Detection of cholesterol bilayer domains in intact biological membranes: Methodology development and its application to studies of eye lens fiber cell plasma membranes.

Four purported lipid domains are expected in plasma membranes of the eye lens fiber cells. Three of these domains, namely, bulk, boundary, and trapped lipids, have been detected. The cholesterol bilayer domain (CBD), which has been detected in lens lipid membranes prepared from the total lipids extracted from fiber cell plasma membranes, has not yet been detected in intact fiber cell plasma membranes. Here, a saturation-recovery electron paramagnetic resonance spin-labeling method has been developed that allows identification of CBDs in intact fiber cell plasma membranes of eye lenses. This method is based on saturation-recovery signal measurements of the cholesterol-analog spin label located in the lipid bilayer portion of intact fiber cell membranes as a function of the partial pressure of molecular oxygen with which the samples are equilibrated. The capabilities and limitations of this method are illustrated for intact cortical and nuclear fiber cell plasma membranes from porcine eye lenses where CBDs were detected in porcine nuclear intact membranes for which CBDs were also detected in lens lipid membranes. CBDs were not detected in porcine cortical intact and lens lipid membranes. CBDs were detected in intact membranes isolated from both cortical and nuclear fiber cells of lenses obtained from human donors. The cholesterol content in fiber cell membranes of these donors was always high enough to induce the formation of CBDs in cortical as well as nuclear lens lipid membranes. The results obtained for intact membranes, when combined with those obtained for lens lipid membranes, advance our understanding of the role of high cholesterol content and CBDs in lens biology, aging, and/or cataract formation.

Extraction, purification and characterization of the crystallin protein of cataractous eye lens nucleus.

The purpose of this study is to separate and identify the crystallin protein present in the nucleus of a human cataractous eye lens. Cataractous lenses were collected from different eye hospitals from patients of different etiologies with ages between 40 and 80 years. Lens nucleus proteins were extracted into four fractions on the basis of their solubility in different media by applying a reported method. These fractions were buffer-soluble proteins (PS), urea-soluble proteins (PU), yellow fraction proteins (PY) and insoluble proteins (PI). All three soluble fractions were subjected to HPLC and GPC analysis. Both HPLC and GPC analysis showed that each fraction contains α-, ß- and γ-crystallins, a major class of protein present in the lenses of vertebrates. Various chromatographic parameters including precision, accuracy and linearity have been evaluated. Studies of water-insoluble crystallins using sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) have demonstrated extreme homogeneity with evidence of major components with molecular masses of 18-70 kDa, similar to the crystallin of the water-soluble portion. The method was found to be suitable for the analysis of various isomers of crystallin protein present in human cataractous eye lens nuclei. The detailed results of the GPC are discussed. This study provides the first HPLC and GPC analysis of a human cataractous eye lens nucleus.

Polymorphisms in sodium-dependent vitamin C transporter genes and plasma, aqueous humor and lens nucleus ascorbate concentrations in an ascorbate depleted setting.

