Characterization of the phosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossification

• Autor(a) principal: Pizauro, João M. [UNESP]
• Data de Publicação: 1995
• Outros Autores: Ciancaglini, Pietro, Leone, Francisco A.
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: Repositório Institucional da UNESP
• Texto Completo: http://dx.doi.org/10.1007/BF01076074; http://hdl.handle.net/11449/224010
• topic: alkaline phosphatase; hydrophobic chromatography; osseous plate; p-nitrophenyl phosphate; phosphatidylinositol-specific phospholipase C; phospholipase C

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 μM, respectively. For pyrophosphate, K0.5 was 128 μM 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 μM) of phosphatidylinositol-specific phospholipase C-released enzyme. Phosphatidylinositol-specific phospholipase C-released alkaline phosphatase was relatively stable at 40°C. However, with increasing temperature from 40-60°C, the enzyme was inactivated rapidly following first order kinetics and thermal inactivation constants varied from 5.08×10-4 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 μM), manganese (K0.5=5 μM) and cobalt ions (K0.5=10.1 μM) restored the activity of Chelex-treated enzyme, demonstrating its metalloenzyme nature. The stimulation of Chelex-treated enzyme by calcium ions (K0.5=653 μM) 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 P1-anchored form of osseous plate alkaline phosphatase is discussed. © 1995 Kluwer Academic Publishers.