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)

The region of the horse cytochrome c molecule recognized by Mab SJL2-4 specific for the denatured form of the protein was located around residues 22-28. Binding studies on antigen pulsed macrophages were also performed. Surprisingly, heme peptide 1-65 was not recognised by Mab when bound on macrophages. This correlates with the incapacity of the same peptide to activate the T-cell clone 2-16. Binding sites on antigen pulsed macrophages varied between 0.5-2 x 10(6) per cell depending on the conditions used. The expression of the antigenic determinant as detected by Mab was also followed under different conditions (chloroquine, trypsin treatment) and time. Kinetics parameters of the antigen-antibody reaction in solution and on antigen bound macrophages were also determined and are dramatically different. This is correlated with a different structure of the peptide in solution and on macrophage cell surface.
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PMID:Antigen presenting cells: detection and quantification of a cytochrome c determinant important for activation of T-cells on bone marrow derived macrophages by using specific anti cytochrome c monoclonal antibody. 245 63

Protease activity present in aerobically grown cells of Pseudomonas perfectomarina, protease apparently copurified with cytochrome c-552, and trypsin achieved a limited proteolysis of the diheme cytochrome c-552. That partial lysis conferred cytochrome c peroxidase activity upon cytochrome c-552. The removal of a 4000-Da peptide explains the structural changes in the cytochrome c-552 molecule that resulted in the appearance of both cytochrome c peroxidase activity (with optimum activity at pH 8.6) and a high-spin heme iron. The oxidized form of the modified cytochrome c-552 bound cyanide to the high-spin ferric heme with a rate constant of (2.1 +/- 0.1) X 10(3) M-1 s-1. The dissociation constant was 11.2 microM. Whereas the intact cytochrome c-552 molecule can be half-reduced by ascorbate, the cytochrome c peroxidase was not reducible by ascorbate, NADH, ferrocyanide, or reduced azurin. Dithionite reduced the intact protein completely but only half-reduced the modified form. The apparent second-order rate constant for dithionite reduction was (7.1 +/- 0.1) X 10(2) M-1 s-1 for the intact protein and (2.2 +/- 0.1) X 10(3) M-1 s-1 for the modified form. In contrast with other diheme cytochrome c peroxidases, reduction of the low-spin heme was not necessary to permit ligand binding by the high-spin heme iron.
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PMID:Cytochrome c peroxidase activity of a protease-modified form of cytochrome c-552 from the denitrifying bacterium Pseudomonas perfectomarina. 253 41

A method of calculating the electrostatic potential energy between two molecules, using finite difference potential, is presented. A reduced charge set is used so that the interaction energy can be calculated as the two static molecules explore their full six-dimensional configurational space. The energies are contoured over surfaces fixed to each molecule with an interactive computer graphics program. For two crystal structures (trypsin-trypsin inhibitor and anti-lysozyme Fab-lysozyme), it is found that the complex corresponds to highly favourable interacting regions in the contour plots. These matches arise from a small number of protruding basic residues interacting with enhanced negative potential in each case. The redox pair cytochrome c peroxidase-cytochrome c exhibits an extensive favourably interacting surface within which a possible electron transfer complex may be defined by an increased electrostatic complementarity, but a decreased electrostatic energy. A possible substrate transfer configuration for the glycolytic enzyme pair glyceraldehyde phosphate dehydrogenase-phosphoglycerate kinase is presented.
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PMID:Investigating protein-protein interaction surfaces using a reduced stereochemical and electrostatic model. 254 Dec 55

A water soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), has been used to crosslink horse heart cytochrome c and trypsin-solubilized bovine liver microsomal cytochrome b5. The reaction was conducted under a variety of solution conditions, and the products were purified by a combination of gel filtration and ion-exchange chromatography. Under all conditions of pH, ionic strength, EDC/protein ratio and reaction time that were studied, multiple 1:1 crosslinked complexes were observed with no evidence of a single, dominant species. Acetate, which is often used as a quencher of such reactions, was found to increase the complexity of the reaction products, presumably through EDC-promoted coupling to cytochrome c. Hydroxylamine treatment of the crosslinked complexes, a procedure frequently used to reverse EDC modification of tyrosyl residues, did not reduce the number of crosslinked components observed. The cytochrome b5 heme group was readily extracted from each of the 1:1 crosslinked complexes by standard techniques, so the crosslinking of heme propionate 7 with Lys79 of cytochrome c that might have been anticipated on the basis of molecular graphics modeling [Salemme, F.R. (1976) J. Mol. Biol. 102, 563-568] was not evident from this analysis. Analysis of HPLC tryptic peptide maps produced from crosslinked complexes revealed reduced specificity of trypsin in hydrolysis of EDC-crosslinked protein-protein complexes and unsatisfactory resolution of crosslinked or branched peptides. Nevertheless, it was possible to demonstrate that residues 52-72 of cytochrome b5, a region predicted to be critical to interaction with cytochrome b5 [Salemme, F.R. (1976) J. Mol. Biol. 102, 563-568] was absent from all peptide maps of 1:1 cytochrome c.cytochrome b5 complexes. Based on these results and a review of the literature involving EDC crosslinking of electron transfer proteins, we conclude that the techniques available for specific protein hydrolysis and separation of crosslinked peptides are not adequate to permit routine unambiguous identification of crosslinking sites in carbodiimide-crosslinked complexes.
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PMID:Crosslinking of cytochrome c and cytochrome b5 with a water-soluble carbodiimide. Reaction conditions, product analysis and critique of the technique. 255 10

