TY - JOUR
T1 - Synthesis and use of 3'-(Azidoiodosalicyl) derivatives of 2',5'- dideoxyadenosine as photoaffinity ligands for adenylyl cyclase
AU - Shoshani, Ilana
AU - Taussig, Ronald
AU - Iyengar, Ravi
AU - Johnson, Roger A.
N1 - Funding Information:
1This work was supported by NIH Research Grants DK38828 to R.A.J., GM 53645 to R.T., and DK 38761 and GM 56508 to R.I. 2To whom correspondence should be addressed. Fax: (516) 444-3432. E-mail: [email protected].
PY - 2000/4/1
Y1 - 2000/4/1
N2 - 3'-[(4-Azidosalicyl)glycyl]-2',5'-dideoxyadenosine (1), 3'-[(4- azidosalicyl)-γ-aminobutyryl]-2',5'-dideoxyadenosine (2), and the 125I- labeled mono- and diiodinated analogs of 1 were synthesized and tested as photoaffinity probes for adenylyl cyclases. Kinetics for inhibition of purified type I enzyme by 1 was noncompetitive with respect to Mn·5'-ATP in the absence of light, implying a P-site mechanism of inhibition. In a UV- dependent manner both 1 and 2 and the iodinated derivative of 1 irreversibly inactivated membrane-bound and purified forms of recombinant type I bovine adenylyl cyclase expressed in ovarian cells of either the fall armyworm (Sf9) or Trichoplasia ni (High Five). Irreversible inactivation was independent of 5'-ATP and was prevented by 2',5'-dideoxyadenosine. Adenylyl cyclase, whether purified from bovine brain or in membranes from High Five cells expressing type I enzyme, when subjected to UV irradiation in the presence of 125I- labeled 1 resulted in radioactive incorporation into protein migrating at ~116 kDa. The cross-linking of 1 and its iodinated derivative with adenylyl cyclase suggests potential for such compounds to be useful in structural studies of adenylyl cyclases or of other proteins for which adenine nucleosides are substrates or allosteric regulators. (C) 2000 Academic Press.
AB - 3'-[(4-Azidosalicyl)glycyl]-2',5'-dideoxyadenosine (1), 3'-[(4- azidosalicyl)-γ-aminobutyryl]-2',5'-dideoxyadenosine (2), and the 125I- labeled mono- and diiodinated analogs of 1 were synthesized and tested as photoaffinity probes for adenylyl cyclases. Kinetics for inhibition of purified type I enzyme by 1 was noncompetitive with respect to Mn·5'-ATP in the absence of light, implying a P-site mechanism of inhibition. In a UV- dependent manner both 1 and 2 and the iodinated derivative of 1 irreversibly inactivated membrane-bound and purified forms of recombinant type I bovine adenylyl cyclase expressed in ovarian cells of either the fall armyworm (Sf9) or Trichoplasia ni (High Five). Irreversible inactivation was independent of 5'-ATP and was prevented by 2',5'-dideoxyadenosine. Adenylyl cyclase, whether purified from bovine brain or in membranes from High Five cells expressing type I enzyme, when subjected to UV irradiation in the presence of 125I- labeled 1 resulted in radioactive incorporation into protein migrating at ~116 kDa. The cross-linking of 1 and its iodinated derivative with adenylyl cyclase suggests potential for such compounds to be useful in structural studies of adenylyl cyclases or of other proteins for which adenine nucleosides are substrates or allosteric regulators. (C) 2000 Academic Press.
KW - 2',5'-dideoxyadenosine
KW - Adenylyl cyclase
KW - Aryl azides
KW - Azidoiodosalicyl derivatives
KW - Photoaffinity ligands
UR - http://www.scopus.com/inward/record.url?scp=0034177087&partnerID=8YFLogxK
U2 - 10.1006/abbi.1999.1690
DO - 10.1006/abbi.1999.1690
M3 - Article
C2 - 10729209
AN - SCOPUS:0034177087
SN - 0003-9861
VL - 376
SP - 221
EP - 228
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 1
ER -