Direct photoaffinity labeling of gizzard myosin with [3H]uridine diphosphate places Glu185 of the heavy chain at the active site.
The active site of chicken gizzard myosin was labeled by direct photoaffinity labeling with [3H]UDP. [3H] UDP was stably trapped at the active site by addition of vanadate (Vi) and Co2+. The extraordinary stability of the myosin.Co2+.[3H]UDP.Vi complex (t1/2 greater than 5 days at 0 degrees C) allowed it to be purified free of extraneous [3H]UDP before irradiation began. Upon UV irradiation, greater than 60% of the trapped [3H]UDP was photoincorporated into the active site. Only the 200-kDa heavy chain was labeled, confirming earlier results (Maruta, H., and Korn, E. (1981) J. Biol. Chem. 256, 499-502) using [3H]UTP. Extensive tryptic digestion of photolabeled myosin subfragment 1 followed by high performance liquid chromatography separations and removal of nucleotide phosphates by treatment with alkaline phosphatase allowed two labeled peptides to be isolated. Sequencing of the labeled peptides and radioactive counting showed that Glu185 was the residue labeled. Since UDP is a "zero-length" cross-linker, Glu185 is located at the purine-binding pocket of the active site of smooth myosin and adjacent to the glycine-rich loop which binds the polyphosphate portion of ATP. This Glu residue is conserved in smooth and nonmuscle myosins and is the same residue identified previously by [3H]UTP photolabeling in Acanthamoeba myosin II (Atkinson, M. A., Robinson, E. A., Appella, E., and Korn, E. D. (1986) J. Biol. Chem. 261, 1844-1848).
Garabedian
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Department of Biochemistry and Biophysics, Washington State University, Pullman 99164-4660.
Yount
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D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D005895:Gizzard, Avian / Q000378:metabolism; D005971:Glutamates; D018698:Glutamic Acid; D006801:Humans; D007231:Infant, Newborn; D007700:Kinetics; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000378:metabolism*; D009218:Myosins / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D012689:Sequence Homology, Nucleic Acid; D014316:Tritium; D014530:Uridine Diphosphate / Q000378:metabolism*
D000345:Affinity Labels; D005971:Glutamates; D010446:Peptide Fragments; D014316:Tritium; D018698:Glutamic Acid; D014530:Uridine Diphosphate; D009218:Myosins