Poole C B, Crane D I
Division of Science and Technology, Griffith University, Brisbane, Queensland, Australia.
Arch Biochem Biophys. 1992 Feb 1;292(2):605-12. doi: 10.1016/0003-9861(92)90038-x.
Peroxisomes from mouse liver were fractionated with Triton X-114, a procedure which yields a detergent phase consisting of proteins containing hydrophobic binding sites, and a nondetergent, or aqueous, phase containing hydrophilic proteins. When this method was applied to peroxisomes from control mice, catalase and fatty acyl-CoA oxidase distributed to the aqueous phase, whereas the integral membrane protein, PMP68, and the bifunctional protein were recovered exclusively in the detergent phase. Urate oxidase distributed intermediate between these two phases. With peroxisomes from mice treated with the peroxisome proliferator clofibrate, the bifunctional protein was recovered in both the detergent and the aqueous phases, and urate oxidase was shifted toward the aqueous phase. Other analyses of the subperoxisomal distribution of the bifunctional protein were consistent with a proportion of this protein being tightly associated with the peroxisomal membrane, or with some other uncharacterized, poorly soluble, component. Sucrose gradient centrifugation of the aqueous phase resulting from Triton X-114 fractionation of peroxisomes revealed that a major proportion of catalase, fatty acyl-CoA oxidase, the bifunctional protein, and other unidentified proteins behaved as if associated under these conditions. In this respect, use of a higher concentration of Triton X-114 for peroxisome fractionation led to the partitioning of some catalase and fatty acyl-CoA oxidase to the detergent phase, indicating the presence of some detergent-accessible hydrophobic binding sites even on these proteins. These data have been interpreted as indicating matrix protein associations in vivo, associations which may be responsive to proliferator treatment.
用 Triton X - 114对小鼠肝脏中的过氧化物酶体进行分级分离，该方法可产生一个由含有疏水结合位点的蛋白质组成的去污剂相，以及一个含有亲水性蛋白质的非去污剂相或水相。当将此方法应用于对照小鼠的过氧化物酶体时，过氧化氢酶和脂肪酰辅酶A氧化酶分布于水相，而整合膜蛋白PMP68和双功能蛋白则仅在去污剂相中回收。尿酸氧化酶分布于这两相之间。对于用过氧化物酶体增殖剂氯贝丁酯处理的小鼠的过氧化物酶体，双功能蛋白在去污剂相和水相中均有回收，且尿酸氧化酶向水相转移。对双功能蛋白的亚过氧化物酶体分布的其他分析表明，该蛋白的一部分与过氧化物酶体膜紧密结合，或与其他一些未鉴定的、难溶的成分结合。对过氧化物酶体进行 Triton X - 114分级分离后得到的水相进行蔗糖梯度离心，结果显示，在这些条件下，大部分过氧化氢酶、脂肪酰辅酶A氧化酶、双功能蛋白和其他未鉴定的蛋白质表现得好像是结合在一起的。在这方面，用过氧化物酶体分级分离时使用更高浓度的 Triton X - 114会导致一些过氧化氢酶和脂肪酰辅酶A氧化酶分配到去污剂相，这表明即使是这些蛋白质上也存在一些可被去污剂接近的疏水结合位点。这些数据被解释为表明体内存在基质蛋白结合，这种结合可能对增殖剂处理有反应。
