STUDIES ON ACETYL-COENZYME A CARBOXYLASE AND INSULIN RECEPTOR (CARBOXYLASE, IGY, INSULIN RECEPTOR)

CHUNG-SEOG SONG, Purdue University

Abstract

Rat liver acetyl-CoA carboxylase can be rapidly isolated by a new procedure which uses avidin-Sepharose affinity chromatography. The isolated enzyme has M(,r) = 260,000; none or very little of the proteolytic products of the carboxylase which are formed in conventional purification procedures are found in our preparations. It is apparent that the previously reported subunit of the carboxylase, with M(,r) = 230,000, is itself the product of proteolysis. The properties of the enzyme produced by our new method are quite different from those of the conventionally prepared enzyme. Our enzyme contains 6 mol of alkali-labile phosphate/mol of subunit, rather than 2 mol; the K(,m) for acetyl-CoA is about 8-fold higher and the specific activity is only about one-fifth of that previously reported. The large amount of phosphate does not appear to cause the low specific activity of the new enzyme preparation, because alkaline phosphatase treatment reduces the number of phosphates/subunit from 6 to 3 mol but does not change the specific activity. Acetyl-CoA carboxylase is activated by physiological concentrations of CoA. Activation of partially purified enzyme by CoA is accompanied by a decrease in the K(,m) for acetyl-CoA from 0.2 mM to about 4 (mu)M, which is the physiological concentration of acetyl-CoA in the cytosol. CoA activation of the purified enzyme is accompanied by an increase in the V(,max), without changing the K(,m) for acetyl-CoA. The K(,m) for acetyl-CoA of the purified enzyme is about 10 to 40 (mu)M. The purification procedure results in a decrease in the K(,m) for acetyl-CoA; under these conditions, CoA activation does not cause further lowering of the K(,m). CoA activation is accompanied by polymerization of the enzyme. However, CoA activation is not causally related to polymerization. There is one CoA binding site/subunit of acetyl-CoA carboxylase. CoA binding at that site is not affected by the presence of citrate, but palmityl-CoA inhibits CoA binding. CoA alone cannot reverse palmityl-CoA inhibition of the carboxylase. Bovine serum albumin and CoA together can activate the palmityl-CoA inhibited enzyme. This indicates that the involvement of bovine serum albumin-like protein, CoA and palmityl-CoA may play a physiologically significant role in the control of acetyl-CoA carboxylase.

Degree

Ph.D.

Subject Area

Anatomy & physiology|Animals

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