[Studies on cyclic nucleotides and protein kinase of lens. II. Endogenous substrate proteins for cyclic AMP dependent protein kinase in bovine lens].
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Animals↗
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Results for “Lens Cortex, Crystalline”
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[Aging of the human lens and the mechanisms of the senile cataract formation--about structural lens crystallin].
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Aged↗
Distribution of membrane phospholipids in the crystalline lens.
PURPOSE: To determine the phospholipid content of specific anatomic regions within the crystalline lens. METHODS: Phospholipid extracts of tissues dissected from 5 sets of 10 rabbit lenses were analyzed by 31P nuclear magnetic resonance spectroscopy. Twenty-nine pathway-specific metabolic indexes were calculated from groups of phospholipids and ratios of phospholipids. RESULTS: Phospholipid levels (mole percent) were determined from the capsule with attached epithelium, the cortex, and the nucleus. Eleven phospholipids were detected with significant regional differences in the lens phospholipid profiles. The levels of phosphatidylcholine (PC), PC plasmalogen-alkylacyl PC, phosphatidylinositol (PI), phosphatidylethanolamine (PE), and diphosphatidylglycerol (DPG), and of the lyso derivatives (lyso PC and lyso PE) were greater in the capsule plus epithelium than in the cortex or the nucleus. Levels of sphingomyelin, phosphatidylserine, and PE plasmalogen (EPLAS) were less in the capsule plus epithelium than in the cortex or the nucleus. PC, PC plasmalogen-alkylacyl PC, EPLAS, and lyso PE had nearly equal amounts in the cortex and the nucleus. PI, lyso PC, and DPG could not be detected in the nucleus. DPG was only detected in the capsule plus epithelium. An unidentified phospholipid at 0.13 ppm was approximately equal in the cortex and the nucleus, but it could not be detected in the capsule plus epithelium. CONCLUSIONS: These differences demonstrate a significant heterogeneity among these anatomic regions of the lens, and differences in the nucleus relative to other regions studied are consistent with those in membranes that less readily undergo transitions from the relatively impermeable lamellar phase to the more permeable hexagonal HII phase.
Animals↗
Changes in calpain II mRNA in young rat lens during maturation and cataract formation.
The purpose of these experiments was to describe the expression of mRNA for calpain II proteolytic enzyme (EC 3.4.22.17) during normal maturation of rat lens and in cataract formation. Quantitative RT-PCR indicated that the concentration of mRNA for calpain II in whole lens was 3-24 times higher than in age-matched rat liver, kidney, lung and brain, and it was at least five times higher than in young human lens. mRNA levels for calpain II were highest in the outer regions of young rat lens at 5 x 10(6) copies microgram-1 total RNA. Early-stage experimental cataract caused increased calpain II mRNA, while mature nuclear cataract showed a 64% loss. In contrast, mRNA levels for GAPDH, beta-actin, and lens-specific structural protein beta A4 remained constant during experimental cataract formation. Unlike the lower and constant levels in rat liver, kidney and lung; calpain II mRNA levels in whole rat lens decreased with age. These data help explain the high enzymatic activity of calpain II in young rat lens, susceptibility of young rat lens to a variety of cataracts showing increased calcium and calpain-induced proteolysis, and low calpain enzyme activity in human lens. Since the up-regulation of calpain II mRNA was more dynamic than either the amounts of calpain II enzyme or proteolysis of crystallins in cortex, resulting proteolytic activity against the bulk of lens proteins seems to be regulated by post-translational factors, such as increased calcium. The precise role of the up-regulation of calpain II mRNA is unknown, but we hypothesize that it may be associated with the initial cataractogenic response in the epithelial cells or peripheral cortical fibers.
Animals↗
Interactions of lens proteins. Concentration dependence of beta-crystallin aggregation.
