Barbara Groth-Vasselli

Interaction of ATP and lens alpha crystallin characterized by equilibrium binding studies and intrinsic tryptophan fluorescence spectroscopy

alpha-Crystallin, the most prevalent protein in vertebrate lenses, is a high molecular weight aggregate composed of alpha A and alpha B subunits. Evidence is presented that ATP, a major phosphorus metabolite of the lens binds to alpha-crystallin extracted from calf lenses. The following parameters were obtained from equilibrium binding studies conducted at 37 degrees C: binding sites per 400 kDa aggregate = 10 and Ka = 8.1 x 10(3) M-1; and an essentially identical Ka of 7.84 x 10(3) M-1 and 22 binding sites were determined for a 850 kDa aggregate. The cooperativity parameter, alpha H, approximates unity which denotes that the binding of ligand is at independent sites. Binding was not significant at 22 degrees C and was absent at 4 degrees C. The specificity of the binding site for ATP was established by intrinsic tryptophan fluorescence spectroscopy. In the presence of increasing concentrations of ATP (0.05-0.3 mM), tryptophan fluorescence decreases in a concentration dependent manner to a minimum of 0.2 mM above which there is a non-linear response. Quenching of fluorescence was not evident with P(i), AMP or ADP. GTP elicited a minimal quenching of fluorescence only at the highest concentration (0.30 mM). Modulation of both supramolecular organization and lens metabolism is predicted as a consequence of ATP/alpha-crystallin binding.

31P NMR studies of the ATP/α-crystallin complex: Functional implications

Abstract Evidence is presented for the binding of ATP to α-crystallin in the lens by 31P NMR spectroscopic measurements. The chemical shift data as well as the T1 and T2 values indicate that Pβ and Pγ of ATP are of prime importance in binding. In addition, it is demonstrated that the association of α-crystallin with purified fiber cell membranes is significantly enhanced by the addition of ATP. These results suggest that ATP modulates the functional behavior of α-crystallin.

Refinement of 3D structure of bovine lens αA-crystallin

Abstract In absence of 3D structures for α -crystallin subunits, α A and α B, we utilized a number of experimental and molecular modeling techniques to generate working 3D models of these polypeptides (Farnsworth et al., 1994. In Molecular Modeling: From Virtual Tools to Real Problems (Eds. Kumosinski, T.F. and Liebman, M.N.) ACS Symposium Series 576, Ch. 9:123–134, 1994, ACS Books, Washington DC). The refinement of the initial bovine α A model was achieved using a more accurate estimation of secondary structure by new/updated methods for analyzing the far UV-CD spectra and by neural network secondary structure predictions in combination with database searches. The spectroscopic study reveals that α -crystallin is not an all β -sheet protein but contains ∼17% α -helices, ∼33% β -structures and ∼50% turns and coils. The refinement of the α A structure results in an elongate, asymmetric amphipathic molecule. The hydrophobic N-terminal domain imparts the driving force for subunit aggregation while the more flexible, polar C-terminal domain imparts aggregate solubility. In our quaternary structure of the aggregate, the monomer is the minimal cooperative subunit. In bovine α A, the highly negatively charged C-terminal domain has three small positive areas which may participate in dimer or tetramer formation of independently expressed C-terminal domains. The electrostatic potential of positive areas is modulated and become more negative with phosphorylation and ATP binding. The refined bovine α A model was used to construct α A models for the human, chick and dogfish shark. A high degree of conservation of the three dimensional structure and the electrostatic potential was observed. Our proposed open micellar quaternary structure correlates well with experimental data accumulated over the past several decades. The structure is also predictive of the more recent data.

α-Crystallin quaternary structure: molecular basis for its chaperone activity

α‐Crystallin, the major protein in all vertebrate lenses, functions as a chaperone. In the present analysis, an ‘open’ micellar structure composed of αA subunits is used to simulate chaperoning of partially heat denatured soluble γ‐crystallin. The interaction is both electrostatic and hydrophobic and satisfies experimental evidence for a 1:1 α/γ molar ratio, a doubling of molecular mass and a minimal increase in the dimensions of the complex [J. Biol. Chem. (1994) 269, 13601–13608; Invest. Opthalmol. Vis. Sci. (1995) 36, 311–321]. These data are also in accord with Farahbaksh et al. [Biochemistry (1995) 34, 509–516]; i.e. the bound γ‐crystallin monomers are not in a central cavity, but are separated by αA subunits.

