Purification and characterization of bone-specific alkaline phosphatase from a human osteosarcoma cell line.
Bone-specific alkaline phosphatase (bone ALP) levels are considered to reflect osteoblastic activity and can therefore be used as a marker of bone formation. However, bone ALP is difficult to distinguish from other ALP isoforms since the kidney, liver, and bone isoenzymes are encoded by the same gene and only differ because of post-translational modification of their carbohydrate side chains. The aim of this study was to purify and separate bone ALP which could be used to raise specific antisera against human bone ALP, from Saos-2, a human osteogenic sarcoma cell line. The procedure involved two steps. The first step, cultivation of 10(5) Saos-2 cells, yielded approximately 1 U ALP. Subsequent butanol extraction achieved 1.82-fold purification. For the second step, separating bone ALP, we used serial lectin affinity chromatography to distinguish between the carbohydrate side chains of the various ALP isoforms. A sample of the butanol extract was fractionated into three peaks (I, II, and III) by concanavalin A. Peaks II and III were subsequently identified as types IIa and IIIb bone ALP using pea lectin and wheat germ agglutinin columns, respectively. The specific activity of bone ALP was measured using commercial kits. Since bone ALP accounted for at least 84% of the total ALP activity after the final separation, this method appears more convenient and reproducible than others using bone or Pagetic sera. The bone ALP purified in this study could be used to raise monoclonal antibodies against bone-specific ALP.
Nakayama
M
M
Department of Obstetrics and Gynecology, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku Yokohama, 236, Japan.
Gorai
I
I
Minaguchi
H
H
Rosenquist
C
C
Qvist
P
P
D000469:Alkaline Phosphatase / Q000032:analysis* / Q000302:isolation & purification* / Q000378:metabolism; D001842:Bone and Bones / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D012685:Sepharose; D012967:Sodium Dodecyl Sulfate; D014407:Tumor Cells, Cultured
D012967:Sodium Dodecyl Sulfate; D012685:Sepharose; D000469:Alkaline Phosphatase