Huang B, Gudi R, Wu P, Harris R A, Hamilton J, Popov K M
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5122, USA.
J Biol Chem. 1998 Jul 10;273(28):17680-8. doi: 10.1074/jbc.273.28.17680.
Pyruvate dehydrogenase phosphatase (PDP) is one of the few mammalian phosphatases residing within the mitochondrial matrix space. It is responsible for dephosphorylation and reactivation of the pyruvate dehydrogenase complex (PDC) and, by this means, is intimately involved in the regulation of utilization of carbohydrate fuels in mammals. PDP is a dimeric enzyme consisting of catalytic and regulatory subunits. The catalytic subunit of PDP is a Mg2+-dependent enzyme homologous to the cytosolic phosphatases of the 2C family. In the present study, we isolated two cDNAs encoding for mitochondrial phosphatases. The first cDNA is highly homologous to the previously identified cDNA encoding for the catalytic subunit of PDP (PDP1). The second cDNA encodes a previously unknown catalytic subunit of PDP (PDP2). The new phosphatase, expressed as the recombinant protein in Escherichia coli, shows strict substrate specificity toward PDC and does not use phosphorylated branched chain alpha-ketoacid dehydrogenase as substrate. Like PDP1, PDP2 is a Mg2+-dependent enzyme, but its sensitivity to Mg2+ ions is almost 10-fold lower than that of PDP1. In contrast to PDP1, PDP2 is not regulated by Ca2+ ions. Instead, it is sensitive to the biological polyamine spermine, which, in turn, has no effect on the enzymatic activity of PDP1. Western blot analysis of PDP extracted from mitochondria isolated from liver and skeletal muscle revealed that PDP1 is predominantly expressed in mitochondria from skeletal muscle, whereas PDP2 is much more abundant in the liver rather than muscle mitochondria. Both isoenzymes are expressed in mitochondria from 3T3-L1 adipocytes, but the level of expression of PDP2 is considerably higher. These observations are consistent with previous findings on the enzymatic parameters of PDP in adipose tissue. Thus, our results provide the first evidence that there are at least two isoenzymes of PDP in mammals that are different with respect to tissue distribution and kinetic parameters and, therefore, are likely to be different functionally.
丙酮酸脱氢酶磷酸酶（PDP）是少数存在于线粒体基质空间中的哺乳动物磷酸酶之一。它负责丙酮酸脱氢酶复合体（PDC）的去磷酸化和再激活，通过这种方式，密切参与哺乳动物碳水化合物燃料利用的调节。PDP是一种由催化亚基和调节亚基组成的二聚体酶。PDP的催化亚基是一种与2C家族胞质磷酸酶同源的Mg2+依赖性酶。在本研究中，我们分离出了两个编码线粒体磷酸酶的cDNA。第一个cDNA与先前鉴定的编码PDP催化亚基（PDP1）的cDNA高度同源。第二个cDNA编码一种先前未知的PDP催化亚基（PDP2）。这种新的磷酸酶在大肠杆菌中表达为重组蛋白，对PDC表现出严格的底物特异性，并且不将磷酸化的支链α-酮酸脱氢酶用作底物。与PDP1一样，PDP2是一种Mg2+依赖性酶，但其对Mg2+离子的敏感性比PDP1低近10倍。与PDP1不同，PDP2不受Ca2+离子调节。相反，它对生物多胺精胺敏感，而精胺对PDP1的酶活性没有影响。对从肝脏和骨骼肌分离的线粒体中提取的PDP进行蛋白质印迹分析表明，PDP1主要在骨骼肌线粒体中表达，而PDP2在肝脏而非肌肉线粒体中含量丰富得多。两种同工酶都在3T3-L1脂肪细胞的线粒体中表达，但PDP2的表达水平要高得多。这些观察结果与先前关于脂肪组织中PDP酶学参数的研究结果一致。因此，我们的结果提供了首个证据，表明哺乳动物中至少存在两种PDP同工酶，它们在组织分布和动力学参数方面存在差异，因此在功能上可能也不同。
