Affinity purification of the C alpha and C beta isoforms of the catalytic subunit of cAMP-dependent protein kinase.
A synthetic peptide of 18 amino acids corresponding to the inhibitory domain of the heat-stable protein kinase inhibitor was synthesized and shown to inhibit both the C alpha and C beta isoforms of the catalytic (C) subunit of cAMP-dependent protein kinase. Extracts from cells transfected with expression vectors coding for the C alpha or the C beta isoform of the C subunit required 200 nM protein kinase inhibitor peptide for half-maximal inhibition of kinase activity in extracts from these cells. An affinity column was constructed using this synthetic peptide, and the column was incubated with protein extracts from cells overexpressing C alpha or C beta. Elution of the affinity column with arginine allowed single step isolation of purified C alpha and C beta subunits. The C alpha and C beta proteins were enriched 200-400-fold from cellular extracts by this single step of affinity chromatography. No residual inhibitory peptide activity could be detected in the purified protein. The purified C subunit isoforms were used to demonstrate preferential antibody reactivity with the C alpha isoform by Western blot analysis. Furthermore, preliminary characterization showed both isoforms have similar apparent Km values for ATP (4 microM) and for Kemptide (5.6 microM). These results demonstrate that a combination of affinity chromatography employing peptides derived from the heat-stable protein kinase inhibitor protein and the use of cells overexpressing C subunit related proteins may be an effective means for purification and characterization of the C subunit isoforms. Furthermore, this method of purification may be applicable to other kinases which are known to be specifically inhibited by small peptides.
Olsen
S R
SR
Graduate Program in Cellular and Molecular Biology, University of Michigan, Ann Arbor 48109.
Uhler
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MD
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity; D000242:Cyclic AMP / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism
D007527:Isoenzymes; D009842:Oligopeptides; D010455:Peptides; D047428:Protein Kinase Inhibitors; C061479:protein kinase inhibitor peptide; C037642:kemptide; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011494:Protein Kinases