Purification and characterization of chicken lipoprotein receptor

(1)用鸡血浆制备的乳糜微粒或VLDL在37℃下体外培养2h后，采用超离心（d<1.019）的方法制备了鸡体内的乳糜微粒残体和VLDL残体。(2)在Bio-Gel A50m柱（1.6 * 50cm）上，乳糜微粒残留物和VLDL残留物的洗脱模式几乎为单峰，粒径小于乳糜微粒和VLDL，因为Bio-Gel A50m对残留物的洗脱与LDL相似，而不是乳糜微粒和VLDL。鸡残体的甘油三酯含量低，胆固醇含量高，Apo B100和Apo A-I含量几乎相当，而Apo C、Apo 52kDa和Apo 20kDa含量低于乳糜微粒或VLDL。(3) ^<125> i标记LDL、乳糜微粒残体和VLDL残体与鸡或大鼠肝膜结合特异性。乳糜微粒、VLDL、VLDL残基或LDL对鸡^<I25> i -标记LDL与鸡肝膜的特异性结合均有70%的抑制作用，而添加乳糜微粒残基对鸡肝膜的特异性结合影响不大。这些结果表明，鸡乳糜微粒残体与不同的LDL受体位点结合。(4)由于过量摄入相应的脂蛋白，鸡原代肝细胞对鸡^< 25> i标记脂蛋白的摄取减少到约>30%。由此可见，鸡对脂蛋白的摄取是由脂蛋白受体介导的。(5)对鸡肝脏脂蛋白受体进行配体印迹分析，发现鸡肝脏中存在3种不同的受体蛋白（145,115和105 kDa）。(6)采用羟基磷灰石层析、低密度脂蛋白亲和层析和Mono q层析三层析纯化了两种鸡肝脂蛋白受体（鸡肝载脂蛋白B受体(CHBR) -1和CHBR-2）， SDS-PAGE鉴定其分子量分别为115kDa和105kDa。在配体印迹上，CHBR-1和CHBR-2都与载脂蛋白B结合。

(I) Chylomicron remnant and VLDL remnant in chickens were prepared by the method using ultracentrifugation (d<1.019) following an in vitro incubation of chylomicron or VLDL prepared from chicken plasma with chicken LPL for 2h at 37ﾟC.(2) The chylomicron remnants and VLDL remnants yielded the almost single peak in the elution pattern on Bio-Gel A50m column (1.6 * 50cm) and the particle size were smaller than chylomicron and VLDL since the elution of remnants by Bio-Gel A50m was similar to LDL rather than chylomicron and VLDL.Chicken remnants had low triglyceride contents with high cholesterol and almost comparable Apo B100 and Apo A-I contents while lower Apo C,Apo 52kDa and Apo 20kDa contents compared to chylomicron or VLDL.(3) ^<125>I-labeled LDL,chylomicron remnant and VLDL remnant bound specificity to chicken or rat liver membranes. The specific binding of chicken ^<I25>I-labeled LDL to chicken liver membrane was inhibited by >70% with chylomicron, VLDL,VLDL remnant or LDL,while the addition of chylomicron remnant had little effect. These results demonstrate that chicken chylomicron remnant bind the site of different receptor of LDL.(4) The uptake of Chicken ^<I25>I-labeled lipoproteins to chicken primary hepatocyte was decreased to about>30% by an excess of the corresponding lipoproteins. It is concluded that the uptake of chicken lipoproteins are mediated by lipoprotein receptors.(5) In ligand blot aunalysis of chicken hepatic lipoprotein receptors, three difrerent receptor proteins (145,115 and 105 kDa) were present in chicken liver.(6) Two kinds of chicken hepatic lipoprotein receptors were purifed by a procedure with three chromatography step on Hydroxyapatite, LDL-affinity and Mono Q.The receptors (chicken hepatic apo B receptor (CHBR) -1 and CHBR-2) were characterized a molecular weight of 115kDa and 105kDa, as estimated on SDS-PAGE.On ligand blotting, both CHBR-1 and CHBR-2 bound to apo B lipoproteins.