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

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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)

Nineteen tryptic peptides produced by cleavage at 18 of the 20 arginyl residues in citraconylated S-carboxymethylcysteinyl-rhodanese have been isolated by a combination of gel filtration and high voltage paper electrophoresis. These Tc fragments account for all of the 293 residues in the parent polypeptide and their partial or complete sequences have been determined by automated and manual Edman degradation. In some cases, sequence analyses were completed by degradation of peptides derived by secondary cleavages of the decitraconylated Tc fragments with trypsin, chymotrypsin, or the protease from Staphylococcus aureus. Automated Edman degradation of intact S-carboxymethylcysteinyl-rhodanese was performed for 60 cycles; the information thus obtained permitted the alignment of seven of the Tc fragments and gave the sequence of the first 79 residues in the polypeptide chain. The Tc peptide at the COOH terminus of rhodanese was placed by virtue of the fact that it contained no arginine. Structural analysis of the Tc peptides provided the sequences surrounding all five of the methionyl residues in the enzyme. One of the methionines was found in a 19-residue Tc fragment which also contained the cysteinyl residue essential for catalysis.
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PMID:The covalent structure of bovine liver rhodanese. NH2-terminal sequence and partial structural analysis of tryptic peptides from the citraconylated protein. 71 36

We have investigated the binding of 125I-staphylococcal enterotoxin-B (SEB) in cultured human proximal tubular cells. We found that the binding of 125I-SEB to PT cells was time and concentration dependent and competitively inhibited by antibody against SEB. Preincubation of cells with trypsin and neuraminidase or with fetuin did not significantly impair the binding of 125I-SEB to such cells. In contrast, treatment with endoglycoceramidase completely inhibited the binding of 125I-SEB to cells. Neutral glycosphingolipids exerted a concentration-dependent inhibition of 125I-SEB binding to such cells, maximum inhibition (96% compared to control) occurred upon incubation of PT cells with neutral glycosphingolipids. Taken together, our studies indicate that SEB specifically binds to a neutral glycosphingolipid in PT cells. In contrast, staphylococcal enterotoxin-A and toxic shock toxin (TST-1) are bound to a protein in such cells.
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PMID:Glycosphingolipids: the putative receptor for Staphylococcus aureus enterotoxin-B in human kidney proximal tubular cells. 132 93

Efficient formation of the cpn60-rhodanese complex can be achieved by mixing unfolded rhodanese with excess cpn60 at low temperature. By employing these conditions, a stable and highly reactivatable complex is formed. The complex is not formed when native enzyme is used. Concentrations of NaCl, as high as 0.75 M, do not have any effect on the formation or disruption of the binary complex. cpn60-bound rhodanese contains an exposed hydrophobic surface, as detected by the binding of the fluorescent reporter, 1-anilinonaphthalene-8-sulfonic acid. The intrinsic fluorescence of cpn60-bound rhodanese reports that the average tryptophan is in an intermediate environment between that found in unfolded and native states. This form of rhodanese has an accessibility to quenching by acrylamide or iodide that is intermediate between the unfolded and native forms of the enzyme. Protease susceptibility studies show that rhodanese bound to cpn60 exhibits a trypsin digestion pattern similar to the native enzyme, although it is more rapidly proteolyzed. The results suggest that the conformation of cpn60-bound rhodanese resembles a native-like conformation, but with increased flexibility. Further, only intact rhodanese or enzyme lacking its N-terminal sequence can interact with cpn60 and form a stable binary complex. The protein fragment corresponding to the rhodanese N-terminal sequence did not form part of a stable complex with cpn60.
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PMID:Characterization of a stable, reactivatable complex between chaperonin 60 and mitochondrial rhodanese. 136 12

For the first time, the enzyme rhodanese has been proteolytically cleaved to give species that most likely correspond to individual domains. This indicates cleavage can occur in the interdomain tether. Further, the conditions for cleavage show that availability of the susceptible bond(s) depends on conformational changes triggered by oxidative inactivation. Rhodanese, without persulfide sulfur (E), was oxidized consequent to incubation with phenylglyoxal, NADH, or hydrogen peroxide. The oxidized enzyme (Eox) was probed using the proteolytic enzymes endoproteinase glutamate C (V8), trypsin, chymotrypsin, or subtilisin. The proteolytic susceptibility of Eox, formed using hydrogen peroxide, was compared with that of E and the form of the enzyme containing transferred sulfur, ES. ES was totally refractory to proteolysis, while E was only clipped to a small extent by trypsin or V8 and not at all by chymotrypsin or subtilisin. Eox was susceptible to proteolysis by all the proteases used, and, although there were some differences among the proteolytic patterns, there was always a band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to Mr = 16,500. This was the only band observed in addition to the parent species (Mr = 33,000) when Eox was digested with chymotrypsin, and conservation of total protein was observed after digestion up to 90 min. No additional species were observable on silver staining, although there was some indication that the band at 16,500 might be a doublet. The results are consistent with the occurrence of a conformational change after oxidation that results in increased exposure and/or flexibility of the interdomain tether which contains residues that meet the specificity requirements of the proteases used.
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PMID:Oxidation increases the proteolytic susceptibility of a localized region in rhodanese. 331 91

