|Molecular Vision 2006;
Received 20 September 2005 | Accepted 16 June 2006 | Published 19 June 2006
NADH binding properties of rabbit lens λ-crystallin
Mikako Oka,2 Kenji Kawai,1 Hajime Obazawa,3 Shizuko
1Department of Ophthalmology, Tokai University School of Medicine, Isehara, Japan; 2Department of Molecular Physiology, Kyoritsu University of Pharmacy, Tokyo, Japan; 3Eye Research Institute of Cataract Foundation, Tokyo, Japan
Correspondence to: Makoto Takehana, PhD, Department of Molecular Physiology, Kyoritsu University of Pharmacy, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512, Japan; Phone: +81-3-5400-2663; FAX: +81-3-5400-2693; email: firstname.lastname@example.org
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.
The eye lens, a transparent and refractive organ that casts a focused image onto the retina, is characterized by the presence of high concentrations of soluble structural proteins called crystallins . Three classes of crystallins, α-, β-, and γ-crystallins, are ubiquitously present in all vertebrates. α-Crystallin is closely related to four Drosophila small heat-shock proteins , and it functions as a molecular chaperone . Recent comparative studies have revealed surprising diversity among the lens crystallins of vertebrates and invertebrates [4,5]. There are more than 10 taxon-specific crystallins in various lineages of vertebrates and invertebrates. These taxon-specific crystallins (enzyme-crystallins) are all either identical or related to metabolic enzymes. Crystallins in the lens seem to have been recruited as structural proteins involved in light refraction from stress-inducible proteins and metabolic enzymes with protective effects by gene sharing or gene duplication [4,5].
The lenses of certain vertebrate species, such as bullfrog, guinea pig, duck, and rabbit, are known to contain high concentrations (0.4-1.5 mM) of reduced pyridine nucleotide, NADPH or NADH . The high nucleotide levels in the lenses appear to be achieved by specific nucleotide binding to taxon-specific enzyme-crystallins related to NAD(P)H/NAD(P)+-dependent oxidoreductases and to produce beneficial effects as near ultraviolet light (UV) filters [6,7]. Although ρ-crystallin, which belongs to a group of aldo-keto reductases, does not exhibit enzyme activity in the bullfrog lens, it does retain NADPH binding capacity . ζ-Crystallin in the guinea pig lens possesses an active NADPH-quinone reductase activity  and specifically binds NADPH . It has been reported that a mutation in the ζ-crystallin gene, which is associated with an autosomal dominant congenital cataract in the guinea pig, produces a mutant form of crystallin that fails to bind NADPH and lacks NADPH-quinone reductase activity . Other examples of enzyme-crystallins with pyridine nucleotide binding that mediate protection against near UV include ε-crystallin/lactate dehydrogenase B of birds [4,12], π-crystallin/glyceraldehyde 3-phosphate dehydrogenase of geckos , and η-crystallin/retinal dehydrogenase of elephant shrews .
λ-Crystallin is a soluble structural lens protein found only in rabbit and hare lenses . The authors have revealed that this enzyme-crystallin shows 30% homology to L-3-hydroxyacyl-CoA dehydrogenase and contains a putative NADH binding site . Recently, this enzyme-crystallin has been reported to be identical to an enzyme, L-gulonate 3-dehydrogenase, which is active in the uronate cycle . We have also found that λ-crystallin is related to NADH-dependent dehydroascorbate (DHA) reductase based on partial enzyme purification and western blot analysis of the enzyme-crystallin [17,18]. In the rabbit lens, however, the reduction of DHA is linked to the glutathione redox cycle by a nonenzymatic reaction between DHA and glutathione . NADH-dependent DHA reductase activity seems to be weak and not so effective for ascorbate regeneration in the rabbit lens . On the other hand, the aforementioned gulonate dehydrogenase and DHA reductase are specific for NAD(H) [16,17]. It has been reported that apparent Km values of rabbit liver gulonate dehydrogenase are 11 μM for NAD+ and 0.21 mM for L-gulonate , Km values of acetoacetate reductase activity of the gulonate dehydrogenase are 0.5 μM for NADH and 8.3 mM for acetoacetate , and Km values of rabbit lens DHA reductase are 4.0 μM for NADH and 5.7 mM for DHA . These kinetic results suggest that λ-crystallin may have a high affinity for NADH. However, NADH binding properties of the enzyme-crystallin remain obscure. In the present investigation, we demonstrate that λ-crystallin, isolated from the rabbit lens soluble fraction by gel filtration and affinity column chromatography, does bind NADH, and that most of the NADH binding of the partially purified enzyme-crystallin is tight and nondialyzable.
