Hydrophobicity of the NADPH binding domain of camel lens zeta-crystallin.
Interaction of camel lens zeta-crystallin with the hydrophobic probe 1-anilinonaphthalene-8-sulfonic acid (ANS) enhanced the ANS fluorescence and quenched the protein fluorescence. Both of these events were concentration-dependent and showed typical saturation curves suggesting specific ANS-zeta-crystallin binding. Quantitative analysis indicated that 1 mole zeta-crystallin bound at most 1 mole ANS. NADPH but not 9,10-phenanthrenequinone (PQ) was able to displace zeta-crystallin-bound ANS. These results suggested the presence of a hydrophobic domain in zeta-crystallin, possibly at the NADPH binding site. alpha-Crystallin as well as NADPH protected zeta-crystallin against thermal inactivation suggesting the importance of this site for enzyme stability. The NADPH:quinone oxidoreductase activity of zeta-crystallin was inhibited by ANS with NADPH as electron donor and PQ as electron acceptor. Lineweaver-Burk plots indicated mixed-type inhibition with respect to NADPH, with a K(i) of 2.3 microM. Secondary plots of inhibition with respect to NADPH indicated a dissociation constant (K'I) of 12 microM for the zeta-crystallin-NADPH-ANS complex. The K(i) being smaller than K'I suggested that competitive inhibition at the NADPH binding site was predominant over non-competitive inhibition. Like ANS-zeta-crystallin binding, inhibition was dependent on ANS concentration but independent of incubation time.
Department of Biochemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia.
M D
MD
Bazzi
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N
Rabbani
A S
AS
Duhaiman
D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D002162:Camelus; D003459:Crystallins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D005453:Fluorescence; D007908:Lens, Crystalline / Q000737:chemistry*; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000037:antagonists & inhibitors; D009249:NADP / Q000737:chemistry*; D013499:Surface Properties; D038226:zeta-Crystallins
D000817:Anilino Naphthalenesulfonates; D003459:Crystallins; D038226:zeta-Crystallins; D009249:NADP; C027132:1-anilino-8-naphthalenesulfonate; D016660:NAD(P)H Dehydrogenase (Quinone)