We have previously reported low concentrations of plasma ascorbate and low dietary vitamin C intake in the older Indian population and a strong inverse association of these with cataract. Little is known about ascorbate levels in aqueous humor and lens in populations habitually depleted of ascorbate and no studies in any setting have investigated whether genetic polymorphisms influence ascorbate levels in ocular tissues. Our objectives were to investigate relationships between ascorbate concentrations in plasma, aqueous humor and lens and whether these relationships are influenced by Single Nucleotide Polymorphisms (SNPs) in sodium-dependent vitamin C transporter genes (SLC23A1 and SLC23A2). We enrolled sixty patients (equal numbers of men and women, mean age 63 years) undergoing small incision cataract surgery in southern India. We measured ascorbate concentrations in plasma, aqueous humor and lens nucleus using high performance liquid chromatography. SLC23A1 SNPs (rs4257763, rs6596473) and SLC23A2 SNPs (rs1279683 and rs12479919) were genotyped using a TaqMan assay. Patients were interviewed for lifestyle factors which might influence ascorbate. Plasma vitamin C was normalized by a log10 transformation. Statistical analysis used linear regression with the slope of the within-subject associations estimated using beta (ß) coefficients. The ascorbate concentrations (µmol/L) were: plasma ascorbate, median and inter-quartile range (IQR), 15.2 (7.8, 34.5), mean (SD) of aqueous humor ascorbate, 1074 (545) and lens nucleus ascorbate, 0.42 (0.16) (µmol/g lens nucleus wet weight). Minimum allele frequencies were: rs1279683 (0.28), rs12479919 (0.30), rs659647 (0.48). Decreasing concentrations of ocular ascorbate from the common to the rare genotype were observed for rs6596473 and rs12479919. The per allele difference in aqueous humor ascorbate for rs6596473 was -217 µmol/L, p < 0.04 and a per allele difference in lens nucleus ascorbate of -0.085 µmol/g, p < 0.02 for rs12479919. The ß coefficients for the regression of log10 plasma ascorbate on aqueous humor ascorbate were higher for the GG genotype of rs6596473: GG, ß = 1460 compared to carriage of the C allele, CG, ß = 1059, CC, ß = 1132, p interaction = 0.1. In conclusion we found that compared to studies in well-nourished populations, ascorbate concentrations in the plasma, aqueous humor and lens nucleus were low. We present novel findings that polymorphisms in SLC23A1/2 genes influenced ascorbate concentration in aqueous humor and lens nucleus.

Properties of membranes derived from the total lipids extracted from the human lens cortex and nucleus.

Human lens lipid membranes prepared using a rapid solvent exchange method from the total lipids extracted from the clear lens cortex and nucleus of 41- to 60-year-old donors were investigated using electron paramagnetic resonance spin-labeling. Profiles of the phospholipid alkyl-chain order, fluidity, oxygen transport parameter, and hydrophobicity were assessed across coexisting membrane domains. Membranes prepared from the lens cortex and nucleus were found to contain two distinct lipid environments, the bulk phospholipid-cholesterol domain and the cholesterol bilayer domain (CBD). The alkyl chains of phospholipids were strongly ordered at all depths, indicating that the amplitude of the wobbling motion of alkyl chains was small. However, profiles of the membrane fluidity, which explicitly contain time (expressed as the spin-lattice relaxation rate) and depend on the rotational motion of spin labels, show relatively high fluidity of alkyl chains close to the membrane center. Profiles of the oxygen transport parameter and hydrophobicity have a rectangular shape and also indicate a high fluidity and hydrophobicity of the membrane center. The amount of CBD was greater in nuclear membranes than in cortical membranes. The presence of the CBD in lens lipid membranes, which at 37°C showed a permeability coefficient for oxygen about 60% smaller than across a water layer of the same thickness, would be expected to raise the barrier for oxygen transport across the fiber cell membrane. Properties of human membranes are compared with those obtained for membranes made of lipids extracted from cortex and nucleus of porcine and bovine eye lenses.

Properties of fiber cell plasma membranes isolated from the cortex and nucleus of the porcine eye lens.

