Photocross-linking from DNPated SH1 in myosin head. I. Cross-linking to the 50-kDa fragment.
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.
D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D004136:Dinitrobenzenes / Q000528:radiation effects*; D008970:Molecular Weight; D009218:Myosins / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000528:radiation effects; D009578:Nitrobenzenes / Q000528:radiation effects*; D010446:Peptide Fragments / Q000378:metabolism*; D010777:Photochemistry; D010782:Photolysis; D011817:Rabbits; D014301:Trinitrobenzenes / Q000494:pharmacology; D014357:Trypsin / Q000378:metabolism
D004136:Dinitrobenzenes; D009578:Nitrobenzenes; D010446:Peptide Fragments; D014301:Trinitrobenzenes; C052926:myosin-dinitrophenyl-sulfhydryl(1); C043308:1,2,4-trinitrobenzene; D014357:Trypsin; D009218:Myosins