Two-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 filamentous actin (F-actin) and ionic conditions strongly favor the formation of F-actin. The existence of unpolymerized actin depends, among other factors, on proteins that bind to G-actin, the so-called G-actin-binding proteins (G-ABPs). We have coupled monomeric actin to divinylsulphone-activated agarose (Mini-Leak) to isolate G-ABPs in human skeletal muscle. Eluted proteins were analyzed by two-dimensional gel electrophoresis (2-DE), which shows that some proteins are selectively retained. Deoxyribonuclease I (DNase I) is known to bind residues at the "pointed end" of actin (subdomains 2 and 4) with a high affinity. When DNase I is bound to the actin Mini-Leak before applying the skeletal muscle extract, the 2-DE gels of the eluted proteins reveals differences when compared to gels of proteins eluted from actin-Mini-Leak and 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.
Coumans
J V
JV
Institute for Biomedical Research, The University of Sydney, Australia. joelle@anatomy.su.oz.au
Humphery-Smith
I
I
dos Remedios
C G
CG
D000042:Absorption; D000199:Actins / Q000378:metabolism; D001665:Binding Sites; D002846:Chromatography, Affinity / Q000379:methods*; D003850:Deoxyribonuclease I / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006801:Humans; D008840:Microfilament Proteins / Q000032:analysis*; D018482:Muscle, Skeletal / Q000737:chemistry*
D000199:Actins; D008840:Microfilament Proteins; D003850:Deoxyribonuclease I