PHOTOCHEMICAL CROSS-LINKING OF THE SKELETAL MYOSIN HEAD HEAVY-CHAIN TO ACTIN SUBDOMAIN-1 AT ARG95 AND ARG28

被引:21
作者
BONAFE, N [1 ]
CHAUSSEPIED, P [1 ]
CAPONY, JP [1 ]
DERANCOURT, J [1 ]
KASSAB, R [1 ]
机构
[1] UNIV MONTPELLIER 1,CNRS,CTR RECH BIOCHIM MACROMOLEC,INSERM,U249,ROUTE MENDE,BP5051,F-34033 MONTPELLIER,FRANCE
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1993年 / 213卷 / 03期
关键词
D O I
10.1111/j.1432-1033.1993.tb17875.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
F-actin specifically substituted with the photocross-linker, p-azidophenylglyoxal, at Arg95 and Arg28 was isolated and characterized. Upon complexation with myosin subfragment-1 (S1) and photolysis at 365 nm, it was readily cross-linked to the Sl heavy chain with a yield of about 13-25%, generating four major actin-heavy-chain adducts with molecular masses in the range 165 - 240 kDa. The elevated Mg2+-ATPase of the covalent complexes displayed a turnover rate of 33 +/- 8 s-1 which is similar to the values reported earlier for other acto-S1 conjugates. The cross-linking between various proteolytic Sl and actin derivatives, combined with the fluorescent and immunochemical detection of the photocross-linked products, indicated that the arylnitrene group on Arg95 was inserted predominantly in the central 50-kDa region, whereas that attached to Arg28 mediated the selective cross-linking of the COOH-terminal 22-21-kDa fragments of the heavy chain, most probably by reacting at or near the connector segment between the 50-kDa and 20-kDa fragments. The rapid photoactivation and cross-linking to Sl of the substituted F-actin, which can be accomplished on a millisecond time scale, may serve to probe the structural dynamics of the interaction of the S1 heavy chain with subdomain-1 of actin during the ATPase cycle.
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页码:1243 / 1254
页数:12
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