Two site-directed mutants of the enzyme rhodanese which replace glutamic acid 17 with either glutamine (E17Q) or with proline (E17P) were produced and purified. Both mutants displayed specific activities similar to the wild type enzyme. E17Q was equivalent to the wild type enzyme in all assayed characteristics, except that the mutant had slightly more solvent exposure of hydrophobic surfaces. Results with E17Q suggest that the charge on Glu17 is not required for helix stabilization, nor is its titration required for the low pH structural transitions seen previously. In contrast, E17P was significantly different from the wild type enzyme. For example, E17P had (a) higher exposure of hydrophobic surfaces in the unperturbed state; (b) considerably lower stability to perturbation by urea; (c) easier exposure of organized hydrophobic surfaces on initial unfolding, even though denaturation to the final disorganized state was the same as for the wild type; (d) the ability to refold without assistants but with lower yields and somewhat slower folding; and (e) similar susceptibility to trypsin and evidence of a new clip site closer to the NH2 terminus. However, E17P and the wild type enzyme had very similar recoveries with chaperonin-assisted refolding, and the chaperonin protein groEL had a very similar ability to suppress unassisted refolding. These results indicate that changes in the NH2-terminal sequence can have dramatic effects on the stability of rhodanese and on its ability to be refolded in the absence of assistants. They further suggest that interactions with chaperonins do not rely exclusively on the detailed conformation at the NH2 terminus. A model that incorporates observations here includes step(s) in which the NH2-terminal sequence folds onto the NH2-terminal domain late in the folding process after the protein had adopted a near native conformation.
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PMID:The folding and stability of rhodanese are influenced by the replacement of glutamic acid 17 in the NH2-terminal helix by proline but not by glutamine. 809 37

When the enzyme rhodanese was partially digested by immobilized trypsin, it retained greater than 50% of its original activity although less than 10% of the undigested enzyme remained. The predominant daughter species were two 31-kDa polypeptides whose amino termini corresponded to either residue 44 or 45 of the enzyme's sequence. Following digestion, charged species were isolated by ion exchange chromatography. Denaturing electrophoresis revealed that a 4-kDa peptide remained associated with the 31-kDa fragment. This 4-kDa peptide appears to correspond to the amino-terminal 45 residues of rhodanese. Further proteolysis gave a 2.5-kDa peptide that dissociated under non-denaturing conditions without apparent change in migration of the 31-kDa fragment on SDS gels. Refolding of undigested, urea-denatured rhodanese restored much of its activity. Similar treatment of rhodanese following limited tryptic digestion resulted in no regain of activity. Refolding of a mixture of intact and digested rhodanese resulted in regain of activity appropriate for the amount of intact rhodanese in the sample, indicating that clipped rhodanese does not inhibit refolding of intact rhodanese. It is concluded that portions of the amino terminus of rhodanese are important in the enzyme's folding, but are not essential for the enzyme's sulfurtransferase activity.
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PMID:Limited tryptic digestion near the amino terminus of bovine liver rhodanese produces active electrophoretic variants with altered refolding. 834 Mar 86

Although the role of nucleotides in the catalytic cycle of the GroESL chaperonin system has been extensively studied, the molecular effects of nucleotides in modulating exposure of sites on GroEL has not been thoroughly investigated. We report here that nucleotides (ATP, ADP, or adenosine 5'-(beta, gamma-imino)triphosphate) in the presence of Mg2+ make the oligomer selectively sensitive to trypsin proteolysis in a fashion suggesting conformational changes in the monomers of one heptameric ring. The site of proteolysis in the monomer that is exposed upon nucleotide binding by the oligomer is in the apical domain (Arg-268). Further, complexes of GroEL with GroES or rhodanese display the same sensitivity to proteolysis, unlike the GroEL-GroES-rhodanese complex, which is protected from proteolysis. The influence of various cations on trypsin proteolysis is investigated to elucidate the differential effects that monovalent and divalent cations have on the oligomeric structure of the chaperonin. These results are discussed in relation to the molecular basis for the chaperonin activity.
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PMID:Ligand-induced conformational changes in the apical domain of the chaperonin GroEL. 855 May 66

