Culture of osteogenic cells from human alveolar bone: a useful source of alkaline phosphatase.
The aim of this study was to obtain membrane-bound alkaline phosphatase from osteoblastic-like cells of human alveolar bone. Cells were obtained by enzymatic digestion and maintained in primary culture in osteogenic medium until subconfluence. First passage cells were cultured in the same medium and at 7, 14, and 21 days, total protein content, collagen content, and alkaline phosphatase activity were evaluated. Bone-like nodule formation was evaluated at 21 days. Cells in primary culture at day 14 were washed with Tris-HCl buffer, and used to extract the membrane-bound alkaline phosphatase. Cells expressed osteoblastic phenotype. The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10.0. This enzyme also hydrolyzes ATP, ADP, fructose-1-phosphate, fructose-6-phosphate, pyrophosphate and beta-glycerophosphate. PNPPase activity was reduced by typical inhibitors of alkaline phosphatase. SDS-PAGE of membrane fraction showed a single band with activity of approximately 120 kDa that could be solubilized by phospholipase C or Polidocanol.
Simão
Ana Maria S
AM
Depto Química, FFCLRP, Universidade de São Paulo, Av. Bandeirantes, 3900, 14040-901, Ribeirão Preto, SP, Brazil.
Beloti
Marcio M
MM
Rosa
Adalberto L
AL
de Oliveira
Paulo T
PT
Granjeiro
José Mauro
JM
Pizauro
João M
JM
Ciancaglini
Pietro
P
D000469:Alkaline Phosphatase / Q000302:isolation & purification* / Q000378:metabolism; D001842:Bone and Bones / Q000166:cytology / Q000201:enzymology*; D003094:Collagen / Q000378:metabolism; D006801:Humans; D010006:Osteoblasts / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*
D003094:Collagen; D000469:Alkaline Phosphatase