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:3.4.21.64 (proteinase K)
4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Some proteases, i.e. trypsin, alpha-chymotrypsin, thermolysin, proteinase K, alpha-amylase, collagenase, and papain were investigated on their effect on isolated zonular fibers. All these enzymes but collagenase were zonulolytic active. An attack on the ground substance of the fibers by substances solving glycosaminoglycans and proteoglycans (hyaluronidase, EDTA, guanidinium chloride, H2O2) showed an increased effect of the enzymes used. These results suggest that the interfibrillar matrix has a protective function on the zonular fibers.
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PMID:[The attack of different proteases on isolated zonular fibers (author's transl)]. 13 75

Compromised neutrophil function has been found in a number of patients with localized juvenile periodontitis (LJP), although the pathogenic mechanism is unknown. Since infection with Actinobacillus actinomycetemcomitans is frequently found in patients with LJP, we have evaluated in vitro the effect of a bacterial extract of A. actinomycetemcomitans on the development of the respiratory burst by neutrophils. Pre-incubation of neutrophils with bacterial extract increased H2O2 induced by FMLP and zymosan in a dose-dependent fashion. Substitution of FMLP for bacterial extract produced similar results. Moreover, FMLP and bacterial extract had an additive effect on superoxide production following phagocytosis of zymosan. In contrast, bacterial extract significantly decreased PMA-stimulated H2O2, but pre-incubation with FMLP instead of bacterial extract failed to decrease PMA-stimulated H2O2. Bacterial extract did not change the percentage of cells activated by FMLP, opsonized zymosan, or PMA. Heat-treated bacterial extract induced effects similar to non-treated extract. Bacterial extract treated with proteinase K or phenol extraction increased FMLP or zymosan stimulated H2O2 equivalent to non-treated bacterial extract. In contrast, proteinase K or phenol extraction abolished the inhibitory effect of bacterial extract on PMA-stimulated H2O2 production. The bacterial extract component(s) that inhibits PMA-stimulated H2O2 is therefore a protein(s), resistant to 56 degrees C, and is not endotoxin. The partially activated state of PMNs exposed to A. actinomycetemcomitans extract, combined with their reduced ability to respond to a protein kinase C-dependent stimulus, may partially explain the abnormalities noted in LJP patients.
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PMID:Neutrophil modulation by Actinobacillus actinomycetemcomitans. II. Phagocytosis and development of respiratory burst. 132 89

We compared the intracellular pathways of the transferrin receptor (TfR) with those of the asialoglycoprotein receptor (ASGPR) and the cation-independent mannose 6-phosphate receptor (MPR)/insulin-like growth factor II receptor during endocytosis in Hep G2 cells. Cells were allowed to endocytose a conjugate of horseradish peroxidase and transferrin (Tf/HRP) via the TfR system. Postnuclear supernatants of homogenized cells were incubated with 3,3'-diaminobenzidine (DAB) and H2O2. Peroxidase-catalyzed oxidation of DAB within Tf/HRP-containing endosomes cross-linked their contents to DAB polymer. The cross-linking efficiency was dependent on the intravesicular Tf/HRP concentration. The loss of detectable receptors from samples of cell homogenates treated with DAB/H2O2 was used as a measure of colocalization with Tf/HRP. To compare the distribution of internalized plasma membrane receptors with Tf/HRP, cells were first surface-labeled with 125I at 0 degrees C. After uptake of surface 125I-labeled receptors at 37 degrees C in the presence of Tf/HRP, proteinase K was used at 0 degrees C to remove receptors remaining at the plasma membrane. Endocytosed receptors were isolated by means of immunoprecipitation. 125I-TfR and 125I-ASGPR were not sorted from endocytosed Tf/HRP. 125I-MPR initially also resided in Tf/HRP-containing compartments, however 70% was sorted from the Tf/HRP pathway between 20 and 45 min after uptake. To study the accessibility of total intracellular receptor pools to endocytosed Tf/HRP, nonlabeled cells were used, and the receptors were detected by means of Western blotting. The entire intracellular TfR population, but only 70 and 50% of ASGPR and MPR, respectively, were accessible to endocytosed Tf/HRP. These steady-state levels were reached by 10 min of continuous Tf/HRP uptake at 37 degrees C. We conclude that 30% of the intracellular ASGPR pool is not involved in endocytosis (i.e., is silent). Double-labeling immunoelectron microscopy on DAB-labeled cells showed a considerable pool of ASGPR in secretory albumin-positive, Tf/HRP-negative, trans-Golgi reticulum. We suggest that this pool represents the silent ASGPR that has been biochemically determined. A model of receptor transport routes is presented and discussed.
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PMID:Relations between the intracellular pathways of the receptors for transferrin, asialoglycoprotein, and mannose 6-phosphate in human hepatoma cells. 254 2

