Joëlle V.F. Coumans and Cristobal G. dos Remedios
Institute for Biological Research and Muscle Research Unit, Department of Anatomy and Histology, The University of Sydney, NSW 2006.
In muscle fibres, actin exists as a mixture of monomeric actin (G-actin) and filamentous actin (F-actin); F-actin being the "functional" form. As cytoplasmic ionic conditions strongly favour the formation of F-actin, the existence of unpolymerised actin depends, among others factors, on proteins that bind to G-actin, the so called G-actin-binding proteins (G-ABP's). Indeed, G-ABP's can bind G-actin and inhibit the formation of F-actin. They have been isolated from different tissues. To isolate G-ABP's in human skeletal muscle, we have coupled carbethoxylated actin to divinylsulphone-activated agarose (Mini-Leak, Kem-En-Tec). This matrix, which reacts with amino, hydroxyl and sulfydryl groups, is recognised for its high coupling efficiency and forms highly stable chemical bonds with different biomolecules (Lihme, et al. (1986), J. Chromatogr. 376, 299-305). Eluted proteins were analysed by two dimensional electrophoresis (2-DE) which shows that some proteins are selectively retained. The precise atomic details of the binding sites for DNase I are known (Kabsch, et al. (1990) Nature 347, 37-44). They involve contact with subdomains 2 and 4 of G-actin. The very high affinity of DNase I for G-actin will allow us to differentiate ABP's which bind at other loci on actin. We have then bound DNase I to the coupled actin before applying the skeletal muscle extract. Comparison of these 2-DE gels with 2-DE gels of proteins eluted from actin-Mini-Leak and DNase I-Mini-Leak affinity columns show differences in the proteins isolated. These differences are analysed and commented.