鱷魚水晶體中，具精氨琥珀酸分解酵素活性之水晶體蛋白研究

Abstract

中文摘要 鱷魚的δ－水晶體蛋白藉由陰離子交換樹脂純化出來後，可以測得比鴨δ－水晶體蛋白高出 3－4 倍的酵素活性。雖然都有精氨琥珀酸分解酵素的催化力，但是由產物抑制的型態來看，它們分屬於不同的 Random Uni－Bi mech－anism。 由二維電泳的圖形來看，鱷魚δ－水晶體蛋白的等電位酸鹼值分佈範圍很大，約由酸鹼值 5 . 2－6 . 0。其 Km 值為 33μM，處於牛肝中的精氨琥珀酸酵素與鴨δ－水晶體蛋白之間。產物抑制型態，包括精氨酸及反丁烯二酸皆對精氨琥珀酸產生競爭性抑制反應。瓜氨酸及琥珀酸也都對精氨琥珀酸產生競爭性抑制反應。值得注意的是琥珀酸不會對於鴨δ－水晶體蛋白進行任何抑制。由此可見在不同種類的δ－水晶體蛋白中，都有一些酵素活性及反應機制上的改變。更進一步的研究，應該可以更詳細地闡明這種酵素水晶蛋白的演化過程和詳細的反應機制。

Abstract: The delta—crystallin of a repitilian species was purified from caiman lens by a single step anion—exchange column chromatagrophy. The obtained crystallins showed about 3 to 4 fold enzymatic activity as compared to duck delta—crystallin under similar assay conditions .The biochemical comparison of delta—crystallins among the caiman and avian species shows some interesting differences in their structural and kinetic properties. Two-dimensional gel electrophoresis of crude extracts and purified proteins showed that caiman, delta—crystallin possesses various 5OkDa subunits with different pI values. Variation of the enzymatic activity with argininosuccinate concentration in the forward reaction gives an apparent Km for the substrate of 33μM . Product inhibition of forward reaction by the two products are both competitive with respect to argininosuccinate. Citrulline and Succinate, the analogues of arginine and fumarate, respectively, are also competitive inhibitors with respect to the substrate. Compared to the non—competitive product inhibition of authentic bovine liver argininosuccinate lyase and •duck delta—crystallin, caiman delta—crystallin exhibited some different enzymatic property. Moreover, succinate can not affect the enzymatic activity of duck delta—crystallin but does inhibit that of caiman’s. These results may suggest different enzymatic activity or mechanisms present in argininosuccinate lyase—like proteins from the avian and reptilian species. And it may provide a clue investigate the evolutionary process and detailed mechanism of this unique enzyme crystallin .