We have confirmed the propensity of fragments of cytochrome c to form complexes that reproduce the structure and, in part, the functionality, of the native protein by preparing four novel complexes. We have used trypsin under three different sets of conditions in sequence to prepare a contiguous two-fragment complex (1-55).(56-104). One of the intermediates is a stable overlapping complex (1-65).(56-104). Conditions for limited acid hydrolysis of peptide bonds in cytochrome c have been developed that optimize the yield of fragments (1-50) and (51-104). These two fragments also form a stable association, as do (1-50) and (56-104). These complexes are potentially useful for the semisynthesis of analogues modified in the region of the cleavage sites, which include a number of highly conserved amino acid residues, and are being used for studies of protein folding, interactions with oxidase, cytochrome c immunogenicity and of artificially induced spontaneous resyntheses between complexing fragments. Like other known two-fragment complexes of cytochrome c, they exhibit normal visible spectra, including the presence of the 695 nm band, indicative of a functional haem crevice. Studies of their biological activities and redox potentials lead to a number of conclusions on structure-function relationships in cytochrome c. Most significantly there is a linear relationship between the logarithm of electron-transfer rates from cytochrome c reductase and redox potential in this series of analogues, indicating that such transfer is thermodynamically controlled. This discovery contributes to our understanding of the interaction of cytochrome and reductase. Since the relationship is obeyed by other types of analogues, except for those that involve modification of the active site of cytochrome c, we have a useful diagnostic for those residues that participate directly in electron transfer.
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PMID:On the relationship between oxidation-reduction potential and biological activity in cytochrome c analogues. Results from four novel two-fragment complexes. 282 30

The interaction between cytochrome c and its heme-free precursor apocytochrome c and chemically prepared fragments of these basic proteins with phosphatidylserine containing model membrane systems was studied by differential scanning calorimetry and carboxyfluorescein release experiments. Addition of apocytochrome c and fragments derived from the N-terminus cause a pronounced and linear decrease of the enthalpy (delta H) of the gel to liquid-crystalline phase transition of dielaidoylphosphatidylserine. In contrast, fragments derived from the C-terminus cause a smaller reduction in delta H; a similar trend was observed for the ability of the fragments to cause an increased carboxyfluorescein release from unilamellar vesicles. In addition, the covalent attachment of the heme at cysteine residues 14 and 17 greatly reduced the ability of both the intact protein and the N-terminal fragments to decrease delta H. Using a protein translocation assay based on large unilamellar vesicles containing enclosed trypsin it was found that at gel state temperatures the ability of apocytochrome c to partially translocate the bilayer (reach the opposite membrane/water interface) was greatly reduced. The implications of these findings for the import mechanism of apocytochrome c in mitochondria are shortly indicated.
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PMID:Influence of heme and importance of the N-terminal part of the protein and physical state of model membranes for the apocytochrome c-lipid interaction. 283 82

Protease susceptibility of homologous proteins in their native conformations was studied. This work aims to establish a broad and quantitative basis for the utilization of protease digestion to analyze the local stability of native proteins. Using high-performance liquid chromatography (HPLC) the time course of the proteolytic degradation of intact proteins was quantitatively traced. Rapid separation of peptide fragments with HPLC made possible the elucidation of sequential digestion originating from the cleavage at a very few sites which are locally unstable in the protein structure. Using four serine proteases, chymotrypsin, trypsin, elastase and subtilisin BPN', we found some common trends in proteolysis for a group of proteins of the cytochrome c family. By comparing of the proteolysis and thermal denaturation with ten homologous cytochromes c extracted from horse, beef, Candida krusei, Saccharomyces cerevisiae, chicken, tuna, pigeon, rabbit, dog and rat, protease susceptibility was related to locally unfolding states intrinsic to the native conformation.
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PMID:Probing stability and dynamics of proteins by protease digestion. I: Comparison of protease susceptibility and thermal stability of cytochromes c. 285 73

