Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Oxidation is known to affect the structure, activity, and rate of degradation of proteins, and is believed to contribute to a variety of pathological conditions. Metal-catalyzed oxidation (MCO) is a primary oxidizing system in many cell types. In this study, the oxidative effects of a MCO system (the Fenton reaction) on the structure of the tryptophan residues of alpha-crystallin were determined. Tandem mass spectrometry (MS/MS) was utilized to identify specific tryptophan and methionine oxidation products in the bovine alpha-crystallin sequence. After oxidative exposure, alpha-crystallin was digested with trypsin, and the resulting peptides were fractionated by reverse-phase HPLC. Structural analysis by mass spectrometry revealed that tryptophan 9 of alphaA- and tryptophan 60 of alphaB-crystallin were each converted into hydroxytryptophans (HTRP), N-formylkynurenine (NFK), and kynurenine (KYN). However, only HTRP and KYN formation were detected at residue 9 of alphaB-crystallin. Oxidation of methionine 1 of alphaA- and methionine 1 and 68 of alphaB-crystallin was also detected. The products NFK and KYN are of particular importance in the lens, as they themselves are photosensitizers that can generate reactive oxygen species (ROS) upon UV light absorption. The unambiguous identification of HTRP, NFK, and KYN in intact alpha-crystallin represents the first structural proof of the formation of these products in an intact protein, and provides a basis for detailed structural analysis of oxidized proteins generated in numerous pathological conditions.
...
PMID:Identification of tryptophan oxidation products in bovine alpha-crystallin. 982 5

The proteasome is a large protease complex that is thought to be responsible for proteolytic removal of damaged proteins. We have previously shown that the level of proteolytic activity due to the proteasome is lower in lens epithelium from human cataractous lenses compared to the activity in epithelium from clear donor lenses. This study aimed to characterize the three main peptidase activities of the proteasome in human lens epithelium with respect to kinetic properties and sensitivity to heat and oxidation. Human lens epithelia were obtained from cataract surgery and analysis performed on pools of epithelial cell cytoplasm. Using the fluorogenic peptide substrates Suc-Leu-Leu-Val-Tyr-AMC (LLVY), Boc-Val-Gly-Arg-AMC (VGR) and Z-Leu-Leu-Glu-betaNA (LLE), Km-values of 56, 678 and 108 micrometers were obtained. All peptidase activities were inhibited by lactacystin, a specific proteasome inhibitor, but at very different rates; with LLVY-hydrolysing activity being the most sensitive (Ki50%=0.15 micrometers). Thermostability was investigated by performing the proteolytic assay at 20 degrees, 37 degrees and 53 degrees C. The trypsin-like activity, as measured by VGR, was completely stable at 53 degrees C for at least 24 hr whereas hydrolysis of LLVY and LLE declined after a few hours at 37 degrees C. Oxidative inhibition was induced by incubation of the samples in 0.5 m m H2O2for 1 or 24 hr. One hour exposure to H2O2caused moderate inhibition of all peptidase activities. The activity could be partially restored by adding 1 m m dithiotreitol, indicating the dependency on intact SH-groups. After 24 hr, peptidase activities were decreased to 25% (LLVY), 73% (VGR) and 44% (LLE) of corresponding control. This inhibition was irreversible for VGR and LLE, but could be partly prevented by the presence of heat shock protein 90 (LLVY and VGR) or alpha-crystallin (LLVY). These data show that the peptidase activities of the human lens proteasome can be modulated by metabolites, such as reactive oxygen species, and by endogenous proteins such as alpha-crystallin and heat shock protein 90.
...
PMID:Differential inhibition of three peptidase activities of the proteasome in human lens epithelium by heat and oxidation. 1037 57

The eye lens protein, betaL-crystallin, aggregates and yields a turbid solution upon refolding from its denatured state. We have observed that the addition of trace amounts of protease results in clearing of this turbidity. Based on this observation, we have developed a simple and rapid method for the detection and assay of proteases. This assay can be performed in the pH range of 6.0-9.0. We could assay the activity of trypsin at a concentration as low as 5 microg/ml.
...
PMID:Detection and assay of proteases using calf lens beta-crystallin aggregate as substrate. 1048 52

Electrospray ionization mass spectrometry (ESI-LC/MS) of tryptic digests of human alphaB-crystallin in the presence and absence of ATP identified four residues located within the core "alpha-crystallin" domain, Lys(82), Lys(103), Arg(116), and Arg(123), that were shielded from the action of trypsin in the presence of ATP. In control experiments, chymotrypsin was used in place of trypsin. The chymotryptic fragments of human alphaB-crystallin produced in the presence and absence of ATP were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Seven chymotryptic cleavage sites, Trp(60), Phe(61), Phe(75), Phe(84), Phe(113), Phe(118), and Tyr(122), located near or within the core alpha-crystallin domain, were shielded from the action of chymotrypsin in the presence of ATP. Chemically similar analogs of ATP were less protective than ATP against proteolysis by trypsin or chymotrypsin. ATP had no effect on the enzymatic activity of trypsin and the K(m) for trypsin was 0.031 mM in the presence of ATP and 0.029 mM in the absence of ATP. The results demonstrated an ATP-dependent structural modification in the core alpha-crystallin domain conserved in nearly all identified small heat-shock proteins that act as molecular chaperones.
...
PMID:ATP and the core "alpha-Crystallin" domain of the small heat-shock protein alphaB-crystallin. 1051 9

