OSSEOUS PLATE ALKALINE-PHOSPHATASE IS ANCHORED BY GPI

Alkaline phosphatase activity was released up to 100% from the membran e by using 0.1 U of phosphatidylinositol-specific phospholipase C from B. thuringiensis. The Mr of solubilized enzyme was 145,000 by Sephacr yl S-300 gel filtration and 66,000 by SDS-PAGE, suggesting a dimeric s tructure. Solubilization of the membrane-bound enzyme with phospholipa se C did not destroy its ability to hydrolyze p-nitrophenyl phosphate (PNPP) (264.3 mu mol min(-1) mg(-1)), ATP (42.0 mu mol min(-1) mg(-1)) and pyrophosphate (28.4 mu mol min(-1) mg(-1)). The hydrolysis of ATP and PNPP by solubilized enzyme exhibited ''Michaelian'' kinetics with K-0.5 = 70 and 979 mu M, respectively. For pyrophosphate, K-0.5 was 1 28 mu M and site-site interactions were observed (n = 1.4). Magnesium ions were stimulatory (K-d = 1.5 mM) but zinc ions were powerful non-c ompetitive inhibitors (K-d = 6.2 mu M) of solubilized enzyme. Treatmen t of solubilized alkaline phosphatase with Chellex 100 reduced the ori ginal PNPPase activity to 5%. Cobalt (K-0.5 = 10.1 mu M), magnesium (K -0.5 = 29.5 mu M) and manganese ions (K-0.5 = 5 mu M) restored the act ivity of the apoenzyme with positive cooperativity, suggesting that ph osphatidylinositol-specific phospholipase C-solubilized alkaline phosp hatase is a metalloenzyme. The stimulation of the apoenzyme by calcium ions (K-0.5 = 653 mu M) was lower than that observed for the other io ns (26%) and exhibited site-site interactions (n = 0.7). Zinc ions had no effect on the apoenzyme of the solubilized enzyme.