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)

Bovine heart mitochondrial cytochrome c oxidase has been treated with trypsin in order to investigate the role of components a, b, and c (nomenclature of Capaldi) in cytochrome c binding, electron transfer, and proton-pumping activities. Cytochrome c oxidase was dispersed in nondenaturing detergent solution (B. Ludwig, N. W. Downer, and R. A. Capaldi (1979) Biochemistry 18, 1401) and treated with trypsin. This treatment inhibited electron transfer activity by 9% when compared to a similarly treated control in a polarographic assay (493 s-1) and had no large effect on the high affinity (Km = 6.1 X 10(-8) M) or low affinity (Km = 2.2 X 10(-6) M) sites of cytochrome c interaction with cytochrome c oxidase. Direct thermodynamic binding experiments with cytochrome c showed that neither the high affinity (1.04 +/- 0.06 mol cytochrome c/mol cytochrome c oxidase) nor the high-plus-low affinity (2.21 +/- 0.15 mol cytochrome c/mol cytochrome c oxidase) binding sites of cytochrome c on the enzyme were perturbed by the trypsin treatment. Control and trypsin-treated enzyme incorporated into phospholipid vesicles (prepared by the cholate dialysis method) exhibited respiratory control ratios of 6.5 +/- 0.7 and 6.3 +/- 0.6, respectively. The vectorial proton translocation activity in the phospholipid vesicles was unaffected by trypsin treatment with proton translocated to electron transferred ratios being equivalent to the control. NaDodSO4-PAGE showed that components a, b, and c were completely removed by the trypsin treatment. [14C]Iodoacetamide labeling experiments showed that the content of component c in the enzyme was depleted by 85% and that greater than 50% of component a was cleaved upon the trypsin treatment. These results suggest that components a, b, and c are not required for maximum electron transfer and proton translocation activities in the isolated enzyme.
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PMID:Characterization of electron transfer and proton translocation activities in trypsin-treated bovine heart mitochondrial cytochrome c oxidase. 300 79

Small amounts (femtomoles) of proteases, as might be present in cell extracts or secretions, were detected using reverse-phase high-performance liquid chromatography. Carboxymethylated lysozyme and cytochrome c were incubated with trypsin and chymotrypsin. Peptide peaks were present in the column elution profiles (as detected by absorbance, 206 nm) from incubations with as little as 0.1 fmol of chymotrypsin and 5 fmol of trypsin. In addition, the disappearance of the substrate peak or the increase in peptide peaks could be quantitated by integrating the areas under the peaks. In this way estimates of relative enzyme concentrations or duration of incubation can be determined. However, when [14C]lysozyme was used as a substrate and the radioactivity of collected peaks was measured, the assay was less sensitive than that using uv absorbance. This finding probably is related to the selective radiolabeling of the substrate, in contrast to uv detection, which should detect all the peptides. The technique reported in this paper should prove to be a sensitive indicator of proteolytic activity in cell or tissue preparations where the use of synthetic ester or amide substrates might lead to erroneous conclusions regarding the nature of the enzymatic activity present. Furthermore, by the collection of the peptides generated, one would have the ability to determine amino acid compositions or sequences and thus ascertain the specificity of enzymatic cleavage.
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PMID:Detection of femtomole quantities of proteases by high-performance liquid chromatography. 300 44

Isolated beef heart cytochrome c oxidase was reconstituted in liposomes by the cholate dialysis method with 85% of the binding site for cytochrome c oriented to the outside. Trypsin cleaved specifically subunit VIa and half of subunit IV from the reconstituted enzyme. The kinetic properties of the reconstituted enzyme were changed by trypsin treatment if measured by the spectrophotometric assay but not by the polarographic assay. It is concluded that subunit VIa and/or subunit IV participate in the electron transport activity of cytochrome c oxidase.
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PMID:Effect of trypsin on the kinetic properties of reconstituted beef heart cytochrome c oxidase. 300 49

The stability of the complex formed between cytochrome c and dimethyl ester heme substituted cytochrome b5 (DME-cytochrome b5) has been determined under a variety of experimental conditions to evaluate the role of the cytochrome b5 heme propionate groups in the interaction of the two native proteins. Interaction between cytochrome c and the modified cytochrome b5 was found to produce a difference spectrum in the visible range that is very similar to that generated by the interaction of the native proteins and that can be used to monitor complex formation between the two proteins. At pH 8 [25 degrees C (HEPPS), I = 5 mM], DME-cytochrome b5 and cytochrome c form a 1:1 complex with an association constant KA of 3 (1) X 10(6) M-1. This pH is the optimal pH for complex formation between these two proteins and is significantly higher than that observed for the interaction between the two native proteins. The stability of the complex formed between DME-cytochrome b5 and cytochrome c is strongly dependent on ionic strength with KA ranging from 2.4 X 10(7) M-1 at I = 1 mM to 8.2 X 10(4) M-1 at I = 13 mM [pH 8.0 (HEPPS), 25 degrees C]. Calculations for the native, trypsin-solubilized form of cytochrome b5 and cytochrome c confirm that the intermolecular complex proposed by Salemme [Salemme, F. R. (1976) J. Mol. Biol. 102, 563] describes the protein-protein orientation that is electrostatically favored at neutral pH.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Electrostatic analysis of the interaction of cytochrome c with native and dimethyl ester heme substituted cytochrome b5. 302 46

