Photocross-Linking from DNPated SH₁ in Myosin Head. I. Cross-Linking to the 50-kDa Fragment

Abstract

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.