Calf Lens Research Articles

The effect of naturally occurring cellular constituents on phase separation and opacification in calf lens nuclear homogenates

We have measured the change in the phase separation temperature, Tc, in calf nuclear homogenate produced by a variety of naturally occurring cellular constituents. For all of the compounds studied, increasing concentrations of test compound were found to lower Tc. Phosphorylated nucleotides had the greatest effect, lowering Tc by 165-305 degrees/mole of test compound. Reduced and oxidized glutathione reduced Tc by 69 and 100 degrees C/mole, respectively. Smaller effects were observed for amino and ascorbic acids and sugars. The pH of the homogenate was also found to affect Tc. In the normal lens, the concentration of each of these constituents is small. Therefore, for each individual component, the cataract associated decrease in concentration is insufficient to produce the large increase in Tc that has been observed during cataractogenesis in some model systems. However, the superposition of the effects of changes in many cellular constituents (including pH and hydration) could possible produce a change in Tc more consistent with the experimentally observed one. In support of this, we have found that combinations of test compound have an additive effect on the Tc of the nuclear homogenate.

Immunological properties of rat lens gamma-crystallins. I. Characterization of the major components.

Rat lens gamma-crystallins were separated by preparative flat bed isoelectric focusing (pH 7-11). The fractions obtained were submitted to analytical isoelectric focusing, fused rocket immunoelectrophoresis and immunodiffusion. The results demonstrate that rat lens gamma-crystallins are electrophoretically and immunologically heterogenous. Evidence is given that there are two immunologically different groups. Rat gamma 1-crystallin, the prominent and immunologically homogeneous part, is identical to calf lens gamma-crystallin. The second group of proteins, gamma 2-crystallins, is heterogeneous and shows only partial identity with gamma 1-crystallin.

Anchorage-independent growth of normal calf lens epithelial cells and effect of SV40 transformation on their growth properties.

Calf lens epithelial cells cultured in vitro show growth properties usually associated with virally transformed fibroblasts. The lens cells are anchorage independent, forming colonies in agar, and show a low requirement for added mitogens. In dense culture they form multilayers and maintain a constant cell number by proliferation and shedding. Strains of lens cells transformed by SV40 virus have been obtained that show similar growth properties to the normal lens epithelium. The major effect of SV40 transformation is to increase the growth rate, final cell density and in vitro life-span of the lens cells and to inhibit the increase in size that occurs after 2-3 weeks of culturing the untransformed cells.

Polypeptide chain pathway in γ-crystallin IIIb from calf lens at 3 Å resolution

Polypeptide chain pathway in γ-crystallin IIIb from calf lens at 3 Å resolution

The structure of γ-crystallin IIIb from calf lens

The structure of γ-crystallin IIIb from calf lens

Coexistence curves for phase separation in the calf lens cytoplasm

We isolate native cytoplasm of calf lens nucleus by centrifuging the lens nuclear homogenate without preliminary dilution. We then dilute the concentrated nuclear cytoplasm using physiological buffer as a solvent to give various solutions ranging from 16 to 42% by dry weight. At each concentration we measure the temperature at which the mixture opacifies due to the effect of a phase separation and directly construct the coexistence curve of the cytoplasm-solvent system. We show that this coexistence curve is depressed when cortical cytoplasm is mixed with the nuclear cytoplasm.

Calf lens phosphofructokinase: Purification by single-step affinity chromatography and partial characterization

Previous studies have established the importance of phosphofructokinase in lens metabolism. However, attempts to purify the enzyme have been hampered by the small quantities available and the very low initial specific activity. We have overcome these problems and have obtained pure lens phosphofructokinase using the single step of affinity chromatography on ATP-agarose. This procedure yields 40–80% of the initial activity with a specific activity of 100–120 U/mg. The purification factor is approximately 20 000. SDS-electrophoresis shows that the subunit molecular weight is 77000. As in rabbit muscle phosphofructokinase, the Stokes' radius of calf lens phosphofructokinase decreases as the enzyme is diluted. At pH 8, stable calf lens phosphofructokinase with Stokes' radii greater than those corresponding to tetrameric rabbit muscle phosphofructokinase are found at low enzyme concentrations. At pH 8 and physiological enzyme concentrations, stable aggregates are formed.

Nutrient transport in a bovine lens epithelial cell line.

