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)

Addition of miscible organic solvents to water increases the solubility of naphthalene. The logarithm of the solubility is linearly dependent on the co-solvent concentration, in an intermediate range. The relative solubilising effects of different solvents correlate well with their known tendency to denature proteins (using literature data for trypsin, cytochrome c, chymotrypsinogen, chymotrypsin, laccase and myoglobin). This is expected if denaturation occurs when the hydrophobic effect has been reduced by a characteristic extent for a given protein. Naphthalene solubility predicts denaturation as well as does the denaturation capacity model.
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PMID:Prediction of denaturing tendency of organic solvents in mixtures with water by measurement of naphthalene solubility. 754 59

Employing a photoaffinity labeling procedure with 8-azido-S-adenosyl-L-[methyl-3H]methionine (8-N3-Ado[methyl-3H]Met), the binding sites for S-adenosyl-L-methionine(AdoMet) of three protein N-methyltransferases [AdoMet:myelin basic protein-arginine N-methyltransferase (EC 2.1.1.23); AdoMet:histone-arginine N-methyltransferase (EC2.1.1.23); and AdoMet:cytochrome c-lysine N-methyltransferase (EC2.1.1.59)] have been investigated. The incorporation of the photoaffinity label into the enzymes upon UV irradiation was highly specific. In order to define the AdoMet binding sites, the photolabeled enzymes were sequentially digested with trypsin, chymotrypsin, and endoproteinase Glu-C. After each proteolytic digestion, radiolabeled peptide from each enzyme was resolved on HPLC first by gradient elution and further purified by an isocratic elution. Retention times of the purified radiolabeled peptides from the three enzymes from the corresponding proteolysis were significantly different, indicating that their sizes and compositions were different. Amino acid composition analysis of these peptides confirmed further that the AdoMet binding sites of these protein N-methyltransferases are quite different.
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PMID:Comparative studies on S-adenosyl-L-methionine binding sites of protein N-methyltransferases, using 8-azido-S-adenosyl-L-methionine as photoaffinity probe. 814 3

Cytochrome b was identified as one of the ubiquinone-binding proteins in bovine heart mitochondrial ubiquinol-cytochrome c reductase by photoaffinity labeling using 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyl[3H]-octyl)-1,4-benzoquinone ([3H]azido-Q). The [3H]azido-Q-labeled cytochrome b protein was purified to homogeneity from the azido-Q-labeled ubiquinol-cytochrome c reductase by a procedure involving Triton X-100 and urea treatment, calcium phosphate column chromatography, acetone precipitation, decanoyl-N-methylglucamide-cholate extraction, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified cytochrome b protein containing 0.5 mol of azido-Q/mol of protein was subjected to reductive carboxymethylation and succinylation prior to digestion by chymotrypsin. Two azido-Q-linked peptides with retention times of 47.1 and 49.0 min were obtained by high performance liquid chromatographic separation. Partial amino-terminal amino acid sequences of these two peptides were determined to be GATVI- and ALVADL-, indicating that these two chymotryptic peptides are from amino residues 142-155 and 326-336. Monospecific polyclonal antibodies against two synthetic ubiquinone-binding peptides, NH2-G-A-T-V-I-T-N-L-L-S-COOH (P-47) and NH2-W-A-L-V-A-D-L-L-T-L-T-W-I-COOH (P-49), were generated in rabbits and purified. Western blotting and enzyme-linked immunosorbent assays showed that the purified antibodies against P-47 reacted with cytochrome b-containing reductases and purified cytochrome b protein. Antibodies against P-47 inhibited activities of succinate-cytochrome c and ubiquinol-cytochrome c reductases only when they were incubated with phospholipid-depleted reductases prior to the replenishment with phospholipid. No inhibition was observed with incubation with phospholipid-containing reductases, indicating that this peptide involved in ubiquinone binding is buried in a phospholipid environment.
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PMID:Ubiquinone binding domains in bovine heart mitochondrial cytochrome b. 829 88

The membranotropic properties of block co-polymers and their protein conjugates were studied by their effect on the rate of oxygen consumption by isolated liver mitochondria and on thymus-derived lymphocytes. The block co-polymers consisted of poly(ethylene oxide) (PoE) [poly(ethylene glycol)] and poly(propylene oxide) (PoP) to give either PoE-PoP or PoE-PoP-PoE. Both types inhibited uncoupled respiration of liver mitochondria in a medium containing glutamate and malate and also of lymphocytes. They also uncoupled respiration in the presence of succinate in K(+)-containing medium and of lymphocytes. A method is described for linking protein to the block polymers to form conjugates. Such conjugates were formed from alpha-chymotrypsin, BSA and cytochrome c, all of which produced similar effects on the respiration of the isolated mitochondria and lymphocytes. The data suggest that both the block co-polymers and their protein conjugates inhibit the NADH dehydrogenase complex and induce a K(+)-conductivity of the mitochondrial inner membrane; the surface activity of the conjugates allows them to pass through the plasma membrane and interact with the mitochondrial inner membrane.
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PMID:The influence of pluronics and their conjugates with proteins on the rate of oxygen consumption by liver mitochondria and thymus lymphocytes. 829 10