A method for assaying the covalent attachment of heme to apoprotein of cytochrome c was developed. 125I-labeled apoprotein was chemically prepared from 125I-labeled yeast cytochrome c (iso-1-cytochrome c). After incubation of 125I-apocytochrome c with yeast mitochondria, the product was extracted with Triton X-100, digested with trypsin in the presence or absence of a reducing agent, and then precipitated in trichloroacetic acid. The resulting precipitates were collected on nitrocellulose membranes and counted for radioactivity. The radioactivity correlated well with the appearance of a heme-containing peptide in the trypsin digested peptide fragments of cytochrome c. This procedure is simpler and faster than the previously reported methods.
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PMID:A simple and rapid assay for heme attachment to apocytochrome c. 303 18

NADPH-cytochrome P-450 reductase in rat testicular microsomal fraction was solubilized by trypsin, and purified to apparent homogeneity in polyacrylamide gel electrophoresis. Molecular weight of the enzyme was estimated to be about 70,000 by SDS-polyacrylamide gel electrophoresis. Km values were estimated as 18 microM for cytochrome c, 17 microM for dichlorophenol indophenol (DCPIP), 50 microM for K3Fe (CN)6 and 1.7 microM for NADPH. The cytochrome c reducing activity of the purified preparation was decreased by tetranitromethane (TNM), a reagent for nitration of tyrosine residues in a protein. The inactivation exhibited pseudo-first-order kinetics. A plot of log kapp vs log [TNM] gave a straight line with slope = 1.05, indicating the reaction of one modifier molecule in the inactivation process. The decrease of the reducing activities for DCPIP and K3Fe(CN)6 by TNM progressed more slowly than that for cytochrome c. The inactivation of cytochrome c reduction was protected completely by 0.1 mM NADP(H) and partially by 0.1 mM DCPIP and cytochrome c. No preventive change of the inactivation by TNM was observed by addition of NAD+ or testosterone. On the other hand, the differential modification by DTNB, TNM and DTT indicated that there were amino acid residues modified by TNM, such as tyrosine residues, at or near the active-site of the NADPH-cytochrome P-450 reductase.
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PMID:Purification of NADPH-cytochrome P-450 reductase from microsomal fraction of rat testes, and its chemical modification by tetranitromethane. 309 39

In a previous publication (Narhi, L. O. and Fulco, A. J. (1986) J. Biol. Chem. 261, 7160-7169) we described the characterization of a soluble 119,000-dalton P-450 cytochrome (P-450BM-3) that was induced by barbiturates in Bacillus megaterium. This single polypeptide contained 1 mol each of FAD and FMN/mol of heme and, in the presence of NADPH and O2, catalyzed the oxygenation of long-chain fatty acids without the aid of any other protein. We have now utilized limited trypsin proteolysis in the presence of substrate to cleave P-450BM-3 into two polypeptides (domains) of about 66,000 and 55,000 daltons. The 66-kDa domain contains both FAD and FMN but no heme, reduces cytochrome c in the presence of NADPH, and is derived from the C-terminal portion of P-450BM-3. The 55-kDa domain is actually a mixture of three discrete peptides (T-I, T-II, and T-III) separable by high performance liquid chromatography. All three contain heme and show a P-450 absorption peak in the presence of CO and dithionite. The major component, T-I (Mr = 55 kDa), binds fatty acid substrate and has an N-terminal amino acid sequence identical to that of intact P-450BM-3, an indication that this domain constitutes the N-terminal portion of the 119-kDa protein. T-II (54 kDa) is the same as T-I except that it is missing the first nine N-terminal amino acids and does not bind substrate. T-III (Mr = 53.5 kDa) has lost the first 15 N-terminal residues and does not bind substrate. Since trypsin digestion of P-450BM-3 carried out in the absence of substrate yields T-II and T-III but no T-I, it appears that 1 or more residues of the first nine N-terminal amino acids of this protein are intimately involved in substrate binding. Although both the heme- and flavin-containing tryptic peptides retain their original half-reactions, fatty acid monooxygenase activity cannot be reconstituted after proteolysis, and the two domains, once separated, show no affinity for each other. In most respects, the reductase domain of P-450BM-3 more closely resembles the mammalian microsomal P-450 reductases than it does any known bacterial protein.
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PMID:Identification and characterization of two functional domains in cytochrome P-450BM-3, a catalytically self-sufficient monooxygenase induced by barbiturates in Bacillus megaterium. 310 60

