FIGURE 8.

Stoichiometric and kinetic analysis of PKA-catalyzed ³²P incorporation by synthetic peptide. Synthetic peptide KKSRSASTISK, corresponding to amino acid residues 445–455 of Kv1.2 and containing the putative PKA site Ser-449, was compared as a substrate to LLRRASLG (Kemptide) (positive control) and KKSRAASTISK (corresponding to the sequence of Kv1.2-S449A mutant, negative control). A, fixed concentrations of each peptide (50 μm) were treated with [γ-³²P]ATP and cPKA (1.0–5.0 μg/ml), and ³²P incorporation was quantified by C̆erenkov counting. Data are the means ± S.E. of three independent experiments carried out in triplicate. B, cPKA activity was determined by quantification of ³²P incorporation into KKSRSASTISK, Kemptide, and KKSRAASTISK peptides during the linear phase of the reaction (t = 1.5 min). Enzyme velocities were plotted against substrate concentration and fitted to the Michaelis-Menten equation to obtain K_m and V_max values.