The concentration-dependence of beta-crystallin aggregation was studied by both high- and low-pressure size exclusion chromatography of calf lens cortical extract (0.5-249 mg ml-1), nuclear extract (0.2-304 mg ml-1) and purified beta-crystallins (0.4-52 mg ml-1). A reversible equilibrium exists between beta H(igh)-crystallins (predominantly hexamers) and a portion of the beta L(ow)-crystallins (predominantly dimers). Association to beta H-crystallin is more extensive in the nucleus than in the cortex. Moreover, at physiological protein concentrations, the weight percentage of beta H-crystallins is greater than that of beta L-crystallins, in both the cortex and the nucleus. beta H-Crystallins can be fully dissociated to beta L-crystallin at low protein concentration. On the other hand, not all of the beta L species are competent to associate to beta H at high concentrations. This association appears to be directly dependent on the presence of beta B1 chains. We therefore propose that the concentration and spatial distribution of beta H-crystallin in vivo is actually regulated by differential synthesis of beta B1 polypeptides.
Animals↗
[Posttranslational deamidation of crystalline lens proteins during animal aging].
The amide content of total proteins and protein fractions (alpha-, beta-, gamma-cristallins and albuminoid) from cortex and nuclear lens zones of cattle has been investigated. The amide content in proteins of cortex and nuclear lens of young animals (1,5-2 years old) is the same. The decrease of the amide content in the proteins of nuclear lenz zone of old animals (6-12 years old) is due to fraction of readily hydrolysed amides. The data obtained prove the posttranslational desamidation of lens proteins at ageing. beta-Cristallines--the proteins with the highest amides content is exposed to posttranslational desamidation most of all.
Aging↗
Covalent change in alpha crystallin in opaque and transparent sections from the same human cataractous lens.
Opaque and transparent regions from the cortex of the same human cataractous lens were removed, and the water soluble proteins from these sections were resolved by isoelectric focusing in the presence of 8M urea, followed by Western blot analysis using antisera to the whole alpha crystallin molecule. Analysis of 18 different sections from 3 different lens demonstrated statistically significant changes in the charge distribution of alpha crystallins between opaque and transparent sections, with the greatest changes occurring in the anterior, posterior and equatorial sections from a 66-year-old cataractous lens. All opaque sections from this lens contained alpha crystallin with more positive charge than the transparent sections from the same lens. This change was present in both the water soluble and the insoluble fractions of the microdissected sections, and could also be demonstrated by probing of the Western blots with antisera made against synthetic peptides corresponding to the N-terminal and C-terminal regions of the molecule. Together, these results demonstrate that during human senile cataractogenesis there is often a covalent modification of the alpha crystallin molecule, which is probably not due to simple proteolysis, and which results in a shift of the alpha crystallin molecule to a more basic form.
Amino Acid Sequence↗
Regional and subcellular distribution of ascorbate free radical reductase activity in the human lens.
This paper reports that the human lens is endowed with a characteristic regional and subcellular distribution of ascorbate free radical (AFR) reductase (EC 1.6.5.4) activity, and it is associated with the soluble fraction of the lens protein. AFR reductase activity in either immature senile cataractous or transparent lens was higher in the cortex, where the level of insoluble protein is lower, than in the nucleus. The high reductase activity and low insoluble protein content of the cortex were comparable between the assayed cataractous and transparent lenses. In the nucleus, in contrast, the reductase activity tended to decrease with an increase in the level of insoluble protein and with the development of nuclear coloration (sclerosis). As for the subcellular distribution of AFR reductase activity, 70-90% of the enzyme activity in the lens was located in the cytosol fraction. Based on the above results, it was proposed that cytosolic free radicals may be involved in oxidation, coloration and aggregation of lens protein in senile cataractogenesis and in lens aging.
Aged↗
Isolation and characterization of a 25K serine proteinase from bovine lens cortex.
