A NONRADIOACTIVE FLUORESCENT METHOD FOR MEASURING PROTEIN-KINASE-C ACTIVITY

The purpose of this study is to report the development of a non-radioa ctive fluorescent peptide assay for measuring protein kinase C activit y (PKC). The assay is based on a glycogen synthase derived fluorescent peptide that is phosphorylated by PKC. Phosphorylation causes the pep tide to migrate toward the anode while the non-phosphorylated peptide migrates toward the cathode during agarose gel electrophoresis. Quanti tation of PKC activity can be accomplished by excision of the appropri ate bands and measuring their relative fluorescence. Using this assay, PKC activity was measured in whole cell homogenates from cultured ren al mesangial cells. The enzyme(s)-substrate system followed Michaelis- Menten kinetics under limited conditions and, therefore, Lineweaver-Bu rk plots were used to obtain Michaelis constant and maximum velocity v alues. An apparent K-M value of 40 mu M was obtained for the fluoresce nt peptide substrate with a control V-max value of 300 pmol/min. Addit ion of phorbol 12-myristate 13-acetate increased V-max to 380 pmol/min .