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The photoaffinity probe 8-azidoadenosine 5'-triphosphate selectively labels the heavy chain of Chlamydomonas 12 S dynein.

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      Chlamydomonas 12 S dynein, which makes up part of the outer arm of the flagellar axoneme, consists of three polypeptides of 330,000, 22,000, and 18,000 daltons. We have used 8-azidoadenosine 5'-triphosphate (8-N3ATP), a photoaffinity analog of ATP, to investigate which of the dynein polypeptides contains the site of ATP hydrolysis. 8-N3ATP is a competitive inhibitor of the hydrolysis of ATP by 12 S dynein and is hydrolyzed by 12 S dynein in an ATP- and vanadate-sensitive fashion, indicating that it binds to the 12 S dynein hydrolytic site in the same way as ATP. When dynein was incubated with [gamma-32P]- or [alpha-32P]8-N3ATP in the presence of UV light to activate the azido moiety, the analog was incorporated into 12 S dynein's heavy polypeptide chain, but not its light chains. The incorporation was UV-dependent, was blocked by addition of ATP or vanadate plus ADP to the reaction mixture, and did not occur in heat-denatured dynein. These results strongly suggest that the hydrolytic site of 12 S dynein is contained in its heavy chain.

      REFERENCES

        • Bradford M.M.
        Anal. Biochem. 1976; 72: 248-254
        • Briggs F.N.
        • Al-Jumaily W.
        • Haley B.
        Cell Calcium. 1980; 1: 205-215
        • Campbell K.P.
        • MacLennan D.H.
        J. Biol. Chem. 1983; 258: 1391-1394
        • Czarnecki J.
        • Geahlen R.
        • Haley B.
        Methods Enzymol. 1979; 56: 642-653
        • Fay R.B.
        • Witman G.B.
        J. Cell Biol. 1977; 75: 286a
        • Gibbons B.H.
        • Gibbons I.R.
        J. Biol. Chem. 1979; 254: 197-201
        • Gibbons I.R.
        Arch. Biol. 1965; 76: 317-355
        • Gibbons I.R.
        J. Cell Biol. 1981; 91: 107s-124s
        • Gibbons I.R.
        • Cosson M.P.
        • Evans J.A.
        • Gibbons B.H.
        • Houck B.
        • Martinson K.H.
        • Sale W.S.
        • Tang W.Y.
        Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 2220-2224
        • Goodenough U.W.
        • Heuser J.E.
        J. Cell Biol. 1982; 95: 798-815
        • Goodno C.C.
        Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 2620-2624
        • Guillory R.J.
        • Jeng S.J.
        Fed. Proc. 1983; 42: 2826-2830
        • Haley B.E.
        • Hoffman J.F.
        Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 3367-3371
        • Hashimoto F.
        • Horigome T.
        • Kanbayashi M.
        • Yoshida K.
        • Sugano H.
        Anal. Biochem. 1983; 129: 192-199
        • Hoppe J.
        • Freist W.
        Eur. J. Biochem. 1979; 93: 141-146
        • Huang B.
        • Piperno G.
        • Luck D.J.L.
        J. Biol. Chem. 1979; 254: 3091-3099
        • Kerlavage A.R.
        • Taylor S.S.
        J. Biol. Chem. 1980; 255: 8483-8488
        • Khatoon S.
        • Atherton R.
        • Al-Jumaily W.
        • Haley B.E.
        Biol. Reprod. 1983; 28: 61-73
        • King M.M.
        • Carlson G.M.
        • Haley B.E.
        J. Biol. Chem. 1982; 257: 14058-14065
        • Kobayashi T.
        • Martensen T.
        • Nath J.
        • Flavin M.
        Biochem. Biophys. Res. Commun. 1978; 81: 1313-1318
        • Maruta H.
        • Korn E.D.
        J. Biol. Chem. 1981; 256: 499-502
        • Murphy D.B.
        • Wallis K.T.
        • Heibsch R.R.
        J. Cell Biol. 1983; 96: 1306-1315
        • Owens J.R.
        • Haley B.E.
        J. Supramol. Struct. 1976; 5: 91-102
        • Pfister K.K.
        • Fay R.B.
        • Witman G.B.
        Cell Motil. 1982; 2: 525-547
        • Piperno G.
        • Huang B.
        • Ramanis Z.
        • Luck D.J.L.
        J. Cell Biol. 1981; 88: 73-79
        • Piperno G.
        • Luck D.J.L.
        J. Biol. Chem. 1979; 254: 2187-2190
        • Piperno G.
        • Luck D.J.L.
        J. Biol. Chem. 1979; 254: 3084-3090
        • Piperno G.
        • Luck D.J.L.
        Cell. 1981; 27: 331-340
        • Piperno G.
        • Luck D.J.L.
        Cell Motil. Suppl. 1982; 1: 95-99
        • Pomerantz A.H.
        • Rudolph S.A.
        • Haley B.E.
        • Greengard P.
        Biochemistry. 1975; 14: 3858-3862
        • Porter M.E.
        • Johnson K.A.
        J. Biol. Chem. 1983; 258: 6575-6581
        • Potter R.L.
        • Haley B.E.
        Methods Enzymol. 1983; 91: 613-633
        • Remillard S.P.
        • Witman G.B.
        J. Cell Biol. 1982; 93: 615-631
        • Roth R.
        • Cassell D.-J.
        Science. 1983; 219: 299-301
        • Satir P.
        • Weis-Steider J.
        • Lebduska S.
        • Nasr A.
        • Avolio J.
        Cell Motil. 1981; 1: 303-327
        • Scheurich P.
        • Schafer H.
        • Dose K.
        Eur. J. Biochem. 1978; 88: 253-257
        • Shimizu T.
        Cell Motil. Suppl. 1982; 1: 107-112
        • Takahashi M.
        • Tonomura Y.
        J. Biochem. (Tokyo). 1978; 84: 1339-1355
        • Tang W.-J.Y.
        • Bell C.W.
        • Sale W.S.
        • Gibbons I.R.
        J. Biol. Chem. 1982; 257: 508-515
        • Tasheva B.
        • Dessev G.
        Anal. Biochem. 1983; 129: 98-102
        • Theurkauf W.E.
        • Vallee R.B.
        J. Biol. Chem. 1982; 257: 3284-3290
        • Tsukita S.
        • Tsukita J.
        • Usukura J.
        • Ishikawa H.
        J. Cell Biol. 1983; 96: 1480-1485
        • Wagenvoord R.J.
        • Van Der Kraan I.
        • Kemp A.
        Biochim. Biophys. Acta. 1977; 460: 17-24
        • Witman G.B.
        Ann. N. Y. Acad. Sci. 1975; 253: 178-191
        • Witman G.B.
        • Minervini N.
        Symp. Soc. Exp. Biol. 1982; 35: 203-224
        • Witman G.B.
        • Johnson K.A.
        • Pfister K.K.
        • Wall J.S.
        J. Submicrosc. Cytol. 1983; 15: 193-197