，分子量为37200。推导的氨基酸序列与MDH1（50%残基同一性）和MDH2（43%残基同一性）的序列密切相关。发现MDH3序列含有羧基末端的SKL三肽，这是许多过氧化物酶体酶的特征，并且使用免疫化学分析来确认MDH3同工酶的细胞器定位。测定以乙酸盐作为碳源生长的细胞中MDH3的水平升高，在这些条件下，MDH3约占细胞总苹果酸脱氢酶活性的10%。染色体MDH3位点的破坏导致细胞在乙酸盐上的生长速率降低，这与该同工酶在酵母乙醛酸途径中的假定功能一致。在一个单倍体菌株中联合破坏MDH1、MDH2和MDH3位点导致无法检测到细胞苹果酸脱氢酶活性。