The organization and physical properties of the lipid bilayer portion of intact cortical and nuclear fiber cell plasma membranes isolated from the eye lenses of two-year-old pigs were studied using electron paramagnetic resonance (EPR) spin-labeling. Membrane fluidity, hydrophobicity, and the oxygen transport parameter (OTP) were assessed from the EPR spectra of precisely positioned spin labels. Intact cortical and nuclear membranes, which include membrane proteins, were found to contain three distinct lipid environments. These lipid environments were termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain (lipids in protein aggregates). The amount of boundary and trapped lipids was greater in intact nuclear membranes than in cortical membranes. The properties of intact membranes were compared with the organization and properties of lens lipid membranes made of the total lipid extracts from the lens cortex or nucleus. In cortical lens lipid membranes, only one homogenous environment was detected, which was designated as a bulk lipid domain (phospholipid bilayer saturated with cholesterol). Lens lipid membranes prepared from the lens nucleus possessed two domains, assigned as a bulk lipid domain and a cholesterol bilayer domain (CBD). In intact nuclear membranes, it was difficult to discriminate the CBD, which was clearly detected in nuclear lens lipid membranes, because the OTP measured in the CBD is the same as in the domain formed by trapped lipids. The two domains unique to intact membranes-namely, the domain formed by boundary lipids and the domain formed by trapped lipids-were most likely formed due to the presence of membrane proteins. It is concluded that formation of rigid and practically impermeable domains is enhanced in the lens nucleus, indicating changes in membrane composition that may help to maintain low oxygen concentration in this lens region.

Instability of the cellular lipidome with age.

The human lens nucleus is formed in utero, and from birth onwards, there appears to be no significant turnover of intracellular proteins or membrane components. Since, in adults, this region also lacks active enzymes, it offers the opportunity to examine the intrinsic stability of macromolecules under physiological conditions. Fifty seven human lenses, ranging in age from 12 to 82 years, were dissected into nucleus and cortex, and the nuclear lipids analyzed by electrospray ionization tandem mass spectrometry. In the first four decades of life, glycerophospholipids (with the exception of lysophosphatidylethanolamines) declined rapidly, such that by age 40, their content became negligible. In contrast the level of ceramides and dihydroceramides, which were undetectable prior to age 30, increased approximately 100-fold. The concentration of sphingomyelins and dihydrosphingomyelins remained unchanged over the whole life span. As a consequence of this marked alteration in composition, the properties of fiber cell membranes in the centre of young lenses are likely to be very different from those in older lenses. Interestingly, the identification of age 40 years as a time of transition in the lipid composition of the nucleus coincides with previously reported macroscopic changes in lens properties (e.g., a massive age-related increase in lens stiffness) and related pathologies such as presbyopia. The underlying reasons for the dramatic change in the lipid profile of the human lens with age are not known, but are most likely linked to the stability of some membrane lipids in a physiological environment.

Physical properties of the lipid bilayer membrane made of cortical and nuclear bovine lens lipids: EPR spin-labeling studies.

The physical properties of membranes derived from the total lipids extracted from the lens cortex and nucleus of a 2-year-old cow were investigated using EPR spin-labeling methods. Conventional EPR spectra and saturation-recovery curves show that spin labels detect a single homogenous environment in membranes made from cortical lipids. Properties of these membranes are very similar to those reported by us for membranes made of the total lipid extract of 6-month-old calf lenses (J. Widomska, M. Raguz, J. Dillon, E. R. Gaillard, W. K. Subczynski, Biochim. Biophys. Acta 1768 (2007) 1454-1465). However, in membranes made from nuclear lipids, two domains were detected by the EPR discrimination by oxygen transport method using the cholesterol analogue spin label and were assigned to the bulk phospholipid-cholesterol domain (PCD) and the immiscible cholesterol crystalline domain (CCD), respectively. Profiles of the order parameter, hydrophobicity, and the oxygen transport parameter are practically identical in the bulk PCD when measured for either the cortical or nuclear lipid membranes. In both membranes, lipids in the bulk PCD are strongly immobilized at all depths. Hydrophobicity and oxygen transport parameter profiles have a rectangular shape with an abrupt change between the C9 and C10 positions, which is approximately where the steroid ring structure of cholesterol reaches into the membrane. The permeability coefficient for oxygen, estimated at 35 degrees C, across the bulk PCD in both membranes is slightly lower than across the water layer of the same thickness. However, the evaluated upper limit of the permeability coefficient for oxygen across the CCD (34.4 cm/s) is significantly lower than across the water layer of the same thickness (85.9 cm/s), indicating that the CCD can significantly reduce oxygen transport in the lens nucleus.