Rat liver Cu,Zn-[35S]thionein and yeast Cu-thionein were subjected to proteolysis in vitro using equilibrium dialysis. The partially copper-loaded vertebrate thionein (2-7 Cu/mol) was affected by different proteases including thermolysin, proteinase K, protease from Streptomyces griseus and lysosomal enzymes. Unlike the 2Cu-thionein the respective 7Cu-thiolate-centred metallothionein was hardly proteolytically digested. In contrast to fully copper-loaded native yeast Cu-thionein both the H2O2-oxidized and the metal-free protein were effectively cleaved in the presence of proteinase K. It is important to realize that the native Cu(I)-thiolate chromophore survives the proteolytic attack. When the copper-sulphur bonding is broken and the same amount of copper is unspecifically bound to the thionein portion, proteolysis proceeds identically with respect to the rate observed in the presence of the apoprotein. The unsuccessful proteolysis of native Cu-thionein is not attributable to a simple copper-dependent inhibition of the proteinases. It is suggested that prior to proteolysis the copper-sulphur clusters must be destroyed.
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PMID:The role of Cu(I)-thiolate clusters during the proteolysis of Cu-thionein. 308 72

DNA-protein and DNA interstrand cross-links were induced in isolated chromatin after treatment with H2O2 and ferrous ethylenediaminetetraacetate (EDTA). Retention of DNA on membrane filters after heating of chromatin in a dissociating solvent indicated the presence of a stable linkage between DNA and protein. Treatment of protein-free DNA with H2O2/Fe2+-EDTA did not result in enhanced filter retention. Incubation of cross-linked chromatin with proteinase K completely eliminated filter retention. Resistance to S1 nuclease after a denaturation-renaturation cycle was used to detect DNA interstrand cross-links. Heating the treated chromatin at 45 degrees C for 16 h and NaBH4 reduction enhanced the extent of interstrand cross-linking. The following data are consistent with, but do not totally prove, the hypothesis that cross-links are induced by hydroxyl radicals generated in Fenton-type reactions: (1) cross-linking was inhibited by hydroxyl radical scavengers; (2) the degree of inhibition of DNA interstrand cross-links correlated very closely with the rate constants of the scavengers for reaction with hydroxyl radicals; (3) cross-linking was eliminated or greatly reduced by catalase; (4) the extent of cross-linking was directly related to the concentration of Fe2+-EDTA. Partial inhibition of cross-linking by superoxide dismutase indicates that superoxide-driven Fenton chemistry is involved. The data indicate that DNA cross-linking may play a role in the manifestation of the biological activity of agents or systems that generate reactive hydroxyl radicals.
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PMID:Deoxyribonucleic acid-protein and deoxyribonucleic acid interstrand cross-links induced in isolated chromatin by hydrogen peroxide and ferrous ethylenediaminetetraacetate chelates. 629 97

In the vagina and endocervix, Neisseria gonorrhoeae must interact with complex microflora. Among these are lactobacilli, which may inhibit the growth of gonococci. Lactobacillus acidophilus, which produce H2O2 (LB+), and L. acidophilus and Lactobacillus casei, which do not produce H2O2 (LB-), were coincubated with catalase-positive and -deficient strains of N. gonorrhoeae. When the incubation medium was maintained at pH 7.3, neither LB+ nor LB- affected gonococcal growth. However, LB+ caused a significant increase in expression of gonococcal catalase, which could be offset by exposure of the bacteria to exogenous catalase. When coincubation medium was at lower pH (4.8-5.0), there was a significant decrease in gonococcal survival and catalase activity, which was only partly reversed by exogenous catalase. Lysates of LB+ also effectively inhibited gonococcal catalase. This inhibition was retained upon heating of the lysate to 100 degrees C for 15 min but was lost with proteinase K treatment. Thus, LB+ may inhibit growth of gonococci by acidification of the environment, secretion of H2O2, and production of protein inhibitors.
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PMID:Effects of H2O2-producing lactobacilli on Neisseria gonorrhoeae growth and catalase activity. 796 15

