ESR and fluorescence studies on the adenine binding site of lectins using a spin-labeled analogue.
The techniques of electron spin resonance (ESR) and fluorescence spectroscopy have been used to study the interaction of a spin-labeled analogue of adenine, N6-(2,2,6,6-tetramethyl-1-oxypiperidin-4-yl)adenine (I), with several plant lectins. While most adenine derivatives enhanced lectin-induced fluorescence of 1,8-anilinonaphthalenesulfonic acid by binding to a separate, adenine-specific site [Roberts, D.D., & Goldstein, I.J. (1982) J. Biol. Chem. 257, 11274-11277], the spin label I caused a decrease in this fluorescence with certain lectins. ESR showed the ligand to interact strongly with lectins from lima bean (Phaseolus lunatus), Dolichos biflorus, and Phaseolus vulgaris (PHA); however, no binding was observed with Griffonia simplicifolia isolectins A4 and B4, soybean agglutinin, or Amphicarpaea bracteata lectins. The spin label was highly immobilized by each of these proteins (2T magnitude of = 68 G). Apparent affinities of the spin label for the lectins decreased in the order lima bean lectin greater than PHA erythroagglutinin greater than PHA leukoagglutinin greater than D. biflorus. Spin-labeled adenine appeared to bind specifically to the adenine binding site of D. biflorus and PHA leukoagglutinin, as demonstrated by total abolition of the ESR spectrum of bound spin label by adenine. PHA erythroagglutinin and lima bean lectin bound the analogue with apparent dissociation constants of 5 X 10(-5) and 3.2 X 10(-5) M, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Maliarik
M
M
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109.
Plessas
N R
NR
Goldstein
I J
IJ
Musci
G
G
Berliner
L J
LJ
D000225:Adenine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism*; D001665:Binding Sites; D003497:Cyclic N-Oxides / Q000138:chemical synthesis* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D007700:Kinetics; D037102:Lectins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013113:Spin Labels / Q000138:chemical synthesis*
D003497:Cyclic N-Oxides; D037102:Lectins; D013113:Spin Labels; C059616:N(6)-(2,2,6,6-tetramethyl-1-oxypiperidin-4-yl)adenine; D000225:Adenine