Meyerson L R, Nesta D
American Cyanamid Company, Medical Research Division, Lederle Laboratories, Pearl River, NY 10965.
Biochem Pharmacol. 1991;41(6-7):995-1000. doi: 10.1016/0006-2952(91)90206-k.
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 particulate 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 +/- 0.19 pmol [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 greater than acetazolamide greater than or equal to benzolamide greater than methazolamide greater than hydrochlorothiazide greater than or equal to 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.
研究了碳酸酐酶（CA）抑制剂[3H]乙酰唑胺（AZ）与可溶性和颗粒性CA的结合情况。实验所用的材料有纯化的CA II、成年大鼠皮质、富含少突胶质细胞和神经元的制剂；培养的小鼠胶质细胞、大鼠C-6胶质瘤细胞、大鼠肝癌细胞和人胶质母细胞瘤细胞。同时，通过孵育过程中pH值的变化来测定相同制剂中的CA酶活性。开发了一种凝胶渗透色谱法来评估[3H]AZ与可溶性CA的结合，而玻璃纤维滤膜真空过滤法用于颗粒性CA的结合。结果表明，[3H]AZ与大鼠皮质可溶性和颗粒性CA制剂存在可饱和的特异性结合。计算机辅助数据分析估计[3H]AZ与大鼠皮质可溶性CA的结合参数为：Bmax = 0.38 ± 0.13 pmol/mg蛋白质，Kd = 34.7 ± 17.5 nM。大鼠皮质颗粒部分的Bmax为2.05 ± 0.28 pmol/mg蛋白质，Kd为107.1 ± 24.2 nM。纯化的牛CA-II与[3H]AZ的结合量为1.15 ± 0.19 pmol [3H]AZ/mg蛋白质，Kd为54.0 ± 3.4 nM。[3H]AZ与可溶性和颗粒性CA结合的最适pH值在6.5至7.5之间。在蛋白质浓度高达1.0 mg/mL时，结合与蛋白质呈线性关系。颗粒部分每单位蛋白质结合的[3H]配体比可溶性部分多3 - 4倍。有趣的是，在人胶质母细胞瘤、大鼠C-6胶质瘤或大鼠肝癌细胞的可溶性或颗粒性部分未观察到可检测到的CA酶活性或[3H]AZ结合。[3H]AZ与其他可溶性酶或蛋白质的结合可忽略不计。在竞争性结合实验中，已有的CA抑制剂对[3H]AZ与大鼠皮质CA结合的抑制作用顺序为：二氯苯酰胺＞乙酰唑胺≥苯并酰胺＞甲唑酰胺＞氢氯噻嗪≥磺胺。[3H]AZ的结合不受其他类别的药理特性试剂影响。[3H]AZ与CA酶分子的结合具有高度特异性和敏感性，可能在体外或原位作为该酶的探针有用。
