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:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cyclophylins are members of a class of proteins with peptidyl-prolyl cis-trans isomerase activity. These enzymes bind the immunosuppressive agent, cyclosporin A (CsA), which acts as a competitive inhibitor. The peptidyl-prolyl cis-trans isomerase from Bacillus subtilis (PPIase) was purified to homogeneity in a 4-step purification procedure, which resulted in a 100-fold protein purification with a yield of 5%. Coomassie blue-stained SDS-PAGE revealed a single band of about 18 kDa. PPIase activity was determined using synthetic peptides as substrates in a 2-step reaction coupled to chymotrypsin. Treatment of Bacillus subtilis PPIase by CsA revealed an inhibition constant of Ki = 175 nM, which differs from cyclophilin of enterobacteria such as E. coli or Salmonella typhimurium and is in the range of human enzymes.
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PMID:Peptidyl-prolyl cis-trans isomerase from Bacillus subtilis. A prokaryotic enzyme that is highly sensitive to cyclosporin A. 151 92

Two new peptidyl-prolyl-cis/trans-isomerases were purified to homogeneity from Fusarium sambucinum and Aspergillus niger. They belong to the class of cyclosporin A binding proteins (cyclophilins) and have molecular masses of about 18 kDa. As has been shown for other cyclophilins, the isomerase activity of the enzymes is inhibited by cyclosporin A in the nanomolar range. Furthermore binding of cyclosporin A prevents proteolytic digestion of the cyclophilin/cyclosporin complexes by the endoproteases GluC, LysC and alpha-chymotrypsin, in contrast to the free cyclophilins, which are readily cleaved by these proteases. We could also observe this protection for cyclophilins from sheep thymus and from the cyclosporin producing fungus Tolypocladium inflatum.
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PMID:Two new cyclophilins from Fusarium sambucinum and Aspergillus niger: resistance of cyclophilin/cyclosporin A complexes against proteolysis. 153 Jun 35

Cyclosporin A (CsA) is an immunosuppressive agent that inhibits the synthesis of lymphokines by T lymphocytes at the level of transcription. A cytoplasmic protein, cyclophilin, is the most thoroughly studied CsA-binding protein, but its ubiquitous presence in cells of all types raises questions about its role in immunosuppression. In an attempt to ascertain the presence of a cell surface receptor, we synthesized two polyvalent macromolecular CsA derivatives, CsA-BBa-ovalbumin and CsA-BBa-aminodextran (CBD), from the product of the photochemical reaction of CsA and 4-benzoylbenzoic acid (CsA-BBa). (i) They inhibited the peptidylprolyl cis-trans isomerase activity of cyclophilin and the synthesis of interleukin 2 by phorbol ester-activated EL-4 cells. (ii) CBD also inhibited interleukin 2 secretion by Con A-activated T-cell-enriched mouse splenocytes. 4-Benzoylbenzoic acid (BBa)-aminodextran and aminodextran were inactive. (iii) Direct binding and competition studies with [3H]CsA indicated that CBD does not enter EL-4 cells (i.e., it acted at the surface). (iv) CBD caused agglutination of EL-4 cells, murine B and T lymphocytes, human thymocytes, and two T-cell hybridomas. Agglutination was inhibited by a monoclonal antibody to CsA and by CsA and CsA-BBa, but not by BBa. No agglutination was seen with BBa-aminodextran or aminodextran. HeLa cells, Vero (monkey kidney) cells, a mouse plasmacytoma, COS cells, and a poorly differentiated B-cell lymphoma were not agglutinated. (v) EL-4 cells failed to be agglutinated after treatment with trypsin or chymotrypsin. Specific agglutination was again possible after incubation for 5 h at 37 degrees C in the absence of enzyme. (vi) CBD covalently linked to crosslinked agarose beads inhibited interleukin 2 production by phorbol ester-stimulated EL-4 cells. No activity was seen if cell-to-bead contact was prevented by a 0.02-microns microporous filter that did not interfere with the passage of CBD. Our findings support the presence of a functional receptor on the surface of selected cells of the immune system.
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PMID:Evidence for a functional receptor for cyclosporin A on the surface of lymphocytes. 158 69