A fully active form of hydroxylamine oxidoreductase from Nitrosomonas has been purified with high recovery and shown by reverse-phase high performance liquid chromatography and N-terminal analysis to contain only a 63-kDa subunit and to lack the 11-kDa protein previously thought to be a second subunit. Based on the previously published values of molecular weight in solution, hydroxylamine oxidoreductase probably has an alpha 2 or alpha 3 oligomeric structure. The enzyme was digested separately with trypsin and chymotrypsin and peptides which contained covalently bound heme were separated by high performance liquid chromatography and their amino acid sequences determined. A total of seven heme-containing peptides of unique amino acid sequence were obtained. Six of these heme-containing peptides clearly contained a single c-heme with optical properties indistinguishable from the tryptic heme-containing peptide from horse heart cytochrome c. No noncovalently bound heme was observed. One of the seven heme-containing peptides (T7) was unusual in that it released 2 amino acid residues after each cycle of the Edman degradation due to a nondisulfide cross-link and exhibited a Soret band that was broadened in both the ferric form at neutral pH and the pyridine ferrohemochrome. Subdigestion of peptide T7 with nonspecific proteases (Pronase, bromelain, or pepsin) resulted in the isolation of two smaller heme-containing peptides of unique sequences. One of these was spectrally identical to the other c-heme containing peptides, whereas the second was still apparently cross-linked, again releasing 2 amino acid residues after each Edman cycle. This second peptide possessed a heme-like chromophore with absorption bands (Soret, alpha and beta) red-shifted about 6 nm relative to the spectrum of c-heme-containing peptides. Thus, hydroxylamine oxidoreductase contains a total of eight covalently bound hemes per subunit, seven of which are c-hemes. The eighth, which is attached to a cross-linked peptide, is probably the unusual P460 heme which is unique to hydroxylamine oxidoreductase and thought to be at the active site.
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PMID:Hydroxylamine oxidoreductase from Nitrosomonas europaea is a multimer of an octa-heme subunit. 832 41

A method using cytochrome c as the substrate of proteinases trypsin, chymotrypsin and subtilisin based on peroxidase effect of the formed haem octapeptide, which is much higher than that of cytochrome c, is described. The octapeptide is degraded by excess hydrogen peroxide and the competition between oxidation of guaiacol catalyzed by the octapeptide and octapeptide degradation leads under experimental conditions to rapid production of a stable colour. Absorption at 476 nm is proportional to proteolytic activity. The method was standardized using the Anson test for proteolytic activity with haemoglobin as a substrate.
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PMID:Proteolytic activity assay based on enhanced peroxidase effect of hydrolyzed cytochrome c. 839 22

A new deconvolution procedure was applied to the analysis of Fourier transform ir spectra of human serum albumin secondary structure in the native state and in states denatured by heat and acid treatment. The deconvolution method is based on the use of the Conjugate Gradient Minimization Algorithm, with the addition of suitable constraints directly obtained by the application to the measured spectrum of the second derivative operator. This method computes central band frequency, bandwidth, and amplitude of the different spectral components of conformation-sensitive amide bands. In the specific case, it was applied to analysis of the amide I band, and the quantitative determination of the different secondary structures (alpha-helix, beta-sheet, beta-turns, and random) was attempted for all the samples examined. The precision of the quantitative determination depends on the amounts of these structures present in the protein. The coefficient of variation is < 10% for values of amide I component > 15%. The accuracy was tested by comparing, by means of linear regression, the results obtained for human serum albumin, hemoglobin, alpha-chymotrypsin, and cytochrome c, using our method, with those obtained by x-ray crystallography and CD; the results obtained by other vibrational spectroscopic approaches were also compared. The fit standard error between x-ray and ir secondary structure values estimated by our method is 2.5% for alpha-helix, 7.16% for beta structures, and 5.1% for other structures (turns and random coils). Quantitative results are given for the secondary structures (alpha-helix, turns, and beta-strands) present in the native state (turns and beta-strands up to now unknown in aqueous solution), together with the percentages of these structures and additional ones (random coils and beta-sheets) formed during denaturization.
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PMID:Determination of the secondary structure of isomeric forms of human serum albumin by a particular frequency deconvolution procedure applied to Fourier transform IR analysis. 872 32

