IDENTIFICATION OF BACTERIAL PERIPLASMIC GLYCINE BETAINE-BINDING PROTEIN AFTER ELECTROPHORESIS AND AFFINITY LABELING

Antibodies were elicited in rabbits againts periplasmic protein obtained by cold osmotic shock from the Gram-negative eubacterium Rhizobium meliloti. When analyzed by crossed immunoelectrophoresis (CIE), the periplasmic proteins gave rise to 20 distinct immunopprecipates corresponding to the same number of bands in polyacrylamide gel electrophoresis (PAGE) under non-denaturing conditions and in SDS-PAGE. The periplasmic glycine betaine-binding protein (GB-BP) was identified by autoradiography after affinity labeling with [14C]glycine betaine in PAGE and in CIE gels. The binding proved to be quite specific to glycine betaine, since the GB-BP was not labeled by choline(a metabolic precursor or glycine betaine in Escherichia coli and Rhizobium meliloti) and 15 distinct l-amino acids, including l-proline which, like glycine betaine is also an osmoprotectant. Affinity labeling of the GB-BP with [14C]glycine betaine after protein separation by PAGE or CIE is a simple and sensitive technique permitting the GB-BP to the unambiguously detected and identified in samples of complex protein mixtures containing down to μg of GB-BP in PAGE and only 0.2 μg in CIE. © 1990.