Pizauro J M, Ciancaglini P, Leone F A
Departamento de Technologia, Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal, UNESP, Brasil.
Braz J Med Biol Res. 1994 Feb;27(2):453-6.
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.
使用来自苏云金芽孢杆菌的0.1 U磷脂酰肌醇特异性磷脂酶C可使碱性磷酸酶活性从膜中释放达100%。通过Sephacryl S - 300凝胶过滤，溶解酶的相对分子质量为145,000，通过SDS - PAGE测定为66,000，表明其为二聚体结构。用磷脂酶C溶解膜结合酶不会破坏其水解对硝基苯磷酸酯（PNPP）（264.3 μmol·min⁻¹·mg⁻¹）、ATP（42.0 μmol·min⁻¹·mg⁻¹）和焦磷酸（28.4 μmol·min⁻¹·mg⁻¹）的能力。溶解酶对ATP和PNPP的水解表现出“米氏”动力学，K0.5分别为70和979 μM。对于焦磷酸，K0.5为128 μM且观察到位点间相互作用（n = 1.4）。镁离子具有刺激作用（Kd = 1.5 mM），但锌离子是溶解酶的强效非竞争性抑制剂（Kd = 6.2 μM）。用Chellex 100处理溶解的碱性磷酸酶可使原始PNPP酶活性降至5%。钴（K0.5 = 10.1 μM）、镁（K0.5 = 29.5 μM）和锰离子（K0.5 = 5 μM）以正协同性恢复脱辅基酶的活性，表明磷脂酰肌醇特异性磷脂酶C溶解碱性磷酸酶是一种金属酶。钙离子（K0.5 = 653 μM）对脱辅基酶的刺激作用低于其他离子（26%）且表现出位点间相互作用（n = 0.7）。锌离子对溶解酶的脱辅基酶无影响。
