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Query: UMLS:C0272170 (SDS)
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Preparations of alpha-crystallin from bovine and human lens exhibited elastase inhibitor activity with a specific activity of 100-250 U mg-1 protein. A washed water-insoluble fraction from bovine, human and cataractous lens nuclei, when solubilized by sonication, gave specific activities of 910, 950 and 1270 U mg-1, respectively. Disaggregation of these water-insoluble fractions in 8.0 M urea, with subsequent reaggregation by urea removal, resulted in a decrease in inhibitor activity. Agarose A-5m gel filtration chromatography after the urea treatment resolved a residual high molecular weight (HMW) fraction and a peak which eluted at the position of water soluble alpha-crystallin. Assays showed that the urea-induced 'alpha-crystallin' peaks from all three preparations had specific activities, equivalent to native alpha-crystallin, whereas the HMW fractions retained their original high specific activity. We conclude that the increased elastase inhibitor activity of the water-insoluble fraction is a property of the aggregate state of the component alpha-crystallin molecules, which is lost upon reaggregation to an 800-kDa alpha-crystallin. Amino acid analysis of the bovine water-insoluble fraction suggested a content of 85-90% alpha-crystallin and 10-15% beta H-crystallin, which was confirmed by SDS-PAGE.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The effect of urea on the aggregate state and elastase inhibitor activity of the water-insoluble fraction from bovine and human lens. 162 42

The phosphorylation of alpha-crystallin B was studied in homogenates of autopsy samples of brain tissue from patients with Alexander's disease, a condition characterized by over-expression of this protein. After incubation in the presence of [gamma-32P]ATP and cAMP the homogenates were analyzed by two-dimensional electrophoresis, (isoelectric focusing followed by SDS-PAGE). Three major polypeptides having the same molecular weight as bovine lens alpha-crystallin B and pIs 7.1, 6.9 and 6.7 were detected in the Coomassie blue stained gels. These three polypeptides were recognized by an alpha-crystallin B-specific antiserum in Western blots. The polypeptides with pIs 7.1 and 6.7 co-migrated in isoelectric focusing gels with bovine lens alpha B and its phosphorylated form alpha Bp, respectively. Radioautography of the two-dimensional gels demonstrated the presence of 32P in the most acidic polypeptide. The results demonstrate the occurrence of alpha B phosphorylation in Alexander's disease brain tissue.
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PMID:Phosphorylation of alpha-crystallin B in Alexander's disease brain. 174 82

Degradation of endogenous lens proteins has been difficult to show under physiological conditions using lens tissue preparations. In contrast, active proteolytic systems in cultured bovine lens epithelial (BLE) cells have been demonstrated previously. BLE cells also contain ubiquitin, a 76 amino-acid polypeptide which is conjugated to proteins in an ATP/Mg(2+)-dependent process prior to their cytosolic proteolysis. In this study, we show that histone H2A, alpha-crystallin, and actin are conjugated to ubiquitin, resulting in higher molecular mass species, which are detected by anti-ubiquitin antibodies. These proteins are also degraded in cell-free assays containing BLE cell supernatants under physiological conditions in an ATP/Mg(2+)-dependent manner. Observation of 125I-labeled proteolytic fragments was made after SDS polyacrylamide gel electrophoresis of the assays. Quantitation of trichloroacetic acid-soluble radiolabeled fragments generated in the presence of ATP/Mg2+ revealed that, with BLE cell supernatant, 25% of the histone H2A was degraded in 3 hr. Proteolysis of alpha-crystallin and actin amounted to 2.3% and 2.9%, respectively. The requirement of ATP/Mg2+ for proteolysis and the observation of ubiquitin conjugation to the same proteins is consistent with the presence of a ubiquitin-dependent proteolytic pathway in BLE cells. Additionally, in this study the BLE cell proteases were even more active on some substrates than the reticulocyte ubiquitin/ATP-dependent proteolytic system.
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PMID:Evidence for ATP and ubiquitin dependent degradation of proteins in cultured bovine lens epithelial cells. 184 31

