Purification and characterization of catalytic fragments of phosphorylase kinase gamma subunit missing a calmodulin-binding domain.

W R Harris,D A Malencik,C M Johnson,S A Carr,G D Roberts,C A Byles,S R Anderson,L M Heilmeyer,E H Fischer,J W Crabb

doi:10.1016/s0021-9258(19)38460-1

Abstract

A catalytic fragment preparation of rabbit muscle phosphorylase kinase produced by limited chymotryptic digestion was isolated and identified as the NH2-terminal region of the gamma subunit by Edman degradation. Mass spectral analysis, gas phase sequence analysis, and amino acid analysis of the active fragment carboxyl-terminal peptides revealed multiple COOH termini generated at residues Tyr290, Arg296, and Phe298 in the gamma subunit sequence. These active fragment species are about 24% smaller than the gamma subunit (Mr 44,673) and range in size from Mr 33,279 to Mr 34,275. The active fragment preparation exhibits a specific activity about 6-fold higher than that of the gamma subunit-calmodulin complex. Calmodulin confers calcium sensitivity to the gamma subunit but has no effect on the enzymatic properties of active fragment. Affinity measurements demonstrated a dissociation constant of 0.7 microM for active fragment binding to dansylcalmodulin, a value about 28-fold weaker than reported for the gamma subunit. These data support the presence of a calmodulin binding domain in the COOH-terminal region of the gamma subunit.

Highlights

19406, the **Department of Biochemistry, University of Washington, Seattle, Washington 98195, and the [(Znstitut ftir Physiologische Chemie, Ruhr
A catalytic fragment preparation of rabbit muscle phosphorylase kinase produced by limited chymotryptic digestion was isolated and identified as the NH2
Gas phase sequence analysis, and amino acid analysis of the active fragment carboxyl-terminal peptides revealed multiple COOH termini generated at residues Tyr2”, Argzse, and Phezs8 in the y subunit sequence

Summary

AND RESULTS’

Limited Proteolysis of Phosphorylase Kinase and Purification of Catalytic Fragments-Phosphorylase kinase (-84 mg) was dialyzed into 50 mM Tris-Cl, 1 mM dithiothreitol, 1 mM EDTA, pH 8.0, and diluted to 0.15 mg/ml in the same buffer. The only HPLC fractions with enzymatic activity contained the y subunit and a component of about 35,000 molecular weight by SDS-PAGE (Fig. 2). CNBr-2 was the more abundant of the two final purification obtained by molecular sieve HPLC (Fig. 3C) COOH-terminal peptides (Table 2). CNBr-1 was matic activity, identical RP-HPLC and SDS-PAGE profiles, cleaved at glutamyl residues with V8 protease, and FABMS and an identical NHp-terminal sequence with the active frag- of the unfractionated digest (Fig. 7C) yielded a significant ment purified entirely by HPLC. These results suggest that the intact y subunit binds calmodulin much more tightly than does the active y fragment or the inactive

Active fragment

Findings

DISCUSSION