title: Characterization of the phosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossification.
abstract: Alkaline phosphatase activity was released up to 100% from the membrane by incubating the rat osseous plate membrane-bound enzyme with phosphatidylinositol-specific phospholipase C. The molecular weight of the released enzyme was 145,000 on Sephacryl S-300 gel filtration and 66,000 on PAGE-SDS, suggesting a dimeric structure. Solubilization of the membrane-bound enzyme with phospholipase C did not destroy its ability to hydrolyse PNPP, ATP and pyrophosphate. The hydrolysis of ATP and PNPP by phosphatidylinositol-specific phospholipase C-released 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 (K0.5 = 1.5 mM) and zinc ions were a powerful noncompetitive inhibitor (Ki = 6.2 microM) of phosphatidylinositol-specific phospholipase C-released enzyme. Phosphatidylinositol-specific phospholipase C-released alkaline phosphatase was relatively stable at 40 degrees C. However, with increasing temperature from 40-60 degrees C, the enzyme was inactivated rapidly following first order kinetics and thermal inactivation constants varied from 5.08 x 10(-4) min-1 to 0.684 min-1. Treatment of phosphatydilinositol-specific phospholipase C-released alkaline phosphatase with Chellex 100 depleted to 5% its original PNPPase activity. Magnesium (K0.5 = 29.5 microM), manganese (K0.5 = 5 microM) and cobalt ions (K0.5 = 10.1 microM) restored the activity of Chelex-treated enzyme, demonstrating its metalloenzyme nature. The stimulation of Chelex-treated enzyme by calcium ions (K0.5 = 653 microM) was less effective (only 26%) and occurred with site-site interactions (n = 0.7). Zinc ions had no stimulatory effects. The possibility that the soluble form of the enzyme, detected during endochondral ossification, would arise by the hydrolysis of the Pl-anchored form of osseous plate alkaline phosphatase is discussed.
authors: Departamento de Tecnologia, UNESP, Brasil.
authors: J M
authors: JM
authors: Pizauro
authors: P
authors: P
authors: Ciancaglini
authors: F A
authors: FA
authors: Leone
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism*; D000818:Animals; D001842:Bone and Bones / Q000201:enzymology*; D008970:Molecular Weight; D010012:Osteogenesis / Q000502:physiology*; D010716:Phosphatidylinositols / Q000378:metabolism*; D051381:Rats; D012995:Solubility; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000378:metabolism*; D014867:Water / Q000737:chemistry
chemical_list: D010716:Phosphatidylinositols; D014867:Water; D000469:Alkaline Phosphatase; D010738:Type C Phospholipases