To investigate the possible role of glycation in the onset of diabetic cataract we used calf lens crystallins as a model. After incubation with reducing sugars, the proteins were investigated by high-pressure gel permeation chromatography, SDS-PAGE and analytical ultracentrifugation. Glucose-6-phosphate incubation resulted in an increase in mean molecular weight of all crystallin fractions and the occurrence of high-molecular weight material, partly formed by disulphide bonds. The glycated crystallins showed a decrease of tryptophan fluorescence and an increase of a specific non-tryptophan fluorescence. This fluorescence was, however, not exclusively associated with the high molecular weight protein, but was present in all protein fractions.
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PMID:Glycation-induced crosslinking of calf lens crystallins. 187 6

Monoclonal antibodies to the bovine alpha A-crystallin were developed and used to probe the relationship between alpha A-crystallin and the bovine lens fiber cell Plasma Membrane-Cytoskeleton Complex (PMCC). Superficial bovine lens cortex was washed by repeated homogenization/centrifugation to remove "soluble protein." The resulting Plasma Membrane-Cytoskeleton Complex was covalently immobilized to inert resin, and extensively buffer washed. SDS PAGE and immunoblot analysis of both the covalently immobilized PMCC and of the sequentially-generated subcellular fractions shows that most of the lens alpha crystallin is "soluble", and readily extracted with physiologic buffers. However, this data also shows that 1) Non-alpha crystallins are progressively and quantitatively extracted from the PMCC with buffer, 2) An irreducible level of non-covalently bound alpha crystallin is achieved which cannot be readily extracted from the PMCC, even with 2 M urea, 1% NP40 or 0.4M KCl. Electron microscope level immunocytochemistry was performed on both the covalently immobilized PMCC, as well as on buffer-extracted thick frozen sections, using monoclonal antibodies to the alpha A-crystallin. The results show a very heavy labelling of both intermediate filaments and beaded filaments, but little or no labelling of fiber cell membranes. The data presented argues that a subfraction of the total alpha A-crystallin is strongly associated with the fiber cell cytoskeleton complex, and constitutes a quantitatively major component of the lens cytoskeleton fraction.
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PMID:Ultrastructural localization of alpha A-crystallin to the bovine lens fiber cell cytoskeleton. 188 28

Lens crystallins from the African ostrich (Struthio camelus) were isolated and characterized. Four crystallin fractions corresponding to alpha-, delta/beta- and beta-crystallins similar to those of duck crystallins were isolated, but epsilon-crystallin was found to be absent. The native molecular masses and subunit structures of the purified fractions were analysed by gel filtration. SDS/PAGE and isoelectric focusing, revealing various extents of heterogeneity in each orthologous crystallin class. An ion-exchange chromatographic method was used for the large-scale preparation of delta-crystallin suitable for structural and enzymic studies. It was unexpectedly found that the purified native delta-crystallin of ostrich lens possessed high argininosuccinate lyase activity, in contrast with chicken delta-crystallin. The c.d. spectra indicated a predominant beta-sheet structure in alpha- and beta-crystallins, and a significant contribution of alpha-helical structure in the delta-crystallin fraction. The estimate of secondary structures from c.d. spectroscopy for each crystallin class bears a resemblance to that of duck crystallins, except that ostrich delta-crystallin possesses much less helical content than duck delta-crystallin. Comparison of crystallin compositions and structures from aquatic and terrestrial birds revealed distinct differences.
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PMID:Ostrich crystallins. Structural characterization of delta-crystallin with enzymic activity. 199 Oct 29