It has been hypothesized that resistance to nonenzymatic deamidation of asparagine and glutamine residues may be an important determinant of protein stability in vivo. As a test of this hypothesis, we analyzed the central region of old human lenses, which contain proteins such as gamma-S crystallin that were synthesized during the fetal-embryonic periods of development. Total protein from the fetal-embryonic region of old human lenses was digested with trypsin, followed by resolution of tryptic fragments containing amidated and deamidated forms using high pressure liquid chromatography-reverse phase chromatography together with synthetic peptide standards and mass spectral analysis. The results demonstrate no detectable deamidation of glutamine 92, glutamine 96, asparagine 143, and glutamine 170 from gamma-S crystallin from old human lenses, consistent with the hypothesis that very long-lived proteins can contain asparagine and glutamine residues that are extremely resistant to in vivo deamidation.
...
PMID:Specific glutamine and asparagine residues of gamma-S crystallin are resistant to in vivo deamidation. 1084 93

Alpha-crystallin possesses a molecular chaperone-like activity that prevents proteins from aggregating; however, the mechanism of this activity is not well known. Here we have taken gamma-irradiated alpha-crystallin and studied the relationship between the decrease in chaperone-like activity and the modifications such as oxidation, isomerization and racemization of amino acids in this molecule. We found that the chaperone-like activity of alpha-crystallin decreased with increasing gamma irradiation. After 4000 Gy gamma irradiation the activity of alpha-crystallin was reduced to 40% of the level of nonirradiated, native alpha-crystallin. The circular dichroism spectrum showed that the secondary structure of the irradiated alpha-crystallin had not changed. However, its tertiary structure appeared to change following more than 1000 Gy irradiation. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis also indicated that cross-linking of alpha-crystallin increased with increasing radiation doses. Irradiated and nonirradiated alpha-crystallin was subjected to trypsin digestion and peptide analysis by reverse-phase high-performance liquid chromatography and mass and sequence analysis. Depending on the radiation dose, Met-1 of alpha A-crystallin was oxidized to methionine sulfoxide. In addition, Asp-151 of alpha A-crystallin was isomerized to the beta-Asp form after irradiation, and racemization of Asp-151 decreased. Thus, the loss of the chaperone-like activity of alpha-crystallin is related to changes in its isomerization, oxidation and racemization.
...
PMID:Correlation between the loss of the chaperone-like activity and the oxidation, isomerization and racemization of gamma-irradiated alpha-crystallin. 1159 64

Recent results indicate that covalent modification of proteins by tryptophan-derived UV filters may explain the age-dependent coloration of human lenses, and play a role in age-related cataract. The sites of attachment of the UV filters to the lens crystallins, however, have not been determined. This study utilized a database of predicted masses of UV filter-modified tryptic peptides to target sites of UV filter attachment. Proteins were isolated from old normal lenses and digested with trypsin at pH 6, in order to preserve the integrity of the sites of modification. Peptides were separated by high-performance liquid chromatography and characterized by mass spectrometry. Major colored and fluorescent peaks in the digest were found to correspond to cysteine-containing peptides in which the sulfur atom of the sidechain was linked to the major UV filter compound, 3-hydroxykynurenine glucoside. Three of the peptides originated from gammaS-crystallin and one from betaB1-crystallin. These results show that a predicted mass database can be used to facilitate the identification of sites of UV filter modification in human lens crystallins. Furthermore, this work represents the first evidence that UV filters bind to specific residues on lens proteins in vivo, and suggests that sulfhydryl groups may be important sites for the attachment of UV filters.
...
PMID:Identifying sites of attachment of UV filters to proteins in older human lenses. 1198 16

Thermal aggregation of betaL-crystallin was higher in the presence of peptide fragments generated from oxidized and trypsin-digested betaL-crystallin compared with thermal aggregation of the control proteins without oxidized betaL-crystallin fragments. Increased aggregation of betaL-crystallin was also observed despite the presence of alpha-crystallin (which has anti-aggregating properties) in the system. Self-aggregation of the oxidized betaL-crystallin fragments per se was not observed under the experimental conditions. Reverse-phase HPLC analysis of the precipitate obtained after heating a mixture of betaL-crystallin and oxidized betaL-crystallin fragments revealed that more than one peptide co-precipitates with betaL-crystallin. Electrospray mass spectrometry analysis of the peptides revealed that the molecular weight(s) of the peptides ranged from 1400-1800. Tandem mass spectrometry and a data base search revealed that two of the peptides originated from betaA4-crystallin (LTIFEQENFLGR, residues 121-132) and betaB3-crystallin (AINGTWVGYEFPGYR, residues 153-167) respectively. Oxidized synthetic peptides representing the same sequence were also found to enhance the aggregation of betaL-crystallin in a manner similar to oxidized lens betaL-crystallin peptides. These data suggest that the polypeptides generated after oxidation and proteolysis of betaL-crystallins interact with denaturing proteins and facilitate their aggregation and light scattering, thus behaving like anti-chaperones.
...
PMID:Identification and properties of anti-chaperone-like peptides derived from oxidized bovine lens betaL-crystallins. 1217 82

