Soluble Lens Proteins Research Articles

Preparation of specific antiserum against Rana esculenta pre-alpha lens crystallin.

Specific antiserum against Rana esculenta lens pre-alpha crystallin was prepared in a rabbit by injecting antigen-antibody precipitate of this crystallin obtained from immunoelectrophoresis of esculenta total soluble lens proteins against homologous antiserum.

Cellulose acetate electrophoresis of soluble lens proteins of different species of mammals; the use of discontinuous buffer systems

1. 1. Cellulose acetate electrophoresis using a Tris-veronal discontinuous buffer system, has been performed with soluble lens proteins of different species of mammals and compared with two conventional continuous buffers. 2. 2. In the four species investigated (mouse, Mus musculus; rat, Rattus norvegicus; bull, Bos taurus; ovine, Ovis aries) a higher migration rate and an improved fractionation were obtained with the discontinuous buffer. 3. 3. The advantages and application to the lens protein research is indicated.

The lens proteins of eastern North American salamanders, and their application to urodelan systematics

The soluble lens proteins of 12 eastern North American salamander species of seven genera ( P. glutinosus, P. cinereus, P. jordani, D quadramaculatus, D. fuscus, D. monticola, D. ochrophaeus, Ps. ruber, G. porphyriticus, E. bislineata, N. viridescens and A. maculatum) were investigated, using cellulose acetate electrophoresis. Each of the species, under the conditions employed, exhibited a characteristic, qualitatively distinct electrophoresis pattern, with the greatest resolution occurring in the γ-crystallin region. It was not possible, however, to distinguish the lens proteins of (a) sub-adults from those of adults (five species), (b) intergrade/subspecies from those of derived species (two species) and (c) a colour-phase from those of normal dorsal colouration (one species). The full species status of all but two of the 12 was confirmed; the separate taxonomic status of D. fuscus and D. ochrophaeus is questionable due to the nearly-identical electrophoresis patterns obtained. Such a discriminative ability in the lens protein system suggests its use as a “phylogenetic fingerprint” in urodelan systematics, with possible application to other problems of speciation in the vertebrates.

Ontogeny and localization of the crystallins during embryonic lens development in Xenopus laevis.

AbstractThe time of appearance and histological localization of the crystallins, especially the lens‐fiber specific γ crystallins, in the normally developing lens of Xenopus laevis were determined by immunofluorescence techniques. Antibodies to total soluble lens proteins as well as to purified γ crystallins were applied to sections through the eye region of X. laevis embryos, Nieuwkoop‐Faber Stages 23–45. Seven stages of lens development, the earliest highly atypical, were recognized in this species. The first positive immunofluorescence reaction for both the anti‐total and anti‐γ crystallin antibodies occurred at Lens Developmental Stage 3. At this time the lens rudiment is irregular and flattened, with a slightly‐condensed central mass of cells. These centrally located cells, probably prospective primary lens fiber cells, are positive for γ crystallins, while both they and surrounding retinally‐oriented lens cells are positive for total lens crystallins. This general immunofluorescence profile continues during lens development, with only the lens fiber areas positive for γ crystallins. No reaction with either antibody type, however, could be detected in the corneally‐oriented external layer or, later, the lens epithelium, until very late in the development of the lens (Nieuwkoop‐Faber Stage 45). The lack of crystallins in this cell type, together with the transitory appearance of a lens vesicle (lens cavity), delayed until after fiber cells have been formed, sets X. laevis morphological and biochemical lens development apart from that of other vertebrates.

Possible roles of near UV light in the cataractous process

Decrease in the in vitro uptake of 14C-AA's by the dogfish lens cortex and in their incorporation into its proteins occurred as a result of lens incubation in a near UV light chamber. Alterations in capsular and fiber cell membrane permeability resulting from near UV irradiation seemed to be the cause of this change. A decreased rate of formation of all soluble lens proteins in maturing mouse lenses also occurred between 16 and 43 weeks of near UV exposure. In addition, the rate of accumulation of lens insoluble proteins was stimulated by near UV exposure for the same length of time. Quantitative changes in the distribution of ten specific soluble proteins were noted. Qualitative changes in sulfonated insoluble lens proteins were also found. The results support the idea that age-related cataractous changes in the lens are accelerated by near UV light.

