2-DIMENSIONAL GEL-ELECTROPHORESIS OF ACTIN-BINDING PROTEINS ISOLATED BY AFFINITY-CHROMATOGRAPHY FROM HUMAN SKELETAL-MUSCLE

In muscle cells actin exists as a mixture of monomeric (G-actin) and f ilamentous actin (F-actin) and ionic conditions strongly favor the for mation of F-actin. The existence of unpolymerized actin depends, among other factors, on proteins that bind to G-actin, the so-called G-acti n-binding proteins (G-ABPs). We have coupled monomeric actin to diviny lsulphone-activated agarose (Mini-Leak) to isolate G-ABPs in human ske letal muscle. fluted proteins were analyzed by two-dimensional gel ele ctrophoresis (2-DE), which shows that some proteins are selectively re tained. Deoxyribonuclease I (DNase I) is known to bind residues at the ''pointed end'' of actin (subdomains 2 and 4) with a high affinity. W hen DNase I is bound to the actin Mini-Leak before applying the skelet al muscle extract, the 2-DE gels of the eluted proteins reveals differ ences when compared to gels of proteins eluted from actin-Mini-leak an d DNase I-Mini-Leak affinity columns. This strategy should detect ABPs which bind to sites other than the DNase I-binding site and some may prove to be novel.