Subunit structure of islet-activating protein, pertussis toxin, in conformity with the A-B model.

Abstract

The subunit structure of islet-activating protein (IAP), pertussis toxin, has been analyzed to study a possibility that this protein is one of the A-B toxins [Gill, D. M. (1978) in Bacterial Toxins and Cell Membranes (Jeljaszewicz, J., & Wadstrom, T., Eds.) pp 291-332, Academic Press, New York]. Heating IAP with 1% sodium dodecyl sulfate caused its dissociation into five dissimilar subunits named S-1 (with a molecular weight of 28 000), S-2 (23 000), S-3 (22 000), S-4 (11 700), and S-5 (9300), as revealed by polyacrylamide gel electrophoresis; their molar ratio in the native IAP was 1:1:1:2:1. The molecular weight of IAP estimated by equilibrium ultracentrifugation was 117 000 which was not at variance with the value obtained by summing up molecular weights of the constituent subunits. The preparative separation of these IAP subunits was next undertaken; exposure of IAP to 5 M ice-cold urea for 4 days followed by column chromatography with carboxymethyl-Sepharose caused sharp separation of S-1 and S-5, leaving the other subunits as two dimers. These dimers were then dissociated into their constituent subunits, i.e., S-2 and S-4 for one dimer and S-3 and S-4 for the other, after 16-h exposure to 8 M urea; these subunits were obtained individually upon further chromatography on a diethylaminoethyl-Sepharose column. Subunits other than S-1 were adsorbed as a pentamer by a column using haptoglobin as an affinity adsorbent. The same pentamer was obtained by adding S-5 to the mixture of two dimers. Neither this pentamer nor other oligomers (or protomers) exhibited biological activity in vivo. Recombination of S-1 with the pentamer at the 1:1 molar ratio yielded a hexamer which was identical with the native IAP in electrophoretic mobility and biological activity to enhance glucose-induced insulin secretion when injected into rats. In the broken-cell preparation, S-1 was biologically as effective as the native IAP; both catalyzed ADP-ribosylation of a protein in membrane preparations from rat C6 glioma cells. In conclusion, IAP is an oligomeric protein consisting of an A (active) protomer (the biggest subunit) and a B (binding) oligomer which is produced by connecting two dimers by the smallest subunit in a noncovalent manner. Rationale for this terminology is discussed based on the A-B model.