Oxindolealanine in age-related human cataracts.

The present study was performed in order to obtain structural and quantitative information regarding the modifications that take place in the human lens as a result of tryptophan oxidation. In particular, the early tryptophan oxidation product, oxindolealanine (OIA) has been detected in lyophilized and hydrolyzed cataractous lenses by mass spectrometry. OIA was confirmed in human cataract samples by observing its ion (m/z 221), fragmentation pattern and absorption spectrum. Quantitative results indicate that there are differences in the amounts of OIA in the nucleus versus the cortex in human cataractous lenses. Expressed as a ratio to the level of phenylalanine (Phe), the nucleus has more than one and a half times greater levels of OIA as compared to the cortex [nucleus=(3.7+/-0.7)x10(-2) versus cortex=(2.3+/-0.3)x10(-2)]. Furthermore, the average value for the OIA/Phe ratio in the calf lens (controls) was (0.8+/-0.2)x10(-2) as compared to (3.7+/-0.7)x10(-2) in human cataractous lens nucleus (p<0.05). The quantitative results correspond to a 4.6-fold increase of OIA in human cataractous lenses. In a separate series of experiments using HPLC with photodiode array (PDA) detection only, the differences in OIA levels in cataract nucleus versus cortex and cataracts versus controls closely matched the LC/MS data. The results suggest that OIA levels are elevated in human cataractous lenses thus providing further evidence to implicate tryptophan oxidation in this process.

Protein-bound UV filters in normal human lenses: the concentration of bound UV filters equals that of free UV filters in the center of older lenses.

PURPOSE: To survey the levels of protein-bound UV filters in the cortices and nuclei of normal human lenses as a function of age and to relate this to the concentration of free UV filters. METHODS: Levels of each of the three kynurenine (Kyn) UV filters, 3-hydroxykynurenine glucoside (3OHKG), Kyn, and 3-hydroxykynurenine (3OHKyn), covalently attached to proteins, were determined by using a newly developed method of reductive capture, after base treatment of the intact lens proteins. RESULTS: The data show that, in the normal lens, each of the three UV filters became bound to proteins to a significant extent only after age 50 and, further, that the levels in the nucleus were much higher than in the cortex. These findings are consistent with the lens barrier that forms in middle age. 3OHKG was present at the highest levels followed by Kyn, with 3OHKyn being attached in the lowest amount. The ratio was 145:4:1 (3OHKG-Kyn-3OHKyn), with a total protein-bound UV filter concentration in the lens nucleus after age 50 of approximately 1300 picomoles/mg protein. This ratio is in agreement with 3OHKG being the most abundant free UV filter in the human lens and 3OHKyn being present in the lowest concentration with free Kyn present in intermediate amounts. CONCLUSIONS: The three Kyn UV filters are bound to the nuclear proteins of all normal lenses over the age of 50. Indeed in the center of older normal lenses, the concentration of UV filters bound to proteins is approximately equal to that of the free filters. Since bound UV filters promote oxidation of proteins after exposure to wavelengths of light that penetrate the cornea, lenses in middle-aged and older individuals may be more prone to photooxidation than those of young people.

Raman spectroscopic evidence for nuclear disulfide in isolated lenses of hyperbaric oxygen-treated guinea pigs.