A lens serine proteinase with trypsin-like specificity has been purified to homogeneity. This is one of two serine proteinases associated with the alpha-crystallin fraction from bovine lens. The purification was accomplished by a combination of isoelectric precipitation, activation to release the proteinase, gel-filtration and affinity chromatography. The purified proteinase showed a single protein band of 25 000 daltons on SDS-PAGE. A single protein band was also seen on non-denaturing gels which correlated with the location of the proteinase activity. The proteinase had a pH optimum between 7.2 and 8.2, was stable between pH 5.8 and 8.6 but was unstable above 40 degrees C upon heating. The enzyme lacked any requirement for metal ions and hydrolyzed arginine, lysine and asparagine substrates. alpha-Crystallin, and especially the B-chain of alpha-crystalline, was rapidly hydrolyzed by the proteinase compared to other lens crystallins. Metallo- and cysteine-proteinase inhibitors had no effect upon the enzyme activity whereas three different serine-proteinase inhibitors completely abolished all activity. A number of protein and peptide trypsin inhibitors also completely inhibited the lens 25K serine proteinase.
Animals↗
Selenite nuclear cataractogenesis: a scanning electron microscope study.
The sequential changes during selenite nuclear cataractogenesis were examined with a scanning electron microscope (SEM) and correlated with slit lamp observations. A posterior opacity, visible with the slit lamp 1-2 days after injection of sodium selenite, was found to consist of masses of vacuoles in the superficial posterior cortex by SEM. 2-3 days post injection, a biomicroscopic refractile ring around the nucleus was represented by SEM abnormalities suggesting membrane damage and possible loss of cytosol in the perinuclear region. All normal structure in this region was lost by 5 days after injection when the central nucleus had become opaque. SEM also showed evidence for damage in areas which were still clear by slit lamp examination. Changes, characteristic of aging, were found near selenite induced damage in peripheral (younger) fibers.
Animals↗
Localization of leucine aminopeptidase in normal hog lens by immunofluorescence and activity assays.
Rabbit antisera to purified bovine lens leucine aminopeptidase (EC 3.4.11.1, LAP) were prepared and shown to be specific for leucine aminopeptidase alone (Taylor et al Cur Eye Res 2:47-56, 1982). When whole hog lens thin sections were treated first with these antisera and then with fluorescein conjugated goat anti-rabbit gamma globulin, greatest fluorescence was observed in the elongating epithelium and outer cortex, and significantly less fluorescence was observed in the lens core. That largest amounts of LAP are present in the epithelium is supported by activity measurements using leucylamide as substrate. The specific activity of LAP in the epithelium/outer cortex is 83 times that of LAP in the inner cortex. The concentration of posttranslationally (postsynthetically) modified and oxidized crystallins is greatest in the core, yet in this zone there is a paucity of aminopeptidase activity, possibly due to age-related degradation of the enzyme. It is possible that LAP is in association with membranes and present in nucleoplasm of rapidly metabolizing, elongating epithelial cells. These observations are discussed in relation to the possible role of proteolysis in cataractogenesis.
Absorption↗
A reassessment of protein synthesis by lens nuclear fiber cells.
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Animals↗
Lipids of human lens fiber cell membranes.
A study of the membranes of human lens fiber cells revealed a very high protein to lipid ratio, which tended to increase with aging and in brunescent cataract. Phospholipids were more abundant than cholesterol, cholesterol esters, and other neutral lipids. With aging and cataract formation, a marked decrease in membranes phospholipid content occurred. Sphingomyelin was present in highest amount. Phosphatidylethanolamine, phosphatidylcholine, lysophosphatidyl-ethanolamine, phosphatidylserine, and phosphatidylglycerol were also present. Cholesterol represented approximately 40% of the total lipids. Saturated and unsaturated fatty acids having 16 to 24 carbons were present. The lipid composition varied according to the portion of the lens examined and the state of the lens. The results do not support a conclusion that lipid peroxidation represents a major mechanism of membrane damage that contributes to cataract formation, since there is no decrease in unsaturated fatty acids with age or in cataractous lenses. We suggest that the aggregation of soluble proteins, and their association with lens membranes, and altered membrane function due to the loss of phospholipids are important processes leading to loss of transparency.
