Hybridization of glyceraldehyde-3-phosphate dehydrogenase in borate

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.