The myosin SH2-50-kilodalton fragment cross-link: location and consequences.
Some of us recently described a new interthiol cross-link which occurs in the skeletal myosin subfragment 1-MgADP complex between the reactive sulfhydryl group "SH2" (Cys-697) and a thiol (named SH chi) of the 50-kilodalton (kDa) central domain of the heavy chain; this link leads to the entrapment of the nucleotide at the active site [Chaussepied, P., Mornet, D., & Kassab, R. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2037-2041]. In the present study, we identify SH chi as Cys-540 of the 50-kDa fragment. The portion of the heavy chain including this residue and also extending to Cys-522 that is cross-linkable to the "SH1" thiol [Ue, K. (1987) Biochemistry 26, 1889-1894] is near the SH2-SH1 region. Furthermore, various spectral and enzymatic properties of the (Cys697-Cys540)-N,N'-p-phenylenedimaleimide (pPDM)-cross-linked myosin chymotryptic subfragment 1 (S-1) were established and compared to those for the well-known (SH1-SH2)-pPDM-cross-linked S-1. The circular dichroism spectra of the new derivative were similar to those of native S-1 complexed to MgADP. At 15 mM ionic strength, (Cys697-Cys540)-S-1 binds very strongly to unregulated actin (Ka = 7 X 10(6) M-1), and the actin binding is very weakly affected by ionic strength. Joining actin with the (Cys697-Cys540)-S-1 heavy chain, using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide, produces different species than does joining unmodified S-1 with actin.(ABSTRACT TRUNCATED AT 250 WORDS)
Chaussepied
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P
Cardiovascular Research Institute, University of California, San Francisco 94143.
Morales
M F
MF
Kassab
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R
D000199:Actins / Q000378:metabolism; D000818:Animals; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003488:Cyanogen Bromide; D008970:Molecular Weight; D015879:Myosin Subfragments; D009218:Myosins / Q000302:isolation & purification / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D000199:Actins; D003432:Cross-Linking Reagents; D015879:Myosin Subfragments; D010446:Peptide Fragments; D013439:Sulfhydryl Reagents; D009218:Myosins; D003488:Cyanogen Bromide