Adult↗
Cloning and characterization of a cow beta crystallin cDNA.
We have isolated and sequenced a beta crystallin cDNA clone derived from the mRNA of cow lenses. Comparisons of the deduced amino acid sequence with the amino acid sequences of the principal beta crystallins of the cow, Beta Bp (1) and the mouse (2,3), confirm the general homology within the beta crystallin family and with the gamma crystallins. This beta crystallin cDNA, designated pBeta25/23, has 96% amino acid homology with the murine beta23 and only 43% amino acid homology with the cow beta Bp. The N-terminal 14 amino acids of the murine beta23, which are composed of a high percentage of hydrophobic amino acid residues, bear no similarity to the predicted amino acid sequence of the cow beta crystallin cDNA clone which we have isolated. The remaining amino acid sequences show greater homology between the mouse and cow beta crystallins than the corresponding alpha and beta globins between the two species. The degree of homology is comparable to that of the alpha crystallins for the cow and mouse. The pBeta25/23 cDNA is interesting for the presence of two potential translation initiation sites which are in phase and, if both are used, would code for two polypeptides of MW 25,100 and 23,200. These correspond to the 25,000 and 23,000 dalton beta crystallins that have been partially sequenced by Berbers et al. (4). This raises the possibility that the beta 25 and the beta23 synthesized in the cow lens are not encoded by two separate genes but may be derived from single mRNA using two translation initiation sites or by post-translational processing of the larger peptide. It is possible also that the two proteins are from two mRNA's derived from a single hnRNA by differential splicing of a intervening sequence at the 5' end of the gene.
Amino Acid Sequence↗
Loss of cytoskeletal proteins and lens cell opacification in the selenite cataract model.
This study of lens protein composition found that some cytoskeletal proteins were degraded during the earliest stages of cataract formation. Cataract was induced in 13-14 day old rats by a single subcutaneous injection of sodium selenite (19 mumol kg-1). By 24 hr after the injection of selenite, the ratio of insoluble to soluble protein increased as lens opacification began. The increase in insoluble protein aggregates was correlated with an accelerated loss of proteins having molecular weights of 42, 55/57 and 235 kDa which reacted with antibodies to the cytoskeletal proteins actin, tubulin/vimentin and spectrin, respectively. We observed the loss of 49, 60 and 90 kDa proteins which were not identified. In the lenses of animals protected from protein aggregation and opacification by administration of 1.5 mmol kg-1 pantethine, the pattern of proteins in SDS-PAGE gels resembled the pattern for proteins from transparent lenses of normal untreated animals and loss of cytoskeletal proteins was prevented.
Actins↗
Colloid osmotic pressure of steer crystallins: implications for the origin of the refractive index gradient and transparency of the lens.
The osmotic behavior of soluble cortical and nuclear steer lens crystallins was characterized by secondary osmometry for several ionic strength and pH conditions. Osmotic pressure versus protein concentration relationships were measured for pressures up to 1.15 x 10(6) dyn cm-2. At low concentrations (< 0.2 g ml-1), the osmotic pressure increased linearly with pressure, whereas for concentrations above 0.2 g ml-1, the pressure rose more sharply, giving progressively larger changes in osmotic pressure with increasing crystallin concentration. At a given ionic strength and applied osmotic pressure, the nuclear proteins attained a higher protein concentration than did the cortical proteins. For example, at the highest osmotic pressure of 1.15 x 10(6) dyn cm-2 at pH 7.6 and 0.1 M ionic strength, the observed protein concentrations were 0.43 g ml-1 for the cortical proteins and 0.52 g ml-1 for the nuclear proteins. For both cortical and nuclear steer crystallins, the pressure rose more steeply with concentration than do pressures for calf crystallins described in the literature. The impact of these developmental differences in osmotic pressure on lens transparency is discussed. Both the nuclear and cortical crystallins exhibited ionic strength-dependent shifts in their pressure-concentration behavior. At 0.02 M ionic strength, higher pressures were observed, whereas at 0.4 M ionic strength lower pressures were observed for a given protein concentration. The crystallins were also found to equilibrate to different protein concentrations at a constant osmotic pressure and 0.1 M ionic strength over a pH range of 4-9, with a maximum concentration around pH 5 for the cortical crystallins and pH 6 for the nuclear crystallins. Thus, the adult bovine cortical and nuclear soluble lens extracts are different in their osmotic properties, reflecting underlying differences in protein composition. The results of the ionic strength and pH experiments suggest that hard-sphere, electrostatic, and Donnan forces contribute to the total colloid osmotic pressure of the lens crystallins. However, near physiologic pH and ionic strength the charges of the proteins are screened to the extent that the colloid osmotic pressure exhibits only minor changes for large changes in ionic conditions. The differences in the osmotic behavior of the cortical and nuclear proteins are consistent with a model where regional variations in the colloid osmotic properties of the proteins across the lens help support the radial refractive index gradient that is present in vertebrate lenses. The importance of a radial concentration gradient of metabolites is also discussed.