Our laboratory treats guinea pigs with hyperbaric oxygen (HBO) as a model for investigating the formation of nuclear cataract. Previous analyses of lens supernatants using this model have shown an increase in disulfide (-SS-) and loss of sulfhydryl (-SH) in the lens nucleus of O(2)-treated animals. In this paper, we have used the non-invasive technique of Raman spectroscopy to confirm these findings in intact, freshly-excised lenses. Guinea pigs were treated 3 times per week with HBO for a total of 50 (4 months of treatment) or 85 (7 months of treatment) times to induce an increased level of lens nuclear light scattering. Intact lenses were analyzed by Raman spectroscopy using a 514.5 nm laser and collecting the scattered light in a 90 degrees geometry. The laser beam was focused either in the lens nucleus or equatorial cortex. Changes in the levels of -SS- (503 cm(-1)) and -SH (2577 cm(-1)) vibrations were measured. Raman spectra were analyzed by fitting Lorentzian profiles to the observed data in the -SS- and -SH regions. -SS- levels in the O(2)-treated nucleus were found to have increased by a factor of 2.1 (p=0.0001) and 2.5 (p=0.001) after 50 and 85 HBO treatments, respectively, compared to age-matched controls. Based on previous biochemical analyses, the -SS- increase was due mainly to the formation of protein disulfide (PSSP) with contribution also from protein/thiol mixed disulfides, but not from oxidized glutathione. -SH levels in the O(2)-treated nucleus decreased by 13% (p=0.007) and 35% (p=0.001) after 50 and 85 HBO treatments, respectively, compared to age-matched controls. No significant increase in -SS- or loss of -SH was observed in the lens cortex of the O(2)-treated guinea pigs. The Raman spectroscopy results rule out the possibility that artifactual production of -SS- and loss of -SH occurred during homogenization of lenses in previous studies. The data provide additional evidence to support a link between O(2), disulfide-crosslinking of lens crystallins in the nucleus, and nuclear cataract.

Connexin 46 and connexin 50 in selenite cataract.

The purpose of this work was to determine if the lens gap junction proteins connexin 46 (Cx46) and connexin 50 (Cx50) were altered with the development of selenite-induced cataract. Cataracts were induced in young Sprague-Dawley rats with a single subcutaneous injection of sodium selenite; age-matched uninjected rats served as controls. Membrane fractions were isolated from homogenates of cortex and nucleus of normal and cataractous lenses by differential and discontinuous sucrose gradient centrifugation. Aliquots of urea-insoluble protein from membrane fractions were analyzed by quantitative densitometry of Western blots probed with antibodies to Cx46 and Cx50. A significant decrease in the more slowly migrating Cx46-reactive band, which represents phosphorylated Cx46, was found in the major membrane fraction of the cortex of cataractous lenses. There was no significant difference in the amounts of either Cx46 or Cx50 associated with selenite cataract in any of the membrane fractions examined. These results suggest that alteration of gap junction function (as evidenced by the change in phosphorylation of Cx46) may be associated with the development of the selenite cataract, but that neither Cx46 nor Cx50 is subject to the well-characterized proteolysis associated with the selenite cataract model.

Aggregation of lens crystallins in an in vivo hyperbaric oxygen guinea pig model of nuclear cataract: dynamic light-scattering and HPLC analysis.

PURPOSE: The role of oxygen in the formation of lens high-molecular-weight (HMW) protein aggregates during the development of human nuclear cataract is not well understood. The purpose of this study was to investigate lens crystallin aggregate formation in hyperbaric oxygen (HBO)-treated guinea pigs by using in vivo and in vitro METHODS: methods. Guinea pigs were treated three times weekly for 7 months with HBO, and lens crystallin aggregation was investigated in vivo with the use of dynamic light-scattering (DLS) and in vitro by HPLC analysis of water-insoluble (WI) proteins. DLS measurements were made every 0.1 mm across the 4.5- to 5.0-mm optical axis of the guinea pig lens. RESULTS: The average apparent diameter of proteins in the nucleus (the central region) of lenses of HBO-treated animals was nearly twice that of the control animals (P < 0.001). Size distribution analysis conducted at one selected point in the nucleus and cortex (the outer periphery of the lens) after dividing the proteins into small-diameter and large-diameter groups, showed in the O2-treated nucleus a threefold increase in intensity (P < 0.001) and a doubling in apparent size (P = 0.03) of large-diameter aggregate proteins, compared with the same control group. No significant changes in apparent protein diameter were detected in the O2-treated cortex, compared with the control. The average diameter of protein aggregates at the single selected location in the O2-treated nucleus was estimated to be 150 nm, a size capable of scattering light and similar to the size of aggregates found in human nuclear cataracts. HPLC analysis indicated that one half of the experimental nuclear WI protein fraction (that had been dissolved in guanidine) consisted of disulfide cross-linked 150- to 1000-kDa aggregates, not present in the control. HPLC-isolated aggregates contained alphaA-, beta-, gamma-, and zeta-crystallins, but not alphaB-crystallin, which is devoid of -SH groups and thus does not participate in disulfide cross-linking. All zeta-crystallin present in the nuclear WI fraction appeared to be there as a result of disulfide cross-linking. CONCLUSIONS: The results indicate that molecular oxygen in vivo can induce the cross-linking of guinea pig lens nuclear crystallins into large disulfide-bonded aggregates capable of scattering light. A similar process may be involved in the formation of human nuclear cataract.

