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)

Assimilatory nitrate reductase from Chlorella is a homotetramer which contains one of each of the prosthetic groups FAD, heme, and molybdenum per subunit. Besides the reduction of nitrate by NADH, nitrate reductase also catalyzes the partial activities NADH:cytochrome c reductase, NADH:ferricyanide reductase, and reduced methyl viologen:nitrate reductase. Incubation of native nitrate reductase with either trypsin, Staphylococcus aureus V8 protease, or a natural inactivator protease from corn results in a loss of NADH:nitrate reductase and NADH:cytochrome c reductase activities but no loss of reduced methyl viologen:nitrate reductase activity. Incubation of nitrate reductase with V8 protease or corn inactivator protease resulted in two different products, each of which retained a different partial activity. Reduced methyl viologen:nitrate reductase activity was associated with a homotetrameric fragment of about 260 kDa which contained heme and molybdenum but no FAD. The molecular mass of native nitrate reductase determined under the same conditions was 375 kDa. NADH:ferricyanide reductase activity was associated with a monomeric species of approximately 30 kDa which contained FAD and the NADH-binding site. These results are consistent with a structure-function model of nitrate reductase which has the following features: FAD/NADH-binding domains exposed on the surface of the molecule, a protease-sensitive hinge region which connects the nitrate-reducing and NADH dehydrogenase moieties, and the quaternary structure maintained via association sites on the heme/molybdenum domain.
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PMID:Functional domains of assimilatory NADH:nitrate reductase from Chlorella. 301 63

The human colon adenocarcinoma cell line HT 29 grows virtually without tight junctions (TJ) under standard culture conditions. Earlier studies have shown that focal TJ (fasciae occludentes) can be rapidly assembled in this cell line under the influence of various proteases. Here we show that focal TJ can be induced in this cell line by a brief treatment with appropriate salt solutions. Induction by ammonium sulfate in Hanks' buffer reached a maximum value after 15 to 30 min. The amount of TJ increased with the salt concentration and reached a plateau value at a concentration of 160 mM ammonium sulfate. The amount and complexity of TJ induced by ammonium sulfate were similar to those in experiments using trypsin as inducing agent as shown by morphometric analysis. At 0 degrees C, no TJ were formed under the influence of the salt. A comparative study of TJ induction using a variety of inorganic and organic salts gave the following results. All alkali sulfates induced TJ, although with different yield. Both calcium and magnesium chloride were potent inducers. Ammonium and sodium salts encompassing a variety of anions covered a wide range from maximum induction (sulfate, citrate) to almost complete absence of induction (nitrate). Sodium chloride did not induce any TJ. It follows that the induction of TJ is a specific effect of individual ionic components of the solution as opposed to a general effect of osmolarity and ionic strength. The data suggest tentatively that antichaotropic but not chaotropic ions have the potential to trigger the formation of TJ in this experimental system.
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PMID:Rapid formation of tight junctions in HT 29 human adenocarcinoma cells by hypertonic salt solutions. 339 Dec 41

Leukotoxic activity in Actinobacillus (Haemophilus) actinomycetemcomitans isolated from patients with rapidly progressive periodontitis (RP), gingivitis (G), and juvenile periodontitis (JP), and several oral bacteria, was determined by observation of morphological changes in polymorphonuclear leukocytes (PMNs). Many A. actinomycetemcomitans isolates yielded both rough-surfaced and umbonate-shaped colonies (A-type), and smooth-surfaced and convex-shaped colonies (B-type), when stock cultures were streaked on agar medium. Both types of cells were identical in terms of Gram stain, cell morphology, sugar fermentation profile, nitrate reduction and cellular fatty acid composition. Sonic extracts were prepared from 32 A. actinomycetemcomitans strains isolated from patients and from 3 American Type Culture Collection (ATCC) strains. Sonic extracts from 8 isolates and 2 ATCC strains induced sphering of PMNs during a 45-50 min period of incubation at 37 C. Extracts from the other oral bacteria had no effects on PMN morphology. The sphered PMNs were found by their fluorochromatic-negative reactions to be damaged cells. The leukotoxic substance was heat-sensitive (56 C, 30 min), trypsin-sensitive and did not induce sphering of PMNs at 4 C. There was no clear correlation between colony type and leukotoxicity. Among 8 leukotoxic strains, 5 were isolates from an RP patient.
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PMID:Leukotoxic activity in Actinobacillus (Haemophilus) actinomycetemcomitans isolated from periodontal disease patients. 361 93

