Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Gamma-crystallins have been implicated in various kinds of cataracts. In order to facilitate studies elucidating the molecular mechanism of cataractogenesis, large quantities of rat recombinant gamma-D-crystallin were produced in E coli. A full length cDNA clone coding for gamma-D-crystallin was isolated from a rat lens lambda gt11 cDNA library using a synthetic oligonucleotide as a probe. The coding region of this cDNA was inserted into a cloning vector pKK233-2 under the control of the trc promoter. The resulting construct, pKKCR91, was transfected into E coli to produce rat gamma-D-crystallin in an amount of 10-15% of the total bacterial proteins. The crystallin produced was purified to an apparent homogeneity as judged by SDS gel electrophoresis. The sequence of the N-terminal 11 amino acids of the purified crystallin was determined, showing that it is completely identical to that predicted from the cDNA sequence. Measurements of the far-UV CD spectra also revealed that recombinant rat gamma-D-crystallin thus produced retains a native conformational structure.
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PMID:High level expression of rat gamma-D-crystallin in Escherichia coli. 784 5

The occurrence of age-related cataract associated with a persistent hyaloid vascular system is the most prominent feature in SAMP9, an inbred strain of Senescence-accelerated Mouse. To examine the cataractogenesis, we analysed protein changes in the process of cataract formation in the lens. The cataractous lenses showed a striking decrease in water-soluble protein content, in contrast to increases in the amount of water insoluble protein. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blots of water-soluble protein in the cataractous lenses showed additional high molecular weight beta-crystallin proteins of about 43 kDa, concomitant with decreased amounts of 29-kDa and 31-kDa beta-crystallins and 21-kDa gamma-crystallin, as compared with findings in normal lenses. Although there was no apparent difference between the patterns of SDS-PAGE of urea-soluble and urea-insoluble proteins isolated from cataractous and normal lenses, slightly increased reactivity of bands around 43 kDa against anti-beta-crystallin antibody was observed in cataractous lenses. The calcium content was elevated and activity of transglutaminase was increased in the cataractous lenses. While the molecular weight of beta-crystallin polymers cross-linked in vitro by exogenous transglutaminase was not completely compatible with those of high molecular weight beta-crystallins observed in the cataractous lenses, these findings do suggest the contribution of this enzyme to production of high molecular weight beta-crystallins and to insolubilization of these proteins in the cataractous lenses in SAMP9.
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PMID:Protein alterations in age-related cataract associated with a persistent hyaloid vascular system in senescence-accelerated mouse (SAM). 785 22

It has previously been reported that alpha crystallin (alpha) a major lens protein composed of alpha A and alpha B subunits, can act as a chaperone interacting with other proteins to prevent heat-induced insolubilization. It is now shown that with gamma (gamma), beta L (beta L), and beta H (beta H), other major crystallin groups, this interaction occurs exclusively with soluble denatured protein. Based on studies primarily conducted with the gamma and the beta L crystallins, there is at least one binding site per alpha monomer (alpha m). This conclusion is derived from the following evidence. The binding of soluble denatured protein to alpha increases in a linear stoichiometric manner until a 1:1 ratio of alpha m to gamma or to the presumed (beta L)m is achieved. This is based upon determination of the apparent molecular weight of the alpha-denatured protein aggregate with a calibrated TSK-G4000 SW column and on the determination of the relative masses from the areas of the aggregate peak and those of the reactants. SDS-polyacrylamide gel electrophoresis confirms that the aggregates contain the presumed components following reaction with either gamma, beta L, or beta H. Studies in which gamma or beta L crystallins have been independently heat denatured indicate that species representing 50% of the gamma population and 30% of beta L population have not heat denatured under the conditions employed. When the abundance of the denatured soluble protein exceeds that of the alpha subunits present in the macromolecular complex, further interaction occurs leading to a loss of solubility of the complex. A small increase in the size of the complex remaining in solution is also observed. The results contribute to understanding earlier observations that soluble native low molecular weight alpha species cannot be found in the inner regions of old human lenses but have shifted to large aggregates containing other crystallin components. The work tentatively suggests that the interaction rates of alpha with denatured soluble crystallins are gamma > beta L > beta H.
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PMID:The chaperone activity of bovine alpha crystallin. Interaction with other lens crystallins in native and denatured states. 790 9

