Kawooya J K, Wells M A, Law J H
Department of Biochemistry, University of Arizona, Tucson 85721.
Biochemistry. 1989 Aug 8;28(16):6658-67. doi: 10.1021/bi00442a019.
The apolipoproteins of insect lipophorin were dissociated in guanidinium chloride and isolated by gel permeation chromatography. Over 98% of the total lipid in lipophorin was associated with apolipophorin I (apoLp-I), thus suggesting this apolipoprotein to be the lipid binding component of the particle. ApoLp-I was delipidated with ethanol/ether and solubilized in buffer that contained radioactive lysophosphatidylcholine ([3H]LPC) above the critical micellar concentration. Sonic irradiation of radioactive phosphatidylcholine ([14C]PC) with [3H]LPC-solubilized apoLp-I at a molar ratio of 318 resulted in reconstituted lipophorin (RLp-I). [3H]LPC was bound to fatty acid free bovine serum albumin and was separated from RLp-I by density gradient ultracentrifugation and gel permeation chromatography. Negatively stained RLp-I particles were quasispherical with an average radium of 55 A, and their overall morphology and secondary structure were similar to those of native hemolymph lipophorin. The RLp-I particle had a rho = 1.137 g/mL, a Mr approximately 5.2 X 10(5), and a [14C]PC:apoLp-I molar ratio of 308. From the compositional analysis, molecular size, trypsinization, and lipolysis with phospholipase A2, we concluded that each RLp-I particle contained one molecule of apoLp-I and a monomolecular layer of [14C]PC. When injected into the hemolymph of adult moths in vivo, RLp-I was loaded with lipid, as judged by a decrease in its density both in the presence and in the absence of adipokinetic hormone. The similarities in morphology and immunology of RLp-I and native lipophorin, together with the ability of RLp-I to load lipid, suggest that reconstituted lipophorins may serve as models to probe lipophorin structure and function.
昆虫脂转运蛋白的载脂蛋白在氯化胍中解离，并通过凝胶渗透色谱法分离。脂转运蛋白中超过98%的总脂质与载脂蛋白I（apoLp-I）结合，因此表明该载脂蛋白是颗粒的脂质结合成分。ApoLp-I用乙醇/乙醚脱脂，并溶解在含有高于临界胶束浓度的放射性溶血磷脂酰胆碱（[3H]LPC）的缓冲液中。以318的摩尔比用[3H]LPC溶解的apoLp-I对放射性磷脂酰胆碱（[14C]PC）进行超声辐照，得到重组脂转运蛋白（RLp-I）。[3H]LPC与无脂肪酸的牛血清白蛋白结合，并通过密度梯度超速离心和凝胶渗透色谱法与RLp-I分离。负染的RLp-I颗粒呈准球形，平均半径为55 Å，其整体形态和二级结构与天然血淋巴脂转运蛋白相似。RLp-I颗粒的ρ = 1.137 g/mL，Mr约为5.2×10(5)，[14C]PC:apoLp-I摩尔比为308。通过组成分析、分子大小、胰蛋白酶消化和磷脂酶A2脂解，我们得出结论，每个RLp-I颗粒包含一个apoLp-I分子和一层[14C]PC单分子层。当体内注射到成年蛾的血淋巴中时，无论有无脂肪动激素，RLp-I的密度都会降低，这表明它被脂质负载。RLp-I与天然脂转运蛋白在形态和免疫学上的相似性，以及RLp-I负载脂质的能力，表明重组脂转运蛋白可作为探究脂转运蛋白结构和功能的模型。
