Purification and Characterization of 3':5'-cyclic GMP-dependent Protein Kinase

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 1976 Nov 1

PMID 186778

Citations 14

Authors

G N Gill

K E Holdy

G M Walton

C B Kanstein

Affiliations

Soon will be listed here.

Abstract

Guanosine 3':5'-cyclic monophosphate (cGMP)-dependent protein kinase has been purified to homogeneity from bovine lung by affinity chromatography and characterized. Partially purified protein kinase, specifically activated by low concentrations of cGMP (22 NM), was adsorbed onto 8-(2-aminoethyl)-amino-adenosine 3':5'-cyclic monophosphate-Sepharose. After washing to remove nonspecific proteins, cGMP-dependent protein kinase was specifically eluted by 0.1 mM cGMP. The purified protein contained cGMP-dependent protein kinase and specific cGMP binding activities. Purification of the holoenzyme was possible because subunit dissociation does not occur upon cyclic nucleotide binding. cGMP-dependent protein kinase holoenzyme has an apparent molecular weight of 150,000 as determined by glycerol density gradient sedimentation. On sodium dodecyl sulfate/polyacrylamide gel electrophoresis, a single protein band of 71,000 molecular weight was observed that suggested the holoenzyme is a dimer composed of subunits of identical molecular weight. cGMP-dependent protein kinase required high concentrations of Mg+2 for optimal activity; a heat-stable protein kinase modulator which inhibited adenosine 3':5'-cyclic monophosphate-dependent protein kinase activity had no effect on the activity of purified cGMP-dependent protein kinase.

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