Mechanism of activation of hepatic branched-chain alpha-ketoacid dehydrogenase by a muscle factor.

H S Paul,S A Adibi

doi:10.1016/s0021-9258(17)44250-5

Abstract

We recently reported that a skeletal muscle factor specifically activates the branched-chain alpha-ketoacid (BCKA) dehydrogenase in liver mitochondria (Paul, H. S., and Adibi, S. A. (1982) J. Biol. Chem. 257, 12581-12588). The evidence suggested that the muscle factor may be a phosphatase or may stimulate the phosphatase in liver mitochondria, thus converting the inactive BCKA dehydrogenase into an active form. In the present study we have investigated these possibilities. The muscle factor did not increase the activity of BCKA dehydrogenase which was previously activated in vitro by preincubation of mitochondria or by the addition of Triton or Ca2+ to the incubation medium. The muscle factor had little or no stimulatory effect on BCKA dehydrogenase when this enzyme was activated in vivo by treatment of rats with streptozotocin or clofibrate. The addition of NaF (an inhibitor of phosphatase) to the incubation medium entirely abolished the stimulatory effect of the muscle factor on BCKA dehydrogenase. Elimination of phosphatase by partial purification of BCKA dehydrogenase resulted in loss of stimulation of the dehydrogenase by the muscle factor. These results suggest that the muscle factor activates the BCKA dehydrogenase in liver mitochondria by stimulating its associated phosphatase.

Highlights

We recently reported that a skeletal muscle factor investigated thesepossibilities and extended our investigation specificallyactivates the branched-chain a-ketoacid of the effect of the muscle factor on the active and inactive (BCKA)dehydrogenase in liver mitochondria
To determine whether the muscle factor has any effect on the activity of BCKA dehydrogenase when it is already activated in uitro, we investigated the effect of the muscle factor on BCKA
Our previous studies have shown that treatment of liver mitochondria with Triton X-100 leads to a Z-3-fold increase in the BCKA dehydrogenase activity (9)

Summary

As compared to themitochondrial BCKA dehydrogenase the partially

This article must dehydrogenase in mitochondria was determined by measuring the be hereby marked “aduertisement” in accordance with 18 release of “CO, from a-keto[l-“Cjisocaproate incubated with mito-. The activity of BCKA dehydrogenase in partially purified enzyme was determined spectrophotometrically at 30 “C by following the reduction of NAD+. Triton X-100, 10 units of dihydrolipoyl dehydrogenase, 0.45 mg of partially purified BCKA dehydrogenase, and 0.20 mM a-ketoisocaproate. Amersham Carp: cr-K&o[l-‘4C]isocaproate was prepared in this laboratory from L-[l-‘4C]leucine by incubating with L-amino acid oxidase as described by Rudiger et al (17). The sodium salt of a-ketoisocaproate, NAD+, coenzime A, thiamin pyrophosphate, dihydrolipoyl dehydrogenase, and bovine serum albumin (Fraction V) were purchased from Sigma. Bovine serum albumin was defatted and dialyzed according to the method of Chen (18).

RESULTS

WITH MUSCLE FACTOR

Effect of the Muscle Factor on BCKA Dehydrogenase When

DISCUSSION