STUDIES ON RAT EPIDIDYMAL FAT TISSUE ACETYL-COENZYME-A CARBOXYLASE PHOSPHATASE

• Main Author: KRAKOWER, GLENN ROBERT
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-1980
• Subjects: Biochemistry
• ISBN: 9798661788023

Acetyl-CoA carboxylase (E.C. 6.4.1.2) from rat epididymal fat tissue is activated by phosphorylase phosphatase, a reaction which is inhibited by rabbit muscle phosphatase inhibitor-1. This activation is accompanied by a corresponding loss of P-32 from the labelled enzyme. Even in the absence of added phosphatase, labelled acetyl-CoA carboxylase undergoes a temperature-dependent activation which is accompanied by a loss of P-32. The endogenous phosphatase which is responsible for this "heat-activation" has been purified by a procedure which utilizes ammonium sulfate precipitation, an optional sucrose gradient centrifugation step, 63-80% ethanol precipitation, and chromatography on Sephadex G-75. The phosphatase, judged pure by SDS gel electrophoresis and gel chromatography of the iodinated enzyme, has a molecular weight of 71,000 and consists of one polypeptide chain. Like many other phosphoprotein phosphatases, it has a broad specificity, acting on fat and liver acetyl-CoA carboxylase, HMG-CoA reductase, glycogen synthase, phosphorylase a, phosphoprotamine, and p-nitrophenyl phosphate. The K(,m)'s for acetyl-CoA carboxylase, phosphorylase a, phosphoprotamine, and p-nitrophenyl phosphate are 1.5 (mu)M, 37 (mu)M, 76 (mu)M, and 1.34 mM, respectively. The phosphatase requires no metal ion for activity and is not inhibited by the rat liver phosphatase inhibitor protein. It is affected differently by small metabolites according to the substrate chosen. We have prepared antiserum to this purified phosphatase and have characterized it by immunoelectrophoresis and by its inactivation of phosphoprotamine phosphatase activity. Insulin activates acetyl-CoA carboxylase by promoting its dephosphorylation. In live rats and in epididymal fat tissue treated with insulin, activation of acetyl-CoA carboxylase is accompanied by increased activities of acetyl-CoA carboxylase phosphatase bound to its substrate. This effect is not blocked by inhibitors of protein or RNA synthesis, and peak activation is seen within 30-45 min of insulin treatment. Increases in acetyl-CoA carboxylase activity are also seen following this activation. Optimal activation is seen between 1 and 3 munits/ml insulin, and the size of the rat makes little difference in the amount of activation. Antibody titration indicates that this increased phosphatase activity is due to increased amounts of phosphatase bound to the carboxylase. This result was confirmed by sucrose density gradient studies which showed increased phosphatase activities in those fractions which contain carboxylase activity. That this effect is due to insulin is shown in experiments without insulin or with insulinomimetic substances. The implications of these results are discussed.