The kinetics of in vivo regulation of mitochondrial respiration by ADP was studied in rat heart, slow-twitch skeletal muscle (soleus) and fast-twitch skeletal muscle (gastrocnemius, plantaris, quadriceps and tibialis anterior) by means of saponin-skinned fibres. Mitochondrial respiratory parameters were determined in the absence and presence of creatine (20 mM), and the effect of proteolytic enzymes (trypsin, chymotrypsin or elastase) on these parameters was investigated in detail. The results of these experiments confirm the observation of Veksler et al. [Veksler, V.I., Kuznetsov, A. V., Anflous, K., Mateo, P., van Deursen, J., Wieringa, B. & Ventura-Clapier, R. (1995) J. Biol. Chem. 270, 19921-19929], who studied muscle fibres from normal and transgenic mice, that the kinetics of respiration regulation in muscle cells is tissue specific. We found that in rat cardiac and soleus muscle fibres the apparent K(m) for respiration regulation was 300-400 microM and decreased to 50-80 microM in the presence of creatine. In contrast, in skinned fibres from gastrocnemius, plantaris, tibialis anterior and quadriceps muscles, this value was initially very low, 10-20 microM, i.e. the same as that is in isolated muscle mitochondria, and the effect of creatine was not observable under these experimental conditions. Treatment of the fibres with trypsin, chymotrypsin or elastase (0.125 micrograms/ml) for 15 min decreased the apparent K(m) for ADP in cardiac and soleus muscle fibres to 40-98 microM without significant alteration of Vmax or the intactness of outer mitochondrial membrane, as assessed by the cytochrome c test. In fibres from gastrocnemius, trypsin increased the apparent K(m) for ADP transiently. The effects of trypsin and chymotrypsin were studied in detail and found to be concentration dependent and time dependent. The effects were characterised by saturation phenomenon with respect to the proteolytic enzyme concentration, saturation being observed above 1 microM enzyme. These results are taken to show that in cardiac and slow-twitch skeletal muscle, the permeability of the outer mitochondrial membrane to adenine nucleotides is low and controlled by a cytoplasmic protein that is sensitive to trypsin and chymotrypsin. This protein may participate in feedback signal transduction by a mechanism of vectorial-ligand conduction. This protein factor is not expressed in fast-twitch skeletal muscle, in which cellular mechanism of regulation of respiration is probably very different from that of slow-twitch muscles.
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PMID:Striking differences between the kinetics of regulation of respiration by ADP in slow-twitch and fast-twitch muscles in vivo. 894 82

Osmolytes are small organic solutes produced by the cells of all organisms (except halobacteria) in high stress situations (e.g. extremes of salt concentration, high temperature, etc.) to stabilize their macromolecules and so conserve biological activity. They do not interact with the macromolecule directly but act by altering the solvent properties in the cellular environment, and so their presence indirectly modifies the stability of proteins. In this paper we examine the effect of a model osmolyte, glycine, on the stabilization of two proteins, chymotrypsin inhibitor 2 and horse heart cytochrome c. We have used NMR to monitor the effect of this osmolyte on amide hydrogen exchange rates, which allows a probe at discrete points within the protein structure. Hydrogen exchange rates of specific backbone amide protons provide information about the localized structural fluctuations that expose these amides to solvent and allow exchange to take place. We find that the presence of a high concentration of glycine osmolyte has a profound stabilizing effect on the proteins studied, which is accompanied by a large reduction of the exchange rate constants of most slowly exchanging amide protons. The spectra indicate that this arises without significant changes in the three-dimensional structure. However, the effects on individual amide protons within a single protein were not uniform, and a wide variation in the magnitude of the effects was observed. This ranged from no apparent change in the exchange rate, to decreases in the exchange rate constant by over 2 orders of magnitude. The osmolyte appears to alter a number of different processes that contribute to the observed exchange rates, and no simple generalization allows prediction of the extent of stabilization at any individual location. The results are discussed in light of the available structures of the proteins studied.
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PMID:Effect of osmolytes on the exchange rates of backbone amide protons in proteins. 948 49

Syncephapepsin is a fungal aspartic proteinase from Syncephalastrum racemosum. By using the property of syncephapepsin after having increased activity at higher temperature, two rapid purification protocols were developed. In the former case, a crude extract was initially diluted fivefold with an activity assay buffer and heated at 50 degrees C overnight. In this situation, syncephapepsin would digest most of the proteins that the crude extract contained. Subsequently, syncephapepsin of the crude extract was precipitated from 50 to 70% of ammonium sulfate and the preparation was then directly applied to the Superdex 200 HR FPLC column. In this manner, syncephapepsin was rapidly purified to apparent homogeneity within 24 h. In this report, an alternative method of purification is also provided. Compared with the procedure mentioned above, the heating step was proceeded after FPLC chromatography through which the same result was obtained. Using cytochrome c and RNase A as substrates, the cleavage sites of both substrates were identified by HPLC peptide mapping. The results showed that syncephapepsin had a broad specificity. Residues recognized to be cleaved were primarily those of trypsin and chymotrypsin and Lys was the most susceptible.
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PMID:Single-column purification of syncephapepsin--an aspartic proteinase from Syncephalastrum racemosum. 953 8


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