A new photopolymerizing reagent, uranyl nitrate, is used for the polymerization of acrylamide gels at low pH. The amount of uranyl nitrate (0.2 mg/ml) required for the polymerization of gels at pH 3.0 is considerably less than that of persulfate (7 mg/ml). Use of this reagent obviates the need for the removal of excess of persulfate by preelectrophoresis. The electrophoretic separation of basic proteins in uranium-polymerized gels showed faster movement and better resolution of proteins and proved the gels to be versatile, uniform, and reproducible. Electrophoresis of trypsin in these gels does not affect the enzymatic activity. The catalyst can also be used for the polymerization of gels containing 3 M urea.
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PMID:Polymerization of acrylamide at acid pH using uranyl nitrate. 370 7

Lyophilized Sigma Type III trypsin has been applied to latent prints two weeks to two months old. This trypsin preparation eliminates the background problems that had been encountered with old prints in a previous study. Zinc chloride treatment of latent prints previously exposed to ninhydrin enhances their detectability upon laser examination. However, it has been reported that the zinc chloride reaction occasionally fails to occur. Accordingly, we have investigated the optimization of this reaction. We find that high humidity and elevated temperature, particularly the former, are needed. Cadmium nitrate, although it produces weaker fluorescence than zinc chloride, may at times be useful. Reaction conditions are much the same as those for zinc chloride.
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PMID:Sensitivity enhancement of ninhydrin-treated latent fingerprints by enzymes and metal salts. 371 25

Purified respiratory nitrate reductase from Escherichia coli is able to use either reduced viologen dyes or quinols as the electron donor and nitrate, chlorate, or bromate as the electron acceptor. When reduced viologen dyes act as the electron donor, the enzyme follows a compulsory-order, "Theorell-Chance" mechanism, in which it is an enzyme-nitrate complex that is reduced rather than the free enzyme. In contrast, if quinols are used as the electron donor, then the enzyme operates by a two-site, enzyme-substitution mechanism. Partial proteolysis of the cytochrome b containing holoenzyme by trypsin results in loss of cytochrome b and in cleavage of one of the enzyme's subunits. The cytochrome-free derivative exhibits a viologen dye dependent activity that is indistinguishable from that of the holoenzyme, but it is incapable of catalyzing the quinol-dependent reaction. The quinol-dependent, but not the viologen dye dependent, activity is inhibited irreversibly by exposure to diethyl pyrocarbonate and reversibly by treatment with 2-n-heptyl-4-hydroxyquinoline N-oxide. We conclude that the holoenzyme has two independent and spatially distinct active sites, one for quinol oxidation and the other for nitrate reduction.
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PMID:Kinetic analysis of respiratory nitrate reductase from Escherichia coli K12. 388 57

Incubation of 2 X 10(-7) M glyceryl trinitrate (GTN) at 37 degrees C with human red blood cells resuspended in saline resulted in a 73.4 +/- 3.5% (S.D.) elimination of GTN after 10 min. The elimination of GTN was accompanied by the appearance of an equimolar amount of the GTN metabolites. The biotransformation of GTN and another organic nitrate, isosorbide dinitrate (ISDN), was examined in more detail using the 25,000 X g supernatant fraction of human red blood cells (RBC-SF). Incubation of 2 X 10(-7) M GTN or ISDN at 37 degrees C with RBC-SF resulted in a 46.3 +/- 7.3% (S.D.) elimination of GTN after 40 min and a 51.8 +/- 5.9% (S.D.) elimination of ISDN after 240 min. The elimination of the parent organic nitrate was accompanied by the appearance of an equimolar amount of metabolites. The biotransformation of ISDN was inhibited completely by pretreatment of the RBC-SF with trypsin, N-ethylmaleimide or heating at 65 degrees C, whereas GTN biotransformation was only inhibited partially by these treatments. Biotransformation of GTN was inhibited partially by pretreatment of the RBC-SF with CO or potassium ferricyanide; these treatments had no effect on ISDN biotransformation. Treatment of the RBC-SF with the combination of N-ethylmaleimide plus CO or trypsin plus CO resulted in complete inhibition of GTN biotransformation. We conclude that ISDN biotransformation by erythrocytes is a sulfhydryl-dependent enzymatic process, whereas the biotransformation of GTN is due to a combination of a sulfhydryl-dependent enzymatic process and an interaction with reduced hemoglobin.
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PMID:Role of hemoglobin in the differential biotransformation of glyceryl trinitrate and isosorbide dinitrate by human erythrocytes. 392 28

