Use of a Diimidoester Cross-Linking Reagent to Examine the Submit Structure of Rabbit Muscle Pyruvate Kinase

Abstract

Rabbit muscle pyruvate kinase, a tetramer of highly similar or identical subunits, is known to dissociate in urea via a dimeric intermediate. To provide additional evidence regarding the structure of the native oligomer, we have treated pyruvate kinase with dimethyl pimelimidate and examined the yields of cross-linked species resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Of four products resolved, predominating species are dimers and tetramers of the pyruvate kinase subunit, while monomers and trimers are present in lesser amounts. These relative yields are consistent with a dimeric structure of the tetramer.In a typical cross-linking reaction, about 86 of the 148 primary amino groups in the pyruvate kinase tetramer are amidinated. Of 53 moles of cross-linking reagent incorporated per mole of tetramer, 38% have reacted monofunctionally, with hydrolysis of the second imidoester group, and 62% have reacted bifunctionally to form intra- and intersubunit cross-links.