In the preceding paper [Maita, T., Miyanishi, T., Matsuzono, K., Tanioka, Y., & Matsuda, G. (1991) J. Biochem. 110, 68-74], we reported the amino-terminal 837-residue sequence of the heavy chain of adult chicken pectoralis muscle myosin. This paper describes the carboxyl terminal 1,097-residue sequence and the linkage of the two sequences. Rod obtained by digesting myosin filaments with alpha-chymotrypsin was redigested with the protease at high KCl concentration, and two fragments, subfragment-2 and light meromyosin, were isolated and sequenced by conventional methods. The linkage of the two fragments was deduced from the sequence of an overlapping peptide obtained by cleaving the rod with cyanogen bromide. The rod contained 1,039 amino acid residues, but lacked the carboxyl-terminal 58 residues of the heavy chain. A carboxyl-terminal 63-residue peptide obtained by cleaving the whole heavy chain with cyanogen bromide was sequenced. Thus, the carboxyl terminal 1,097-residue sequence of the heavy chain was completed. The linkage of subfragment-1 and the rod was deduced from the sequence of an overlapping peptide between the two which was obtained by cleaving heavy meromyosin with cyanogen bromide. Comparing the sequence of the adult myosin thus determined with that of chicken embryonic myosin reported by Molina et al. [Molina, M.I., Kropp, K.E., Gulick, J., & Robbins, J. (1987) J. Biol. Chem. 262, 6478-6488], we found that the sequence homology is 94%.

Chicken gizzard myosin was modified with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)-ethylenediamine (IAEDANS) in the presence of ATP and in 0.15 M KCl, where the myosin assumed 10S conformation. From the tryptic digest of the modified myosin, a fluorescent fragment (24 kilodaltons) was isolated by gel filtration on a Sephadex G-100 column followed by chromatography on a CM 52 column. The amino acid sequence of the fragment was analyzed by conventional methods, and was: (S,Z)K-P-L-S-D-D-E-K-F-L-F-V-D-K-N-F-V-N-N-P-L-A-Q-A-D-W-S-A-K-K- L-V-W-V-P-S-E-K-H-G-F-E-A-A-S-I-K-E-E-K-G-D-E-V-T-V-E-L-Q-E-N-G-K-K- V-T-L-S-K-D-D-I-Q-K-M-N-P-P-K-F-S-K-V-E-D-M-A-E-L-T-C-L-N-E-A-S-V-L- H-N-L-R-E-R-Y-F-S-G-L-I-Y-T-Y-S-G-L-F-C-V-V-I-N-P-Y-K-Q-L-P-I-Y-S-E-K-I- I-D-M-Y-K-G-K-K-R-H-E-M-P-P-H-I-Y-A-I-A-D-T-A-Y-R-S-M-L-Q-D-R-E-D-Q- S-I-L-C-T-G-E-S-G-A-G-K-T-E-N-T-K-K-V-I-Q-Y-L-A-V-V-A-S-S-H-K-G-K. The amino-terminus was blocked, and the fragment was assigned as an amino-terminal part of the heavy chain of gizzard myosin. Position 127 was occupied by epsilon-N-trimethyllysine. Trp-130 of rabbit skeletal myosin heavy chain, which was reported to cross-link to an azide derivative of ATP by Okamoto and Yount (Proc. Natl. Acad. Sci. U.S. 82, 1575-1579 (1985], was replaced by glutamine in gizzard myosin. Cys-93 of the fragment is the amino acid residue whose reaction with IAEDANS alters the ATPase activity of gizzard myosin (Onishi, H. (1985) J. Biochem. 98, 81-86).

The sequence of the NH2-terminal 830 amino acid residues of chicken cardiac ventricular muscle myosin subfragment-1 (S-1) was determined. S-1 was obtained by limited chymotryptic digestion, and cleaved into three characteristics fragments (23, 41, and 22 kDa fragments) by limited tryptic digestion. These fragments were isolated by gel filtration on a Sephadex G-100 column, followed by cation-exchange chromatography on a CM-52 column and reverse-phase HPLC. The isolated fragments were sequenced completely. Peptides overlapping the 23 and 41 kDa fragments and also overlapping the 41 and 22 kDa fragments were obtained by cleaving S-1 with cyanogen bromide, and sequenced completely. We also obtained a minor fragment, the 20 kDa fragment, in addition to the three characteristic fragments. Amino acid compositions of the cyanogen bromide peptides of the 20 kDa fragment indicated that a portion of S-1 heavy chains had lost their COOH-terminal 21 residues during limited tryptic digestion. Methylated amino acid residues were found at four positions: epsilon-N-monomethyllysine at position 32, epsilon-N-trimethyllysine residues at 127 and 549, and 3-N-methylhistidine at 754.

The kinetic relation between the photoinactivation and photooxidation of mitomycin C in the presence of riboflavin was investigated. The photoinactivation was tested for lambda-phage induction in Escherichia coli K-12 (lambda) cells and colony formation of E. coli Bs-1 cells. Mitomycin C lost its phage-inducing and antibiotic activities when the antibiotic was irradiated in vitro with visible light in the presence of riboflavin. The loss of phage-inducing activity followed a Stern-Volmer type equation with respect to the dose of irradiation, and the inactivation constant was evaluated to be 0.96X10(-4)m2/J. The initial rate of decay of mitomycin C by photooxidation in the presence of riboflavin obeyed first order kinetics, and its cross section was estimated to be 2.51X10(-6)m/J independent of the intensity of incident light. The cross section for photooxidation was found to be proportional to the inactivation constant. These results suggest that the photoinactivation of mitomycin C is caused by its photooxidation. In order to rationalize this conclusion, a mechanism of photooxidation was proposed and reactions in vivo of the photoproduct were discussed in relation to the inactivation.