During the past year, we have reported the isolation and characterization of a low molecular mass (less than 5000 Da) complex (SAP), having profound immunosuppressive activity, found in the plasma of patients with major thermal injuries. Our continued studies have revealed that non-cytotoxic inhibition of neutrophil chemotaxis by SAP is paralleled by erythrocyte hemolysis by the same compound. Both neutrophil inhibitory and hemolytic activities appear to be a function of the peptide portion of SAP (determined by sensitivity to trypsin and pronase), which is augmented by the presence of sialic acid in the complex (activity eliminated by the addition of neuraminidase). The lipid portion of SAP, which is critical to its neutrophil immunosuppressive activity, does not appear to participate in erythrocyte hemolysis. Hemolytic activity of SAP is not due to protease activity, and does not appear to be receptor dependent. However, it is completely dependent upon the presence of calcium. The hemolytic activity of SAP appears to be abrogated by the addition of cerium nitrate, leading to a hypothesized relationship of SAP to the immunological activity of "burn toxin", previously described in detail (Schoenenberger, G.A. (1975) Monogr. Allergy 9, 72-139).
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PMID:Hemolysis and suppression of neutrophil chemotaxis by a low molecular weight component of human burn patient sera. 782 80

In vitro formation of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate reductase (NADPH: nitrate oxido-reductase, EC 1.6.6.2) has been attained by using extracts of the nitrate reductase mutant of Neurospora crassa, nit-1, and extracts of either photosynthetically or heterotrophically grown Rhodospirillum rubrum, which contribute the constitutive component. The in vitro formation of NADPH-nitrate reductase is characterized by the conversion of the flavin adenine dinucleotide (FAD) stimulated NADPH-cytochrome c reductase, contributed by the N. crassa nit-1 extract from a slower sedimenting form (4.5S) to a faster sedimenting form (7.8S). The 7.8S NADPH-cytochrome c reductase peak coincides in sucrose density gradient profiles with the NADPH-nitrate reductase, FADH(2)-nitrate reductase and reduced methyl viologen (MVH)-nitrate reductase activities which are also formed in vitro. The constitutive component from R. rubrum is soluble (both in heterotrophically and photosynthetically grown cells), is stimulated by the addition of 10(-4) M Na(2)MoO(4) and 10(-2) M NaNO(3) to cell-free preparations, and has variable activity over the pH range from 3.0 to 9.5. The activity of the constitutive component in some extracts showed a threefold stimulation when the pH was lowered from 6.5 to 4.0. The constitutive activity appears to be associated with a large molecular weight component which sediments as a single peak in sucrose density gradients. However, the constitutive component from R. rubrum is dialyzable and is insensitive to trypsin and protease. These results demonstrate that R. rubrum contains the constitutive component and suggests that it is a low molecular weight, trypsin- and protease-insensitive factor which participates in the in vitro formation of NADPH nitrate reductase.
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PMID:In vitro formation of nitrate reductase using extracts of the nitrate reductase mutant of Neurospora crassa, nit-1, and Rhodospirillum rubrum. 427 Apr 47

In vitro complementation of the soluble assimilatory nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-nitrate reductase was attained by mixing cell-free preparations of certain Neurospora nitrate reductase mutants: induced nit-1 (uniquely possessing inducible NADPH-cytochrome c reductase) with (a) uninduced or induced nit-2 or nit-3, or (b) uninduced wild type. The complementing activity of induced nit-1 is soluble while that of nit-2, nit-3, and wild type is particulate but not of mitochondrial origin. All fractions are inactivated by heat or trypsin. The NADPH-nitrate reductase enzymes formed in the above three complementing mixtures are similar to the wild-type enzyme in sucrose density gradient profiles, molecular weight, substrate affinity, sensitivity to inhibitors and temperature, but show different ratios of associated enzyme activities. The data suggest that nitrate reductase consists of at least two protein subunits: a nitrate-inductible subunit as reflected by inductible NADPH-cytochrome c reductase, and a constitutive protein which is activated (as indicated by the appearance of flavine adenine dinucleotide, reduced form (FADH(2))- and reduced methyl viologen-nitrate reductase activities) when it combines with the inductible subunit.
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PMID:Formation of assimilatory nitrate reductase by in vitro inter-cistronic complementation in Neurospora crassa. 439 54


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