Lenses and preparation of the lens soluble fraction
Rabbit lenses were obtained from freshly enucleated eyes of Japanese albino rabbits (about 6-20 months old) sacrificed with overdoses of anesthetics. The lenses were kept frozen at -80 °C until use. All animal procedures were in accordance with the ARVO resolution on animals and ophthalmic research.
Lenses were homogenized in 5-10 times their weight of 0.1 M KCl, 10 mM K-phosphate (pH 7.2) in a glass homogenizer on ice, and the soluble fraction was separated from the insoluble fraction by centrifugation at 15,000x g for 1 h at 4 °C.
Isolation of λ-crystallin by column chromatography
All chromatography was carried out at 4 °C. As reported previously , the λ/βL1-crystallin fraction with DHA reductase activity was separated from the rabbit lens soluble fraction by gel filtration on a column of Sephadex G-75 superfine or Sephacryl S-200HR (Amersham Biosciences, Piscataway, NJ). The enzyme-crystallin fraction was pooled, concentrated, and dialyzed against 30 mM Na-phosphate, 1 mM EDTA, pH 6.1. The dialyzed protein solution was then applied to a column (1.0 cm in diameter x 7.5 cm in length) of Affi-Gel blue gel (Bio-Rad Laboratories, Hercules, CA) equilibrated with the 30 mM phosphate buffer including EDTA. The column was washed with 40 ml of the same buffer. DHA reductase activity was not found in the wash buffer, and more than 90% of protein in the λ/βL1-crystallin fraction was bound to the affinity column. λ-Crystallin bound on the column was eluted with a linear gradient of 0-1 mM NADH plus 0-1 M NaCl in 40 ml of the buffer at a flow rate of approximately 30 ml/h. The enzyme-crystallin with DHA reductase activity was found at about 0.25 mM NADH, 0.25 M NaCl. A solution of the enzyme-crystallin thus isolated was thoroughly dialyzed twice against 50 volumes of 10 mM K-phosphate (pH 7.2) to remove NADH and NaCl used for the chromatography.
Assay of DHA reductase activity
DHA reductase activity was determined by spectrophotometric measurement of the oxidation rates of 100 μM NADH at 340 nm in the presence of 500 μM DHA, using a method described previously in the literature . Protein concentration was assayed by the bicinchoninic acid method  using bovine serum albumin as standard.
SDS-PAGE and western blotting
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on slab gels of 5-20% acrylamide according to the method of Laemmli . After electrophoresis, the gels were either stained for protein with Coomassie brilliant blue or subjected to western blotting. Proteins in the gels were transferred to nitrocellulose membranes for western blotting, and the resulting blots were immunostained with antiserum to recombinant λ-crystallin, as reported previously . The antiserum used was strongly and specifically reactive with λ-crystallin from the rabbit lens, and partial degradation proteins of the enzyme-crystallin were also weakly detectable .
Removal and extraction of tightly bound NADH in λ-crystallin
Removal of tightly bound NADH in the enzyme-crystallin was performed by stirring a solution containing 0.47 mg protein/ml in 10 mM K-phosphate (pH 7.2) with a 0.6% suspension of washed charcoal (Norit A; SERVA Electrophoresis, Heidelberg, Germany) for 4 min at 4 °C, according to the method of De Vijlder and Slater . The charcoal used was eliminated by centrifugation at 10,000x g for 5 min at 4 °C with a 13 mm Millex-HV syringe-driven filter (0.45 μm; Millipore, Bedford, MA).