Eosinophil-directed chemotactic inhibitory factors (ECIF) from T lymphocytes of complete Freund's adjuvant-treated guinea pigs were recovered in two separate molecular weight (MW) fractions: the one is eluted near bovine serum albumin (MW about 70,000), and in pH range between 7.0 and 7.5 by chromatofocusing column, whereas the other is near cytochrome c (MW 12,500), and in pH range between 5.0 and 5.5. It was, however, found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoadsorption that the high molecular ECIF is probably an aggregated form of the low molecular ECIF. It was subsequently confirmed that both the ECIF had similar physicochemical properties: sensitive to heating at 56 or 80 degrees C sensitive to enzyme treatment with trypsin and neuraminidase, sensitive in acid but not alkaline condition, and bind to peanut agglutinin-or Limulus polyphemus agglutinin-coupled agarose beads. The inhibitory activity of ECIF was suppressed by previous treatment of eosinophils with D-galactose and sialic acid. ECIF activity was specifically absorbed by eosinophils; the absorption was inhibited by pretreatment of eosinophils with D-galactose and sialic acid. Previous treatment of eosinophils with beta-galactosidase and neuraminidase led the cells not to respond to both ECIF. It was thus suggested that the inhibition by ECIF is receptor-mediating phenomenon, and that ECIF and ECIF receptors on eosinophils contain galactose and sialic acid residues which may play an essential role for the biological activity.
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PMID:The regulation of tissue eosinophilia. IV. Purification and properties of eosinophil-directed chemotactic inhibitory factors from complete Freund's adjuvant-treated guinea pigs. 348 46

Rabbit polyclonal antibody was raised to a chemically synthesized nonapeptide (Trp-Ala-Glu-Trp-Cys-Gly-Pro-Cys-Lys) corresponding to the active-site sequence of Escherichia coli thioredoxin. The antiserum efficiently inhibited thioredoxin activity in the standard thioredoxin reductase/NADPH coupled assay. This inhibition was blocked by preincubation of the antiserum with the nonapeptide. Tight association of the E. coli thioredoxin to the active-site antibody required SDS denaturation. These results suggest that thioredoxin reductase (NADPH: oxidized-thioredoxin oxidoreductase, EC 1.6.4.5) alters the conformation of thioredoxin sufficiently to permit binding to the antibody. The antiserum bound to plant and liver thioredoxins. Bovine pancreatic trypsin inhibitor, whose active site (Gly-Pro-Cys-Lys) is homologous to that of thioredoxin, also competes for the active-site antibody. This result led to experiments showing that thioredoxin can inhibit the digestion of cytochrome c by trypsin. The ability of thioredoxin to act as a trypsin inhibitor analogue provides a rationale for thioredoxin's resistance to digestion by trypsin.
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PMID:The catalytic active site of thioredoxin: conformation and homology with bovine pancreatic trypsin inhibitor. 370 71

Rat liver nuclear thyroid hormone receptor was subjected to limited trypsin digestion, and the tryptic fragment of the 3,5,3'-triiodo-L-thyronine (T3)-receptor complex was characterized. Rat liver nuclear thyroid hormone receptor is an asymmetrical protein with Stokes radius of 34 A, sedimentation coefficient of 3.4 S, and molecular weight of 49,000. A globular T3-receptor complex with Stokes radius of 22 A, sedimentation coefficient of 2.8 S, and molecular weight of 26,000 was obtained by tryptic digestion. This fragment had no DNA binding activity, whereas undigested receptor showed significant DNA binding activity. Addition of undigested receptor to the tryptic fragment did not restore DNA binding activity of digested receptor, nor did mixing inhibit DNA binding activity of undigested receptor complex. Undigested receptor bound to core histones, and this activity was stronger than with other proteins tested (H1 histone, cytochrome c, and ovalbumin). The tryptic fragment of receptor maintained core histone binding activity comparable to that of undigested receptor. The tryptic fragment had affinity for T3 comparable to undigested receptor as assessed by Scatchard analysis and the same rate for dissociation of [125I]T3 from receptor. The tryptic fragment of the T3-receptor complex was more stable than undigested receptor at 43 degrees C. Digestion of receptor unoccupied by T3 caused a significantly larger loss of T3 binding capacity than did digestion of T3-occupied receptor, suggesting a protective effect of T3 on a second trypsin-sensitive site on the receptor, which, when cut, destroys T3 binding activity.
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PMID:Separation of DNA binding domain from hormone and core histone binding domains by trypsin digestion of rat liver nuclear thyroid hormone receptor. 378 33


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