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)

In order to clarify the physicochemical property of tissue plasminogen activator (TA), tissue extracts of human paranasal mucous membrane and pig heart were studied by the biochemical techniques. The studies by gel filtration revealed that two plasminogen activators of different molecular weight were present in the extract of human paranasal mucous membrane. The existence of tissue plasminogen activator with a low molecular weight (LMW-TA) has not previously been reported. This molecular weight of this compound was lower than that of cytochrome c. On the other hand, the molecular weight of tissue plasminogen activator from pig heart (PH-TA) was similar to that of ovalbumin, about 46,000 daltons, as estimated by Sephadex G-150 gel filtration. From the physicochemical property of LMW-TA, it is suggested that the LMW-TA from paranasal mucous membrane with chronic sinusitis was produced by proteases, i.e. trypsin-like enzyme, present in the mucous membrane with chronic inflammation.
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PMID:Differences in physicochemical properties between tissue plasminogen activators from human paranasal mucous membrane and pig heart. 719 7

Treatment of intact pigeon erythrocytes with trypsin or alpha-chymotrypsin does not alter the isoproterenol-dependent adenylate cyclase activity in plasma membranes prepared after proteolysis. However, both proteases affect adenylate cyclase activity when isolated membranes are digested. Thus, the proteases probably act at the cytoplasmic side of the membranes. This conclusion is supported by the finding that proteases are able to inhibit NADH cytochrome c oxidoreductase, an enzyme located on the inner face of the plasma membrane. In isolated membranes, trypsin inhibits adenylate cyclase. Chymotrypsin (2.5 microgram/ml, 10 min, 37 degrees C) activates adenylate cyclase about 3-fold when the enzyme activity is measured with NaF, guanosine 5'-(beta, gamma-imino)-triphosphate, or guanosine 5'-(beta, gamma-imino)-triphosphate and isoproterenol. Chymotrypsin also activates adenylate cyclase in membranes pretreated with cholera toxin. Activation by chymotrypsin is not expressed when adenylate cyclase is assayed with 5 mM Mn2+ without guanine nucleotides or fluoride. However, the chymotryptic activation is expressed when guanosine 5'-(beta, gamma-imino)-triphosphate is present together with Mn2+. We conclude that interaction of the guanine nucleotide regulatory subunit with the catalytic subunit of adenylate cyclase is required for expression of chymotryptic activation.
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PMID:The site of alpha-chymotryptic activation of pigeon erythrocyte adenylate cyclase. 737 11

The presence of cytochromes b5, P-450 and P-420 and activities of NADH- and NADPH-cytochrome c redutases were determined in plasma membranes isolated from microvilli of the chick and rat intestinal epithelium and erythrocyte membranes from chick, rat and man. The results are compared with the amounts of these components found in microsomal fractions from intestinal epithelium and in nuclear membranes from chick erythrocytes. Plasma membranes from intestinal microvilli and from erythrocytes contained significant amounts of NADH-cytochrome c reductase activity and of a pigment spectrophotometrically indistinguishable from rat liver microsomal cytochrome b5. In addition, cytochrome b5 fragments were prepared from the membranes by limited trypsin digestion and consisted of two to four components with Mr values in the range 10 000-13 500. In low-temperature difference spectra, the presence of a second cytochrome was noted which was similar to cytochrome P-420. Cytochrome P-450 and NADPH-cytochrome c reductase activities were not detected in plasma membrane fractions in significant concentrations but were present in the corresponding endomembrane fractions. These findings in highly purified, well defined plasma membrane fractions, in which contamination by endomembranes is minimal, strengthen the evidence for the existence of cytochrome-containing redox systems in plasma membranes of various cells and suggest that such redox components are general components of the cell surface. Possible functions and origins of these redox components in plasma membranes are discussed.
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PMID:Plasma membranes from intestinal microvilli and erythrocytes contain cytochromes b5 and P-420. 740 43