Fate of human lens soluble protein during cataractogenesis

The possibility that whole soluble proteins leak from human normal and nuclear cataractous lenses in vivo and in vitro was studied. No appreciable leakage of proteins out of these lenses in vivo could be detected. However, when human lenses were kept in TC199 incubation medium for 24 hr, 0·5 and 1·8 mg of protein were leached into the medium by normal and grade D nuclear sclerotic (brunescent) cataractous lenses, respectively. Cataractous lenses leak in this situation in positive relationship to the severity of cataract. The leakage may occur as a result of damaged lens capsules or a medium incompatible with lens epithelial cell metabolism. The general conclusion reached is that conversion of soluble proteins to insoluble forms represents the manner in which soluble protein levels are depressed in the senile cataractous lens, rather than leakage through the capsule into the aqueous humor.

Soluble lens proteins of mutant stock mice in cataract development

Changes in soluble proteins of the maturing cataract were studied in mice of the mutant stocks: ocular retardation (gene symbol or ); white ( Mi wh ); fidget ( fi ); and dominant cataract-Fraser ( Cat Fr ). Crystallin fractions of the normal lens were first identified by polyacrylamide gel electrophoresis. In the course of cataract development in mice of the studied mutant stocks, a diminution or loss of some crystallin fractions was observed. The maturation of secondary cataracts in or/or, Mi wh / Mi wh , and fi/fi mice was accompanied by a significant increase in serum albumin, which preceded visible lens opacity. In agar electrophoresis, albumin and α-crystallin were found to migrate together, simulating quantitative maintenance of α-crystallin in the maturing cataract. Another serum protein, electrophoretically similar to transferrin, also penetrated into the lens during cataract development. Maturation of primary nuclear cataract in Cat Fr / Cat Fr mice was not accompanied by increased penetration of serum proteins into the lens. Apparently, penetration of serum proteins into the lens participates in the pathogenesis of secondary subcapsular cataracts, while primary nuclear cataracts have a different etiology.

Soluble lens proteins in rats on different sugar diets

The soluble lens proteins of rats maintained on normal rat chow, and on diets containing 40% cane sugar, lactose or galactose, were studied by means of paper electrophoresis, agar gel electrophoresis, immunoelectrophoresis, gel filtration chromatography on Sephadex G-100, and ultracentrifugation. Saccharose feeding did not disturb the distribution of the soluble lens proteins analyzed by all of these methods. Lactose, a cataractogenic sugar, given to the animals during the time needed to develop cataracts with a galactose diet, produced only minor changes. In galactose cataracts, important modifications in the soluble lens proteins were found by each one of the methods used. In this condition, a fraction with a higher anodic mobility than the normal α-crystallin group was found on paper and agar gel electrophoresis. On immunoelectrophoresis the extra fraction showed a reaction of identity with one of the normal arcs of the α-crystallin group. Other modifications of the immunoelectrophoretic pattern were also found. The methods which investigate the molecular size, showed a decrease in the low molecular weight proteins, and an increase of the high molecular weight proteins. The possibility of a common feature in different types of cataracts is discussed.

Reversible binding of soluble lens proteins to lens fibre ghosts

Soluble lens proteins can interact with lens fibre membranes (the urea-insoluble fraction of lens homogenate) and thus become water-insoluble. This protein binding capacity of the urea-insoluble material is considerable, the amount of soluble protein bound being, under some conditions, comparable to that of the urea-soluble protein fraction of albuminoid. It is suggested that some fractions of the total water-soluble lens proteins (probably “aged” α-crystallins) are bound preferentially by the fibres membranes. These observations are relevant to the problem of the origin of albuminoid.

