INVESTIGATIONS ON THE QUATERNARY STRUCTURE AND FUNCTION OF OLIGOMERIC PROTEINS BY HYBRIDIZATION WITH VARIANTS PRODUCED BY CHEMICAL MODIFICATION

Abstract

ABSTRACT. A relatively homogeneous electrophoretic variant with the same quaternarystructure as the native enzyme can be prepared by reaction of limited amounts of succinic anhydride with an oligomeric protein. The modification of approximately 40% of the lysyl residues of E. coli alkaline phosphatase or rabbit muscle aldolase will give such variants. These variants are useful for hybridization experiments in order to investigate subunit structure and subunit interactions. Hybridization of native and succinyl aldolase results in three hybrid species consistent with a tetrameric structure. The succinyl derivative of alkaline phosphatase was hybridized with another chemical variant produced by chemical modification of the tyrosine residues of alkaline phosphatase with tetranitromethane. Since both the succinyl and nitrotyrosyl derivatives have dimeric structures and differ in charge, one hybrid species was detected on electrophoresis. The succinyl nitrotyrosyl hybrid was isolated by anion exchange chromatography and then converted to a succinyl aminotyrosyl hybrid by reduction of the nitrotyrosyl residues with sodium dithionite. The activities of the two hybrids agreed with values predicted, assuming that the subunits of alkaline phosphatase function independently. These results are not consistent with mechanisms involving negative homotropic interactions during the rate limiting step of the alkaline phosphatase reaction.