The Regulation of Glycogen Metaabolism

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Abstract
Inhibitor-1 is a protein which inhibits phosphorylase phosphatase only when it has been phosphorvlated by cyclic-AMP-dependent protein kinase [Huang, F. L. and Glinsmann. W. H. (1976) Eur. J. Biochem. 70, 419–426]. Inhibitor-1 was purified by a heat treatment at 90°C, precipitation with ammonium sulphate, chromatography on DEAE-cellulose, gel filtration on Sephadex G-100, and finally recromatography of the phosp0horylated protein on DEAE-cellulose. The protein was purified 4000-fold and 1.5 mg per 1000 g muscle was obtained in seveven days corresponding to an overall yield of 15–20%. The purified protein was in a state approaching homogeneity as judged by the criteria of polyacrylamide-gel electrophoresis and ultracentrifugal analysis. The concentration of inhibitor-1 in vivo was calculated to be 1.5 μMM, which is at least as high as the concentrationof phosphorylase phosphatase. The amino acid composition of inhibitor-1 showed several unusual features. Glutamic acid and proline accounted for nearly one third of the residues, tyrosine, tryptophan and cysteine were absent, and the content of aromatic amino acids was very low. The molecular weight measured by sedimentation equilibraium centrifugation was 19 200 and by amino acid analysis was 20 800. These values were lower than the mol. wt of 26000 determined empirically by gel electrophoresis in the presence of sodium dodecyl sulphate, and much lower than the apparent molecular weitht of 60000 estimated by filtraation on Sephadex G-100. The gel filtration behaviour, stability to heating at 100°C and amino acid composition suggest than inhibitor-1 may possess little ordered structure. The phosphorylated form of inhibitor-1contained close to one molecule of covalently bound phosphate per mole of protein, which is consistent with previous finding of a unique decapeptide sequence at the site of phosphorylation, Ile-Arg-Arg-Arg-Arg-Pro-Thr(p-Pro-Ala-Thr-[Cohen, P., Rylatt, D. B. and Nimmo, G. A. (1977) FEEBS Lett. 76, 182–186]. The phosphorylated form of inhibitor-1 inhibited phosphorylase activity (0.02 U) by 50% at a concentration of only 7.0 nM in the standard assay, but the phosphorylated decapeptide was 1000–2000 times less effective as an inhibitor.

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