A bovine calf lens epithelial cell line (CLE-1) that synthesizes crystallin has been established in culture and some of its transport properties have been characterized using both cells and membrane vesicles derived from them. The membrane vesicles fractionate with high recovery of plasma membrane markers, showing a 40-fold purification of 5'-AMPase and a 20-fold decrease in the specific activity of the mitochondrial marker enzyme succinic dehydrogenase relative to a cell homogenate. Transport sites demonstrated higher specific activity than has been seen in vesicles from cell lines studied previously. The uptake of alpha-amino isobutyric acid (AIB) (an alanine analog) by CLE-1 cells is stimulated four- to fivefold by Na+ and exhibits a Km of 5.4 mM with a Vmax of 50 pmoles/min.microgram of cell protein. The uptake of leucine was not Na+ stimulatable. The uptake of AIB by the cells was reduced by 43% at confluence. Thus, the cell density dependent behavior of the uptake of the alanine amino acid family in CLE-1 is similar to that of various fibroblast cells. The Na+ caused a threefold stimulation of AIB uptake in the membrane vesicles, while vesicular uptake of leucine was unaffected by Na+. The uptake of adenine, guanine, uridine, and guanosine was also tested in these vesicles. The substrates were rapidly accumulated, came to a steady state distribution within 1-2 minutes, and were recovered as the unaltered compounds after uptake.

Preparation and characterization of native lens cell cytoplasm.

We have developed an ultracentrifugation procedure for isolating fractions of native cortical and nuclear cytoplasm from calf lens without dilution and free of cell membranes. The concentration of these isolated cytoplasmic fractions spans the physiological range (20 to 50%). Some fractions of the nuclear cytoplasm phase-separate at low temperature giving rise to opacification while other fractions do not phase-separate. The fractions of cortical cytoplasm do not phase-separate at any temperature. Both cortical and nuclear cytoplasm were studied using quasielastic light scattering spectroscopy, electron microscopy, SDS-polyacrylamide gel electrophoresis and isoelectric focusing. We found that the structural elements in the cortical cytoplasm were very similar to those in the nuclear cytoplasm.

Immunological characterization of calf lens γ-crystallins, separated by preparative isoelectric focusing

Calf gamma-crystallins were separated by preparative flat-bed isoelectric focusing and characterized according to their immunological properties, molecular weights and isoelectric points. Immunologically 4 different groups of gamma-crystallins (gamma 1a-1d-crystallin) could be detected. The main immunological component, the gamma 1b-crystallin corresponds to two polypeptides with molecular weights of 20,500 and 22,000. The two minor components, the gamma 1c- and gamma 1d-crystallin, are immunologically partial identical with the main component gamma 1b-crystallin. One of these minor components, the gamma 1d-crystallin, has a MW of 29,000. The 4th antigen (gamma 1a-crystallin), which has a MW of 21,000, seems to have only remote relationship with the major gamma-crystallin component.

Gap junction dynamics: reversible effects of hydrogen ions.

Reversible crystallization of intramembrane particle packings is induced in gap junctions isolated from calf lens fibers by exposure to 3 x 10(-7) M or higher [H+] (pH 6.5 or lower). The changes from disordered to crystalline particle packings induced by low pH are similar to those produced in junctions of intact cells by uncoupling treatments, indicating that H+, like divalent cations, could be an uncoupling agent. The freeze-fracture appearance of both control and low pH-treated gap junctions is not altered by glutaraldehyde fixation and cryoprotective treatment, as suggested by experiments in which gap junctions of both intact cells and isolated fractions are freeze-fractured after rapid freezing to liquid N2 temperature according to Heuser et al. (13). In junctions exposed to low pH, the particles most often form orthogonal and rhombic arrays, frequently fused with each other. A number of structural characteristics of these arrays suggest that the particles of lens fiber gap junctions may be shaped as tetrameres.

Gap junction dynamics: reversible effects of divalent cations.

Reversible changes in gap junction structure similar to those previously seen to parallel electrical uncoupling (9, 33, 34) are produced by treating with Ca++ or Mg++ gap junctions isolated in EDTA from calf lens fibers. The changes, characterized primarily by a switch from disordered to crystalline particle packings, occur at a [Ca++] of 5 x 10(-7) M or higher and a [Mg++] of 1 x 10(-3) M or higher and can be reversed by exposing the junctions to Ca++- and Mg++-free EGTA solutions. Similar changes are obtained in junctions of rat stomach epithelia incubated at 37 degrees C in well-oxygenated Tyrode's solutions containing a Ca++ ionophore (A23187). Deep etching experiments on isolated lens junctions show that the true cytoplasmic surface of the junctions (PS face) is mostly bare, suggesting that the particles may not be connected to cytoskeletal elements. A hypothesis is proposed suggesting a mechanism of particle aggregation and channel narrowing based on neutralization of negative charges by divalent cations or H+.

A comparison of aerobic and anerobic photolysis of lens protein

The soluble protein fraction from the cortex of calf lens (ca. 2·5 mg/ml in phosphate buffer, pH 7·4) was photolyzed under both aerobic and anerobic environments. The photolyzed protein was kept at ambinet temperatures, and filters were employed to exclude light Upon photolysis, human lens protein from 14- and 72-year-old normal lenses undergo a photodestruction similar to that found in calf lens protein. But old human lens protein does not give a similar increase of non-tryptophan fluorescence, suggesting that a pre-existing modification prevented its formation.