The kinetic properties and substrate specificity of two well-characterized peptidyl prolyl cis-trans isomerases (PPIases), cyclophilin and the FK-506 binding protein (FKBP), have been previously examined [Fischer, G., Bang, H., Berger, E., & Schellenberger, A. (1984) Biochim. Biophys. Acta 791, 87-97; Harrison, R.K., & Stein, R.L. (1990) Biochemistry 29, 1684-1689; Albers, M.W., Walsh, C.T., & Schreiber, S. L. (1990) J. Org. Chem. 55, 4984-4986]. The chymotrypsin-coupled enzymatic assay employed in these studies suffers from two serious shortcomings. Due to the low equilibrium population of the X-cis-Pro-Phe-pNA isomer (the PPIase substrate), in conjunction with the low solubility of p-nitroaniline generated by chymotrypsin hydrolysis, substrate concentrations in the saturating region are not experimentally attainable. Secondly, the uncatalyzed cis-trans isomerization obscures the interpretation of the initial velocity. As a result of these limitations, the steady-state kinetic parameters (Km,Kcat) have not been determined. Here we introduce an improved version of the spectrophotometric assay and report for the first time the Michaelis constants and turnover numbers for both PPIases with established substrates. The improvements in the experimental conditions originate in a medium-induced increase in the equilibrium population of the cis X-Pro conformer and in conducting the assay at 0 degrees C to suppress the uncatalyzed thermal isomerization. In addition, we present a rigorous mathematical model of the spectrophotometric progress curves that accounts for the contributions of the residual background rate.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Determination of kinetic constants for peptidyl prolyl cis-trans isomerases by an improved spectrophotometric assay. 205 21

The prokaryotic peptidyl-prolyl cis-trans-isomerase called "rotamase", a homolog of the human cyclophilin, has been identified in Escherichia coli. The E. coli rotamase, a product of the gene we suggest be called "rot," has been purified to homogeneity after cloning of the gene by the polymerase chain reaction and its overexpression in E. coli. Based on the chymotrypsin-coupled assay using the tetrapeptide substrate succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, the purified protein has rotamase activity identical to human cyclophilin with a catalytic efficiency close to the upper diffusional limit (kcat/Km approximately 1.0 x 10(7) M-1 x S-1 at 10 degrees C). Unlike the human cyclophilins, however, the E. coli rotamase is not significantly inhibited by the immunosuppressant drug cyclosporin A. By spheroplast fractionation of cells harboring the expression vector for the complete rot gene, the rotamase is located in the periplasm, where it could function in refolding of secreted proteins.
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PMID:Peptidyl-prolyl cis-trans-isomerase from Escherichia coli: a periplasmic homolog of cyclophilin that is not inhibited by cyclosporin A. 219 Feb 12

Cyclophilin, a specific cytosolic binding protein responsible for the concentration of the immunosuppressant cyclosporin A by lymphoid cells, was purified to homogeneity from bovine thymocytes. Cation-exchange high-performance liquid chromatography resolved a major and minor cyclophilin species that bind cyclosporin A with a dissociation constant of about 2 X 10(-7) moles per liter and specific activities of 77 and 67 micrograms per milligram of protein, respectively. Both cyclophilin species have an apparent molecular weight of 15,000, an isoelectric point of 9.6, and nearly identical amino acid compositions. A portion of the NH2-terminal amino acid sequence of the major species was determined. The cyclosporin A-binding activity of cyclophilin is sulfhydryl dependent, unstable at 56 degrees C and at pH 4 or 9.5, and sensitive to trypsin but not to chymotrypsin digestion. Cyclophilin specifically binds a series of cyclosporin analogs in proportion to their activity in a mixed lymphocyte reaction. Isolation of cyclophilin from the cytosol of thymocytes suggests that the immunosuppressive activity of cyclosporin A is mediated by an intracellular mechanism, not by a membrane-associated mechanism.
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PMID:Cyclophilin: a specific cytosolic binding protein for cyclosporin A. 623 8

The prolyl isomerase activity of cyclophilins is traditionally measured by an assay in which prolyl cis/trans isomerization in a chromogenic tetrapeptide is coupled with its isomer-specific cleavage by chymotrypsin. Two variants of mitochondrial cyclophilin with substitutions in the presumed active site (R73A and H144Q) are inactive in the protease-coupled assay, but show almost wild-type activity in an assay that is based on the catalysis of a proline-limited protein folding reaction. This prolyl isomerase assay is preferable, both because coupling with proteolysis is avoided and because an intact protein instead of a short peptide is used as a substrate. Possibly, some earlier conclusions about the catalytic mechanism and the involvement of the prolyl isomerase activity in the cellular function of immunophilins may need reevaluation.
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PMID:Cyclophilin active site mutants have native prolyl isomerase activity with a protein substrate. 930 34