Several mechanisms have been proposed for the way in which glucose and its metabolites cause cataract, retinopathy and other complications of diabetes, the most convincing being glycation. Glycation, the reaction of sugars with free amino groups of proteins, is one of a variety of non-enzymic post-translational modifications. The aim of the present study was to identify some of the most reactive proteins in the lens when incubated under physiological conditions. Fresh intact bovine lenses were incubated with [14C]glucose in a conventional tissue-culture medium with added antibiotics. After 3 and 6 days of incubation, the water-soluble proteins were separated by size-exclusion chromatography. Glycated proteins from the water-soluble fractions were separated by using a sugar affinity column (Affi-Gel 601). Then the radioactive fractions were identified on SDS/polyacrylamide gels. In addition, the whole bovine lenses were incubated with 10 mM fructose and glucose for 3 and 6 days. The glycated proteins from the water-soluble fractions in parallel with the radioactive fractions were separated by affinity chromatography, and were identified further by amino-acid sequencing. A progressive uptake of radioactive label showed that the majority of proteins incorporating both glucose and fructose were water-soluble fractions. Chromatography and SDS/polyacrylamide gel results showed that alpha- and gamma-crystallin and some proteins of a mean molecular mass of 36-37 kDa incorporated sugars early during incubation. After 6 days of incubation, more crystallins were glycated compared with 3 days, in particular beta-crystallin. Affinity-chromatography results indicated that proteins with subunit masses of 36 kDa and 20 kDa were possibly radiolabelled at an early stage. The purified glycated proteins following incubation with both glucose and fructose, which corresponded to 20 kDa and 36 kDa bands on SDS/polyacrylamide gels, were sequenced by Edman degradation. N-terminal sequences of both 20 kDa bands were Gly-Lys-Ile-Thr, characteristic of gamma-crystallins, but the N-termini of both 36 kDa bands were blocked. Further sequencing after digestion of 36 kDa bands with trypsin and running on HPLC revealed that the glucose sample gave the peptide sequences as Gly-Glu-Tyr-Pro-Asp-Tyr-Gln-Gln and Tyr-Glu-Leu-Pro-Asn-Tyr-Arg, which match with bovine gammaIIIb-crystallin. The peptide sequence Tyr-Glu-Leu-Pro-Asn-Tyr-Arg is only present in the published sequence of bovine gammaIIIb-crystallin and not in any other type of gamma-crystallin. The fructose sample gave the peptide sequences Ile-Thr-Phe-Tyr-Glu-Asp-Arg, Arg-Gly-Asp-Tyr-Pro-Asp-Tyr-Gln-Gln-Trp, Gln-Tyr-Leu-Leu-Arg and Val-Val-Asp-Leu-Tyr, which all matched with bovine gammaIIIa-crystallin. The sequence Val-Val-Asp-Leu-Tyr only appears in the sequence of bovine gammaIIIa-crystallin. gammaIII-Crystallin is the most susceptible lens protein to glycation. The primary target of glucose is gammaIIIb-crystallin, whereas that of fructose is gammaIIIa-crystallin. The early glycation of gammaIII-crystallin by glucose and fructose could result in structural alterations, leading to aggregation of crystallin and eventually cataract formation.
...
PMID:Gamma III-crystallin is the primary target of glycation in the bovine lens incubated under physiological conditions. 1280 41

This study investigates post-translational modification of proteins of bovine lens with aging (3 year old vs. 6 month old cows). After water-soluble proteins were submitted to gel and ion exchange chromatography, betaH-crystallin, a subunit of beta-crystallin, and modified materials were isolated. These materials were then submitted to two dimensional polyacrylamide gel electrophoresis (2D-SDS PAGE) to detect and isolate the new spots. Results for lens proteins from 3 year old animals were compared to those from 6 month old animals. All spots were digested in gel with trypsin and the molecular masses of tryptic digests were measured by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOFMS). Peptides peaks obtained from mass mapping were identified using the protein database of the MS-Fit program in the Protein prospector program of the University of California, San Francisco. We found that two post translational modifications of betaH-crystallin, acetylation and phosphorylation occurred with aging.
...
PMID:Post-translational modification of betaH-crystallin of bovine lens with aging. 1464 77


<< Previous 1 2 3 4 5 6 Next >>

---