Actin-binding proteins in mouse C2 myoblasts and myotubes: a combination of affinity chromatography and two-dimensional gel electrophoresis.
This paper analyzes proteins expressed in a mouse muscle precursor cell line (C2 myoblasts) and compares them with those observed in differentiated myotubes from the same cell line. We observed hundreds of proteins in myoblasts using IPG two-dimensional gel electrophoresis but this 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 actin-binding protein (DNase I) which was bound to the actin Mini-Leak column to block specific sites on actin. Actin-binding proteins in homogenates of myoblasts or myotubes were passed through the affinity columns and eluted under high salt conditions. The Mini-Leak affinity medium itself appeared to have little ability to bind proteins. Our two-dimensional (2-D) gels identified a small number of proteins and we are currently focusing our attention on a particular protein spot which could correspond to cofilin. Comparison of myoblast and myotube proteins using affinity chromatography shows no qualitative, clearly identifiable differences but the analysis is still in progress. These findings are discussed in relation to reports in which the myoblast-myotube transformation was associated with the up-regulation or de novo synthesis of more than ten proteins.
Coumans
J V
JV
Institute for Biomedical Research, Department of Anatomy and Histology, The University of Sydney, Australia. joelle@anatomy.usyd.edu.au
dos Remedios
C G
CG
D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D003850:Deoxyribonuclease I; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D051379:Mice; D008840:Microfilament Proteins / Q000032:analysis*; D009132:Muscles / Q000737:chemistry* / Q000166:cytology
D008840:Microfilament Proteins; D003850:Deoxyribonuclease I