Studies on guanosine 3' :5'-monophosphate-dependent protein kinase; isolation and characterization of catalytic fragment
Inoue, M.
Kobe Journal of Medical Sciences 24(3): 125-140
1978
ISSN/ISBN: 0023-2513 PMID: 216847 Document Number: 136974
The mechanism of action of the cyclic nucleotide on GMP (cyclicGMP)-dependent protein kinase (protein kinase G) was studied in extracts of Bombyx mori pupae. Protein kinase G was not dissociated by cGMP into catalytic and regulatory subunits established for cAMP-dependent protein kinase (protein kinase A). Limited proteolysis of protein kinase G with trypsin resulted in the formation of catalytic and cGMP-binding fragments; the MW of these fragments were approximately 3.4 .times. 104 and 3.6 .times. 104, respectively (the MW of native protein kinase G was 1.4 .times. 105). The catalytic fragment produced was fully active in the absence of cyclic nucleotides, and did not require high concentration of Mg2+ (50-100 mM) and a sulfhydryl compound, both of which were necessary for the maximal activation of native protein kinase G. In spite of such kinetic differences the catalytic fragment and native enzyme showed identical catalytic properties as judged by the fingerprint procedure. The catalytic fragment was not inhibited by the cGMP-binding fragment, nor by the regulatory subunit of protein kinase A. The heat stable protein inhibitor of protein kinase A was also inactive for the catalytic fragment. The cGMP-binding fragment was unable to regulate the catalytic subunit of protein kinase A. The catalytic and cGMP-binding sites on protein kinase G are evidently clearly separable. Protein kinase G may be distinguishable from protein kinase A by comparing the response to cyclic nucleotide.