Rhodopsin kinase: substrate specificity and factors that influence activity.
Rhodopsin kinase was prepared from bovine retinas by the method of Sitaramayya [Sitaramayya, A. (1986) Biochemistry 25, 5460] with some minor modifications. The enzyme is able to phosphorylate bovine rhodopsin in the disk membrane, rhodopsin from other species, and rhodopsin solubilized in mild detergent (dodecyl maltoside). Rhodopsin kinase can phosphorylate synthetic peptides containing the appropriate sequences from bovine rhodopsin; however, the Km values for these peptides are about 3 orders of magnitude higher than that for rhodopsin or ATP. Some peptides from the cytosolic surface of rhodopsin inhibit the phosphorylation. These results suggest that more than one region of rhodopsin is involved in the interaction of rhodopsin of the kinase. Mg2+ is required for the Mg-ATP complex as shown by the observation that (ethylenedinitrilo)tetraacetic acid inhibits kinase activity. Second, free Mg2+ above the concentration required to complex all of the ATP present activates the kinase. Third, higher concentrations of Mg2+ yield Mg-ATP-Mg instead of Mg-ATP and therefore inhibit the kinase activity. Other physiologically important cations such as Ca2+, Na+, and K+ reduce the activity of the kinase, probably by forming a metal ion-ATP complex, thereby reducing the concentration of Mg-ATP. 5'-[p-(Fluorosulfonyl)benzoyl]adenosine (FSO2BzAdo), an inhibitor of kinases and ATPases, inhibits rhodopsin kinase according to pseudo-first-order kinetics. The relationship between the first-order constant and the concentration of FSO2BzAdo is hyperbolic. This indicates that a reversible complex between the ATP analogue and the enzyme is formed prior to the covalent attachment of the analogue to rhodopsin kinase.(ABSTRACT TRUNCATED AT 250 WORDS)
Palczewski
K
K
Department of Ophthalmology, University of Florida, Gainesville 32610-0284.
McDowell
J H
JH
Hargrave
P A
PA
D000107:Acetylation; D000818:Animals; D002412:Cations; D002417:Cattle; D005136:Eye Proteins; D051606:G-Protein-Coupled Receptor Kinase 1; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D010786:Photoreceptor Cells / Q000201:enzymology*; D011494:Protein Kinases / Q000378:metabolism*; D012243:Rhodopsin / Q000302:isolation & purification / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000201:enzymology*; D013379:Substrate Specificity
D002412:Cations; D005136:Eye Proteins; D010446:Peptide Fragments; D012243:Rhodopsin; D011494:Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1; D008274:Magnesium