Photoaffinity labeling of scallop myosin with 2-[(4-azido-2-nitrophenyl)amino]ethyl diphosphate: identification of an active site arginine analogous to tryptophan-130 in skeletal muscle myosin.
The ADP photoaffinity analogue 2-[(4-azido-2-nitrophenyl)amino]ethyl diphosphate (NANDP) was used to photolabel the ATP binding site of scallop myosin. Approximately 1 mol of NANDP per mol of myosin was trapped at the active site by complexation with vanadate and manganese. ADP, but not AMP, inhibited trapping of NANDP. The trapped NANDP photolabeled up to 37% of the myosin upon UV irradiation. Papain subfragment-1 prepared from the photolabeled myosin was digested with trypsin, and the major photolabeled tryptic peptides were isolated by reversed-phase HPLC. The amino acid sequence of the major labeled peptide was X-Leu-Pro-Ile-Tyr-Thr-Asp-Ser-Val-Ile-Ala-Lys, where X represents the photolabeled amino acid Arg128. Previously, Trp130 of rabbit skeletal muscle myosin has been shown to be photolabeled by NANDP [Okamoto, Y., and Yount, R. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1575-1580]. Scallop and rabbit skeletal muscle myosin display a high degree of sequence similarity in this region with Arg128 in an equivalent position as Trp130. These results suggest that the composition of the purine binding site is analogous in both myosins and that Arg and Trp play a similar role in binding ATP, despite the marked differences of their side chains.
Kerwin
B A
BA
Department of Biochemistry and Biophysics, Washington State University, Pullman 99164-4660.
Yount
R G
RG
D000244:Adenosine Diphosphate / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000302:isolation & purification; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000032:analysis*; D001386:Azides; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D008974:Mollusca / Q000378:metabolism*; D009132:Muscles / Q000737:chemistry* / Q000201:enzymology; D009218:Myosins / Q000032:analysis* / Q000302:isolation & purification; D010455:Peptides / Q000302:isolation & purification; D010782:Photolysis; D011983:Receptors, Purinergic / Q000737:chemistry / Q000187:drug effects; D014364:Tryptophan / Q000032:analysis*
D000345:Affinity Labels; D001386:Azides; D010455:Peptides; D011983:Receptors, Purinergic; D000244:Adenosine Diphosphate; D014364:Tryptophan; D001120:Arginine; C044793:N-(4-azido-2-nitrophenyl)-2-aminoethyl diphosphate; D000251:Adenosine Triphosphatases; D009218:Myosins