Cortical opacity, calcium concentration and fiber membrane structure in the calf lens

We studied the dependence of calf lens transparency on the concentration of calcium in bathing solutions using turbidimetric methods and scanning electron microscopy. The calf lens is transparent in concentrations of calcium between 0·5 and 1·3 m m which correspond to the levels of calcium found inside the normal transparent lens. The calf lens cortex opacifies in concentrations of calcium which are either above or below the levels of calcium found in the normal lens. Scanning electron microscope studies show that lens fiber membranes are disrupted at both high and low calcium concentrations but normal membrane structure is maintained in calcium concentrations between 0·5 and 1·3 m m . These results quantitatively demonstrate the relationship between cortical opacity, fiber membrane structure and calcium concentrations.

Structure of γ-crystallin IIIb from calf lens at 5 Å resolution

Structure of γ-crystallin IIIb from calf lens at 5 Å resolution

Studies on the assembly of the rat lens capsule. Biosynthesis of a cross-linked collagenous component of high molecular weight.

1. Intact rat lenses in tissue culture synthesize hydroxy[3H]proline-containing polypeptides of apparent mol.wt. approx. 180000, which become assembled into aggregates of higher molecular weight with time. 2. Both the 180000-mol.wt. species and the aggregates are components of the deoxycholate-insoluble base-membrane matrix. 3. Formation of the high-molecular-weight aggregate is accompanied by the biosynthesis of the reducible hydroxylysine-derived cross-link hydroxylysino-5-oxo-norleucine. 4. Hydroxylysino-5-oxonorleucine and dehydrohydroxylysinonorleucine are the major reducible cross-links present in intact foetal and 1-month-old calf lens capsules.

Phase diagram for cell cytoplasm from the calf lens

We have determined the phase diagram for cytoplasmic homogenate isolated from the cells of calf lenses. In the intact lens, this material is associated with the reversible opacification known as cold cataract. The coexistence curve we find resembles that found in concentrated high polymer solutions. Two coexisting phases are found below the transition temperature. Both these phases have similar protein compositions but different net protein concentrations. The constituent proteins have the following molecular weights: 17,000, 20,000, 22,000, 26,000, 28,000, 31,000, 36,000, 52,000 and 55,000 daltons. Scanning electron microscopy shows that the phase separation in the cytoplasmic homogenate is accompanied by a subtle structural reorganization which consists of increased spatial density fluctuations of dimensions comparable to the wavelength of light.

An immunochemical study of the different proteins in the beta and gamma crystallin families

The crystallins isolated from calf lens cortex were separated by gel filtration on Bio-gel A 0·5 m columns. Immunoelectrophoretic and immunodiffusion analysis of these crystallins demonstrated complete separation of the α-crystallins (fraction 1), various β-crystallins (fractions 2, 3 and 4) and γ-crystallins (fractions 5 and 6). The presence of at least seven different β-crystallin proteins (in terms of different genetic products) were established. The high molecular weight β-crystallins (fraction 2) contained proteins I and II, which were absent from the low molecular weight β-crystallins (fraction 4). Conversely, the low molecular weight β-crystallins contained protein VII, which was absent from the high molecular weight fraction, β-Crystallins III, V and VI were present in both of these factions. The medium molecular weight β-crystallins (fraction 3) contained proteins III through VII. Both the high (fraction 5) and the low (fraction 6) molecular weight γ-crystallins contained proteins I and II. The use of Bio-gel A 0·5 m columns yielded better separation of α-from β-crystallins than did the Sephadex G-200 columns. This difference may have resulted from more efficient dissociation of α-β crystallin complexes on the Bio-gel A 0·5 m than on the Sephadex G-200.

Scanning Electron Microscopy of Opaque and Transparent States in Reversible Calf Lens Cataracts

The microstructure of transparent and opaque calf lenses was studied using scanning electron microscopy. Cells in opaque lenses are very similar to cells in transparent lenses. This demonstrates that

Light scattering and reversible cataracts in the calf and human lens

By using measurements of the intensity of light scattered from intact calf lenses, we have determined a number of reagents that induce nuclear and cortical opacification at body temperature. Diffusion of buffered saline solutions of glycerol, other glycols, urea, guanidine hydrochloride or glycine into the lens reverses the opacity of all the reagent-induced cataracts. Similar findings are obtained with lens homogenates, which have gel-like properties as determined from viscosity measurements. A 50 % (by volume) glycerol or 5 M urea solution clarifies human pathologic cataractous lenses by reducing the opacification due to light scattering. These findings suggest that it may be possible, in principle, to reverse human lens cataracts chemically in situ . The scattering of laser light from quasi-periodic lattice of normal lens cells produces a regular diffraction pattern containing many Bragg spots whose positions are those predicted from the basis vectors of the cellular lattice. The intensity of the Bragg reflexions increases greatly when cataracts are formed in the calf and human lens, and falls greatly when the lenses are clarified. The spatial variation in the scattered light intensity of the Bragg spots and between these spots contains detailed information on the structure of the scattering elements associated with opacification.