Characterization of a new substrate for protein kinase C: assay by continuous fluorometric monitoring and high performance liquid chromatography.
A synthetic peptide derived from the phosphorylation site in the beta-subunit of phosphorylase kinase (RTKRSGSVYEPLKI) is an efficient substrate for rat brain protein kinase C: Km = 18 +/- 2 microM and Vmax = 2.1 +/- 0.1 mumol/min/mg. The phosphorylation of the peptide, which occurs at Ser7, can be followed by four independent procedures. 1. Standard measurement of 32P incorporation. 2. Reverse phase HPLC in a gradient system containing 0.1 M ammonium sulfate in the stationary phase. 3. Continuous fluorometric monitoring of the changes in intrinsic peptide fluorescence. 4. Continuous fluorometric determination of NADH oxidation in a coupled enzyme assay.
Zhao
Z H
ZH
Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331.
Malencik
D A
DA
Anderson
S R
SR
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D007700:Kinetics; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D010455:Peptides / Q000138:chemical synthesis*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D013050:Spectrometry, Fluorescence / Q000379:methods; D013379:Substrate Specificity
D010455:Peptides; D009243:NAD; D011493:Protein Kinase C