3H]Acetazolamide binding to carbonic anhydrase in normal and transformed cells

Abstract

The binding of [ 3H]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 [ 3H]AZ binding to soluble CA, while glass fiber filter vacuum filtration was used for paniculate CA binding. Saturable specific binding of [ 3H]AZ to rat cortical soluble and particulate CA preparations was demonstrated. Computer-assisted data analysis estimated the binding parameters of [ 3H]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 [ 3H]AZ/mg protein with a K d of 54.0 ± 3.4 nM. The pH optima for [ 3H]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 [ 3H]ligand per unit protein than the soluble fraction. Interestingly, no detectable CA enzyme activity or [ 3H]AZ binding was observed in the soluble or particulate fractions of human glioblastoma, rat C-6 glioma or rat hepatoma cells. Binding of [ 3H]AZ to other soluble enzymes or proteins was negligible. In competition binding experiments, a rank order of inhibition of [ 3H]AZ binding to rat cortical CA by established CA inhibitors was: dichlorphenamide > acetazolamide ⩾ benzolamide > methazolamide > hydrochlorothiazide ⩾ sulfanilamide. [ 3H]AZ binding was not affected by other classes of pharmacologic characterizing agents. The binding of [ 3H]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.