NADH tightly bound to protein was extracted by treatment with 0.1% SDS in 10 mM K-phosphate (pH 7.2) for 20 min at room temperature or by heat treatment for 5 min at 70 °C. After the treatment, extracts were collected as filtrates of low molecular weight components by centrifugal ultrafiltration at 2,500x g for 15-45 min at 25 °C using an Ultrafree-CL filter (UFC4 LGC 25; 10,000 NMWL; Millipore).
Binding of added NADH to λ-crystallin
Binding of added NADH to the enzyme-crystallin was analyzed using a simple ultrafiltration method, which, as shown by Sophianopoulos et al. , is theoretically equivalent to equilibrium dialysis. NADH was added at concentrations of 5-100 μM (Ct) to 0.4-2.0 mg protein/ml of the crystallin solutions in 50 mM K-phosphate (pH 7.2), and the mixtures were allowed to stand for about 5 min at room temperature. Subsequently, small aliquots of free NADH were collected as filtrates by pressure-driven ultrafiltration at 2-4 atmospheres for about 5 min at room temperature using an Ultrafree-PF filter (UFP1 LGC 24; 10,000 NMWL; Millipore) or centrifugal ultrafiltration at 2,500x g for about 5 min at 25 °C using an Ultrafree-CL filter (UFC4 LGC 25; 10,000 NMWL; Millipore). There was little detectable binding of either NADH or protein to the filter devices. Concentrations of free NADH (Cf) were determined from absorbances of the filtrates at 340 nm. Quantities (nmol NADH/mg protein) of NADH additionally bound to protein were calculated by subtracting Cf from Ct values.
Results & Discussion
Isolation of λ-crystallin
In the present study, λ-crystallin was partially purified by a two-step isolation procedure comprising gel filtration and Affi-Gel blue affinity column chromatography. We chose affinity chromatography since Affi-Gel blue gel was previously shown to be useful in the isolation of proteins containing the dinucleotide fold (as per the supplier's instruction manual) . The λ/βL1-crystallin fraction with DHA reductase activity was separated from the rabbit lens soluble fraction by gel filtration, using a method reported elsewhere , following which the major λ-crystallin fraction was co-isolated with DHA reductase by affinity column chromatography of the λ/βL1-crystallin fraction (Figure 1A). Western blot analysis of the crude and partially purified samples using an antiserum to recombinant λ-crystallin revealed strong immunoreactivity for a 33 kDa protein, the main subunit of λ-crystallin , while a few protein bands with molecular weights of approximately 25-30 kDa were weakly detectable (Figure 1B,C). On the basis of densitometric analysis of the SDS-PAGE gels (Figure 1B), the purity of the 33 kDa protein was calculated to reach 88% (the average of two experiments) in the partially purified enzyme-crystallin, whereas the 33 kDa protein and the lower molecular weight proteins accounted for 34% and 64% (the average of two experiments) of the λ/βL1-crystallin fraction, respectively. As described previously , the majority of the lower molecular weight proteins are probably posttranslationally generated by proteolytic and/or nonenzymatic cleavage of the main 33 kDa protein. Moreover, small amounts of β-crystallin were also detected in the same molecular mass region.
Tightly bound NADH in λ-crystallin
To characterize whether λ-crystallin bound NADH tightly even though the enzyme-crystallin had undergone several chromatographic, ultrafiltration, and dialysis steps during its isolation, absorption spectra of the prepared crystallin samples were measured. The enzyme-crystallin samples had an absorption maximum at 340 nm (Figure 2A,B), suggesting the presence of NADH bound tightly to the protein. The ratio of absorbance at 340 nm to 280 nm increased from 0.03 in the λ/βL1-crystallin fraction to 0.12 in the partially purified enzyme-crystallin. Although the bound substance was not removed by dialysis or ultrafiltration, the cofactor was removed by charcoal treatment (Figure 2C). It was also extracted by 0.1% SDS (Figure 2D) or 70 °C heat treatment (data not shown). Thus, these results revealed that the cofactor binding was noncovalent. The absorption spectrum of the extracts was generally consistent with that of an authentic NADH sample (Figure 2D), except that the absorbance ratios at 205 and 260 nm to 340 nm were a little larger in the extracts compared to the authentic NADH. This larger absorption in the shorter wavelength region of the extracts might reflect coexistence of small amounts of NAD+ and any foreign UV-absorbing contaminants. Furthermore, we confirmed that at 340 nm, the absorption band of the partially purified enzyme-crystallin and the extracts obtained by the heat treatment was eliminated by the enzymatic action of NADH-specific oxidoreductase, its own DHA reductase, or lactate dehydrogenase (from chicken heart; Wako Pure Chemical Industries, Osaka, Japan; unpublished).
