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• W2076243509 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 Bmax = 0.38 ± 0.13 pmol/mg protein and Kd = 34.7 ± 17.5 nM. The rat cortical particulate fraction Bmax was 2.05 ± 0.28 pmol/mg protein with a Kd of 107.1 ± 24.2 nM. Purified bovine CA-II bound 1.15 ± 0.19pmol [3H]AZ/mg protein with a Kd 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." @default.
• W2076243509 created "2016-06-24" @default.
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• W2076243509 date "1991-03-01" @default.
• W2076243509 modified "2023-09-24" @default.
• W2076243509 title "[3H]Acetazolamide binding to carbonic anhydrase in normal and transformed cells" @default.
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• W2076243509 doi "https://doi.org/10.1016/0006-2952(91)90206-k" @default.
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