Determination of cyclic AMP-dependent protein kinase subunits by an immunoassay reveals a different subcellular distribution of the enzyme in rat parotid than does determination of the enzyme activity.
The distribution of cyclic cAMP-dependent protein kinase in subcellular fractions of rat parotid was determined by two independent biochemical methods, measurement of kinase catalytic activity or by quantitation of the catalytic and regulatory subunits in an enzyme-linked immunosorbent assay using monospecific antibodies. The major amount (85%) of the catalytic activity was found associated with the 100,000 g soluble fraction, whereas only 1/3 of the total catalytic subunit was demonstrated in the soluble fraction by the immunoassay. The immunoassay results furthermore indicated that approximately 50% of the total cellular protein kinase was associated with the extranuclear particulate fraction and that the predominant form of the kinase in the particulate fractions was the type II isoenzyme. The reasons for the differences in the distribution of the protein kinase demonstrated by the two methods were examined. Incomplete extraction of membrane-bound protein kinase and the influence of membrane localized ATPases on activity measurements were, at least in part, responsible for the low percentage of kinase activity measured in the particulate fractions. These results emphasize that the precise quantitation of protein kinase subunits merits investigation by more than one method. For the parotid, the finding that approximately 2/3 of the total catalytic subunit may be particulate associated provides additional evidence that cyclic AMP could be involved in membrane mechanisms of hormone-regulated secretion.
Schwoch
G
G
Lohmann
S M
SM
Walter
U
U
Jung
U
U
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay; D007118:Immunoassay; D008297:Male; D009206:Myocardium / Q000201:enzymology; D010306:Parotid Gland / Q000201:enzymology*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D011494:Protein Kinases / Q000032:analysis*; D051381:Rats; D011919:Rats, Inbred Strains; D013347:Subcellular Fractions / Q000201:enzymology*
D000255:Adenosine Triphosphate; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases; D000251:Adenosine Triphosphatases