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Journal of Histochemistry and Cytochemistry, Vol. 47, 545-550, April 1999, Copyright © 1999, The Histochemical Society, Inc.

ARTICLE

Localization of Carbonic Anhydrase in Living Osteoclasts with Bodipy 558/568-modified Acetazolamide, a Thiadiazole Carbonic Anhydrase Inhibitor

Kristen D. Brubakera, Fei Maoc, and Carol V. Gaya,b
a Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania
b Department of Poultry Science, Pennsylvania State University, University Park, Pennsylvania
c Molecular Probes, Inc., Eugene, Oregon

Correspondence to: Carol V. Gay, 108 Althouse Laboratory, University Park, PA 16802.

We describe the synthesis of Bodipy 558/568-modified acetazolamide, a fluorescent inhibitor of carbonic anhydrase and its use to localize the enzyme in living cells. The modified acetazolamide, with its specific sulfonamide group intact, labeled cells at concentrations as low as 10-9 M, with a minimal loading time of 5 min. The staining was decreased by 57.4% by preincubating cells with unaltered acetazolamide (1:100) or with trifluoromethane sulfonamide, 6-ethoxyzolamide, and 5-(3-hydroxybenzoyl)-thiophene-2-sulfonamide. The efficacy of the inhibitor was unchanged by the fluorescent label, as determined by an acridine orange assay that detects acidification of osteoclasts, the cell model used in this study. This compound should prove to be useful for studying carbonic anhydrase in many organisms because of the high degree of conservation of the active site of this enzyme. (J Histochem Cytochem 47:545–550, 1999)

Key Words: osteoclasts, carbonic anhydrase, acetazolamide, fluorescent microscopy


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