The presence of a 9-kDa gamma D-crystallin fragment among water-soluble (WS) and water-insoluble (WI) proteins of human lenses was investigated using individual site specific antibodies to the N- and C-terminal regions of the molecule. The polyclonal antibodies were raised against nonapeptides corresponding to the N- and C-terminal ends and are referred to as anti-9-kDa-N and anti-9-kDa-C antibodies respectively. On Western blot analysis of WS and WI proteins from lenses of donors of different ages, the WS9-kDa species showed immunoreactivity to both the anti-9-kDa-N and anti-9-kDa-C antibodies whereas WI 9 kDa species showed immunoreactivity to only the anti-9-kDa-N antibody. This suggested that possible modification had occurred at the C-terminal region of the WI 9-kDa polypeptide. The 9-kDa species of WS, water-soluble-high-molecular-weight (WS-HMW), water-insoluble-urea-soluble (WI-US) and water-insoluble-urea-insoluble (WI-UI) protein fractions was purified by preparative SDS-PAGE separation followed by HPLC on a C-18 column. Two forms of 9-kDa species were isolated from the WS proteins; one associated with the gamma-crystallin, immunoreactive to both the antibodies, and the other associated with high-molecular-weight protein, immunoreactive to only the anti-9-kDa-N antibody. In contrast, only one form of the 9-kDa species, immunoreactive to the anti-9-kDa-N antibody could be detected in the WI-US and WI-UI protein fractions.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Covalent modification at the C-terminal end of a 9 kDa gamma D-crystallin fragment in human lenses. 792 97

Bovine lens alpha-crystallin inhibited both porcine pancreatic elastase (PPE) and human neutrophil elastase (HNE), but not in the same manner. PPE was immediately inhibited with a stoichiometry of 10 moles of PPE inhibited per mole of alpha-crystallin. The inhibition was markedly decreased by the addition of even low levels of salts. The inhibition was transient, as PPE activity returned to normal with a t1/2 of 30 min even in low salt. HNE required a short preincubation to show maximum inhibition with a stoichiometry of approximately one mole of HNE inhibited per mole of alpha-crystallin. The inhibition of HNE was only slightly decreased by the addition of 0.1 M salt, and HNE activity returned slowly exhibiting a t1/2 of 30 hrs under these conditions. The inhibition of each enzyme by alpha-crystallin was evaluated by Dixon plots giving Ki values of 1.5 nM for PPE and 0.25 nM for HNE. DFP-trypsin was able to compete with PPE for binding to alpha-crystallin and cause the release of PPE already bound to alpha-crystallin. The inhibition of HNE, however, was unaffected by the addition of DFP-trypsin. A mixture of HNE and alpha-crystallin in 0.1 M NaCl was incubated at 25 degrees C for 6 hours. Aliquots showed a slow, continuous cleavage of the alpha-crystallin subunits by SDS-PAGE, but little or no increase in HNE activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A comparison of the inhibition of porcine pancreatic elastase and human neutrophil elastase by alpha-crystallin. 795 8

Restriction of dietary calorie intake is associated with life extension and with the delay of age-related disorders. Preliminary studies demonstrated that by feeding the Emory mouse a diet restricted by 21% in calories cataract and insolubilization of protein could also be delayed. To observe the effects of calorie restriction over prolonged portions of adulthood, Emory mice were fed the control diet (C) or a diet restricted by 40% in calories (R). Feeding the R diet was associated with delayed formation or progress of cataract over virtually the entire second half of life. At 11 months of age, bilateral grade 5 cataracts were present in 17% and 2% of C and R lenses, respectively. At 22 months of age, bilateral grade 5 cataracts were present in 90% and 18% of C and R lenses, respectively. The distribution of alpha-, beta-, and gamma- crystallins in the water-soluble, urea-soluble, and SDS-soluble fractions indicates more similarities than differences between C and R lenses with a specific grade of cataract or of a given age. However, there were significant and abrupt (after grade 4 cataract) losses of particular gamma-crystallins; gamma-crystallins which were not prominent at earlier stages became the major gamma-crystallin moieties. Losses of alpha-crystallins were also noted upon cataract formation or aging in most of the fractions. Aggregates including gamma- and alpha-crystallins also accumulate faster in the C group.
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PMID:Cataract incidence and analysis of lens crystallins in the water-, urea- and SDS-soluble fractions of Emory mice fed a diet restricted by 40% in calories. 813 32

