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)

Primary cultured rat epidermal keratinocytes were used as an experimental model to detect oxidant-mediated adverse effects of dithranol (anthralin), a widely used antipsoriasis drug with tumor-promoting and skin-irritating properties. Keratinocytes were isolated and prepared from the skin of neonatal rats by a trypsin flotation method. Highly proliferative monolayer cells cultured in a serum-free medium were exposed to the test compound at concentrations (5-100 microM) used therapeutically for the treatment of skin disorders. Cytotoxicity was evaluated by changes in plasma membrane integrity (lactate dehydrogenase leakage), lysosomal function (neutral red uptake), and mitochondrial metabolic activity (reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, MTT). Exposure of keratinocytes to dithranol produced time- and concentration-related toxic responses. MTT reduction was found to be a more sensitive endpoint of cytotoxicity, showing significant toxic effects at 2 hr, while significant leakage of lactate dehydrogenase did not result until 6 hr. Oxygen consumption in keratinocytes and isolated mitochondria showed a similar pattern after exposure to dithranol. Increased cyanide-insensitive respiration was also noted. Oxidative stress, measured by superoxide anion-dependent reduction of nitroblue tetrazolium, occurred before dithranol produced cytotoxicity in the keratinocyte cultures. Superoxide formation, which increased with time after dithranol exposure, was detected both extracellularly and intracellularly and was inhibited by the addition of superoxide dismutase. Dithranol-induced cell injury was partially prevented by treatment with superoxide dismutase, and greater protection was shown by concurrent treatment with superoxide dismutase plus catalase. These findings suggest that the superoxide anion and hydrogen peroxide may be involved in the cytotoxicity of dithranol and that a culture system of rat keratinocytes may be useful in evaluating the mechanism of toxicity of dermatotoxicants.
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PMID:Dithranol-induced cytotoxicity in primary cultures of rat epidermal keratinocytes. I. The role of reactive oxygen species. 184 45

Trypsin and Zymosan induce an increase in respiration activity of cultivated endothelial cells. These effects are not due to the production of oxygen radicals. The effect of trypsin will be explained by interfering with the cellular homeostasis of calcium. The stimulated respiration after incubation with zymosan may be connected with the increased phagocytosis.
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PMID:[The action of trypsin and zymosan on the respiration of cultivated endothelial cells]. 185 31

The naturally occurring peptidyl protease inhibitor leupeptin (N-acetyl-L-leucyl-L-leucyl-L-argininal) has been prepared labeled with 13C at the argininal carbonyl. 13C chemical shift data for the trypsin-leupeptin inhibitor complex in the pH range 3.0-7.6 reveal the presence of two pH-dependent covalent complexes, suggestive of two interconverting diastereomers at the new asymmetric tetrahedral center created by covalent addition of Ser195 to either side of the 13C-enriched aldehyde of the inhibitor. At pH 7 two signals are observable at delta 98.8 and delta 97.2 (84:16 ratio), while at pH 3.0 the latter signal predominates. In the selective proton 13C-edited NOE spectrum of the major diastereomer at pH 7.4, a strong NOE is observed between the hemiacetal proton of the inhibitor and the C2 proton of His57 of the enzyme, thus defining the stereochemistry of the high pH complex to the S configuration in which the hemiacetal oxygen resides in the oxyanion hole. pH titration studies further indicate that the 13C chemical shift of the S diastereomer follows a titration curve with a pKa of 4.69, the magnitude of which is consistent with direct titration of the hemiacetal oxygen. Similar pH-dependent chemical shifts were obtained by using CPMAS 13C NMR, providing evidence for the existence of the same diastereomeric equilibrium in the solid state.
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PMID:Diastereotopic covalent binding of the natural inhibitor leupeptin to trypsin: detection of two interconverting hemiacetals by solution and solid-state NMR spectroscopy. 191 68