Ligand-induced conformational changes of GroEL alone and with bound rhodanese, citrate synthase, or dihydrofolate reductase were studied by limited proteolysis. Similar digestion patterns of GroEL, with or without bound substrate polypeptide, were obtained in the absence and presence of the chaperonin ligands, K+, Mg2+, or ATP. The rates of formation and degradation of the six produced proteolytic fragments were significantly different, however. Strikingly, only with Mg2+/ATP or K+/Mg2+/ATP an additional fragment of approximately 25 kDa was generated during digestion of GroEL alone or with bound rhodanese or dihydrofolate reductase, but not with bound citrate synthase. Most of the trypsin-sensitive sites in GroEL were localized in the flexible apical domain, which contains the putative polypeptide-binding region. Our data indicate that subtle structural changes in the trypsin-sensitive regions of GroEL occur as a result of the binding of the chaperonin ligands. However, these structural changes are influenced by the GroEL substrate polypeptides.
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PMID:Ligand-induced conformational changes of GroEL are dependent on the bound substrate polypeptide. 866 87

The enzyme rhodanese was investigated for the conformational transition associated with its urea unfolding. When rhodanese was treated with 0 or 3 M urea, the activity was not significantly affected. 4.25 M urea treatment led to a time-dependent loss of activity in 60 min. Rhodanese was completely inactivated within 2 min in 6 M urea. The 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid fluorescence intensity was not significantly increased during 0, 3, and 6 M urea equilibrations, and the fluorescence was dramatically increased with 4.25 M urea, indicating that hydrophobic surfaces are exposed. After 0 and 3 M urea equilibration, rhodanese was not significantly proteolyzed with trypsin. Treatment with 4.25 M urea led to simultaneous formation of major 12-, 15.9-, 17-, and 21.2-kDa fragments, followed by progressive emergence of smaller peptides. The N termini of the 17- and 21.2-kDa bands were those of intact rhodanese. The N terminus of the 15.9-kDa band starts at the end of the interdomain tether. The 12-kDa band begins with either residue 183 or residue 187. The size and sequence information suggest that the 17- and 15.9-kDa bands correspond to the two domains. The 21.2- and 12-kDa bands appear to be generated through one-site tryptic cleavage. It is concluded that urea disrupts interaction between the two domains, increasing the accessibility of the interdomain tether that can be digested by trypsin. The released domains have increased proteolytic susceptibility and produce smaller peptides, which may represent subdomains of rhodanese.
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PMID:Domain separation precedes global unfolding of rhodanese. 1055 74

Mutation of all nonessential cysteine residues in rhodanese turns the enzyme into a form (C3S) that is fully active but less stable than wild type (WT). This less stable mutant allowed testing of two hypotheses; (a) the two domains of rhodanese are differentially stable, and (b) the chaperonin GroEL can bind better to less stable proteins. Reduced temperatures during expression and purification were required to limit inclusion bodies and obtain usable quantities of soluble C3S. C3S and WT have the same secondary structures by circular dichroism. C3S, in the absence of the substrate thiosulfate, is cleaved by trypsin to give a stable 21-kDa species. With thiosulfate, C3S is resistant to proteolysis. In contrast, wild type rhodanese is not proteolyzed significantly under any of the experimental conditions used here. Mass spectrometric analysis of bands from SDS gels of digested C3S indicated that the C-terminal domain of C3S was preferentially digested. Active C3S can exist in a state(s) recognized by GroEL, and it displays additional accessibility of tryptophans to acrylamide quenching. Unlike WT, the sulfur-loaded mutant form (C3S-ES) shows slow inactivation in the presence of GroEL. Both WT and C3S lacking transferred sulfur (WT-E and C3S-E) become inactivated. Inactivation is not due to irreversible covalent modification, since GroEL can reactivate both C3S-E and WT-E in the presence of GroES and ATP. C3S-E can be reactivated to 100%, the highest reactivation observed for any form of rhodanese. These results suggest that inactivation of C3S-E or WT-E is due to formation of an altered, labile conformation accessible from the native state. This conformation cannot as easily be achieved in the presence of the substrate, thiosulfate.
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PMID:Active rhodanese lacking nonessential sulfhydryl groups contains an unstable C-terminal domain and can be bound, inactivated, and reactivated by GroEL. 1243 28


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