Osseous plate alkaline phosphatase is anchored by GPI.
Alkaline phosphatase activity was released up to 100% from the membrane by using 0.1 U of phosphatidylinositol-specific phospholipase C from B. thuringiensis. The M(r) of solubilized enzyme was 145,000 by Sephacryl S-300 gel filtration and 66,000 by SDS-PAGE, suggesting a dimeric structure. Solubilization of the membrane-bound enzyme with phospholipase C did not destroy its ability to hydrolyze p-nitrophenyl phosphate (PNPP) (264.3 mumol min-1 mg-1),ATP (42.0 mumol min-1 mg-1) and pyrophosphate (28.4 mumol min-1 mg-1). The hydrolysis of ATP and PNPP by solubilized enzyme exhibited "Michaelian" kinetics with K0.5 = 70 and 979 microM, respectively. For pyrophosphate, K0.5 was 128 microM and site-site interactions were observed (n = 1.4). Magnesium ions were stimulatory (Kd = 1.5 mM) but zinc ions were powerful non-competitive inhibitors (Kd = 6.2 microM) of solubilized enzyme. Treatment of solubilized alkaline phosphatase with Chellex 100 reduced the original PNPPase activity to 5%. Cobalt (K0.5 = 10.1 microM), magnesium (K0.5 = 29.5 microM) and manganese ions (K0.5 = 5 microM) restored the activity of the apoenzyme with positive cooperativity, suggesting that phosphatidylinositol-specific phospholipase C-solubilized alkaline phosphatase is a metalloenzyme. The stimulation of the apoenzyme by calcium ions (K0.5 = 653 microM) was lower than that observed for the other ions (26%) and exhibited site-site interactions (n = 0.7). Zinc ions had no effect on the apoenzyme of the solubilized enzyme.
Pizauro
J M
JM
Departamento de Technologia, Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal, UNESP, Brasil.
Ciancaglini
P
P
Leone
F A
FA
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001413:Bacillus thuringiensis / Q000302:isolation & purification; D017261:Glycosylphosphatidylinositols / Q000737:chemistry*; D006132:Growth Plate / Q000201:enzymology*; D007700:Kinetics; D051381:Rats; D010738:Type C Phospholipases / Q000378:metabolism
D017261:Glycosylphosphatidylinositols; D000469:Alkaline Phosphatase; D010738:Type C Phospholipases