The biological and physical properties of albumin and nitroxides make them attractive candidates as special purpose MRI contrast agents which could be used to study the intravascular compartment or specific targets in tissues. In this study, albumin-nitroxide complexes were prepared by reduction and alkylation of the disulfide bonds of the protein and characterized by electron spin resonance and ultraviolet absorption spectroscopy. An average of six nitroxides were bound covalently to each molecule of human serum albumin. The water proton relaxivity of the protein-bound nitroxide (at 20 MHz and 37 degrees C) was 4-fold greater than that of the free nitroxide. The digestion of the nitroxide-albumin complexes by cells or by trypsin decreased the relaxivity of the nitroxide-protein complex. The rate of reduction of albumin-bound nitroxide by cells was much slower than that of the free nitroxide but still was oxygen-sensitive (2-3-fold increase in the rate of reduction in the absence of oxygen).
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PMID:Potential of albumin labeled with nitroxides as a contrast agent for magnetic resonance imaging and spectroscopy. 196 24

Hb Fukutomi [alpha 126(H9)Asp----Val], a new hemoglobin variant with a high affinity for oxygen (log P50 = 0.201 at pH 7.0, normal = 1.087) was detected in a Japanese male during a screening survey. The amino acid substitution is located in the alpha 1 beta 1 contact of the tetramic molecule. Like other variants of the high oxygen affinity type Hb Fukutomi showed increased resistance to auto-oxidation. The tryptic peptide alpha T-13, that usually forms part of the trypsin-resistant core of the alpha chain, was cleaved off by trypsin in the new variant and released into the soluble fraction.
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PMID:Hb Fukutomi [alpha 126(H9)Asp----Val]: a new hemoglobin variant with high oxygen affinity. 207 32

Two nitrate reductases, nitrate reductase A and nitrate reductase Z, exist in Escherichia coli. The nitrate reductase Z enzyme has been purified from the membrane fraction of a strain which is deleted for the operon encoding the nitrate reductase A enzyme and which harbours a multicopy plasmid carrying the nitrate reductase Z structural genes; it was purified 219 times with a yield of about 11%. It is an Mr-230,000 complex containing 13 atoms iron and 12 atoms labile sulfur/molecule. The presence of a molybdopterin cofactor in the nitrate reductase Z complex was demonstrated by reconstitution experiments of the molybdenum-cofactor-deficient NADPH-dependent nitrate reductase activity from a Neurospora crassa nit-1 mutant and by fluorescence emission and excitation spectra of stable derivatives of molybdoterin extracted from the purified enzyme. Both nitrate reductases share common properties such as relative molecular mass, subunit composition and electron donors and acceptors. Nevertheless, they diverge by two properties: their electrophoretic migrations are very different (RF of 0.38 for nitrate reductase Z versus 0.23 for nitrate reductase A), as are their susceptibilities to trypsin. An immunological study performed with a serum raised against nitrate reductase Z confirmed the existence of common epitopes in both complexes but unambiguously demonstrated the presence of specific determinants in nitrate reductase Z. Furthermore, it revealed a peculiar aspect of the regulation of both nitrate reductases: the nitrate reductase A enzyme is repressed by oxygen, strongly inducible by nitrate and positively controlled by the fnr gene product; on the contrary, the nitrate reductase Z enzyme is produced aerobically, barely induced by nitrate and repressed by the fnr gene product in anaerobiosis.
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PMID:Purification and further characterization of the second nitrate reductase of Escherichia coli K12. 213 7

Oxidant-induced injury is associated with breakdown of interstitial collagen and the accumulation of inflammatory cells in the lungs. In previous studies, we demonstrated that phagocyte accumulation is mediated, in part, by chemotactic factors generated from damaged collagen. To determine if alveolar macrophages also mediate the migration of polymorphonuclear leukocytes (PMN) into the lungs, we examined the release of chemotactic factors from alveolar macrophages treated with native or synthetic collagenous peptides. These included fragments of bovine collagen digested with bacterial collagenase (CG) or cyanogen bromide (CB) as well as small molecular weight synthetic polypeptides containing proline (Pro), glycine (Gly), and hydroxyproline (Hyp), the major amino acids that comprise collagen. We found a dose- and time-related generation of PMN chemotactic activity by collagen peptide-treated macrophages. The maximum activity was released 72 h after pretreatment of macrophages for 1 to 3 h with 0.1 to 1 microM CG-, CB-, or (Pro-Pro-Gly)5-peptides. The native peptides derived from CG-digested collagen were more active than synthetic peptides containing Pro and Gly. Neither trypsin digests of bovine serum albumin nor synthetic peptides containing Hyp stimulated chemotactic factor release from macrophages. The alveolar macrophage-derived chemotactic factor was found to lose activity when dialyzed and after heat or trypsin treatment. The release of PMN-activating factors by collagen peptide-treated macrophages was also examined. Alveolar macrophage-conditioned medium was found to stimulate PMN production of reactive oxygen intermediates as well as elastase and gelatinase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Activation of neutrophils by factors released from alveolar macrophages stimulated with collagen-like polypeptides. 216 Feb 56

