Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human lens proteins were digested with trypsin or V8 protease, and the resulting peptides resolved on a C18 reverse phase column. Fractions from this column were probed with polyclonal antiserum made against the whole alpha crystallin molecule. Peptides in the seropositive fraction were purified to homogeneity, then characterized by mass spectral analysis and partial Edman degradation. The tryptic and V8 digests contained only one seropositive peptide that was derived from the C-terminal region of the alpha-A molecule. To determine the exact boundaries of the epitope, various size analogues of this region were synthesized and probed with anti-alpha serum. Together, these studies demonstrate that the major continuous epitope of the alpha-A chain includes the sequence KPTSAPS, corresponding to residues 166-172 of the human alpha-A crystallin chain.
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PMID:Identification of a major continuous epitope of human alpha crystallin. 137 46

In an effort to elucidate the molecular changes which take place in the human lens with the onset of nuclear cataract, the urea-insoluble protein fraction, solubilized with dithiothreitol, was digested with trypsin. Tryptic peptides separated by HPLC, were examined by both mass spectrometry and Edman degradation. A pentapeptide Gly-Glu-Tyr-Pro-Arg which is contained within the beta-crystallin sequence was isolated. This finding provides direct evidence that beta-crystallin is present in the urea-insoluble protein fraction which is known to be characteristic of human nuclear cataract lenses.
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PMID:Direct approach to identification, at the molecular level, of modified proteins in human nuclear cataractous lenses: beta-crystallin is a component of the urea-insoluble protein fraction. 147 78

The amino acid sequence of bovine gamma II-crystallin has been verified by a combination of electrospray and fast atom bombardment mass spectrometry. The molecular weight of gamma II, isolated by gel filtration and ion exchange chromatography, was determined to be 20,967 +/- 3 by electrospray mass spectrometry. Another aliquot of gamma II was completely digested by trypsin in a medium of 20% CH3CN and 0.1 M Tris, pH 8.2. The tryptic peptides were separated by reversed phase HPLC and identified by their molecular weights, as determined by fast atom bombardment mass spectrometry (FABMS). The identification of each peptide was confirmed by digesting the peptide further to give new peptides whose molecular weights were also determined by FABMS and related to the proposed amino acid sequences. The data from both types of mass spectrometric analyses were consistent with the sequence previously proposed by Hay et al. (J. Biol. Chem. 1987, 146, 332-338), including threonine at position 119. The FAB mass spectrum of one HPLC fraction suggested that disulfide bonding between Cys 18 and Cys 22 was present in at least half the protein preparation. Whether the Cys 18/Cys 22 disulfide bond was present in native gamma II or was produced during isolation or enzymic digestion could not be determined from these studies. Samples that had been stored for several weeks showed that several of the cysteines had become disulfide bonded. These studies illustrate the power of mass spectrometric techniques to accurately confirm the primary structure of proteins and to identify post-translational modifications.
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PMID:Mass spectrometric analysis of the structure of gamma II bovine lens crystallin. 154 37

Previous studies have demonstrated that alpha-crystallin binds specifically, in a saturable manner, to lens membrane. To determine the region of the alpha-crystallin molecule that might be involved in this binding, native alpha-crystallin from the bovine lens has been treated by limited digestion with trypsin, to produce alpha-A molecules with an intact C-terminal region, and a nicked N-terminal region. Compared to intact alpha-crystallin, trypsin-treated alpha-crystallin binds less avidly to lens membrane, suggesting that the N-terminal region of the alpha-A molecule may play a key role in the recognition between lens membrane and crystallin.
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PMID:Involvement of the N-terminal region in alpha-crystallin-lens membrane recognition. 193 66

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.
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PMID:Immunochemical characterization of the major low molecular weight polypeptide (10K) from human cataractous lenses. 246 75

One possible route to cataract formation may be via the carbamoylation of lens proteins due to increased concentrations of cyanate in the body resulting from uraemia associated with renal failure and with severe diarrhoea. Carbamoylation of gamma-II-crystallin, which is found in the lens core, could alter the surface charge network of the molecules, resulting in aggregation, increased light-scattering and hence cataract. We have attempted to locate the site(s) of carbamoylation in gamma-II-crystallin. gamma-II-Crystallin was isolated by gel chromatography and ion-exchange chromatography. gamma-II-Crystallin was then carbamoylated by incubation with potassium [14C]cyanate, followed by citraconylation and digestion with trypsin to give peptides that were separated by high-resolution ion-exchange chromatography. The amino acid compositions of the radioactive peptides were compared with the expected peptide composition for gamma-II-crystallin. The radioactive peptide compositions, which agreed with the theoretical peptides, all matched with the N-terminal region of gamma-II-crystallin and had in common the presence of the N-terminal glycine residue. It appears that the alpha-amino group of the N-terminal glycine was the main site of carbamoylation. This site forms part of the charge network on the surface of gamma-II-crystallin.
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PMID:Site of carbamoylation of bovine gamma-II-crystallin by potassium [14C]cyanate. 259 Jan 75