The authors isolated the circulating immune complexes in experimental lens-induced uveitis by PEG-6000 precipitation and molecular filtration in two peaks. ELISA showed that Peak I was a fraction of antigens comprising four components of soluble lens crystallin, of which gamma-crystallin was the most abundant, but no soluble antigens of the retina; Peak II was the antibodies corresponding to Peak I. SDS electrophoresis demonstrated the presence of over 10 antigen-antibody complexes. The results proved that the toxic complexes of experimental lens-induced uveitis was the lens crystallin immune complexes, and further suggested that gamma-crystallin immune complexes induced experimental uveitis as well as the alpha- and beta-crystallin immune complexes.
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PMID:[Analysis of the circulating immune complexes in experimental lens-induced uveitis]. 206 Apr 8

In soluble extracts of bird lenses, crystallin subunits of apparent sizes between 30kDa and 40kDa show considerable variability in both abundance and mobility in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). This is only partly due to the taxon-specific distribution of lactate dehydrogenase-B (LDH-B)/epsilon-crystallin. Here we show that, in spite of high conservation in primary sequence, beta B1-crystallin subunits of avian lenses have a markedly slower migration in SDS PAGE than those of mammals and that the apparent subunit size of beta B1-crystallin varies among birds. This may be at least partly due to unusual post-translational modifications, since beta B1-crystallins of adult birds react strongly in a glycan detection procedure. No other crystallins in birds or mammals share this strong reaction. Modification of beta B1-crystallin could have interesting consequences for the formation of high molecular weight beta-crystallin aggregates in bird lenses.
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PMID:Anomalous behavior of beta B1-crystallin subunits from avian lenses. 207 Jun 40

1. Water-soluble crystallins from cortex and nucleus of both young and adult camel lenses were fractionated by gel filtration into high molecular weight aggregate (HMW-aggregate), alpha-low, beta-high, beta-low, gamma-high and gamma-low protein fractions. 2. Crystallins were characterized by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF). Changes related to age and lens parts were compared. 3. The major changes in crystallin distribution included an increase in HMW-aggregate (alpha-high), beta-high, and gamma, and a decrease in alpha-low and beta-low, when comparing lenses in the direction of the nucleus and in the direction of increasing age. 4. Similar changes were detected comparing either cortex and nucleus of the same lens or whole lenses of different age.
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PMID:Age-dependent variations in the camel lens crystallins. 225 78

Calf alpha- and gamma-crystallin were photolyzed in 1-2 mg ml-1 aqueous solutions, using both laser and conventional UV radiation in the 297-320 nm wavelength region. Gamma-crystallin solutions became highly turbid upon UV irradiation, while alpha-crystallin developed no turbidity when irradiated under identical conditions. The photolyzed solutions were analyzed by SDS-PAGE. These gels revealed loss of normal 20 kDa polypeptide, and formation of higher molecular weight peptides, in both alpha- and gamma-crystallin, presumably as a result of photocross-linking reactions and/or protein insolubilization. Thus, although both crystallins underwent photocross-linking, significant turbidity production only occurred in gamma-crystallin. Some possible explanations for these differences are proposed, with one possibility being that most photocross-links in alpha-crystallin occur between subunits of the 1000-kDa oligomer, while in gamma-crystallin the cross-links occur between 20-kDa monomer units. Hence, cross-linking in alpha-crystallin does not affect the average size of particles in solution (or the turbidity), while cross-linking in gamma-crystallin results in a significant increase in average particle size with concomitant increase in turbidity. Another possible explanation is that UV-irradiated gamma-crystallin becomes insoluble (due to charge changes resulting in non-covalent aggregation) while alpha-crystallin does not. Other differences in the photochemical behavior of alpha- vs. gamma-crystallin were noted--gamma-crystallin photolysis rate was about 50% greater than alpha-crystallin. Alpha-crystallin photolysis yielded strong NFK-like fluorescence, while gamma-crystallin did not. One similarity was that photolyzed alpha- and gamma-crystallin lost amino acids His and Trp at about the same rate.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mechanisms of photochemically produced turbidity in lens protein solutions. 226 77


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