Characterization of a Cytosolic Protein (P36) Isolated from Pig Brain by Benzodiazepine‐Affinity Chromatography
Characterization of a Cytosolic Protein (P36) Isolated from Pig Brain by Benzodiazepine‐Affinity Chromatography
复制标题
通过苯二氮卓亲和色谱法表征从猪脑中分离的胞浆蛋白 (P36)
作者：
E. Kirkness;A. Turner
在1012 S-Sepharose柱上进行苯二氮卓亲和色谱，可检测并纯化猪大脑皮层胞质组分中的结合蛋白（P36）。纯化的P36在一个步骤中富集超过3，500倍，并以50- 60%的效率回收。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析纯化的制备物，证明了Mr 36，000的单一多肽。Stokes半径（3.44nm）和沉降系数（4.43S）表明纯化的P36是二聚体蛋白。对P36的氨基酸组成的分析揭示了相对高含量的疏水性氨基酸、缬氨酸和亮氨酸。免疫印迹的几个猪组织制备的抗血清提出了对纯化的P36表现出大约相等的富集P36在大脑皮质，小脑，肾上腺。在肾脏和肝脏中观察到较少的富集，而许多其他组织显示无免疫反应性。γ-氨基丁酸/苯二氮卓受体复合物与P36无免疫交叉反应。在纯化P36制备物中未检测到对[3 H]Ro 15-4513、[3 H]氟硝西泮或[3 H] PK 11195的高亲和力结合活性。然而，γ-氨基丁酸/苯二氮卓受体反向激动剂，甲基-和乙基-β-咔啉-3-羧酸酯，在相对低的浓度下抑制P36与1012 S-琼脂糖结合的能力表明P36表现出一定程度的结合特异性。
Benzodiazepine‐affinity chromatography, on a column of 1012S‐Sepharose, resulted in the detection and purification of a binding protein (P36) from the cytosolic fraction of pig cerebral cortex. Purified P36 was enriched over 3,500‐fold in a single step and was recovered with an efficiency of 50–60%. Analysis of the purified preparation by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis demonstrated a single polypeptide of Mr 36,000. TheStokes radius (3.44 nm) and sedimentation coefficient (4.43S) indicated that purified P36 is a dimeric protein. Analysis of the amino acid composition of P36 revealed a relatively high content of the hydrophobic amino acids, valine and leucine. Immunoblotting of several pig tissue preparations with an antiserum raised against purified P36 demonstrated approximately equal enrichment of P36 in cerebral cortex, cerebellum, and adrenal glands. Lesser enrichment was observed in kidney and liver, whereas a number of other tissues displayed no immunoreactivity. The γ‐aminobutyrate/benzodiazepine receptor complex and P36 showed no immunological cross‐reactivity. High‐affinity binding activity for [3H]Ro 15–4513, [3H]flunitrazepam, or [3H]PK11195 was not detected in preparations of purified P36. However, the ability of the γ‐aminobutyrate/benzodiazepine receptor inverse agonists, methyl‐ and ethyl‐#bT‐carboline‐3‐carboxylate, to inhibit the binding of P36 to 1012S‐Sepharose at relatively low concentrations indicates that P36 exhibits a degree of binding specificity.
大鼠脑和肾中 [3H]Ro 5-4864 的特异性高亲和力结合位点。
DOI：
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发表时间：
1983
期刊：
The Journal of pharmacology and experimental therapeutics
影响因子：
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作者：
Schoemaker,H;Boles,RG;Horst,WD;Yamamura,HI
通讯作者：
Yamamura,HI