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J. Biol. Chem., Vol. 278, Issue 34, 31891-31894, August 22, 2003

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Chemical Identification of Serine 181 at the ATP-binding Site of Myosin as a Residue Esterified Selectively by the Fluorescent Reagent 9-Anthroylnitrile^*

Toshiaki Hiratsuka   and Tsuyoshi Katoh ¶

From the Departments of Chemistry and ^¶Biochemistry, Asahikawa Medical College, Asahikawa 078-8510, Japan

The esterification reagent 9-anthroylnitrile (ANN) reacts with a serine residue in the NH₂-terminal 23-kDa peptide segment of myosin subfragment-1 heavy chain to yield a fluorescent S1 derivative labeled by the anthroyl group (Hiratsuka, T. (1989) J. Biol. Chem. 264, 18188–18194). The labeling was highly selective and accelerated by nucleotides. In the present study, to determine the exact location of the labeled serine residue, the labeled 23-kDa peptide fragment was isolated. The subsequent extensive proteolytic digestion of the peptide fragment yielded two labeled peptides, a pentapeptide and its precursor nonapeptide. Amino acid sequence and composition analyses of both labeled peptides revealed that the anthroyl group is attached to Ser-181 involved in the phosphate binding loop for ATP (Smith, C. A., and Rayment, I. (1996) Biochemistry 35, 5404–5417). We concluded that ANN can esterify Ser-181 selectively out of over 40 serine residues in the subfragment 1 heavy chain. Thus ANN is proved to be a valuable fluorescent tool to identify peptides containing the phosphate binding loop of S1 and to detect the conformational changes around this loop.

Received for publication, March 28, 2003 , and in revised form, May 16, 2003.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Chemistry, Asahikawa Medical College, Midorigaoka higashi 2-1-1-1, Asahikawa 078-8510, Japan. Fax: 81-166-68-2782; E-mail: toshiaki{at}asahikawamed.ac.jp.

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