Two-dimensional gel electrophoresis of actin-binding proteins isolated by affinity chromatography from human skeletal muscle

被引:15
作者
Coumans, JVF [1 ]
HumpherySmith, I [1 ]
dosRemedios, CG [1 ]
机构
[1] UNIV SYDNEY,INST BIOMED RES,SYDNEY,NSW 2006,AUSTRALIA
关键词
actin; actin-binding proteins; psoas; divinylsulphone-activated agarose (Mini-Leak); two-dimensional polyacrylamide gel electrophoresis;
D O I
10.1002/elps.1150180709
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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. fluted 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.
引用
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页码:1079 / 1085
页数:7
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