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

J Biol Chem. 1990 Jul 15;265(20):11740-5.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Calmodulin / metabolism*
  • Chromatography, High Pressure Liquid
  • Cyanogen Bromide
  • Kinetics
  • Macromolecular Substances
  • Mass Spectrometry
  • Molecular Sequence Data
  • Muscles / enzymology
  • Peptide Fragments / isolation & purification
  • Phosphorylase Kinase / isolation & purification*
  • Phosphorylase Kinase / metabolism
  • Rabbits

Substances

  • Calmodulin
  • Macromolecular Substances
  • Peptide Fragments
  • Phosphorylase Kinase
  • Cyanogen Bromide