Purification, molecular properties and ontogeny of carbonic anhydrase isozymes. Evidence for A, B and C isozymes in avian and mammalian tissues.
Purification, molecular properties and ontogeny of carbonic anhydrase isozymes. Evidence for A, B and C isozymes in avian and mammalian tissues.
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碳酸酐酶同工酶的纯化、分子特性和个体发育。
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发表时间：
1977
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通讯作者：
R. Holmes
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作者：
R. Holmes
用亲和层析法从绵羊和鸡的红色骨骼肌中分离出碳酸氢酶A，从鸡肠和红细胞中分别分离出高纯度的碳酸氢酶B和C。用十二烷基硫酸钠/聚丙烯酰胺凝胶电泳法和Sephadex凝胶过滤法分别测定绵羊碳酸酐酶A的亚基和天然分子量均约为34000。相比之下，来自鸡肉的碳酸氢酶A、B和C的相对分子质量由十二烷基硫酸钠电泳法测定为30000。绵羊肌肉碳酸酐酶A的氨基酸组成不同于B和C同工酶，尤其是碱性氨基酸含量，A同工酶的氨基酸含量显著高于B和C同工酶(38cf。24-29/摩尔)。绵羊碳酸酐酶A的锌含量为0.81g原子锌/摩尔。当A：B：C=1：6：47时，鸡碳酸酐酶同工酶的比活力差异显著。此外，这些同工酶对磺胺的亲和力也不同。鸡碳酸酐酶A对乙酰唑胺抑制的敏感性约为鸡C同工酶的100倍。对猫的个体发育研究表明，碳酸酐酶C是中期胚胎动物中唯一存在的酶，碳酸酐酶A在晚期出现在肌肉中，碳酸酐酶B在出生后2天内出现在盲肠中。
这一证据与之前提出的在哺乳动物和鸟类中编码碳酸氢酶的第三个基因座的建议是一致的。该同工酶(A)是一种单体锌金属酶，在催化效率、氨基酸组成、绵羊体内相对分子质量、个体形成时间、磺胺抑制作用和组织分布等方面不同于广泛研究的碳酸酐酶B和C同工酶。
Carbonic anhydrase A from sheep and chicken red skeletal muscles and carbonic anhydrases B and C from chicken intestine and red cells respectively have been isolated in a high state of purity by affinity chromatography. The subunit and native molecular weights of sheep carbonic anhydrase A determined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and Sephadex gel filtration respectively were both approximately 34000. In contrast, carbonic anhydrases A, B and C from chicken exhibited a molecular weight determined by sodium dodecyl sulphate electrophoresis of 30000. The amino acid composition of sheep muscle carbonic anhydrase A was distinct from the B and C isozymes, particularly in basic amino acid content, which was significantly higher for the A isozyme (38 cf. 24–29/mol). The zinc content of sheep carbonic anhydrase A was found to be 0.81 g atom Zn/mol. The specific activities of chicken carbonic anhydrase isozyme differed significantly according to the ratio A:B:C = 1:6:47. In addition, these isozymes differed in their affinities for sulphonamide. Chicken carbonic anhydrase A was approximately 100 times less sensitive to acetazolamide inhibition than the chicken C isozyme. Ontogenetic studies in cats showed carbonic anhydrase C to be the only form of the enzyme present in mid-term fetal animals, with carbonic anhydrase A appearing in muscle in the late stages, and carbonic anhydrase B appearing in caecum within 2 days of birth.
The evidence is consistent with previous proposals for a third locus encoding carbonic anhydrase in mammalian and avian species. This isozyme (A) is a monomeric, zinc metalloenzyme, which differs from the extensively studied B and C isozymes of carbonic anhydrase in terms of catalytic efficiency, amino acid composition, molecular weight (in sheep), time of ontogenic appearance, sulphonamide inhibition and tissue distribution.