3-磷酸甘油醛脱氢酶在硼酸盐中的杂交

Hybridization of glyceraldehyde-3-phosphate dehydrogenase in borate.

作者信息

Suzuki K, Hibino K, Imahori K

出版信息

J Biochem. 1976 Jun;79(6):1287-95. doi: 10.1093/oxfordjournals.jbchem.a131182.

DOI:10.1093/oxfordjournals.jbchem.a131182

Abstract

Conditions for the hybridization of glyceraldehyde-3-phosphate dehydrogenase (GPD) [EC 1.2.1.12] in the presence of dilute borate were examined with horseshoe crab and rabbit GPDs. Hybridization was strongly dependent upon pH, borate concentration, and temperature. The optimum medium for hybridization was 10mM Tris-HCl-1 mM 2-mercaptoethanol-1mM EDTA containing 10-20 mM borate, pH 8.5-9.0. Hybridization was performed by incubation of two electrophoretically distinct GPDs at 30 degrees for 3-15 hr in the above medium at a protein concentration of 1-2 mg/ml. The time course of hybridization was analyzed under the optimized conditions. Symmetrical A2B2-type hybrid appeared only 5 min after incubation for 1 hr. Hybridization of GPDs from 7 different species was examined under the optimal conditions. Hybridization was detected with rabbit-horseshoe crab, yeast-rabbit, yeast-chicken, and chicken-horseshoe crab combinations. Subunit-subunit interaction, the mechanism of hybridization, and the structure of GPD are discussed based on the results obtained.

摘要

在稀硼酸盐存在的情况下，对鲎和兔的甘油醛-3-磷酸脱氢酶（GPD）[EC 1.2.1.12]的杂交条件进行了研究。杂交强烈依赖于pH值、硼酸盐浓度和温度。杂交的最佳介质是含有10-20 mM硼酸盐、pH 8.5-9.0的10 mM Tris-HCl-1 mM 2-巯基乙醇-1 mM EDTA。通过在上述介质中，于30℃下以1-2 mg/ml的蛋白质浓度孵育两种电泳不同的GPD 3-15小时来进行杂交。在优化条件下分析杂交的时间进程。孵育1小时后仅5分钟就出现了对称的A2B2型杂种。在最佳条件下检测了7种不同物种的GPD的杂交情况。根据所得结果讨论了亚基-亚基相互作用、杂交机制和GPD的结构。