It has been shown that the induction of cytolytic activity in macrophages cause remarkable metabolic and morpho-structural shifts in effector cells, such as: 1) an expression of trypsin-sensitive receptors responsible for macrophage binding to malignant cells; 2) some differences in macrophage sensitivity to the cAMP system modifiers dependent on the activation inducer nature; 3) an enhanced production of oxygen metabolites directly involved in oxygen-dependent cytotoxicity of macrophages; 4) augmentation of lysosomal contents and activation of lysosomal enzymes. The inhibitor analysis enabled us to identify the main lytic components acting in the process of cytolytic functioning of macrophages of different origin and activation.
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PMID:[The cytolytic function of mononuclear phagocytes. I. The dependence of the mechanisms regulating and realizing the cytolytic function of macrophages on their origin and the mode of activation]. 217 Nov 73

The ability of various reactive oxygen species and serine proteases to activate latent collagenase (matrix metalloproteinase-1) purified from human neutrophils was examined. Latent 70-75 kD human neutrophil collagenase (HNC) was efficiently activated by known non-proteolytic activators phenylmercuric chloride (an organomercurial compound) and gold thioglucose (Au(I)-salt). Corresponding degree of activation was achieved by reactive oxygen species including hypochlorous acid (HOCl), hydrogen peroxide (H2O2) and hydroxyl radical generated by hypoxanthine/xanthine oxidase (HX/XAO). The presence of trace amounts of iron and EDTA were necessary and even enhanced H2O2 induced activation of latent HNC. This activation could be abolished by an iron chelator desferrioxamine and a hydroxyl radical scavenger mannitol. HOCl induced activation of latent HNC was not affected by desferrioxamine and mannitol. Thus, these compounds do not inhibit the active/activated form of HNC. Latent HNC could also be activated by trypsin and chymotrypsin but not by plasmin and plasma kallikrein. The ability of mannitol and desferrioxamine to inhibit the H2O2-induced activation of HNC suggests the transition metal dependent Fenton reaction to be responsible for localized and/or site-specific generation of hydroxyl radical/hydroxyl radical -like oxidants to act as the activating oxygen species. Our results support the ability of myeloperoxidase derived HOCl to act as a direct oxidative activator of HNC and further suggest the existence of a new/alternative oxidative activation pathway of HNC involving hydroxyl radical.
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PMID:Activation of latent human neutrophil collagenase by reactive oxygen species and serine proteases. 217 13

Fibronectin (FN), a glycoprotein present in the plasma and the extracellular matrix, has been shown to enhance adherence-related functions of polymorphonuclear leukocytes (PMNs). In this study we investigated the effects of FN on the activation of human PMNs in suspension by soluble stimuli, as determined by the generation of superoxide radicals (respiratory burst). FN (up to 100 micrograms/ml) did not directly stimulate the PMN respiratory burst assessed using a sensitive assay, luminol-dependent chemiluminescence (CL). Low FN concentrations (Up to 25 micrograms/ml) caused a dose-dependent enhancement of the CL induced by two chemoattractants. N-formyl-methionyl-leucyl-phenylalanine (FMLP) and platelet-activating factor (Paf), and also by phorbol myristate acetate (PMA), a known protein kinase C activator. Higher FN concentrations were less effective. The potentiation involved both initial rate and total CL responses and was more active on extracellular than intracellular generation of oxygen radicals. FN potentiation persisted after cell washing and was abolished by treatment of FN with trypsin. Measurement of the respiratory burst using the cytochrome c reduction assay confirmed that FN enhanced both the initial rate and total amount of superoxide anion generated by FMLP-stimulated PMNs. These data indicate that FN facilitates the respiratory burst of chemoattractant-stimulated PMNs and suggest that FN can prepare PMNs in suspension for amplified biological functions induced by soluble inflammatory stimuli.
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PMID:Priming effect of fibronectin on respiratory burst of human neutrophils induced by formyl peptides and platelet-activating factor. 217 7


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