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1.
Membrane from individual cataractous and normal human lenses has been prepared by discontinuous sucrose gradient centrifugation. A sensitive solid-phase radioimmunoassay has been developed to quantitate the levels of alpha, beta, and gamma crystallins associated with membrane obtained from each sucrose gradient interface. The results of this analysis demonstrate significant levels of all three crystallins associated with purified membrane. Although the relative percentage of each membrane-associated crystallin does not change during aging and/or cataractogenesis of the human lens, membrane from cataractous lenses in characterized by greater total amounts of crystallins associated with purified membrane obtained from denser interfaces of the discontinuous sucrose gradient.  相似文献   

2.
High molecular weight and low molecular weight fractions were obtained from total soluble proteins of human cataractous and normal lens nuclei. These fractions were analyzed using a solid phase radioimmunoassay that employed monospecific antisera to alpha, beta and gamma crystallins. Relative to the high molecular weight fraction from normal lens nuclei, the high molecular weight fraction from cataractous lens nuclei showed decreased binding to antisera specific for beta and gamma crystallins. These results demonstrate that the polypeptides of the high molecular weight fraction comprise a special class of polypeptides that have preferentially undergone covalent and/or structural changes during the process of human cataractogenesis.  相似文献   

3.
It has been suggested that the presence of high molecular weight protein aggregates in the lens can lead to light scattering and a consequent loss of transparency. We have measured the concentration of aggregates having a molecular weight greater than approximately 150 × 106 g/mole present in the soluble fraction of both aging normal and cataractous human lenses. This protein population is approximately 5% of the total soluble protein in lenses up to age 75 and increases to 10–15% in lenses aged greater than 75 years and in cataracts. The amino acid composition of the heavy molecular weight soluble aggregates of normal lenses is different in leucine content from alpha, beta and gamma crystallins and different in tyrosine content from beta and gamma crystallins. The aggregates from cataractous lenses show a greater increase in leucine and greater decrease in tyrosine. An unidentified component is present in the cataractous aggregates. Furthermore, calcium, an ion which increases in concentration in cataractogenesis, induces alpha crystallin to aggregate and induces the heavy molecular weight fraction to aggregate. This aggregation is irreversible by dialysis or chelation.  相似文献   

4.
In order to characterize possible disulfide-linked interactions between lens fiber cell membranes and crystallins, two-dimensional diagonal electrophoresis has been used in combination with Western blot analysis. When these blots were probed with monospecific antisera against alpha, beta and gamma crystallins, membrane from five individual normal lenses showed no disulfide-bonded components. Membrane from 13 individual cataractous human lenses showed no disulfide-bonded alpha crystallin, but did show significant amounts of disulfide-bonded beta crystallin in four out of the 13 lenses studied, and significant amounts of disulfide-bonded gamma crystallin in 10 out of the 13 lenses studied. Together, these studies demonstrate that intermolecular disulfide bonding of crystallins to purified fiber cell membranes is found only in cataractous lenses, and that the predominant polypeptide species involved in this interaction is gamma crystallin.  相似文献   

5.
Water soluble protein from human lenses at various ages are separated by Sephadex gel chromatography. The results show that there are major three gamma-crystallins (gamma 1, gamma 2, gamma 3) in human lenses. In normal lenses the total contents of the three gamma-crystallins in water soluble fraction of lens proteins keep stable as age increases. However, among the three gamma-crystallins, gamma 1 increase and gamma 2 decrease markedly, and very little change with gamma 3 are found as age increases. In nuclear cataractous lenses, all three gamma-crystallins decrease as compared with the normal lenses of the same age. The variety of synthesis of each gamma-crystallin is mainly responsible for the changes of their quantities. It is postulated that the total low molecular weight crystallin contents in water soluble proteins of the human lens is related to the transparency of lens. The structural characterization of gamma 3-crystallin is responsible for the relative constant as age increases.  相似文献   