Cigarette smoking is a main source of cyanide in human body, which can be taken as a risk factor of cataract formation. In this study, combined gas chromatography and mass spectrum (GC/MS) was used to determine the amino acid hydantoin after the incubation of soluble human lens gamma-crystallins with cyanate. The carbamylated amino acids obtained by this procedure are alanine and glycine, which are N-terminal amino acids of gamma-crystallin, and leucine. The aggregate, which can be observed in carbamylated gamma 1-crystallin on SDS-PAGE, may be related to the formation of disulfide and non-disulfide covalent bonds, and it seems that gamma 2 and gamma 3-crystallins can not be aggregated to any great extend. The results in this study indicate that the GC/MS is an effective method for analyzing the carbamylation of lens proteins; gamma-crystallin may play a very important role in the formation of cataract associated with accumulation of cyanate in human body, such as heavy smoking.
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PMID:Carbamylation of human lens gamma-crystallins: relevance to cataract formation. 816 8

The interaction of human and bovine alpha-crystallins with bovine lens membranes was evaluated using binding curves and Scatchard plots constructed from scans of SDS-PAGE gels and/or from the association of [14C]-leu alpha-crystallin with the membranes. No differences were observed for total bovine, normal human 19 and 88 year old and cataractous alpha-crystallins. In each case, interaction takes place through two distinct processes, a) a high affinity (Kd = 1 x 10(-8) M) binding with low capacity (25 mg alpha-crystallin/g membrane protein) and b) partitioning (Kp = 0.25 l/g membrane protein). Loss of the high-affinity binding component was observed for bovine nuclear alpha-crystallin. Contrary to previous reports, it is concluded that cataract formation does not affect the ability of human alpha-crystallins to interact with bovine lens membranes. Reanalysis of previously published data supports this conclusion.
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PMID:On the interaction of alpha-crystallin with membranes. 819 71

Human fetal gamma 2-crystallin solution was irradiated by ultra-violet (UV) (lambda 365 nm) at varying time, and analysed by light scattering, SDS-PAGE with DACM, which could specifically combine to free sulphydryl, as well as tryptophan fluorescence and its quenching with acrylamide. The results showed that light scattering of gamma 2-crystallin solution increased after 24 hour irradiation; SDS-PAGE reveled formation of dimer which did not have free sulphydryl and low molecular weight peptide of gamma 2-crystallin. The tryptophan fluorescence of gamma 2-crystallin decreased, and acrylamide fluorescence quenching effect did not show significant change. It suggested that human fetal gamma 2-crystallin photooxidized by UV could aggreate by disulfide and degrated to low molecular weight peptide, and its tryptophan were also oxidized in some degree.
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PMID:[Study on human gamma-crystallins: IV. Photooxidation of fetal gamma 2-crystallin by ultra-violet irradiation]. 827 96

1. The use of an Ultrogel AcA 54 gel-filtration column separates camel lens cortex low molecular weight proteins into four peaks containing beta s-, gamma 1-, gamma 2- and gamma 3-crystallins. 2. The molecular weight of beta s-crystallin corresponded to 29 kDa on SDS-PAGE and showed three major bands between pH 5.85 and 8.45 on isoelectric focusing. In addition, as compared to gamma-crystallins it has a lower degree of homology in amino acid composition, a low sulfhydryl content and a blocked N-terminal amino acid. 3. gamma 1-, gamma 2- and gamma 3-crystallins appeared homogenous on SDS-PAGE and their molecular weights were recorded as 23, 22 and 21 kDa. The isoelectric points of the gamma-crystallin fractions ranged from pH 6.55 to 8.60 and they were found to have an unmodified glycine at the N-terminal end. 4. The three camel gamma-crystallin fractions were similar in molecular weight, isoelectric points, amino acid composition, sulfhydryl concentration and N-terminal amino acid.
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PMID:Purification and some properties of low molecular weight crystallins from camel lens (Camelus dromedarius). 829 57


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