[H-3] ACETAZOLAMIDE BINDING TO CARBONIC-ANHYDRASE IN NORMAL AND TRANSFORMED-CELLS

The binding of [H-3]acetazolamide (AZ), a carbonic anhydrase (CA) inhibitor, to soluble and particulate forms of CA was investigated. Sources for the assays were purified CA II, adult rat cortical, oligodendrocyte and neuronal enriched preparations; cultured murine glial cells, rat C-6 glioma, rat hepatoma and human glioblastoma cells. CA enzyme activity in the same preparations was also assayed by following change in pH during incubation. A gel permeation chromatographic method was developed to assess [H-3]AZ binding to soluble CA, while glass fiber filter vacuum filtration was used for particulate CA binding. Saturable specific binding of [H-3]AZ to rat cortical soluble and particulate CA preparations was demonstrated. Computer-assisted data analysis estimated the binding parameters of [H-3]AZ to soluble rat cortical CA to be B(max) = 0.38 +/- 0.13 pmol/mg protein and K(d) = 34.7 +/- 17.5 nM. The rat cortical particulate fraction B(max) was 2.05 +/- 0.28 pmol/mg protein with a K(d) of 107.1 +/- 24.2 nM. Purified bovine CA-II bound 1.15 +/- 0.19 pmol [H-3]AZ/mg protein with a K(d) of 54.0 +/- 3.4 nM. The pH optima for [H-3]AZ binding to soluble and particulate CA was between 6.5 and 7.5. Binding was linear with respect to protein up to 1.0 mg/mL. The particulate fraction bound 3-4 times more [H-3]ligand per unit protein than the soluble fraction. Interestingly, no detectable CA enzyme activity or [H-3]AZ binding was observed in the soluble or particulate fractions of human glioblastoma, rat C-6 glioma or rat hepatoma cells. Binding of [H-3]AZ to other soluble enzymes or proteins was negligible. In competition binding experiments, a rank order of inhibition of [H-3]AZ binding to rat cortical CA by established CA inhibitors was: dichlorphenamide > acetazolamide greater-than-or-equal-to benzolamide > methazolamide > hydrochlorothiazide greater-than-or-equal-to sulfanilamide. [H-3]AZ binding was not affected by other classes of pharmacologic characterizing agents. The binding of [H-3]AZ to the CA enzyme molecule is highly specific and sensitive and may prove useful in vitro or in situ as a probe for this enzyme.