Assays were carried out to determine the trypsin inhibitor activity present in the water-soluble and water-insoluble fractions of human lenses of various ages. Little change was seen in the inhibitor activity of the water-soluble protein fraction. When this fraction was chromatographed on an Agarose A-1.5 m column, however, the inhibitor activity was increasingly associated with the high molecular weight (HMW) protein fraction with age. A gradual increase in water-insoluble inhibitor was seen up to age 60, which correlated with the increase in protein in this fraction. After age 60, a marked increase in the water-soluble inhibitor activity was observed. In 80-90-yr old lenses, 1 mg of water-insoluble protein was able to inhibit 200 micrograms of crystallin trypsin by 50%. Similar assays on a collection of cortical and brunescent cataracts also showed very high levels of water-insoluble inhibitor activity. In most cases, these values were higher than those for the age-matched control lenses. Fractionation of the water-insoluble proteins showed that the bulk of the activity remained with the urea-insoluble fraction in cataractous lenses. A low molecular weight trypsin inhibitor was isolated from the water-soluble and water-insoluble fractions of human lenses. An age-dependent increase in this inhibitor was observed by activity measurements and electrophoretic analysis.
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PMID:The effects of aging and cataract formation on the trypsin inhibitor activity of human lens. 292 Jul 82

Protein-mixed disulfides (PSSG) were formed by interaction of glutathione disulfide (GSSG) with lens crystallins. Total water-soluble crystallins and alpha-crystallin purified on a Sephacryl S-200 column were separately incubated with 0, 2, 4, and 8 mM (final concentrations) GSSG overnight and then dialyzed to remove unbound GSSG and GSH. Either TPCK-treated trypsin or TLCK-treated alpha-chymotrypsin were added to about 200 micrograms crystallin samples and incubated for 20 min at room temperature. Reactions were terminated by boiling in SDS-mercaptoethanol-Tris (pH 6.8) solution and subjected to electrophoresis on 10% polyacrylamide slab gels. Comparison of SDS-PAGE patterns of proteolysis with or without GSSG treatment showed that GSSG at a concentration of 2 mM or higher reduced or abolished proteolysis of alpha-crystallin by trypsin but not by alpha-chymotrypsin. The protective effect of GSSG was greater with alpha-crystallin than with beta-crystallins. Addition of alpha-crystallin-mixed-disulfide to an assay system in which trypsin was hydrolyzing N-alpha-benzoyl-DL-arginine-P-anilide (BAPNA) inhibited the tryptic activity. Direct addition of GSSG or native alpha-crystallin had no significant inhibitory effect on trypsin. Based on these results, it is speculated that alpha-crystallin glutathione mixed-disulfide appears to become resistant to trypsin probably by non-competetive inhibition of the enzyme.
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PMID:Resistance of alpha-crystallin-glutathione mixed-disulfide to tryptic digestion. 301 92

One of the major lens-structural proteins, alpha-crystallin, is a multimeric protein containing 40 subunits of approx. 20 kDa each. There are two subunit types with distinct but similar structures. This protein was capable of inhibiting trypsin, chymotrypsin and elastase, but had no effect on thrombin or kallikrein. Complete inhibition was not observed, but rather plateau levels of inhibition were obtained in each case. Maximum inhibition was observed at a ratio of 1 mol of alpha-crystallin for every 9-10 mol of trypsin. alpha-Crystallin also inhibited the labeling of the active site of trypsin by [3H]diisopropyl fluorophosphate (DFP). Greater than 90% inhibition of DFP labeling was observed at a ratio of 1 mol of alpha-crystallin for every 7-8 mol of trypsin. Both trypsin and [3H]DFP-labeled trypsin formed a complex with alpha-crystallin, as demonstrated by gel-filtration chromatography. The active site of trypsin when bound to alpha-crystallin was still capable of reacting with p-nitrophenyl p-guanidobenzoate and soybean trypsin inhibitor, but was inaccessible to alpha 1-antitrypsin. These data suggest that alpha-crystallin acts as a multivalent modified inhibitor which is consistent with the proposed quaternary structure of alpha-crystallin.
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PMID:The binding and inhibition of trypsin by alpha-crystallin. 349 15

Leakage of lens proteins from a hypermature cataract can result in a characteristic glaucoma that is associated with the invasion of the anterior chamber by monocytes. We hypothesized that the lens proteins themselves might account for the monocyte response. A sonicated lens induced concentration-dependent migration of monocytes in a Boyden chamber assay system. Checkerboard analysis indicated that the movement was directed rather than merely random. Relative to a control chemoattractant, N-formyl-methionyl-leucyl-phenylalanine, the lens induced monocyte migration more potently than neutrophil migration. The ability to induce migration was markedly reduced by incubating the lens with either trypsin or papain. Chemotactic activity was readily demonstrable in lenses from young donors without cataracts. Separation of lens proteins by gel filtration with high-performance liquid chromatography indicated that the chemotactic activity was most consistently associated with the gamma crystallin fraction. The chemotactic activity of lens proteins may contribute to the pathogenesis of phacolytic glaucoma or the uveitis resulting from retained cortical material after cataract extraction.
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PMID:Chemotactic activity of lens proteins and the pathogenesis of phacolytic glaucoma. 367 92


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