Purification and characterization of a tartrate-resistant acid phosphatase from human osteoclastomas.
Tartrate-resistant acid phosphatase is one of the major enzymes produced and secreted by osteoclasts. To obtain sufficient enzyme for biochemical characterization, we have purified this enzyme from human osteoclastomas by sequential chromatography on SP-Sephadex, CM-Sephadex, hydroxylapatite, Sephadex G-150 and concanavalin A-Sepharose. The purification over the original tumour extract was about 2000-fold, with a yield of 10%. The enzyme appeared to be homogeneous when assessed by SDS/polyacrylamide-gel electrophoresis. Both gel filtration and SDS/polyacrylamide-gel electrophoresis indicated an Mr of about 30,000. The reduced and alkylated enzyme consists of two subunits with Mrs of 15,000 and 17,500. The N-terminal amino acid sequence of both subunits indicates that there is a high degree of identity between the osteoclastoma enzyme and similar enzymes purified from spleen and uterus. Using 4-methylumbelliferyl phosphate as substrate, the specific activity of the purified enzyme was 387 units.mg-1, and the Km was 284 microns. The pH optimum was 5.7. Unlike similar enzymes purified from human and bovine bone, osteoclastoma acid phosphatase is not activated by reducing agents (2-mercaptoethanol or ascorbic acid). The enzyme contains 4.8 mol of Fe2+/3+, 0.3 mol of Mn2+ and 1.7 mol of Mg2+ per mol of enzyme. Although the enzyme loses 50% of its activity in the presence of EDTA, it is not inhibited by the iron chelator 1,10-phenanthroline. However, the enzyme is activated to a small extent by Mn2+ and Mg2+. Using a variety of substrates and inhibitors, we demonstrate that there are differences between the osteoclastoma acid phosphatase and the enzyme purified from other sources.
Hayman
A R
AR
Department of Histopathology, St. George's Hospital Medical School, London, U.K.
Warburton
M J
MJ
Pringle
J A
JA
Coles
B
B
Chambers
T J
TJ
D000135:Acid Phosphatase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D001859:Bone Neoplasms / Q000201:enzymology*; D005870:Giant Cell Tumors / Q000201:enzymology*; D006801:Humans; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D013644:Tartrates
D013644:Tartrates; D000135:Acid Phosphatase