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  • Title: Both the 25-kDa and 50-kDa domains in myosin subfragment 1 are close to the reactive thiols.
    Author: Lu RC, Moo L, Wong AG.
    Journal: Proc Natl Acad Sci U S A; 1986 Sep; 83(17):6392-6. PubMed ID: 2944108.
    Abstract:
    The thiol-specific photoactivatable reagent benzophenone-4-iodoacetamide can be incorporated into myosin subfragment 1 (S1), accompanied by an increase of Ca2+-ATPase and the loss of K+-ATPase activities, a characteristic property of S1 when reactive sulfhydryl 1 (SH-1) is modified. After trypsin cleavage, 25-kDa, 50-kDa, and 20-kDa fragments were found upon NaDodSO4/polyacrylamide gel electrophoresis of the unphotolyzed sample, whereas only the 50-kDa fragment and a 45-kDa fragment appeared in the photolyzed sample, indicating that the NH2-terminal 25-kDa fragment was crosslinked to the COOH-terminal 20-kDa fragment via SH-1. When photolysis was carried out in the presence of Mg2+ and ATP or Mg2+ and adenosine 5-[beta, gamma-imido]triphosphate (AdoPP[NH]P), a 70-kDa band, attributable to a crosslinked (50 kDa + 20 kDa) species, was also observed. This suggests that the conformational change induced by nucleotide binding reduces the distance between the 50-kDa region and the label on SH-1. Similar results were obtained when labeling and photolysis were carried out on trypsin-nicked S1, in which the 25-kDa, 50-kDa, and 20-kDa fragments are held together noncovalently. Further, when labeling with benzophenone-4-iodoacetamide was carried out in the presence of Mg-ATP, which increases the reactivity of another thiol, presumably SH-2, both 45-kDa and 70-kDa species were formed upon photolysis in the absence of ATP, suggesting that SH-2 is close to the 50-kDa region. More of the 70-kDa species was formed, at the expense of the 45-kDa species, when photolysis was carried out in the presence of Mg-ATP. Partial heat denaturation preferentially reduced the crosslinking between the reactive thiols and the 50-kDa region.
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