Photocross-linking from DNPated SH1 in myosin head. I. Cross-linking to the 50-kDa fragment

• Published in: Journal of biochemistry (Tokyo) Vol. 101; no. 3; p. 661
• Main Authors: Muno, D, Sutoh, N, Sekine, T
• Format: Journal Article
• Language: English
• Published: England 01-03-1987
• Subjects: Amino Acid Sequence; Animals; Chickens; Dinitrobenzenes - radiation effects; Molecular Weight; Myosins - analogs & derivatives; Myosins - metabolism; Myosins - radiation effects; Nitrobenzenes - radiation effects; Peptide Fragments - metabolism; Photochemistry; Photolysis; Rabbits; Trinitrobenzenes - pharmacology; Trypsin - metabolism
• ISSN: 0021-924X
• DOI: 10.1093/jb/101.3.661

When DNP-SH1-myosin, selectively dinitrophenylated at SH1 by 1,2,4-trinitrobenzene, was irradiated with a high-pressure mercury lamp equipped with a UV cut filter, a new 220-kDa band called the X-band appeared right above the heavy chain band (200 kDa) on SDS-PAGE (Laemmli). The time course of the X-band formation was composed of two phases, the initial one being rapid, and the second slow. Immune reaction experiments using antibodies specific for heavy or light chains indicated that the X-band in the initial phase contained heavy chain alone, but no light chains. Such an extra band (106 kDa) was also observed in the initial phase of photolysis of DNP-SH1-Subfragment-1 (heavy chain: 96 kDa) obtained from DNP-SH1-myosin. Trypsinolysis of the 106-kDa product generated a 83-kDa band. N-Terminal sequence analysis and the amino acid composition of the band revealed that the X-band is an intraheavy chain cross-linking product between the 20- and the 50-kDa fragments. This presents a striking contrast to the other cross-linking from SH1 using benzophenone-4-iodoacetamide which reacted with the 25-kDa fragment alone (Lu, R.C. et al. (1986) Proc. Natl. Acad. Sci. U.S. 83, 6392-6396). Based upon the result obtained, the spatial arrangement of the three tryptic domains around SH1 is discussed.