Ca2+ sensitivity of Ca2+-dependent protein kinase activities toward intrinsic proteins in synaptosomal membrane fragments from rat cerebral tissue.
The Ca2+ and calmodulin sensitivity of endogenous protein kinase activity in synaptosomal membrane fragments from rat brain was studied in medium containing Ca2+ plus EGTA using a modified computer programme to calculate free Ca2+ concentrations that took into account the effect of all competing cations and chelators. The Ca2+-dependent phosphorylation of 10 major polypeptide acceptors with Mr values ranging from 50 to 360 kilodaltons required calmodulin in reactions that were all equally sensitive to Ca2+; half-maximal phosphorylation required a free Ca2+ concentration of 45 nM and maximal phosphorylation approximately 110 nM. The significance of these values in relation to published data on the intracellular concentration of free Ca2+ in the nervous system is discussed. One acceptor of 45 kilodaltons was phosphorylated in a Ca2+-dependent reaction that did not require calmodulin. This polypeptide appeared to correspond to the B-50 protein, an established substrate of the lipid-dependent protein kinase C. Further study of this phosphorylating system showed that the reaction was only independent of calmodulin at saturating concentrations of Ca2+; at subsaturating concentrations (in the range 50-130 nM), a small but significant stimulation of the enzyme by calmodulin was demonstrated. The possible significance of this finding is discussed.
Gower
H
H
Rodnight
R
R
Brammer
M J
MJ
D000818:Animals; D001921:Brain / Q000648:ultrastructure*; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D016704:Synapsins; D013570:Synaptic Membranes / Q000378:metabolism*; D013574:Synaptosomes / Q000648:ultrastructure*
D002147:Calmodulin; D009419:Nerve Tissue Proteins; D016704:Synapsins; D011494:Protein Kinases; D002118:Calcium