Glyceraldehyde 3-phosphate dehydrogenase isolated from rabbit and lobster muscles has been previously reported to contain tightly bound NAD+, which is removable by charcoal treatment but not by dialysis [22,25,26]. Moreover, the dissociation constant (Kd) of the nondialyzable NAD+ binding is roughly estimated as less than 5 nM . Therefore, the Kd for the tight NADH binding of λ-crystallin might also be less than 5 nM. By assuming that the molecular extinction coefficient of bound NADH at 340 nm was identical to that (6200 M-1cm-1 ) of free NADH, the quantity of tightly bound NADH in the partially purified enzyme-crystallin was determined to be 20.5 nmol/mg protein (the average of two experiments), suggesting a stoichiometry of 0.7 mol of the nucleotide/mol of the 33 kDa protein. The NADH content in the partially purified enzyme-crystallin was 3-6 times more than in the λ/βL1-crystallin fraction. These results indicate that the tight binding of NADH to the enzyme-crystallin required the 33 kDa protein.
Additional NADH binding to λ-crystallin
Binding titration of λ-crystallin by added NADH or NADPH was further studied. Saturated titration curves for NADH were obtained in the λ/βL1-crystallin fraction and the partially purified enzyme-crystallin (Figure 3, red and blue lines). The nucleotide binding was also specific for NADH, because little of it was observed for NADPH (Figure 3, green line). From Scatchard plots of the titration data, the maximum capacity and Kd of the additional NADH binding were determined to be 13.7 nmol/mg protein and 2.3 μM in the λ/βL1-crystallin fraction, and to be 11.9 nmol/mg protein (about half the tightly bound NADH content) and 6.4 μM in the partially purified enzyme-crystallin. The maximum binding capacity was 15% higher in the crude crystallin fraction than in the partially purified enzyme-crystallin. The two Kd values for the additional NADH binding were more than 400 times that for the tight NADH binding. It is assumed from the aforedescribed analysis of binding titration that although a proportion (about 30%) of the 33 kDa protein (the main subunit of the enzyme-crystallin) and most of the 25-30 kDa proteins (the minor cleaved subunits) cannot tightly bind NADH, their proteins still possess the ability to bind the nucleotide loosely. It is conceivable that partial degradation of the enzyme-crystallin weakens the strength of NADH binding. But, why does the binding affinity of a part of the native λ-crystallin also become looser? It is possible that the lower affinity binding reflects structural changes in the nucleotide binding sites. Negative cooperativity among the cofactor binding sites, in which the binding of one molecule makes it more difficult for the next molecule to bind, is observed in homotetrameric glyceraldehyde 3-phosphate dehydrogenase . Since λ-crystallin exists as a homodimer or a tetramer , such negative cooperativity may also occur in this enzyme-crystallin.
From the present study, we conclude that λ-crystallin can play a sufficiently important role as a NADH binding protein to retain high levels of this nucleotide in the rabbit lens. Moreover, it is known that the rabbit lens contains not only NADH (about 0.4 mM), but also NAD+ (about 1.1 mM) at high concentrations . λ-Crystallin probably binds NAD+ more loosely than does NADH, as suggested by the kinetic analysis of gulonate dehydrogenase/acetoacetate reductase. which is identical to the enzyme-crystallin . The elevation of both the reduced and oxidized nucleotides indicates that they may participate in a redox cycle, linked to glucose metabolism, in the lens . However, further investigations are needed to elucidate the NAD+ binding properties of the enzyme-crystallin and the redox cycling of these nucleotides in the lens.
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