Isolation and lipid characterization of cholesterol-enriched fractions in cortical and nuclear human lens fibers.

PURPOSE: Human lens membranes contain unusually high levels of cholesterol and sphingolipids, lipids known to segregate into liquid-ordered domains. The current study was conducted to pursue the determination and characterization of these domains in membranes of clear and cataractous human lenses. METHODS: Cortical and nuclear regions of aged clear and cataractous lenses were obtained. After lysis with Triton X-100 at 4 degrees C and sucrose linear-density centrifugation, sedimenting and nonsedimenting fractions (when present) were collected. Phospholipids were analyzed by (31)P-nuclear magnetic resonance (NMR) and mass spectrometry. Caveolae and raft markers were tested by Western blot analysis. RESULTS: Only samples from clear lenses exhibited a nonsedimenting band. Phospholipid contents were comparable for sedimenting fractions of clear and cataractous membranes. Cholesterol to phospholipid molar ratios in light-density bands were nearly 7, three times greater than in sedimenting fractions. The portion of total cholesterol present in nonsedimenting fractions increased from 5.5% in the cortex to 14% in the nucleus. Two lysophospholipids comprising approximately 10% of all phospholipids in total membranes were undetectable in nonsedimenting fractions. Caveolin-1 was enriched in these fractions. CONCLUSIONS: Phospholipid compositional differences between lighter and heavier fractions from clear lenses were relatively minor and could not, alone, account for the substantial enrichment of cholesterol in the lighter fractions. Specific proteins, such as caveolin-1, must recruit cholesterol and induce clustering. Undetectable amounts of light-density domains in cataractous membranes suggest either disruption of these aggregates and thus the function of proteins within them, possibly relevant to lens transparency, and/or greater density of these clusters due to stronger binding of insoluble crystallins to membranes.

Identification of a novel fluorophore, xanthurenic acid 8- O -beta-D-glucoside in human brunescent cataract.

We have identified the chemical structure of a novel protein-unbound fluorescent glucoside (Fl-Glc), found to be far more abundant in the human brunescent cataractous lens nuclei than in non-brunescent ones. Our earlier experiments showed that long-term incubation of the protein-free filtrate of non-brunescent cataractous nuclei generated increasing amounts of a particular yet to be characterized fluorophore (Fl-X). High performance liquid chromatography (HPLC) analyses revealed Fl-X and Fl-Glc to be identical. HPLC-electrospray ionization-mass spectrometry (HPLC-ESI-MS) disclosed the molecular weights (MW) of Fl-X and its beta-glucosidase-digest (Fl-X-aglycon) to be 367 and 205, respectively. Fl-X-aglycon and authentic xanthurenic acid (MW = 205) not only eluted at exactly the same retention time on HPLC but also revealed their protonated ions at the same m/z of 206.1 by positive ion analysis on HPLC-ESI-MS. These results suggest that Fl-X ( = Fl-Glc) is a beta-glucoside of xanthurenic acid. Fl-Glc was finally identified as xanthurenic acid 8- O -beta- D -glucoside because the retention times of both completely agreed with three kinds of HPLC conditions.

