Erythrocyte ghost cell-alkaline phosphatase: construction and characterization of a vesicular system for use in biomineralization studies.
Alkaline phosphatase is required for the mineralization of bone and cartilage. This enzyme is localized in the matrix vesicle, which plays a role key in calcifying cartilage. In this paper we standardize a method to construction a resealed ghost cell-alkaline phosphatase system to mimic matrix vesicles and examine the kinetic behavior of the incorporated enzyme. Polidocanol-solubilized alkaline phosphatase, free of detergent, was incorporated into resealed ghost cells. This process was time-dependent and practically 50% of the enzyme was incorporated into the vesicles in 40 h of incubation, at 25 degrees C. Alkaline phosphatase-ghost cell systems were relatively homogeneous with diameters of about 300 nm and were more stable when stored at -20 degrees C. Alkaline phosphatase was completely released from the resealed ghost cell-system using only phospholipase C. These experiments confirm that the interaction between alkaline phosphatase and the lipid bilayer of resealed ghost cell is exclusively via glycosylphosphatidylinositol (GPI) anchor of the enzyme. An important point shown is that an enzyme bound to resealed ghost cell does not lose the ability to hydrolyze ATP, pyrophosphate and p-nitrophenyl phosphate (PNPP), but the presence of a ghost membrane, as a support of the enzyme, affects its kinetic properties. Moreover, calcium ions stimulate and phosphate ions inhibit the PNPPase activity of alkaline phosphatase present in resealed ghost cells.
Ierardi
Daniela F
DF
Departamento de Qui;mica, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, USP, Avenida Bandeirantes 3.900, 14040-901 SP, Ribeirão Preto, Brazil.
Pizauro
João M
JM
Ciancaglini
Pietro
P
D000469:Alkaline Phosphatase / Q000378:metabolism*; D000818:Animals; D001842:Bone and Bones / Q000648:ultrastructure; D002113:Calcification, Physiologic; D004910:Erythrocyte Membrane / Q000201:enzymology*; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D008854:Microscopy, Electron; D051381:Rats
D017261:Glycosylphosphatidylinositols; D000469:Alkaline Phosphatase