Extraction, Purification And Gene Cloning Of Alkaline Phosphatase And Effects Of Calcium Hydroxide On Its Activity And Gene Expression In Pulp Cells

Alkaine phosphatase (ALP)is a ubiquitous enzyme expressed at high levels in cells of mineralizing tissues. There are three kinds of ALP. i.e. intestinal. placental and tiver/bone/kidney type (L/B/K) ALP. Dental pulp is rich in ALP, of which the nature is similar to L/B/K-ALP. As ALP commonly exists in calcifying area, research on the structure, function and expression of dental pulp ALP will be helpful to clarify the mineralization mechanisms and the repairing of pulp and dentine.Calcium hydroxide is one of the best pulp capping agent as it has specific functions of accelerating the repairing process of dental pulp and formation of dentinal bridge. The mechanism of it is up to now uncertain. Recent investigations showed that pulp cells had the ability of differentiation and migration after pulp irritation. So. the study of the effects of calcium hydroxide on pulp cells will shed new light on the mineralization mechanisms.In our study, ALP of canine dental pulp was extracted with n-butanol and purified 24.6—folds using DEAE-cellulose chromatography and Sephadex G-200 gel filtration, to a final specific activity of 27units/mg protein. On polvacrvlamide gel electrophoresis (PAGE), the rough and purified enzymes gave only one band. The pH optimum for the enzyme was found to be 10. 1 with p-nitrophenyl phosphate (p-NPP) at 5mM. The saturation of substrate p-NPP was proven to be 6mM. On SDS-PAGE. the enzyme gave two bands with Mw. as 64500 and 15900 respectively. It was shown that the enzyme could be stimulated by Mg2+ and inhibited by canion chelator EDTA. In the abscence of Mg2+, both Zn2+ and...