Nitric oxide synthase (NOS) catalyzes the NADPH-dependent, Ca2+/calmodulin-dependent formation of NO and citrulline from L-arginine and molecular oxygen. The localization of the heme-binding consensus sequence in the NH2-terminal half of NOS and of the binding sequences for nucleotides (FMN and FAD) in the COOH-terminal half suggests a bidomain structure. In addition, the presence of a putative calmodulin-binding sequence between the heme- and flavin-binding domains of the enzyme suggests a role for calmodulin in modulating a spatial orientation of these domains that is required for catalytic activity. First, to determine the effects of calmodulin and the functionality of the separated domains, Ca2+/calmodulin binding-induced conformational changes in NOS were measured by fluorescence quenching, from which a binding constant of approximately 1 nM for calmodulin was calculated. Second, electron transport to various artificial acceptors was measured. The addition of Ca2+/calmodulin increased cytochrome c reduction from 10-15-fold while stimulating the rate of 2,6-dichlorophenolindophenol and ferricyanide reduction only slightly, if at all. Calmodulin stimulation of NOS results in NADPH-mediated cytochrome c reduction, which is sensitive to superoxide dismutase, and the reduction of acetylated cytochrome c, which is only weakly reducible by unstimulated NOS. Thus, this stimulated activity is presumably superoxide anion-mediated. Third, limited proteolysis of NOS in the absence of calmodulin resulted in a time-dependent increase in cytochrome c reductase activity, which was not inhibitable by superoxide dismutase, and a decrease in catalysis of NO formation. SDS-polyacrylamide gel electrophoresis analysis of the tryptic digest demonstrated the formation of approximately 89- and approximately 79-kDa fragments. Sequence analysis of the peptides confirmed that trypsin cleaves the enzyme in the putative calmodulin-binding region beginning with Ala728. This region was protected from proteolysis by the addition of Ca2+/calmodulin. The separated NH2-terminal domain exhibited the characteristic spectrum of bound heme, while the COOH-terminal domain showed the characteristic spectrum of bound flavins. Other cleavage patterns were obtained in the presence of calmodulin. The data demonstrate that the heme- and flavin-binding domains of NOS can be isolated in functionally intact forms.
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PMID:Evidence for a bidomain structure of constitutive cerebellar nitric oxide synthase. 751 50

Macrophage NO synthase (NOS) is a dimeric enzyme comprising two identical 130 kDa subunits and contains iron protoporphyrin IX (heme), tetrahydrobiopterin, FAD, FMN, and calmodulin. We have carried out limited proteolysis to locate the domains involved in prosthetic group binding and subunit interaction. Trypsin cleaved the subunits of dimeric macrophage NOS at a single locus, splitting the enzyme into two fragments whose denatured molecular masses were 56 and 74 kDa. The smaller fragments remained dimeric in their native form (112 kDa), contained heme and tetrahydrobiopterin, and could bind L-arginine, CO, or imidazole. In contrast, the larger fragments were monomeric in their native form, contained FAD, FMN, and CAM, and bound NADPH. Although neither purified fragment alone or in combination catalyzed NO synthesis from L-arginine, the flavin-containing fragment did catalyze cytochrome c reduction at a rate that was equivalent to that of native dimeric NOS. These results indicate that trypsin cuts macrophage NOS into two domains that can exist and function independently of one another. The domain that binds heme, H4biopterin, and substrate is also responsible for maintaining the NOS dimeric structure, while the domain containing FAD, FMN, and CAM is not required for subunit interaction. This suggests a structural model for macrophage NOS in which the subunits align in a head-to-head manner, with the oxygenase domains interacting to form a dimer and the reductase domains existing as independent extensions.
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PMID:Macrophage NO synthase: characterization of isolated oxygenase and reductase domains reveals a head-to-head subunit interaction. 753 45

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

Cytochrome c oxidase of the thermophilic bacterium, PS3, was treated with trypsin. The hydrophilic domain of 26 kDa can be easily cleaved off from the hydrophobic anchor domain at the N-terminal region of subunit II, but remains attached to the rest of the enzyme upon gel-filtration in the presence of 0.2% lauroyl sarcosinate. The separation occurred in the presence of 5 M urea in addition to 0.2% lauroyl sarcosinate. After relatively prolonged proteolysis, that induced severe activity decay, and subunit I fragmentation, the 26 kDa fragment of subunit II can be easily isolated from the rest, suggesting that this fragment with cytochrome c and CuA interacts with subunit I. The separated fragment showed absorption spectra due to CuA and cytochrome c. Reconstitution of the cytochrome oxidase activity occurred on addition of the 26 kDa fragment to the proper gel-filtration chromatographic fraction.
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PMID:Preparation and characterization of the hydrophilic CuA-cytochrome c domain of subunit II of cytochrome c oxidase from thermophilic bacillus PS3. 762 17