The distribution of the soluble proteins in the lenses of some marine vertebrates

1. 1. The soluble proteins from the lenses of one marine mammal, four body fishes and one cartilaginous fish were examined by free electrophoresis, zone electrophoresis and sedimentation. 2. 2. Electrophoretic distribution of the soluble lens proteins were characteristic for the two classes of fishes examined. 3. 3. The electrophoretic distribution of the soluble whale lens proteins do not resemble those of the fishes; but do resemble those of the mammals. 4. 4. Sedimentation analyses of revealed two typical patterns for the bony fishes, while the cartilaginous fish and whale each had distinctive patterns. 5. 5. A cold precipitating fraction in the shark lens is noted.

Differences in the soluble lens proteins from tadpole and adult bullfrogs

Soluble proteins from the whole lens of Rana catesbeiana at tadpole and adult stages were examined by cellulose acetate electrophoresis. The soluble proteins isolated from the periphery (cortex) and the core (nucleus) of the adult lens were also examined. Electrophoresis for 2 hours showed distinct quantitative differences between the profiles of tadpole and adult frog stages, while a similarity was noted between the whole tadpole lens and the nucleus of the adult lens. Electrophoresis for 50 minutes revealed distinct alpha crystallin fractions, differing in electrophoretic mobility and relative protein content, in adult nucleus, in adult cortex, and in tadpole lens preparations.

Study of the soluble lens proteins from different amphibian species

Soluble lens proteins from five species of amphibia have been studied by zone electrophoresis and other immunochemical methods. Their patterns, as revealed by electrophoresis, do not differ markedly though the numbers of bands and subunits vary. The γ-crystallin appears to be the predominant lens protein in all the species. Immunodiffusion tests showed a reaction of complete identity of lens antigens of different species with respect to Xenopus laevis lens antiserum; while X. laevis, Triturus crislatus, Ambystoma mexicanum lens antigens revealed partial identity when tested against Rana esculenta and Bufo bufo lens antisera.

Effects of urea on solubility and electrophoretic characteristics of protein from the eye lens nucleus in bigeye and skipjack tunas, and in menpachi

1. 1. A solution of urea was sought which would solubilize the albuminoid present in the eye lens nucleus without degrading the soluble lens proteins or producing undesirable effects in electrophoretic patterns. 2. 2. Tests on extracts of macerated, pooled lens nuclei from bigeye and skipjack tunas, and from menpachi, revealed that the most effective extracting solution for albuminoid is composed of 0·14 g% NaCl, 0·1 M urea, with 3 drops of merthiolate (10 −5) (preservative) to make up 50 ml. 3. 3. The solubility characteristics of the nuclear lens proteins reflected the closer relationship between the two tunas than between either tuna and the menpachi.

Studies on the structural proteins of the human lens

In the aging and cataractous human lens, soluble protein decreases and insoluble protein increases in concentration. This change is more marked in the cataractous than in the aging normal lens. Of the soluble lens proteins, γ-crystallin decreases and α-crystallin increases in both aging and cataract formation, and this change is more pronounced in the cataract. In the human, as in bovine lenses, α-crystallin and albuminoid appear to be more closely related than any of the other fractions. Senile normal lenses and senile cataracts resemble each other as seen in their amino acid analyses, E11 values and sulfhydryl content. In the human lens, albuminoid appears to derive from α-crystalling mostly (as in bovine lens), not from γ-erystallin as in the rat and dogfish.

Lens Soluble Proteins: Correlation with the Cytological Differentiation in the Young Adult Organ of the Chick

THE lens of the eye is an outstanding example of cellular differentiation. At any moment the organ shows the complete sequence of its cellular ontogeny from epithelial cells to equatorial, cortical and nuclear fibre cells1,2.

DEAE-cellulose chromatographic studies on water soluble lens proteins of various animals.

DEAE-cellulose chromatographic studies on water soluble lens proteins of various animals.

