Tartrate-resistant acid phosphatase from human bone: purification and development of an immunoassay.
Tartrate-resistant acid phosphatase (TRAP) was purified 20,000-fold to apparent homogeneity from human bone. The purified enzyme consisted of one 32 kd subunit, which was cleaved by beta-mercaptoethanol into two subunits of 15 kd and 20 kd, as shown by sodium dodecyl sulfide-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. The purified enzyme was identified by N-terminal amino acid sequencing, and it was shown to be homologous with previously purified TRAPs from other sources. We developed a polyclonal antiserum against the purified enzyme in mice. In immunohistochemistry, the antiserum recognized osteoclasts from human bone and alveolar macrophages from human lung tissue, but no cells from human spleen tissue. It also stained osteoclasts from rat bone cells cultured on bovine bone slices. Purified TRAP could be inhibited by vanadate and molybdate, but not by tartrate, and it was activated 2-fold by beta-mercaptoethanol. The glycoprotein structure of human bone TRAP was analyzed, and it was shown to contain only high-mannose type carbohydrates. We used the polyclonal antibody to develop a competitive fluorescence immunoassay for measuring serum TRAP concentrations. According to the assay, children have higher serum TRAP concentrations than adults, and postmenopausal women have higher concentrations than premenopausal women. Postmenopausal women also have higher serum TRAP concentrations than postmenopausal women on estrogen replacement therapy.
Department of Anatomy, University of Oulu, Finland.
J
J
Halleen
T A
TA
Hentunen
J
J
Hellman
H K
HK
Väänänen
D000135:Acid Phosphatase / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D014408:Biomarkers, Tumor / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D002648:Child; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005270:Femur Head / Q000166:cytology / Q000378:metabolism; D006023:Glycoproteins / Q000032:analysis / Q000378:metabolism; D006622:Hip Prosthesis; D006801:Humans; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D016676:Macrophages, Alveolar / Q000166:cytology / Q000378:metabolism; D008358:Mannose / Q000378:metabolism; D008623:Mercaptoethanol / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism; D015663:Osteoporosis, Postmenopausal / Q000097:blood / Q000378:metabolism; D011233:Precipitin Tests; D051381:Rats; D016622:Silver Staining; D013154:Spleen / Q000166:cytology; D000071681:Tartrate-Resistant Acid Phosphatase
D014408:Biomarkers, Tumor; D006023:Glycoproteins; D007527:Isoenzymes; D008623:Mercaptoethanol; D000135:Acid Phosphatase; C000605511:Acp5 protein, mouse; D000071681:Tartrate-Resistant Acid Phosphatase; D008358:Mannose