Comparison of non-tryptophan fluorophores in protein-free extract of brunescent and non-brunescent human cataract.

PURPOSE: To investigate whether any of the fluorophores in the human lens nuclei might be responsible for human brunescent cataract formation. METHODS: Human lens nuclei (non-brunescent, from 13; brunescent, from 8) were obtained after extracapsular cataract extraction in nondiabetic patients. Protein-free extract, prepared by filtrating the water-soluble fraction of each nucleus through a centrifugal ultrafilter (molecular weight < 5,000), was analytically separated by high-performance liquid chromatography. RESULTS: No significant differences between non-brunescent and brunescent nuclei were observed in the concentrations (mean +/- SD) of 3-hydroxykynurenine O-beta-glucoside (0.67 +/- 0.38 vs. 0.85 +/- 0. 62 micromol/g wet weight), 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-glucoside (4.1 x 10(5) +/- 2.9 x 10(5) vs. 6.3 x 10(5) +/- 5. 0 x 10(5) area unit/g wet weight), and kynurenine (0.016 +/- 0.011 vs. 0.029 +/- 0.021 micromol/g wet weight). A novel fluorophore that has not been identified so far was significantly present more in brunescent than in non-brunescent nuclei (brunescent: 1.5 x 10(5) +/- 1.0 x 10(5) vs. non-brunescent: 2.6 x 10(3) +/- 6.3 x 10(3) area unit/g wet weight, P <.01). Digestion of the protein-free extract with beta-glucosidase eliminated the peak corresponding to the novel unidentified fluorophore. CONCLUSION: The present results imply a novel protein-unbound fluorophore, presumably a beta-glucoside, might possibly be involved in brunescent cataract formation.

Direct evidence for immiscible cholesterol domains in human ocular lens fiber cell plasma membranes.

The molecular structure of human ocular lens fiber cell plasma membranes was examined directly using small angle x-ray diffraction approaches. A distinct biochemical feature of these membranes is their high relative levels of free cholesterol; the mole ratio of cholesterol to phospholipid (C/P) measured in these membranes ranges from 1 to 4. The organization of cholesterol in this membrane system is not well understood, however. In this study, the structure of plasma membrane samples isolated from nuclear (3.3 C/P) and cortical (2.4 C/P) regions of human lenses was evaluated with x-ray diffraction approaches. Meridional diffraction patterns obtained from the oriented membrane samples demonstrated the presence of an immiscible cholesterol domain with a unit cell periodicity of 34.0 A, consistent with a cholesterol monohydrate bilayer. The dimensions of the sterol-rich domains remained constant over a broad range of temperatures (5-20 degrees C) and relative humidity levels (31-97%). In contrast, dimensions of the surrounding sterol-poor phase were significantly affected by experimental conditions. Similar structural features were observed in membranes reconstituted from fiber cell plasma membrane lipid extracts. The results of this study indicate that the lens fiber cell plasma membrane is a complex structure consisting of separate sterol-rich and -poor domains. Maintenance of these separate domains may be required for the normal function of lens fiber cell plasma membrane and may interfere with the cataractogenic aggregation of soluble lens proteins at the membrane surface.

Evidence for the participation of alpha B-crystallin in human age-related nuclear cataract.

