N-myristoylation of recoverin enhances its efficiency as an inhibitor of rhodopsin kinase.
Recoverin, a recently identified member of the EF-hand superfamily of Ca(2+)-binding proteins, is capable to inhibit rhodopsin phosphorylation by rhodopsin kinase at high but not at low free [Ca2+]. The N-terminal glycine residue of retinal recoverin is heterogeneously acylated with myristoyl or related N-acyl group. To clarify the role of the N-terminal acylation of recoverin in its inhibitory action upon rhodopsin phosphorylation, we compared the efficiency of myristoylated and non-myristoylated forms of recombinant recoverin as inhibitors of rhodopsin kinase activity. We have found that rhodopsin phosphorylation by purified rhodopsin kinase, which does not depend on free [Ca2+] in the absence of recoverin, is regulated by Ca2+ in the presence of both forms of the recombinant protein. EC50 values for Ca2+ are the same (2 microM) for the myristoylated and non-myristoylated forms; the Hill coefficients of 1.7 and 0.9, respectively, indicate that the effect is cooperative with respect to Ca2+ only for myristoylated recoverin. In the presence of Ca2+, both forms of recoverin taken at saturated concentrations cause an almost equal inhibition of rhodopsin phosphorylation. However, the inhibitory action of the myristoylated form occurs at much lower its concentrations than that of the non-myristoylated form (EC50 are 0.9 and 6.5 microM, respectively).
Department of Enzymology, A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russian Federation.
I I
II
Senin
A A
AA
Zargarov
A M
AM
Alekseev
E N
EN
Gorodovikova
V M
VM
Lipkin
P P
PP
Philippov
D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D002417:Cattle; D005136:Eye Proteins; D051606:G-Protein-Coupled Receptor Kinase 1; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D051597:Hippocalcin; D008074:Lipoproteins; D008565:Membrane Proteins / Q000378:metabolism; D009226:Myristates / Q000494:pharmacology*; D009419:Nerve Tissue Proteins; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; D051603:Recoverin; D012160:Retina / Q000378:metabolism; D012243:Rhodopsin / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D005136:Eye Proteins; D008074:Lipoproteins; D008565:Membrane Proteins; D009226:Myristates; D009419:Nerve Tissue Proteins; D047428:Protein Kinase Inhibitors; D051603:Recoverin; D051597:Hippocalcin; D012243:Rhodopsin; D011494:Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1; D002118:Calcium