Differential effects of phospholipids on skeletal alkaline phosphatase activity in extracts, in situ and in circulation

Abstract

Human skeletal alkaline phosphatase (ALP) purified from human bone was subject to competitive inhibitions by phospholipids including cephalins, lecithins, and phosphatidylinositol. K i values ranged from 0.7 to 1.5 m m, at pH 9.5. As previously shown, the enzyme was subject to uncompetitive inhibition by imidazole. The inhibitory phospholipids potentiated this effect, and altered the nature of the imidazole inhibition, from uncompetitive to mixed type, suggesting that imidazole was bound more efficiently to the enzyme-phospholipid complex than to the enzyme-substrate complex. No interactions were observed between phospholipids and other uncompetitive inhibitors of ALP. The skeletal ALP activity of cultured chick calvarial cells was assayed both in situ and in extracts. Like the extracted human isoenzyme, the extracted chick ALP was subject to competitive inhibition by cephalin ( K i = 0.3 mM at pH 9.3) and an inhibitory interaction between cephalin and imidazole, but the same isoenzyme showed neither effect in situ. The value of K m,PNPP at pH 9.5 for chick skeletal ALP was 1.5 m m in extracts and 7.1 m m in situ. When embryonic chick bones were cultured in vitro, skeletal ALP activity was released into the serum-free medium. Unlike the same isoenzyme extracted from the bones, the ALP activity in the medium was not inhibited by cephalin and showed no inhibitory interaction between cephalin and imidazole. Similarly, human serum ALP activities were not as sensitive to phospholipid inhibition as the same isoenzymes extracted from tissues. Human skeletal ALP extracted from bone was inhibited by cephalin, but the skeletal isoenzyme in Pagetic serum was not, suggesting that the potential for phospholipid interaction was altered during or after release from osteoblast cell membranes. The observation that extracted human skeletal ALP lost its potential for inhibition by phospholipids after treatment with phospholipase C further suggests that ALP activity may be released from cells during membrane turnover.