The aim of this study was to determine if the unusual coloured species characteristic of age-related nuclear cataract could be localised to specific residues of the crystallins. The insoluble, crosslinked and coloured cataract protein fraction (CPF) was isolated from cataract human lenses. Using a combination of tryptic digestion, gel filtration and multiple reversed phase high performance liquid chromatography (RP-HPLC), coloured peaks were isolated and subjected to amino acid sequence analysis. With these techniques, it was hoped to identify and locate the modified residues. Sequence information was obtained on 16 'coloured' peptides. Many of the peptides were found to be derived from alpha B-crystallin. When redundancies are taken into account, six distinctive peptides were found to be derived from alpha B-crystallin; one from beta B1-crystallin, two from beta A3/A1-crystallin and three from gamma S-crystallin. Three sites of possible crystallin residue isomerisation to modification were detected in the alpha B- and beta A3/beta A1-crystallins, including probable asp isomerisation at residues 25 and 36 in alpha B-crystallin. Since the CPF is unique to nuclear cataract lenses, these data suggest that alpha-crystallin, and alpha B-crystallin in particular, may be implicated in the cataract process. This finding supports that of a recent study on cataract proteins using pronase digestion [Chen YC, Reid GE, Simpson RJ, Truscott RJW. Exp Eye Res 1997;65:835.]

Copper, lead, cadmium and calcium in cataractous lenses.

Lenticular copper, lead, cadmium and calcium levels were measured using atomic absorption spectrophotometry in 9 normal and 37 cataractous human lenses. The nuclear parts of the lenses were used for the study in both groups. The lenticular deposition of these four elements in cataract was statistically significantly. In the cataractous group, males had higher levels of cadmium compared to females. No lead was detected in normal lenses. The calcium content was significantly higher in cortical cataract whereas the other elements were not correlated with the types of cataract.

[A study of coloring in human lens nucleus--association of four inorganic elements and dielectric behavior with nuclear color].

We classified senile cataractous lenses before surgery into five grades on the basis of their nuclear color and analyzed the extracted nuclei on wet weight, water content (weight %), inorganic element content (sodium, potassium, calcium and magnesium), and dielectric behavior. With increase in the grade, water content (p < 0.01) and potassium content (p < 0.05) significantly decreased, and wet weight (p < 0.01) and sodium (p < 0.05), calcium (p < 0.01) and magnesium (p < 0.001) content significantly increased. In dielectric behavior, limiting conductivity at low frequencies significantly decreased with increase in the grade (p < 0.05), and limiting permittivity at high frequencies showed a trend to decrease, but it was not significant (p = 0.052). These results indicate that changes in water content and in inorganic element content are associated with advancement in color grade, suggesting a close relation between the color grade and the membrane functions of lens fibers. In the nuclei with high color grade, we concluded that part of the active transport systems of the fiber membranes was damaged, although the electrical barrier of the membranes was retained.

Analysis of small GTP-binding proteins of the lens by GTP overlay assay reveals the presence of unique GTP-binding proteins associated with fiber cells.

Low molecular weight GTP-binding proteins are molecular switches which are thought to play pivotal roles in cell growth, differentiation, cytoskeletal organization and vesicular trafficking. In this study, members of this family of proteins have been identified and characterized in the eye lens, for the first time. [alpha 33P]GTP blot overlay assays of monkey and human lens water soluble and membranous insoluble fractions revealed the presence of specific GTP-binding proteins in the range of 20-30 kDa (small GTPases) in both fractions, with much higher amounts in the membranous insoluble fraction. In the insoluble fraction, in addition to 20-30 kDa GTPases, there are three distinct GTP-binding proteins, ranging from 33-45 kDa. The small GTPases (20-30 kDa) were present throughout the lens in epithelium, cortex and nucleus, while the 33-45 kDa GTP-binding protein bands were exclusively associated with the cortex and nucleus (fiber cells). Analysis of lens fractions by two-dimensional electrophoresis, immunoprecipitation using monoclonal and sequence specific polyclonal antibodies and C3 exoenzyme mediated ADP-ribosylation demonstrated the presence of Ras, Rap, Rho, Rac, Rab and several other small GTPases. The 33-45 kDa GTP-binding proteins that are associated with lens fiber cells appear to be distinct from the small GTPases and from heterotrimeric GTPases, and were not detected in brain or heart tissue. The presence of different complements of GTP-binding proteins in lens fibers and epithelial, cells suggests their involvement in important regulatory functions, possibly related to cell growth, differentiation and organization of the cytoskeleton.