A comparison of structural relationships among α-crystallin, human Hsp27, γ-crystallins and βB2-crystallin

Abstract The 3D structures of α-crystallin, a major eye lens protein, and related small heat shock proteins are unresolved. It has been assumed that α-crystallin is primarily a β-sheet globular protein similar to γ-crystallin (Siezen and Argos, Biochim. Biophys. Acta, 1983, 748, 56–67) containing sequence repeats in its two domains (Wistow, FEBS Lett. 1985, 181, 1–6). Positional flexibility of amino acid residues and far UV-circular dichroism spectroscopy were used to investigate structural relationships among these proteins. The utility of flexibility plots for predicting protein structure is demonstrated by the excellent correlation of these plots with the known 3D X-ray structures of β/γ-crystallins. Similar analyses of α-crystallin subunits, αA and αB, and human heat shock protein 27 show that the C-terminal domains and connecting segments of these proteins are very similar while the N-terminal domains have significant structural differences. Unlike β/γ-crystallins, both Hsp27 and α-crystallin subunits are asymmetrical with highly flexible C-terminal domains. Flexibility is considered essential for protein functional activity. Therefore, the C-terminal region may play an active role in α-crystallin and small heat shock protein function. Differences in flexibility profiles and estimated secondary structure distribution in α-crystallin by three recent/updated algorithms from far UV-CD spectra support our predicted 3D structure and the concept that α-crystallin and members of β/γ-superfamily are structurally dissimilar.

Investigation of lens glycolytic enzymes: Species distribution and interaction with supramolecular order

Distribution of several glycolytic enzymes in the lenses of different vertebrate species and their organization in the calf lenses were studied. Though no general pattern of enzyme activities in different species is discernible, high activities of TPI followed, in decreasing order, by GAPDH, enolase, PK, LDH and aldolase appear to be more common. Our observation on the unusually high activities of aldolase in the pig, enolase in the sheep and LDH in the duck lens are interesting in view of the already known dual function of LDH as an enzyme and a structural protein (epsilon-crystallin) in duck. Controlled treatment with detergents Brij-58 and Triton X-100 caused distinctly differential purturbations in the lens cells. In spite of fiber membrane disruption and partial actin dissolution by Brij-58, no significant increase in the release of glycolytic enzymes compared to control was observed. This suggests that none of the enzymes existed as a completely soluble and freely diffusible fraction. But treatment with a strong detergent (Triton X-100) caused the release of higher amounts of enzymes suggesting either a direct or indirect interaction with the cytomatrix components. Aldolase appears to be maximally bound in the cytosol followed by TPI, GAPDH, LDH and PK in decreasing order. Although thin lens slices were incubated with the detergents for a total period of 40 min and the loss of fiber architecture and organization confirmed by light microscopy, in the Triton X-100 treated tissues less than 25% of the total activity of any enzyme except TPI appeared in the bathing medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphorus and proton magnetic resonance spectroscopic studies on the relationship between transparency and glucose metabolism in the rabbit lens

We report the results of a series of nuclear magnetic resonance (NMR) experiments designed to investigate the relationship between particular aspects of glucose metabolism and cataract formation in the rabbit lens. The glucose metabolism of the rabbit lens incubated in TC-199 medium containing 5.5 mM glucose, in glucose-deficient medium, and in modified Earles medium containing 5.5 mM glucose devoid of NaCl, is examined in conjunction with the assessment of lens transparency. Significant age-dependent differences in the phosphorus metabolite profile and in hexosemonophosphate shunt flux, as measured by NMR, were observed in lenses incubated in TC-199 medium containing 5.5 mM glucose. Incubation in glucose-deficient medium for 8 hr results in significant increases in the levels of inorganic phosphate and phosphomonoesters, and decreases in ATP and L-alpha-glycerolphosphate. These levels regain near-normal values after 24 hr incubation in control medium containing 5.5 mM glucose. By contrast, shunt flux is three times the basal level during the recovery period. Lens clarity, as assessed by slit lamp micrography, was maintained throughout the duration of the experiment. Incubation of adult and juvenile lenses for 18 hr in Earles medium (pH 7.4 or 9.2) containing 5.5 mM glucose, and no NaCl, results in uniform lenticular opacification within 18 hr and changes in ultrastructure of the epithelial and cortical lens fiber cells. No statistically significant change in the NMR visible phosphorus metabolite profile or intralenticular pH is observed for the adult rabbit lens relative to a lens incubated under control conditions. For the juvenile rabbit lens, small, but statistically significant differences in the levels of dinucleotide and uridinediphosphoglucose were observed. Shunt flux, in contrast, is increased two-fold. These results demonstrate that the NMR visible phosphorus metabolite profile of the lens does not necessarily correlate with transparency, and that hexosemonophosphate shunt activity provides a sensitive measure of prior or current lenticular stress.

