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Purification and characterization of the beta-adrenergic receptor kinase.

Publication ,  Journal Article
Benovic, JL; Mayor, F; Staniszewski, C; Lefkowitz, RJ; Caron, MG
Published in: J Biol Chem
July 5, 1987

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.

Duke Scholars

Published In

J Biol Chem

ISSN

0021-9258

Publication Date

July 5, 1987

Volume

262

Issue

19

Start / End Page

9026 / 9032

Location

United States

Related Subject Headings

  • Tissue Distribution
  • Rod Cell Outer Segment
  • Receptors, Adrenergic, beta
  • Protein Kinases
  • Molecular Weight
  • Manganese
  • Magnesium
  • Lung
  • Kinetics
  • Isoenzymes
 

Citation

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Benovic, J. L., Mayor, F., Staniszewski, C., Lefkowitz, R. J., & Caron, M. G. (1987). Purification and characterization of the beta-adrenergic receptor kinase. J Biol Chem, 262(19), 9026–9032.
Benovic, J. L., F. Mayor, C. Staniszewski, R. J. Lefkowitz, and M. G. Caron. “Purification and characterization of the beta-adrenergic receptor kinase.J Biol Chem 262, no. 19 (July 5, 1987): 9026–32.
Benovic JL, Mayor F, Staniszewski C, Lefkowitz RJ, Caron MG. Purification and characterization of the beta-adrenergic receptor kinase. J Biol Chem. 1987 Jul 5;262(19):9026–32.
Benovic, J. L., et al. “Purification and characterization of the beta-adrenergic receptor kinase.J Biol Chem, vol. 262, no. 19, July 1987, pp. 9026–32.
Benovic JL, Mayor F, Staniszewski C, Lefkowitz RJ, Caron MG. Purification and characterization of the beta-adrenergic receptor kinase. J Biol Chem. 1987 Jul 5;262(19):9026–9032.

Published In

J Biol Chem

ISSN

0021-9258

Publication Date

July 5, 1987

Volume

262

Issue

19

Start / End Page

9026 / 9032

Location

United States

Related Subject Headings

  • Tissue Distribution
  • Rod Cell Outer Segment
  • Receptors, Adrenergic, beta
  • Protein Kinases
  • Molecular Weight
  • Manganese
  • Magnesium
  • Lung
  • Kinetics
  • Isoenzymes