Purpose: The present investigation aims to evaluate the NADH binding ability of λ-crystallin, a taxon-specific enzyme-crystallin, in the rabbit lens. Methods: A λ/βL1-crystallin fraction was separated from the rabbit lens soluble fraction by gel filtration and the enzyme-crystallin was partially purified by subsequent affinity column chromatography. Analysis of NADH bound to the λ-crystallin preparation was performed using spectrophotometric and enzymological methods. Binding of added NADH to the enzyme-crystallin preparation was also analyzed using a simple ultrafiltration method, which was theoretically equivalent to equilibrium dialysis, to study additional NADH binding to the protein. Results: The prepared λ-crystallin samples clearly exhibited an absorption maximum at 340 nm, even though they were thoroughly dialyzed. This was due to the presence of nondialyzable NADH bound tightly to the protein. The bound NADH was removed by charcoal treatment, and extracted by 0.1% SDS or 70 °C heat treatment. A dissociation constant (Kd) of less than 5 nM indicated tight binding of NADH. The quantity of bound NADH in the 88% purified 33 kDa enzyme-crystallin was estimated to be 20.5 nmol/mg protein, suggesting a stoichiometry of 0.7 mol of the nucleotide/mol of the 33 kDa protein. Additional looser binding of added NADH to λ-crystallin was observed in both the λ/βL1-crystallin fraction (including the full-length 33 kDa protein: 34%; 25-30 kDa proteins, most of which might be generated by cleavage of the 33 kDa protein: 64%) and the partially purified enzyme-crystallin. It was assumed from the analysis of binding titration that some (about 30%) of the 33 kDa protein and most of the lower molecular weight proteins still possessed the ability to loosely bind NADH. Kd values of their lower affinity binding were determined to be 2 or 6 μM. Conclusions: From the present study, we conclude that λ-crystallin plays a sufficiently important role as a NADH binding protein to maintain high levels of this nucleotide in the rabbit lens.
|出版ステータス||Published - 2006 6 19|
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