NADH binding properties of rabbit lens λ-crystallin

[摘要] Purpose: The present investigation aims to evaluate the NADH bindingability of λ-crystallin, a taxon-specific enzyme-crystallin, inthe rabbit lens.Methods: A λ/βL1-crystallin fraction was separated fromthe rabbit lens soluble fraction by gel filtration and theenzyme-crystallin was partially purified by subsequent affinity columnchromatography. Analysis of NADH bound to the λ-crystallinpreparation was performed using spectrophotometric and enzymologicalmethods. Binding of added NADH to the enzyme-crystallin preparation wasalso analyzed using a simple ultrafiltration method, which wastheoretically equivalent to equilibrium dialysis, to study additionalNADH binding to the protein.Results: The prepared λ-crystallin samples clearly exhibitedan absorption maximum at 340 nm, even though they were thoroughlydialyzed. This was due to the presence of nondialyzable NADH boundtightly to the protein. The bound NADH was removed by charcoaltreatment, and extracted by 0.1% SDS or 70 °C heat treatment. Adissociation constant (Kd) of less than 5 nM indicated tight binding ofNADH. The quantity of bound NADH in the 88% purified 33 kDaenzyme-crystallin was estimated to be 20.5 nmol/mg protein, suggesting astoichiometry of 0.7 mol of the nucleotide/mol of the 33 kDa protein.Additional looser binding of added NADH to λ-crystallin wasobserved in both the λ/βL1-crystallin fraction (including thefull-length 33 kDa protein: 34%; 25-30 kDa proteins, most of which mightbe generated by cleavage of the 33 kDa protein: 64%) and the partiallypurified enzyme-crystallin. It was assumed from the analysis of bindingtitration that some (about 30%) of the 33 kDa protein and most of thelower molecular weight proteins still possessed the ability to looselybind NADH. Kd values of their lower affinity binding were determined tobe 2 or 6 μM.Conclusions: From the present study, we conclude thatλ-crystallin plays a sufficiently important role as a NADH bindingprotein to maintain high levels of this nucleotide in the rabbit lens.

[学科分类] 生物化学/生物物理