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大鼠脑(去小脑)突触膜在含二硫苏糖醇和胰酶抑制剂的Tris中用CHAPS溶脱。将溶脱液先后通过自制的阿片类配体10b和10cd 2个亲和层析柱,均用纳洛酮溶液洗脱。洗脱液再经过麦胚凝集素亲和层析和N-乙酰葡萄糖胺洗脱。进一步用制备型粒状凝胶等电聚焦电泳纯化和浓缩,所用凝胶为SG200,在pH5和pH7.8各有一蛋白质峰。淋冼所得样品用银染法测蛋白质含量,并用RRA测与~3H-伊托啡结合的活性,结果表明二者都是OPR,纯化程度达80 000倍以上。分子量用凝胶过滤法测定,pH5和pH7.8样品中的OPR分子量分别为52kD和42kD。pH5中OPR经用与~3H-羟基芬太尼结合的活性鉴定为μ-型受体。
Rat brain (degenerative cerebellar) synaptic membrane was eluted with CHAPS in Tris containing dithiothreitol and trypsin inhibitor. The lysate was passed through a home-made opioid 10b and 10cd 2 affinity column, both eluted with naloxone solution. The eluate was then eluted through wheat germ agglutinin affinity chromatography and N-acetylglucosamine. Further purification and concentration were carried out using preparative, granular gel isoelectric focusing electrophoresis using a gel of SG200 with one protein peak at pH5 and pH7.8. The samples from the lavage were assayed for protein content by silver staining and the binding activity to ~ 3H-Itoenin was determined by RRA. The results showed that both were OPR with over 80,000-fold purification. The molecular weight was determined by gel filtration and the OPR molecular weights in pH5 and pH7.8 samples were 52 kD and 42 kD, respectively. OPR at pH 5 was identified as a μ-type receptor by its activity in combination with ~ 3H-hydroxy fentanyl.