Brouwer M, Whaling P, Engel D W
Environ Health Perspect. 1986 Mar;65:93-100. doi: 10.1289/ehp.866593.
The physiological function of copper(I)-metallothionein is not well understood. The respiratory function of hemocyanin, a copper(I)-containing respiratory protein found in the hemolymph of many invertebrates, has been known a long time. However, the mechanism by which Cu(I) is inserted into the oxygen-binding site of apohemocyanin is completely unknown. This investigation tests the hypothesis that copper(I)-metallothionein may act as a Cu(I) donor to apohemocyanin. To this end, copper-binding proteins and hemocyanin were purified from the digestive gland and hemolymph of the American lobster, Homarus americanus. In the presence of beta-mercaptoethanol, the copper-binding proteins can be resolved into three components on DEAE-cellulose. The first two have been characterized as metallothioneins, based on their high cysteine content and lack of aromatic amino acid residues. The cysteine content of the third component is half of that of components I and II. In the absence of beta-mercaptoethanol the three proteins elute as a single protein complex during ion-exchange chromatography. Components I and II show a strong tendency to polymerize, a process that is accompanied by the loss of protein-bound copper. The purified proteins are not capable of transferring Cu(I) to the active sites of completely copper-free apohemocyanin. They are capable, however, of transferring Cu(I) to active sites of hemocyanin containing reduced amounts of Cu(I), suggesting that the conformational state of hemocyanin is the determining factor in the Cu(I) transfer mechanism.
铜(I)-金属硫蛋白的生理功能尚未完全明确。血蓝蛋白是一种含铜(I)的呼吸蛋白,存在于许多无脊椎动物的血淋巴中,其呼吸功能早已为人所知。然而,铜(I)插入脱辅基血蓝蛋白氧结合位点的机制却完全未知。本研究检验了一个假说,即铜(I)-金属硫蛋白可能作为脱辅基血蓝蛋白的铜(I)供体。为此,从美洲龙虾(美洲螯龙虾)的消化腺和血淋巴中纯化了铜结合蛋白和血蓝蛋白。在β-巯基乙醇存在的情况下,铜结合蛋白在DEAE-纤维素上可分离为三个组分。基于其高半胱氨酸含量和缺乏芳香族氨基酸残基,前两个组分已被鉴定为金属硫蛋白。第三个组分的半胱氨酸含量是组分I和II的一半。在没有β-巯基乙醇的情况下,这三种蛋白质在离子交换色谱中作为单一蛋白质复合物洗脱。组分I和II表现出强烈的聚合倾向,这一过程伴随着蛋白质结合铜的损失。纯化后的蛋白质无法将铜(I)转移至完全不含铜的脱辅基血蓝蛋白的活性位点。然而,它们能够将铜(I)转移至含铜(I)量减少的血蓝蛋白的活性位点,这表明血蓝蛋白的构象状态是铜(I)转移机制的决定性因素。