Ortwerth B J, Olesen P R
Mason Institute of Ophthalmology, University of Missouri, Columbia 65212.
Exp Eye Res. 1992 Jan;54(1):103-11. doi: 10.1016/0014-4835(92)90074-3.
Alpha-crystallin exhibits variable inhibition of several members of the chymotrypsin family of proteinases. Complete inhibition of elastase was obtained by the addition of either alpha-crystallin or a sonicated preparation of the water-insoluble fraction from bovine lens. Little or no inhibition was seen, however, with either beta-crystallin or bovine serum albumin under the same conditions. Complete binding of elastase was demonstrated by Sephadex G-100 gel filtration chromatography, and a direct correlation between binding and inhibition was obtained. This observation permitted us to do a Scatchard analysis of the inhibition data. Scatchard plots for the binding of elastase gave a biphasic response suggesting two separate binding sites. These sites had Kd values of 15 and 40 nM for alpha-crystallin and 6 and 42 nM for the bovine water-insoluble fraction. Similarly, a Dixon plot exhibited a Ki value of 3 nM and was consistent with non-competitive inhibition. One mole of alpha-crystallin (8 x 10(5) Da), or an equivalent amount of water-insoluble protein, bound from 13 to 19 mol of elastase which were about equally divided between the higher and lower affinity sites. Saturation studies confirmed 20 and 16 elastase binding sites per 8 x 10(5) Da for alpha-crystallin and water-insoluble protein, respectively. DFP-elastase was capable of binding to alpha-crystallin suggesting that a proteolytic cleavage was not required for complex formation. Stability measurements showed a linear return to 60% of the original activity over a 30-min period. Therefore, the interaction between elastase and alpha-crystallin resembles that of a heterologous protease:inhibitor complex in both binding and stability.
α-晶状体蛋白对胰凝乳蛋白酶家族的几种蛋白酶表现出不同程度的抑制作用。通过添加α-晶状体蛋白或牛晶状体水不溶性部分的超声处理制剂，可实现对弹性蛋白酶的完全抑制。然而，在相同条件下，β-晶状体蛋白或牛血清白蛋白几乎没有抑制作用。通过Sephadex G - 100凝胶过滤色谱法证明了弹性蛋白酶的完全结合，并获得了结合与抑制之间的直接相关性。这一观察结果使我们能够对抑制数据进行Scatchard分析。弹性蛋白酶结合的Scatchard图呈现双相反应，表明存在两个独立的结合位点。这些位点对α-晶状体蛋白的解离常数（Kd）值分别为15和40 nM，对牛水不溶性部分为6和42 nM。同样，Dixon图显示抑制常数（Ki）值为3 nM，与非竞争性抑制一致。一摩尔α-晶状体蛋白（8×10⁵Da）或等量的水不溶性蛋白结合13至19摩尔弹性蛋白酶，这些弹性蛋白酶在高亲和力和低亲和力位点之间大致平均分配。饱和研究证实，每8×10⁵Da的α-晶状体蛋白和水不溶性蛋白分别有20和16个弹性蛋白酶结合位点。二异丙基氟磷酸酯（DFP）处理的弹性蛋白酶能够与α-晶状体蛋白结合，这表明复合物形成不需要蛋白水解切割。稳定性测量显示，在30分钟内活性线性恢复至原始活性的60%。因此，弹性蛋白酶与α-晶状体蛋白之间的相互作用在结合和稳定性方面类似于异源蛋白酶 - 抑制剂复合物。