Aging↗
High-performance gel permeation chromatography of bovine eye lens proteins in combination with low-angle laser light scattering. Superior resolution of the oligomeric beta-crystallins.
Calf lens extracts were subjected to high-performance gel permeation chromatography on TSK GEL G4000 SW and G3000 SW columns (fractionation range: 5 x 10(6)-10(4) daltons) and resolved into thirteen crystallin fractions: HM-, alpha-, six beta H-, two beta L-, beta S- and two gamma-crystallins. Molecular weights were determined using a low-angle laser light scattering detection system. The weight average and number average molecular weights for cortical alpha-crystallin, 860,000 and 740,000, respectively, reveal a polydispersity factor of 1.16 for this heterogeneous protein. The eight different beta-crystallin fractions could be found with practically all possible oligomeric structures from dimers to aggregates larger than dodecamers. Different structures are found for the predominant beta H-crystallin fractions, viz., hexamers and pentamers, in the extracts from cortex and nucleus. Additional identification of the fractions by sodium dodecyl sulphate gel electrophoresis and isoelectric focusing in the presence of urea also indicated that semi-preparative application of this high-performance technique is possible. The co-elution of putative cytoskeletal proteins with some beta-crystallins was remarkable; moreover, co-elution of FM-crystallin with beta S-crystallin is discussed. A 23,000-dalton fraction, mainly found in the cortical region, most likely corresponds to the 24,000-dalton gamma-crystallin preparation obtained from cattle lens cortices. It is questioned whether the similarities between this fraction and beta S-crystallin are merely coincidental.
Animals↗
Effect of chronic near-ultraviolet radiation on the gray squirrel lens in vivo.
The effects of ambient exposure to near-ultraviolet (near-UV) radiation (300-400 nm) on the ocular lens of the diurnal squirrel (Sciurus carolinensis) are reported. Gray squirrels lived in cages illuminated for 12 hr a day with near-UV light (6 mW/cm2, 365 nm) for 1 yr. The non-UV-exposed controls were housed separately. In the lenses of UV-exposed animals, anterior pole changes occurred. Central epithelial cells swelled, disappeared, or underwent proliferation. A band of disoriented degenerating fiber cells was seen in the midcortex, with a degree of liquefaction. When lens protein compartments were separated by centrifugation, water-insoluble but urea-soluble fractions were enhanced in the outer and inner cortex and the nucleus. Both high-performance liquid chromatography and polyacrylamide gel electrophoresis revealed that proteins mainly in the midcortex and nucleus were altered considerably. Evidence of a loss of sulfhydryl compounds (by chemical and Raman spectroscopic analyses) and an increase of protein-thiol mixed disulfides (chemically) was also observed. These data prove that repetitive ambient exposure of diurnal animals to near-UV radiation at subsolar levels damages the lens by interfering with the maintenance of epithelial cells and altering the structural proteins; some of this may be due to the conversion of sulfhydryls to mixed disulfides.
Animals↗