Calcium activation of mitochondrial glycerol phosphate dehydrogenase restudied.
Calcium activation of mitochondrial glycerol phosphate dehydrogenase restudied.
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重新研究了线粒体磷酸甘油脱氢酶的钙激活。
DOI：
10.1006/abbi.1996.0049
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Brown,LJ
中科院分区：
文献类型：
--
作者：
MacDonald,MJ;Brown,LJ
编码大鼠睾丸和胰岛线粒体磷酸甘油脱氢酶(MGPD)的基因(EC1.1.99.5)是磷酸甘油穿梭的关键酶，最近被克隆和测序，发现含有钙调素样钙结合序列，从而解释了在高等真核生物的许多组织中广泛观察到的钙激活现象。从Western分析判断，MGPD的活性和蛋白似乎在大鼠的睾丸和胰岛中最为丰富。已知MGPD位于线粒体膜内。在生理浓度的磷酸甘油(75μM)下，Triton X-100溶解的睾丸MGPD的活性在0.1~0.2 5μM游离钙存在时达到最大值。钙(10−6-10−5M)使MGPD的Km值从2.5mM磷酸甘油(胰岛MGPD)和3.2mM甘油磷酸(睾丸MGPD)降至0.4mMGPD。钙调素抑制剂不能阻止睾丸和胰岛MGPD的钙激活，这与MGPD具有能介导自身被钙激活的区域是一致的。45钙覆盖实验表明，MGPD是睾丸线粒体膜上的主要钙结合蛋白。水变图表明，成熟的MGPD蛋白有三个跨膜螺旋。第一个膜跨区与FAD位点重合，因此该位点被放置在膜内。亲水性分析表明，钙结合区和可能的甘油磷酸结合部位位于暴露于细胞液环境中的膜外。这与早期的生化证据是一致的，该证据表明这些部位位于膜外(M.Klingenberg，EUR)。《生物化学》第13卷，247-252页，1970年)。提示细胞内钙离子对MGPD活性有调节作用。由于胰岛β细胞的电活动、细胞内钙离子、糖酵解和胰岛素释放同时发生振荡，MGPD的活性也可能波动，并允许磷酸甘油穿梭参与糖酵解振荡。
cDNAs which encode the rat testis and pancreatic islet mitochondrial glycerol phosphate dehydrogenase (mGPD) (EC 1.1.99.5), the key enzyme of the glycerol phosphate shuttle, were recently cloned and sequenced and found to contain calmodulin-like calcium-binding sequences, thus explaining the widely observed calcium activation of the enzyme from many tissues of higher eukaryotes. mGPD activity and protein, as judged from Western analysis, appear to be most abundant in testis and pancreatic islets in the rat. mGPD is known to be located within the inner mitochondrial membrane. At a physiologic concentration of glycerol phosphate (75 μM), half maximal activity of Triton X-100-solubilized testis mGPD was seen in the presence of 0.1–0.25 μMfree calcium. Calcium (10−6–10−5M) lowered theKmof mGPD from 2.5 mMglycerol phosphate (islet mGPD) and 3.2 mMglycerol phosphate (testis mGPD) to 0.4 mMglycerol phosphate. Calcium activation of mGPD from both testis and islets was not prevented by calmodulin inhibitors, which is consistent with mGPD possessing regions that can mediate its own activation by calcium.45Calcium overlay experiments, in which proteins were separated by SDS–polyacrylamide gel electrophoresis, blotted onto nitrocellulose membranes, and probed with45Ca, showed that mGPD is a major calcium-binding protein in testis mitochondrial membranes. A hydropathy plot suggested that the mature mGPD protein has three transmembrane helices. The first membrane-spanning region coincides with the FAD site and thus this site is placed within the membrane. The hydropathy analysis indicated that the calcium-binding region and the putative glycerol phosphate-binding site lie outside the membrane exposed to the cytosolic environment. This is consistent with earlier biochemical evidence which indicated that these sites are situated outside the membrane (M. Klingenberg,Eur. J. Biochem.13, 247–252, 1970). This suggests that cytosolic calcium can regulate mGPD activity. Since simultaneous oscillations in electrical activity, cytosolic calcium, glycolysis, and insulin release occur in the pancreatic beta cell, mGPD activity might also fluctuate and allow the glycerol phosphate shuttle to participate in glycolytic oscillations.