Specific receptors for alpha 2-macroglobulin (alpha 2M) are found on the plasma membrane of macrophages (M phi s), one of antigen presenting cells. So far, a receptor-mediated effective uptake by M phi of foreign antigens which were linked to alpha 2M has been shown to provoke a remarkable increase in the proliferation of T lymphocytes and in the production of antibodies in vitro. Such results encouraged us to develop a new type of vaccine using a receptor-mediated antigen delivery and incorporation system based on alpha 2M and its receptor interaction. In this report, we applied the system to experimental animals. Yeast cytochrome c was used as an antigen to see if the system worked in vivo as well as in vitro. Cytochrome c was conjugated to alpha 2M through the action of trypsin and intraperitoneally administered to mice. The titer induced in mice was measured by enzyme linked immunosorbent assay (ELISA). The production of antibodies against cytochrome c was significantly increased when the protein was given in conjugated forms with alpha 2M.
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PMID:A receptor-mediated antigen delivery and incorporation system. Administration of alpha 2-macroglobulin-cytochrome c conjugate induced high concentrations of antibodies against cytochrome c in mice. 768 69

Methods and procedures for the construction and operation of a microscale immobilized protease (trypsin) reactor are described. Optimization of reactor efficiency with regard to reactor flowrate, length, and temperature as well as sample concentration has been examined, and a novel system for sample preconcentration is presented. The structural analysis of a standard protein (cytochrome c) by a combination of trypsin microreactor digestion followed by off-line electrospray ionization MS analysis is demonstrated at the 10-pmol level. The application of these techniques for the rapid confirmation (1-2 h) of a genetic variant (hemoglobin E) including MS/MS analysis of the variant peptide is included.
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PMID:Microscale immobilized protease reactor columns for peptide mapping by liquid chromatography/mass spectral analyses. 771 77

Recently we have proposed and presented evidence suggesting the existence of a "bi-trans-membrane" electron transport chain, located at the contact sites between outer and inner mitochondrial membranes, which can be utilized to promote either the oxidation of exogenous NADH in the presence of catalytic amounts of added cytochrome c or the reduction of exogenous cytochrome c supported by the oxidation of respiratory substrates present inside the mitochondria. Here we show that the oxidation of exogenous NADH is accompanied by a net alkalinization of the incubation medium preceded by a transient acidification phase. In oxygen-pulse experiments, the alcohol oxidation (induced by the addition of alcohol dehydrogenase) was used to mimic a cytosolic source of reducing equivalents. Oxygen pulses promote an acidification-alkalinization proton cycle which is insensitive to antimycin and myxothiazol inhibitory effect, is stimulated by valinomycin, inhibited by trypsin-aprotinin complex, abolished by the protonophore carbonyl cyanide-p-trifluoromethoxy phenylhydrazone (FCCP), and is absent or at least inverted (alkalinization-acidification cycle) in broken mitochondria. The oxidation of cytosolic substrates, mediated by the bi-trans-membrane electron transport chain, does not involve endogenous cytochrome c and is associated with a vectorial proton translocation from the inside to the outside of the mitochondria. In the out-->in electron transport pathway the components involved appear to be cytosolic reduced substrates-->NADH produced by cytosolic dehydrogenases activity-->NADH-cytochrome b5 oxidoreductase complex leaning out the external side of the external membrane-->exogenous cytochrome c-->cytochrome oxidase of contact sites-->molecular oxygen. The possible components of the in-->out pathway are matrix respiratory substrates-->primary dehydrogenases of the matrix-->Complexes I, II, and III of the respiratory chain present in the inner membrane-->NADH-cytochrome b5 oxidoreductase system of the external membrane-->exogenous cytochrome c-->additional cytosolic electron acceptors or, alternatively, cytochrome oxidase of contact sites. The two pathways can be considered a bi-trans-membrane electron channeling system which, at the level of bridges set up by the contact points between the outer and the inner mitochondrial membrane, may represent a link between the redox processes occurring inside with those present outside the mitochondrion.
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PMID:Proton translocation linked to the activity of the bi-trans-membrane electron transport chain. 777 4


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