The cytoplasmically made subunits 2 (beta) and 3 (gamma) of the H+-ATPase from mammalian mitochondria are synthesized in vitro as larger polypeptides. In contrast, pre-cytochrome c could not, on the basis of its molecular weight, be distinguished from the mature polypeptide. This was shown by programming a reticulocyte lysate with rat heart RNA and immunoprecipitating the labeled translation products with polypeptide-specific antibodies. When a translated lysate containing the precursor to the beta-subunit was incubated with isolated rat spleen mitochondria, it was converted to the mature subunit and was no longer susceptible to externally added trypsin. The conversion to the mature form occurred in the absence of protein synthesis. This post-translational maturation process of the beta-subunit was more efficient when carried out with spleen or liver mitochondria than with heart or kidney mitochondria. The converse relative efficiency was observed when the processing of the precursor to ornithine carbamyltransferase by these mitochondria was examined. These results indicate that mitochondria do not discriminate against tissue-specific mitochondrial proteins. In addition, the observed varying degrees of efficiency of mitochondria from different tissues in importing and processing these two precursors suggest that the activity of precursor(s)-specific translocation-maturation systems varies between different types of mitochondria.
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PMID:Differential import and processing of the precursors to F1-ATPase beta-subunit and ornithine carbamyltransferase by liver, spleen, heart and kidney mitochondria. 286 Sep 3

The reagent 1-ethyl-3-(3-[14C]trimethylaminopropyl)carbodiimide (ETC) was used to identify specific carboxyl groups on the cytochrome bc1 complex (ubiquinol-cytochrome c reductase, EC 1.10.2.2) involved in binding cytochrome c. Treatment of the cytochrome bc1 complex with 2 mM ETC led to inhibition of the electron transfer activity with cytochrome c. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that both the cytochrome c1 heme peptide and the Mr = 9175 "hinge" peptide were radiolabeled by ETC. In addition, a new band appeared at a position consistent with a 1:1 cross-linked cytochrome c1-hinge peptide species. Treatment of a 1:1 cytochrome bc1-cytochrome c complex with ETC led to the same inhibition of electron transfer activity observed with the uncomplexed cytochrome bc1, but to decreased radiolabeling of the cytochrome c1 heme peptide. Two new cross-linked species corresponding to cytochrome c-hinge peptide and cytochrome c-cytochrome c1 were formed in place of the cytochrome c1-hinge peptide species. In order to identify the specific carboxyl groups labeled by ETC, a purified cytochrome c1 preparation containing both the heme peptide and the hinge peptide was dimethylated at all the lysines to prevent internal cross-linking. The methylated cytochrome c1 preparation was treated with ETC and digested with trypsin and chymotrypsin, and the resulting peptides were separated by high pressure liquid chromatography. ETC was found to label the cytochrome c1 peptides 63-81, 121-128, and 153-179 and the hinge peptides 1-17 and 48-65. All of these peptides are highly acidic and contain one or more regions of adjacent carboxyl groups. The only peptide consistently protected from labeling by cytochrome c binding was 63-81, demonstrating that the carboxyl groups at residues 66, 67, 76, and 77 are involved in binding cytochrome c. These residues are relatively close to the heme-binding cysteine residues 37 and 40 and indicate a possible site for electron transfer from cytochrome c1 to cytochrome c.
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PMID:Identification of the binding site on cytochrome c1 for cytochrome c. 298 91

The activation of 14C-labeled estradiol by "true" and "pseudo" peroxidases to form conjugates and other products was compared in four model systems using H2O2, glutathione, Mn2+ or irradiated riboflavin. Albumin was used as acceptor except in the glutathione system. The binding of estradiol to glutathione in the presence of the true peroxidases, lacto- or uterine peroxidase (no H2O2 added), was also examined and the conditions shown to differ from those required with the pseudoperoxidases, microperoxidase or trypsin-digested cytochrome c. The conjugates were purified by chromatography after elution from Amberlite XAD-2 and the relative amounts of these products assessed by autoradiography. The ratio of steroid to glutathione in the main water-soluble metabolite formed with lactoperoxidase was found to be approx 1:1 in a double label experiment with [14C]estradiol and [3H]glutathione. It was also shown, using estradiol labeled with 3H in different positions of the steroid molecule, that lactoperoxidase acts non-specifically in catalyzing the formation of glutathionyl conjugates as indicated by the release of 3H2O. The possible role of peroxidase and glutathione in the metabolism of estrogens and in the formation of artifactual products is discussed.
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PMID:Metabolism of estradiol by true and pseudoperoxidases. 299 58


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