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An improved method for exchanging troponin subunits in detergent skinned rat cardiac fiber bundles.

Abstract：

:We describe a method for the removal of endogenous troponin (Tn) complex from bundles of detergent-treated cardiac fibers. After 70 min treatment with cTnT-cTnI most of the endogenous Tn complex was removed from fiber bundles. Complete reconstitution of the Tn complex was achieved by reconstituting with cardiac troponin C (cTnC) in fully relaxing conditions. Ca(2+)-dependent maximum force of the fibers treated with cTnT-cTnI or cTnT-cTnI(33-211), which was used to aid in the visualization of the troponin exchange, decreased to 85-90% of the force developed by fibers before the treatment. SDS-PAGE analysis of the cTnT-cTnI(33-211) and the cTnT(77-289)-cTnI(33-211) treated fiber bundles demonstrated that 70-80% of the endogenous Tn subunits were removed. After reconstitution with cTnC, approximately 80-85% of the Ca(2+)-regulated force was restored in cTnT-cTnI/cTnI(33-211) treated fibers. Our results demonstrate that by minimizing the prolonged exposure of skinned cardiac fiber bundles to rigor conditions, successful exchange of all three subunits of the Tn complex can be accomplished with minimal loss of function.

journal_name

Biochem Biophys Res Commun

journal_title

Biochemical and biophysical research communications

authors

Chandra M,Kim JJ,Solaro RJ

doi

10.1006/bbrc.1999.1341

subject

Has Abstract

pub_date

1999-09-16 00:00:00

pages

219-23

issue

1

eissn

0006-291X

issn

1090-2104

pii

S0006-291X(99)91341-4

journal_volume

263

pub_type

杂志文章

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