ACTIN-BINDING PROTEINS IN MOUSE C2 MYOBLASTS AND MYOTUBES - A COMBINATION OF AFFINITY-CHROMATOGRAPHY AND 2-DIMENSIONAL GEL-ELECTROPHORESIS

This paper analyzes proteins expressed in a mouse muscle precursor cel l line (C2 myoblasts) and compares them with those observed in differe ntiated myotubes from the same cell line. We observed hundreds of prot eins in myoblasts using IPG two-dimensional gel electrophoresis but th is number is greatly reduced using Mini-Leak (divinylsulfone-activated agarose) affinity chromatography. Two kinds of affinity columns were prepared. One contained a chemically modified monomeric actin bound to the affinity matrix. The second matrix contained a high-affinity acti n-binding protein (DNase I) which was bound to the actin Mini-Leak col umn to block specific sites on actin. Actin-binding proteins in homoge nates of myoblasts or myotubes were passed through the affinity column s and eluted under high salt conditions. The Mini-Leak affinity medium itself appeared to have little ability to bind proteins. Our two-dime nsional (2-D) gels identified a small number of proteins and we are cu rrently focusing our attention on a particular protein spot which coul d correspond to cofilin. Comparison of myoblast and myotube proteins u sing affinity chromatography shows no qualitative, clearly identifiabl e differences but the analysis is still in progress. These findings ar e discussed in relation to reports in which the myoblast-myotube trans formation was associated with the up-regulation or de novo synthesis o f more than ten proteins.