6.
The lens crystallins were analyzed in normal dogs and Miniature offSchnauzer dogs with congenital cataract formation. There was an increase in the relative proportions of alpha and beta L-crystallin and a decrease in the beta H and gamma-crystallin with increasing age in the noncataractous lens. These trends were advanced in the age-matched cataractous lenses. "Advanced aging" trends were also noted in various polypeptide components of beta-crystallin. Specifically, the appearance of a 29K band as well as a reversal of the 26K to 27.6K ratio occurred at an earlier age in the cataractous lens than in the clear lens. Three subunits of approximately 19K, 20K, and 21.5K were present on SDS-PAGE for alpha-crystallin from the cataractous lens as opposed to only two of 19K and 21.5K from the clear lens. However, if the protein was not heated following resolubilization in buffer containing 2% SDS and 5% 2-mercaptoethanol, only two subunits of 20K and 21.5K were evident in both clear and cataractous lenses. The electrophoretic behavior observed for both alpha and gamma-crystallins did not appear to be age related.  相似文献   

7.
Polyclonal antisera to whole crystallins and to synthetic peptides corresponding to various sequences of these crystallins have been used to probe Western blots that contain a low molecular weight component of approximately 10,000 daltons found in the water-soluble fractions from human cataractous lenses. This 10K component binds only to antiserum made against human gamma crystallin. Incubation of human cataractous lens homogenates with alpha chymotrypsin or trypsin will produce low molecular components of similar molecular weight, and identical specificity of binding to the gamma crystallin antiserum. Together, these results suggest that the gamma crystallins constitute a class of macromolecules that are susceptible to in vivo proteolysis during cataractogenesis of the aged human lens.  相似文献   

8.
Individual crystallins, urea-soluble and urea-insoluble proteins were isolated from the nucleus and cortex of types I-IV cataractous lenses and normal lenses. The levels of protein sulphydryls (P-SH), disulphides (S-S), as well as surface (F-SH) and buried (S-SH) in these proteins were determined by reaction with 5, 5'-dithiotris- (2-nitrobenzoic acid) or performic acid oxidation followed by amino acid analysis. During nuclear colour development there is a progressive decrease in the sulphydryl content of the crystallins. In the nuclei of advanced cataractous lenses, the P-SH decreases to 10% of the levels found in the normal nucleus. Similar but smaller changes take place in the cortex. No specific changes were found between the crystallins, with the exception of beta S crystallin. The cysteine remains constant in all lens types suggesting no higher oxidation products are formed. There is a significant shift in the distribution of cysteine in the nucleus of type III and IV lenses. Urea-insoluble proteins are the predominant species, accounting for about 70% of the total cysteine pool. This is consistent with the accumulation of modified insoluble polypeptides during senile nuclear cataract formation.  相似文献   

9.
Lens crystallins were isolated from the homogenates of mammalian eye lenses derived from three different species by gel permeation chromatography and characterized by SDS-gel electrophoresis, isoelectric focusing, amino acid analysis and N-terminal sequence analysis. Five fractions corresponding to HM alpha-, alpha-, beta H-, beta L- and gamma-crystallins were obtained for the crystallins from these phylogenetically distant species. The native molecular masses for these purified fractions and their polypeptide compositions were determined by gel filtration and SDS-gel electrophoresis respectively, revealing the typical subunit compositions for each classified crystallin. The gel pattern of gamma-crystallins from the marmot lens appeared to be more complex than those of gibbon and deer lenses. Comparison of the amino acid contents of each orthologous class of mammalian crystallins with those of evolutionarily distant species still exhibited similarity in their amino acid compositions. The charge heterogeneity of each crystallin fraction can be detected by isoelectric focusing under denaturing conditions. N-terminal sequence analysis of the crystallin fractions revealed that all fractions except that of gamma-crystallin are N-terminally blocked. Extensive sequence similarity between mammalian gamma-crystallin polypeptides were found, which suggested the close relatedness of gamma-crystallins amongst different species of mammals and also established the heterogeneous nature of this multigene family.  相似文献   

10.
This paper presents results of a comparative study of the HMW proteins and crystallins from 9900 g supernatant fraction of cataractous lens homogenate between undernourished and well-nourished subjects. The proportion of HMW protein from cataractous lenses obtained from undernourished subjects was markedly reduced, the proportion of alpha-crystallin was increased and the proportions of beta- and gamma-crystallins were similar as compared to well-nourished subjects. Values of insoluble/soluble protein ratios were significantly higher in the former group compared to the latter. These results suggest that the process of lens protein insolubilization occurs earlier or faster in undernourished subjects.  相似文献   

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