The differences in Ca(2+)-sensitivity of protein kinase C in platelets from Wistar Kyoto rat and stroke-prone spontaneously hypertensive rat.
Thrombin-induced phosphorylation of 47 kDa protein (P47) in platelets, a substrate of protein kinase C (PKC), was defective in stroke-prone spontaneously hypertensive rats (SHRSP) (Hypertens. 14, 304-315, 1989). Platelet PKC from SHRSP and Wistar Kyoto rats (WKY) was partially purified and Ca(2+)-sensitivity of PKC activity was examined to understand the defect in the protein phosphorylation. When the platelets from SHRSP and WKY were homogenized in a buffer containing 10 mM EGTA and 2 mM EDTA, approx. 80% of PKC was in the cytosol fraction. PKC in this fraction was purified by DE52 and hydroxyapatite column chromatography. Both platelet PKC preparations contained only PKC-alpha, and there was no significant difference in the Ca(2+)-dependency of activity between them. When the platelets from SHRSP and WKY were homogenized in a buffer containing 10 microM CaCl2, 90% of PKC was found to be bound to the membrane. PKC was extracted from the membrane with a buffer containing 2.5 mM EGTA and 2.5 mM EDTA, and purified by DE52 column chromatography. PKC from WKY platelets eluted as a single peak whereas that from SHRSP platelets eluted as two peaks (peak 1 and peak 2). Ca(2+)-sensitivity of peak 1 PKC was much lower than that of WKY PKC. In contrast, the Ca(2+)-sensitivity of peak 2 PKC appeared to be slightly higher than that of WKY PKC. The specific activity of peak 2 PKC was 4% to 5% of that of peak 1 and WKY PKC. These results suggest that there are two different types of PKC, normal and low Ca(2+)-sensitive in SHRSP platelets. Defective P47 phosphorylation in SHRSP platelets might be attributable to the occurrence of this low Ca(2+)-sensitive PKC.
Ikeda
M
M
University of Shizuoka, Graduate School of Health Sciences, Japan.
Onda
T
T
Tomita
I
I
Tomita
T
T
D000818:Animals; D016718:Arachidonic Acid / Q000008:administration & dosage / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology*; D001798:Blood Proteins / Q000378:metabolism; D002118:Calcium / Q000097:blood*; D002561:Cerebrovascular Disorders / Q000097:blood* / Q000201:enzymology; D002850:Chromatography, Gel; D004305:Dose-Response Relationship, Drug; D010750:Phosphoproteins; D010766:Phosphorylation; D011493:Protein Kinase C / Q000097:blood*; D051381:Rats; D011918:Rats, Inbred SHR; D011921:Rats, Inbred WKY
D001798:Blood Proteins; D010750:Phosphoproteins; C039077:platelet protein P47; D016718:Arachidonic Acid; D011493:Protein Kinase C; D002118:Calcium