INTERACTION OF DNA WITH BOVINE LENS ALPHA -CRYSTALLIN : ITS FUNCTIONAL IMPLICATIONS

Abstract Under normal conditions, lens aggregates of α -crystallin subunits, α A and α B, are found in the cytoplasm. However, during stress in nonlenticular tissues, α B translocates to the nucleus. A sequence study revealed that both subunits share a consensus sequence with other DNA binding proteins. These observations prompted us to investigate DNA binding with α -crystallin by UV-mediated photo-crosslinking. The data show that both single and double stranded DNA crosslink mainly with tetramers of α -crystallin subunits. The formation of tetramers appears to modify α -crystallin interactive properties and, therefore, its induction may have functional significance. These observations suggest that α -crystallin may have a nuclear function which includes DNA binding.

Morphological studies of an ion-dependent perinuclear cataract model

The perinuclear region of the rabbit lens is susceptible to alterations in the ionic composition of incubation medium. Rabbit lenses and a comparable cell type, red blood cells, were stressed during ex vivo incubations in isotonic modified Earles medium with 131 mM NaCl replaced by either 232 mM sucrose or 131 mM choline chloride at pH 7.2 (normal) or 9.2. Our parallel NMR study revealed that these experimental media maintain normal intracellular pH and phosphorus metabolite levels. The present study demonstrates that lens transparency, normal fiber cell ultrastructure and volume were maintained in either sodium chloride or choline chloride containing media at normal or elevated pH. Similarly, normal morphology, mean cell volume (MCV) and mean cell hemoglobin concentration (MCHC), 86.8 +/- 0.03 micron 3 and 33.2 +/- 1.0 g dl-1, respectively, were maintained in red blood cells in either sodium chloride or choline chloride containing media. In sodium chloride deficient media at both normal and elevated pH the lens developed a nuclear cataract based on slit-lamp examination; however, SEM examination showed that fiber cell morphological abnormalities were confined to a narrow band, 50 micron wide, in the perinuclear region of the transition zone. Damage consisted of ruptured cell membranes and an absence of identifiable interdigitations with the combination of sodium chloride deficiency and elevated pH. The major abnormality produced during incubation in sodium chloride deficient medium at normal pH was the presence of numerous smooth-surfaced cellular protrusions along the vertices of the perinuclear fiber cells. In addition, the sodium chloride deficient medium, pH 9.2, produced a volume loss both in the lens and RBC (4.5 +/- 1.5% and 5.6 +/- 1.1%, respectively). The sodium chloride deficient medium, pH 7.4, produced no volume loss in the lens or red blood cells (MCV 86.0 +/- 0.05 micron 3). Further studies indicated that the cataract induced by sodium chloride deficiency (pH 9.2) is irreversible. The mechanism for perinuclear opacification due to ion deficiency remains to be elucidated.

Electrostatic parameters of the theoretical quaternary structure of bovine α-crystallin

By changing the ionic strength, the effects of charge modification on the electrostatic properties of our predicted ‘open’ micellar quaternary structure composed of bovine αA subunits were determined. The electrostatic potential values (o) at 6 arbitrary points surrounding the protein and at all atomic sites of the protein were calculated using the non-linear Poisson-Boltzmann equation. The effective charge (q) of our predicted aggregate ranged from 16 at 0.0022 M to 45 at 0.1472 M ionic strengths. The variation of potential (o), as well as charge, is a hyperbolic function of ionic strength (R2, 0.901). Plotting the inverse charge (l/q) against inverse ionic strength (1/I) it is possible to calculate maximum charge (qmax) (-48) at saturation. This value is close to previously reported experimental (50±5), and our theoretical charge (45), values at physiological ionic strength (0.145 M). These data indicate that maximal repulsion among the α-crystallin aggregates occurs at or near physiological ionic strength. Also, half-maximal charge (qmax/2) at 0.003-0.004 M indicates a transition state at very low ionic strength. The calculated volume available for the mobile solvent in our quaternary structure is -70%. These data are in good agreement with experimental values for bovine α-crystallin in solution reported by Xia et al. (Biophys. J., 1994; 66: 861-872). This agreement provides support for our predicted models of α-crystallin and a level of confidence in the reliability of the theoretical calculations. Since an ionic gradient exists between the lens cortical and nuclear regions, the modification of charge on α-crystallin by varying ionic strength could contribute to the function of α-crystallin as a modulator of lens supermolecular order during fiber cell maturation.