Calcium-independent phospholipid/diolein-dependent phosphorylation of a soluble ovarian Mr 80,000 substrate protein: biochemical characteristics.
Soluble ovarian extracts were incubated with protein kinase effectors in the presence of [gamma 32P]ATP and proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Autoradiograms revealed phosphorylation of an ovarian Mr = 80,000 substrate in the presence of EGTA ([ethylenebis(oxyethylenenitrilo)]tetraacetic acid), phosphatidylserine and 1,2-diolein. In contrast to a classical response pattern to C-kinase effectors, the ovarian Mr = 80,000 phosphorylation was inhibited by 2 x 10(-7) M or greater free Ca2+. The ovarian Mr = 80,000 substrate was distinguished from the myristoylated acidic Mr = 80,000 C-kinase substrate of brain tissue on the basis of heat stability and phosphorylative response to effectors. Phosphorylation of the exogenous substrate myelin basic protein by DEAE-resolved ovarian kinase showed the variant effector dependence, maximal in the presence of EGTA, phosphatidylserine and 1,2-diolein. Finally, the effect of Ca2+ on ovarian Mr = 80,000 [32P]phosphate content could not be accounted for by post-phosphorylation activities, or by DEAE-resolvable or hydroxylapatite-resolvable inhibitory activities.
Department of Cell, Molecular and Structural Biology, Northwestern University Medical School, Chicago, IL 60611.
E T
ET
Maizels
J B
JB
Miller
R E
RE
Cutler
V
V
Jackiw
E M
EM
Carney
L
L
Kern
M
M
Hunzicker-Dunn
D000818:Animals; D001921:Brain / Q000201:enzymology / Q000378:metabolism; D002118:Calcium / Q000502:physiology; D004075:Diglycerides / Q000502:physiology; D005260:Female; D008970:Molecular Weight; D004676:Myelin Basic Protein / Q000378:metabolism; D010053:Ovary / Q000201:enzymology / Q000378:metabolism*; D010743:Phospholipids / Q000502:physiology; D010750:Phosphoproteins / Q000378:metabolism*; D011494:Protein Kinases / Q000145:classification / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D004075:Diglycerides; D004676:Myelin Basic Protein; D010743:Phospholipids; D010750:Phosphoproteins; D011494:Protein Kinases; D002118:Calcium; C013965:diolein