Purification and functional characterization of beta-adrenergic receptor kinase expressed in insect cells.
The beta-adrenergic receptor kinase mediates agonist-dependent phosphorylation of beta-adrenergic receptors, which is thought to represent the first step of homologous desensitization. We have expressed bovine and human beta ARK1 in Sf9 cells and purified them to apparent homogeneity in milligram quantities. The Km-values of the enzyme were 3.8 microM for rhodopsin and 22 microM for ATP; the Vmax-value was 9.9 mol phosphate/mol beta ARK/min. These data indicate that the two recombinant kinases were at least as active as preparations previously obtained from bovine brain. There were no differences in the functional activity of human and bovine beta ARK.
Laboratory of Molecular Biology, University of Munich, Max-Planck-Institute of Biochemistry, Martinsried, Germany.
P
P
Söhlemann
M
M
Hekman
C
C
Buchen
J S
JS
Elce
M J
MJ
Lohse
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002460:Cell Line; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases; D006801:Humans; D007700:Kinetics; D009036:Moths; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012243:Rhodopsin / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000378:metabolism; D014162:Transfection; D051552:beta-Adrenergic Receptor Kinases
D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D012243:Rhodopsin; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases