Differential solubilization of osteoblastic alkaline phosphatase from human primary bone cell cultures.
Mineralization of cartilage and bone requires alkaline phosphatase activity. In order to study the enzymatic properties of bone alkaline phosphatase in bone disease and more particularly in patients with osteoporosis and osteoarthritis, we investigated the solubilization of alkaline phosphatase from primary bone cell cultures derived from human bone explants. To study the release of alkaline phosphatase from membranes, several detergents at a concentration above the critical micellar concentration and cholesterol were used. Solubilized alkaline phosphatase was characterized by enzymatic activity and electrophoresis analysis. Almost all the alkaline phosphatase was solubilized using non-ionic detergent as n-octylglucoside and hecameg. In comparison with initial membranous activity, the solubilized activity was increased by a factor, i.e. 2 +/- 0.05 (SEM, n = 3) (with n-octylglucoside), i.e. 2.1 +/- 0.05 (SEM, n = 3) (with Hecameg). With an ionic detergent (sodium dodecylsulfate), zwitterionic detergent ((cholamido propyl) dimethylammonio 1 propane sulfonate) and cholesterol, a fraction of alkaline phosphatase was resistant to solubilization. Electrophoresis studies showed that released alkaline phosphatase was a glycosylphosphatidylinositol protein (amphipatic form) with 140 kDa as apparent molecular weight. A hydrophilic form was obtained by treatment with a specific lipase. This study showed differential solubilization of osteoblastic alkaline phosphatase from human primary bone cell cultures. Better extractibility and higher activation of this membrane anchored enzyme were obtained with non-ionic detergents.
Radisson
J
J
Laboratoire de Physico-chimie Biologique, CNRS URA 1535, Université Claude Bernard Lyon I, France.
Angrand
M
M
Chavassieux
P
P
Roux
B
B
Azzar
G
G
D000469:Alkaline Phosphatase / Q000378:metabolism*; D002478:Cells, Cultured; D002784:Cholesterol / Q000494:pharmacology; D003902:Detergents; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006801:Humans; D010006:Osteoblasts / Q000201:enzymology*; D012995:Solubility
D003902:Detergents; D017261:Glycosylphosphatidylinositols; D002784:Cholesterol; D000469:Alkaline Phosphatase