| Abstract: | In this paper we report the detection of phospholipase C activity in cultured human skin fibroblasts by a rapid, sensitive method. Sonicates of fibroblasts were incubated with L-3-phosphatidyl-[U-14C]-inositol and the incubation mixture extracted with chloroform/methanol. The solvent components were then separated into 2 phases by the addition of 2 M KCl. Phospholipase C activity, determined from the amount of [14C] in the aqueous phase, agreed well with the enzyme activity assessed by other methods. The optimum pH for the enzyme was 7.0 and the enzyme was found to be dependent on Ca2+ and deoxycholate for optimal activity. The demonstration of phospholipase C activity by this method in cultured skin fibroblasts provides a useful means with which to study, in human tissues, the physiological control of this enzyme and its derangements in disease states in a controlled fashion. |