Cyclophilin and FK506 binding protein (FKBP) accelerate cis-trans peptidyl-prolyl isomerization and bind to and mediate the effects of the immunosuppressants cyclosporin A and FK506. The normal cellular functions of these proteins, however, are unknown. We altered the active sites of FKBP12 and mitochondrial cyclophilin from the yeast Saccharomyces cerevisiae by introducing mutations previously reported to inactivate these enzymes. Surprisingly, most of these mutant enzymes were biologically active in vivo. In accord with previous reports, all of the mutant enzymes had little or no detectable prolyl isomerase activity in the standard peptide substrate-chymotrypsin coupled in vitro assay. However, in a variation of this assay in which the protease is omitted, the mutant enzymes exhibited substantial levels of prolyl isomerase activity (5-20% of wild-type), revealing that these mutations confer sensitivity to protease digestion and that the classic in vitro assay for prolyl isomerase activity may be misleading. In addition, the mutant enzymes exhibited near wild-type activity with two protein substrates, dihydrofolate reductase and ribonuclease T1, whose folding is accelerated by prolyl isomerases. Thus, a number of cyclophilin and FKBP12 "active-site" mutants previously identified are largely active but protease sensitive, in accord with our findings that these mutants display wild-type functions in vivo. One mitochondrial cyclophilin mutant (R73A), and also the wild-type human FKBP12 enzyme, catalyze protein folding in vitro but lack biological activity in vivo in yeast. Our findings provide evidence that both prolyl isomerase activity and other structural features are linked to FKBP and cyclophilin in vivo functions and suggest caution in the use of these active-site mutations to study FKBP and cyclophilin functions.
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PMID:Functions of FKBP12 and mitochondrial cyclophilin active site residues in vitro and in vivo in Saccharomyces cerevisiae. 936 68

Assembly of the human immunodeficiency virus type 1 (HIV-1) first occurs on the plasma membrane of host cells where binding is driven by strong electrostatic interactions between the N-terminal matrix (MA) domain of the structural precursor polyprotein, Gag, and the membrane. MA is also myristylated, but the exact role this modification plays is not clear. In this study, we compared the protein oligomerization and membrane binding properties of Myr(+) and Myr(-) Gag(MA) expressed in COS-1 cells. Sedimentation studies in solution showed that both the myristylated Gag precursor and the mature MA product were detected in larger complexes than their unmyristylated counterparts, and the myristylated MA protein bound liposomes with approximately 3-fold greater affinity than unmyristylated MA. Aromatic residues near the N-terminal region of the MA protein were more accessible to chymotrypsin in the unmyristylated form and, consistent with this, an epitope in the N-terminal region was more exposed. Moreover, the cyclophilin binding site in the CA domain downstream of MA was more accessible in the unmyristylated Gag protein, while the Tsg101 binding site in the C-terminal region was equally available in the unmyristylated and myristylated Gag proteins. Taken together, our results suggest that myristylation promotes assembly by inducing conformational changes and facilitating MA multimerization. This observation offers a novel role for myristylation.
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PMID:Role of myristylation in HIV-1 Gag assembly. 1276 22

The seed proteome of two traditional maize inbred lines (pb269 and pb369) contrasting in grain hardness and in preferable use for bread-making was evaluated. The pb269 seeds, of flint type (i.e., hard endosperm), are preferably used by manufacturers, while pb369 (dent, soft endosperm) is rejected. The hypothesis that the content and relative amounts of specific proteins in the maize flour are relevant for such discrimination of the inbred lines was tested. The flour proteins were sequentially extracted following the Osborne fractionation (selective solubilization), and the four Osborne fractions were submitted to two-dimensional electrophoresis (2DE). The total amount of protein extracted from the seeds was not significantly different, but pb369 flour exhibited significantly higher proportions of salt-extracted proteins (globulins) and ethanol-extracted proteins (alcohol-soluble prolamins). The proteome analysis allowed discrimination between the two inbred lines, with pb269 demonstrating higher heterogeneity than pb369. From the 967 spots (358 common to both lines, 208 specific to pb269, and 401 specific to pb369), 588 were submitted to mass spectrometry (MS). Through the combined use of trypsin and chymotrypsin it was possible to identify proteins in 436 spots. The functional categorization in combination with multivariate analysis highlighted the most discriminant biological processes (carbohydrate metabolic process, response to stress, chitin catabolic process, oxidation-reduction process) and molecular function (nutrient reservoir activity). The inbred lines exhibited quantitative and qualitative differences in these categories. Differences were also revealed in the amounts, proportions, and distribution of several groups of storage proteins, which can have an impact on the organization of the protein body and endosperm hardness. For some proteins (granule-bound starch synthase-1, cyclophilin, zeamatin), a change in the protein solubility rather than in the total amount extracted was observed, which reveals distinct in vivo associations and/or changes in binding strength between the inbred lines. Our approach produced information that relates protein content, relative protein content, and specific protein types to endosperm hardness and to the preferable use for "broa" bread-making.
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PMID:Two traditional maize inbred lines of contrasting technological abilities are discriminated by the seed flour proteome. 2370 Oct 26


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