Studies upon the Sulfhydryl Groups of Calf Lens α-Crystallins

Abstract α-Crystallin, one of the major soluble lens proteins, is composed of approximately 50 subunits. Each subunit contains but one sulfhydryl group. However, owing to the packing arrangement of the subunits in the aggregate macromolecule, a large fraction of the sulfhydryl groups is inaccessible to p-hydroxymercuribenzoate (HMB) at neutral pH and 20°. The sulfhydryl groups are exposed by increasing temperature. Studies at different temperatures of the reactivity of the —SH groups toward HMB suggest that α-crystallin may exist in at least three different stable aggregate forms. At any temperature between 20 and 34°, 71% of the —SH groups of the α-crystallin preparations α1 from the periphery of the lens (α1P) and the α1 from the nucleus of the lens (α1N) react with HMB. When the number of HMB-reactive —SH groups in these fractions is plotted against temperatures between 34 and 50° a linear increase is obtained until, at 50°, all sulfhydryl groups are exposed. The α2 preparation from the periphery of the lens (α2P) has 54% of its sulfhydryl groups available to HMB in the 20–34° range. A linear increase with temperature in the number of HMB-reactive —SH groups can be observed to 70° at which point all —SH groups are exposed. Of the —SH groups of the α2 preparation from the nucleus of the lens (α2N) 46% react with HMB at 20° and a linear increase with temperature to 70° is observed. All of these reactions are reversible. Any of the aggregate forms described above can be reversibly transformed to the other conformations by alteration in pH or salt concentration, or by addition of low concentrations of urea. The effect of pH upon the availability of the —SH groups to HMB with increasing temperature suggests that an ionizable group with a pK of approximately 9.6 may be involved in the unmasking of the —SH groups of all the α preparations. If any of the α-crystallin macromolecular forms are reversibly deaggregated, the reaggregated material appears to be packed in the α2N form on the basis of its temperature-sulfhydryl group reactivity relationship. However, treatment with low concentrations of urea will cause re-establishment of the initial conformation. Studies with oxidized material indicate that only 35% of the —SH groups in the aggregate molecule can form disulfide bonds, although in the deaggregated state all of the —SH groups are capable of forming disulfides. At 25° few of the HMB-reactive —SH groups of α1N and α1P and none of the groups of α2P and α2N appear to form disulfide bonds. The data suggest that there are no exposed —SH groups on the outer surface of the α-crystallin macromolecules, and that the —SH groups are immobilized by the aggregate structure, so that most of them are incapable of forming disulfide groups.

Optimal conditions for immunoelectrophoresis of some soluble bovine lens antigens

It was demonstrated that the conventional conditions for immunoelectrophoresis of lens antigens with respect to pH, temperature and specific conductance of buffer may not be adequate. The best results were obtained with a Tris-phosphate buffer with a specific conductance equivalent to that of 0·025 m KCl, at 4°C with a pH of 6·0–7·5. The optimum conditions, however, varied depending upon which lens antigen was being studied. Immunization of rabbits to bovine lens antigens was considerably more successful when relatively purified protein fractions were utilized rather than the total soluble lens protein fraction.

Soluble Lens Proteins of Some Scombroid Fishes

Soluble Lens Proteins of Some Scombroid Fishes

Separation and characterization of the lens proteins of the amphibian, Rana pipiens.

AbstractThe soluble lens proteins (α, β, and γ crystallins) of the adult American grass frog, R. pipiens, were investigated, using DEAE‐cellulose ion‐exchange chromatography and disc electrophoresis on polyacrylamide gels. Column chromatography of whole adult lens proteins, with a NaH2PO4‐Na2HPO4‐NaCl buffer elution program, revealed six fractions. The first to be eluted was identified as γ crystallin, the predominant lens protein of this organism; the second and third as β crystallins; and the fourth, fifth, and sixth as a crystallins. Disc electrophoresis of these fractions on polyacrylamide gels of appropriate porosity confirmed the identity of these proteins as separated by ion‐exchange chromatography. Rechromatography of the isolated γ crystallin fraction of whole lens protein, employing a tris‐HCl buffer elution system, resolved the γ crystallin into four distinct components. These individual γ crystallins exhibited different electrophoretic mobilities, as judged by their behavior in disc electrophoresis. This precludes the possibility that the fractions obtained are artifacts of the elution process, and confirms the existence of four γ crystallins in the lens of adult R. pipiens, as has been reported for mammalian (bovine) embryonic and adult γ crystallins. The phylogenetic and development implications of these findings are discussed.