Wu Q, Delamere N A, Pierce W
Department of Pharmacology and Toxicology, University of Louisville, Kentucky 40292, USA.
Invest Ophthalmol Vis Sci. 1997 Sep;38(10):2093-102.
To measure the activity of membrane-associated carbonic anhydrase (CA) in cultured rabbit nonpigmented epithelial (NPE) cells, determine its identity and its sensitivity to extracellular trypsin, and compare the ability of acetazolamide and a cell-impermeant dextran-bound CA inhibitor to change cytoplasmic pH.
Studies were conducted using a cell line derived from rabbit NPE. The cells were lysed and separated into soluble and insoluble fractions by differential centrifugation. CA activity in these fractions was determined using a CO2 hydration assay. In studies with intact cells, a membrane-impermeable high-molecular-weight dextran-bound inhibitor (DBI) was synthesized and used to selectively bind and inhibit the extracellular-facing membrane-bound CA. Measurements of CA activity in intact red blood cells were conducted to confirm DBI remains extracellular. Acetazolamide, a membrane-permeable CA inhibitor, was used to inhibit total CA activity. Intracellular pH was determined using the pH-dependent absorbance of the fluorescent dye BCECF-AM.
A low-speed pellet enriched with plasma membrane material accounted for 22.3 +/- 6.1% (n = 18) of the total CA activity in the cultured NPE. When intact cells were exposed to trypsin-EDTA, a 28% reduction of membrane-associated CA activity was observed; DBI inhibited this CA activity loss. Cytosolic CA activity was inhibited by 0.2% sodium dodecyl sulfate (SDS). In contrast, membrane-associated CA was SDS resistant, a characteristic of the CA-IV isozyme. By Western blot, CA-IV immunoreactive polypeptide was detected in the cultured cells and also in native rabbit and porcine ciliary epithelium. Inhibition of total CA activity with acetazolamide and inhibition of extracellular-facing membrane-associated CA with DBI caused an identical intracellular pH decrease in intact NPE cells.
Expression of the CA-IV isozyme could account for the significant fraction of CA activity in the cultured NPE, which is membrane associated and SDS resistant. Sensitivity to tryptic hydrolysis suggests the membrane-associated CA partially faces extracellularly. As judged by responses to an extracellular CA inhibitor, the membrane-associated CA has a functional role in maintaining cytoplasmic pH.
测量培养的兔非色素上皮（NPE）细胞中膜相关碳酸酐酶（CA）的活性，确定其身份及其对细胞外胰蛋白酶的敏感性，并比较乙酰唑胺和一种细胞不可渗透的葡聚糖结合CA抑制剂改变细胞质pH的能力。
使用源自兔NPE的细胞系进行研究。细胞经裂解后通过差速离心分离为可溶性和不可溶性部分。使用二氧化碳水合测定法测定这些部分中的CA活性。在对完整细胞的研究中，合成了一种膜不可渗透的高分子量葡聚糖结合抑制剂（DBI），并用于选择性结合和抑制面向细胞外的膜结合CA。对完整红细胞中的CA活性进行测量以确认DBI仍位于细胞外。使用膜可渗透的CA抑制剂乙酰唑胺抑制总CA活性。使用荧光染料BCECF-AM的pH依赖性吸光度测定细胞内pH。
富含质膜材料的低速沉淀占培养的NPE中总CA活性的22.3±6.1%（n = 18）。当完整细胞暴露于胰蛋白酶-EDTA时，观察到膜相关CA活性降低28%；DBI抑制了这种CA活性损失。细胞溶质CA活性被0.2%十二烷基硫酸钠（SDS）抑制。相比之下，膜相关CA对SDS具有抗性，这是CA-IV同工酶的一个特征。通过蛋白质印迹法，在培养的细胞以及天然兔和猪睫状体上皮中检测到CA-IV免疫反应性多肽。用乙酰唑胺抑制总CA活性以及用DBI抑制面向细胞外的膜相关CA导致完整NPE细胞中的细胞内pH下降相同。
CA-IV同工酶的表达可能是培养的NPE中CA活性的重要组成部分，其与膜相关且对SDS具有抗性。对胰蛋白酶水解的敏感性表明膜相关CA部分面向细胞外。根据对细胞外CA抑制剂的反应判断，膜相关CA在维持细胞质pH方面具有功能性作用。
