Purification and characterization of the beta-adrenergic receptor kinase.
The beta-adrenergic receptor kinase (beta-ARK) is a recently discovered enzyme which specifically phosphorylates the agonist-occupied form of the beta-adrenergic receptor (beta-AR) as well as the light-bleached form of rhodopsin. beta-ARK is present in a wide variety of mammalian tissues. The kinase can be purified from bovine cerebral cortex to greater than 90% homogeneity by sequential chromatography on Ultrogel AcA34, DEAE-Sephacel, CM-Fractogel, and hydroxylapatite. This results in an approximately 20,000-fold purification with an overall recovery of 12%. The purified kinase has an Mr approximately 80,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Several findings indicate that this peptide contains the beta-ARK activity. First, on hydroxylapatite chromatography the enzyme activity coelutes with the Mr approximately 80,000 protein as revealed by Coomassie-Blue staining. Second, under phosphorylating conditions the Mr approximately 80,000 protein is phosphorylated. Finally, the Mr approximately 80,000 protein specifically interacts with reconstituted agonist-occupied beta-AR. Kinetic parameters of the enzyme for beta-AR are Km = 0.25 microM and Vmax = 78 nmol/min/mg whereas for rhodopsin the values are Km = 6 microM and Vmax = 72 nmol/min/mg. The Km value of the enzyme for ATP is approximately 35 microM using either beta-AR or rhodopsin as substrate. Receptor phosphorylation by beta-ARK is effectively inhibited by Zn2+, digitonin and a variety of salts. The availability of purified beta-ARK should greatly facilitate studies of its role in receptor desensitization.
D000818:Animals; D002417:Cattle; D002540:Cerebral Cortex / Q000201:enzymology; D002850:Chromatography, Gel; D006224:Cricetinae; D005136:Eye Proteins; D051606:G-Protein-Coupled Receptor Kinase 1; D007527:Isoenzymes / Q000302:isolation & purification*; D007700:Kinetics; D008168:Lung / Q000032:analysis; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008970:Molecular Weight; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000201:enzymology; D014018:Tissue Distribution
D005136:Eye Proteins; D007527:Isoenzymes; D011943:Receptors, Adrenergic, beta; D008345:Manganese; D011494:Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1; D008274:Magnesium