膳食蛋白质摄入量对大鼠支链酮酸脱氢酶活性的影响。酶复合物的免疫化学分析。

Effects of dietary protein intake on branched-chain keto acid dehydrogenase activity of the rat. Immunochemical analysis of the enzyme complex.

作者信息

Miller R H, Eisenstein R S, Harper A E

机构信息

Department of Nutritional Sciences, College of Agricultural and Life Sciences, University of Wisconsin-Madison 53706.

出版信息

J Biol Chem. 1988 Mar 5;263(7):3454-61.

PMID:3343251

Abstract

Polyclonal antibodies directed against the dihydrolipoyl transacylase (E2) and alpha subunit of branched-chain alpha-keto acid decarboxylase (E1 alpha) components of the bovine branched-chain keto acid dehydrogenase complex were shown to cross-react with the E2 and E1 alpha polypeptides of the enzyme complex of different rat tissues. Phosphorylation of the branched-chain keto acid dehydrogenase complex resulted in inhibition of enzyme activity concomitant with phosphate incorporation into the E1 alpha polypeptide. Phosphorylation of E1 alpha slowed its rate of migration through sodium dodecyl sulfate-polyacrylamide gels. This permitted resolution of the phosphorylated and unphosphorylated forms of E1 alpha on immunoblots. Liver and skeletal muscle mitochondria were prepared from rats consuming 6, 20, or 50% casein diets. The enzyme complex in mitochondria was measured by radioisotopic enzyme assay and immunoassay. Liver branched-chain keto acid dehydrogenase was 25% active in rats consuming 6% casein diets; whereas in rats consuming 20 or 50% casein diets, the liver enzyme was 82 or 100% active, respectively. Branched-chain keto acid dehydrogenase of muscle was 10, 13, and 22% active, respectively, in rats consuming 6, 20, and 50% casein diets. The amount of protein consumed by rats did not affect the total amount of the enzyme complex per unit of mitochondrial protein as measured by either the radioisotopic assay (enzyme activity) or the immunoassay. However, the protein intake of rats did affect activity of the enzyme kinase in liver. Liver branched-chain keto acid dehydrogenase kinase was more active in rats consuming 6% casein than in those fed chow or 50% casein diets. The amount of protein consumed by rats thus influences the enzyme activity in liver and muscle by affecting the reversible phosphorylation mechanism and not by induction of branched-chain keto acid dehydrogenase.

摘要

针对牛支链酮酸脱氢酶复合体中二氢硫辛酰转乙酰基酶（E2）和支链α-酮酸脱羧酶α亚基（E1α）成分的多克隆抗体，被证明能与不同大鼠组织的酶复合体中的E2和E1α多肽发生交叉反应。支链酮酸脱氢酶复合体的磷酸化导致酶活性受到抑制，同时磷酸盐掺入E1α多肽中。E1α的磷酸化减缓了其在十二烷基硫酸钠-聚丙烯酰胺凝胶中的迁移速率。这使得在免疫印迹上能够分辨出E1α的磷酸化和未磷酸化形式。从食用6%、20%或50%酪蛋白饮食的大鼠中制备肝脏和骨骼肌线粒体。通过放射性同位素酶法和免疫分析法测定线粒体中的酶复合体。食用6%酪蛋白饮食的大鼠肝脏支链酮酸脱氢酶活性为25%；而食用20%或50%酪蛋白饮食的大鼠，肝脏酶活性分别为82%或100%。食用6%、20%和50%酪蛋白饮食的大鼠肌肉中支链酮酸脱氢酶活性分别为10%、13%和22%。通过放射性同位素分析法（酶活性）或免疫分析法测量，大鼠摄入的蛋白质量不影响每单位线粒体蛋白中酶复合体的总量。然而，大鼠的蛋白质摄入量确实影响肝脏中酶激酶的活性。食用6%酪蛋白的大鼠肝脏支链酮酸脱氢酶激酶比食用普通食物或50%酪蛋白饮食的大鼠更活跃。因此，大鼠摄入的蛋白质量通过影响可逆磷酸化机制而非诱导支链酮酸脱氢酶来影响肝脏和肌肉中的酶活性。