All methods described in the literature that allow quantitative measurements of protein expression at the cell surface are applicable to subsets of surface-exposed proteins only. We developed a new method, involving 3,3'-diaminobenzidine (DAB) cytochemistry, which allowed determination of cell-surface expression of all plasma membrane proteins measured, in at least three different cell lines. Adherent cells were first brought into suspension by proteinase K and EDTA treatment at 0 degrees C removing many, but not all, surface-exposed proteins. Subsequently, horseradish peroxidase (HRP) was linked by means of its glycosyl residues to specific cell-surface-exposed sugar moieties using the multivalent lectin concanavalin A (ConA). The suspended cells were encapsulated by polymerized DAB, a process that was catalysed by plasma membrane-bound HRP. After cell lysis, and removal of nuclei and most of the DAB polymer by centrifugation, proteins were analysed by SDS-PAGE. Surface proteins encapsulated by non-pelleted DAB polymer were retained on top of the stacking gel. After 125I-labelling the cell surface, protease-resistant 125I-labelled proteins could be quantitatively coupled to DAB polymer. This process was completely dependent on the presence of ConA, HRP, DAB and H2O2. Surface 125I-labelled beta-Na+,K(+)-ATPase was resistant to proteinase K but could be completely removed using DAB cytochemistry. Intracellular ConA binding proteins were not affected. Other intracellular proteins, including endosomal asialoglycoprotein receptor and cation-independent mannose 6-phosphate/insulin-like growth factor II receptor were also not affected.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A novel method for measuring protein expression at the cell surface. 812 1

The kinetics of frank DNA strand breaks and DNA base modifications produced by Cu(II)/ascorbate/H2O2 were simultaneously determined in purified human genomic DNA in vitro. Modified bases were determined by cleavage with Escherichia coli enzymes Nth protein (modified pyrimidines) and Fpg protein (modified purines). Single-stranded lesion frequency before (frank strand breaks) and after (modified bases) Nth or Fpg protein digestion was quantified by neutral glyoxal gel electrophoresis. Dialysis of EDTA-treated genomic DNA purified by standard proteinase K digestion/phenol extraction was necessary to remove low molecular weight species, probably transition metal ions and metal ion chelators, which supported frank strand breaks in the presence of ascorbate + H2O2 without supplemental copper ions. We then established a kinetic model of the DNA-damaging reactions caused by Cu(II) + ascorbate + H2O2. The principal new assumption in our model was that DNA base modifications were caused exclusively by DNA-bound Cu(I) and frank strand breaks by non-DNA-bound Cu(I). The model was simulated by computer using published rate constants. The computer simulation quantitatively predicted: (1) the rate of H2O2 degradation, which was measured using an H2O2-sensitive electrode, (2) the linearity of accumulation of DNA strand breaks and modified bases over the reaction period, (3) the rate of modified base accumulation, and (4) the dependence of modified base and frank strand production on initial Cu(II) concentration. The simulation significantly overestimated the rate of frank strand break accumulation, suggesting either that the ultimate oxidizing species that attacks the sugar-phosphate backbone is a less-reactive species than the hydroxyl radical used in the model and/or an unidentified hydroxyl radical-scavenging species was present in the reactions. Our experimental data are consistent with a model of copper ion-DNA interaction in which DNA-bound Cu(I) primarily mediates DNA base modifications and nonbound Cu(I) primarily mediates frank strand break production.
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PMID:Cupric ion/ascorbate/hydrogen peroxide-induced DNA damage: DNA-bound copper ion primarily induces base modifications. 885 37

An extracellular serine proteinase purified from cultures of a psychrotrophic Vibrio species (strain PA-44) belongs to the proteinase K family of the superfamily of subtilisin-like proteinases. The enzyme is secreted as a 47-kDa protein, but under mild heat treatment (30 min at 40 degrees C) undergoes autoproteolytic cleavage on the carboxyl-side of the molecule to give a proteinase with a molecular mass of about 36 kDa that apparently shares most of the enzymatic characteristics and the stability of the 47-kDa protein. In this study, selected enzymatic properties of the Vibrio proteinase were compared with those of the related proteinases, proteinase K and aqualysin I, as representative mesophilic and thermophilic enzymes, respectively. The catalytic efficiency (kcat/Km) for the amidase activity of the cold-adapted enzyme against succinyl-AAPF-p-nitroanilide was significantly higher than that of its mesophilic and thermophilic counterparts, especially when compared with aqualysin I. The stability of the Vibrio proteinase, both towards heat and denaturants, was found to be significantly lower than of either proteinase K or aqualysin I. One or more disulfide bonds in the psychrotrophic proteinase are important for the integrity of the active enzyme structure, as disulfide cleavage, either by reduction with dithiothreitol or by sulfitolysis, led to a loss in its activity. Under the same conditions, aqualysin I was also partially inactivated by dithiothreitol, but the activity of proteinase K was unaffected. The disulfides of either proteinase K or aqualysin I were not reactive towards sulfitolysis, except under denaturing conditions, while all disulfides of the Vibrio proteinase reacted in absence of a denaturant. The reactivity of the disulfides of the proteins as a function of denaturant concentration followed the order: Vibrio proteinase > proteinase K > aqualysin I. The same order of reactivity was also observed for the inactivation of the enzymes by H2O2-oxidation, as a function of temperature. The order of reactivity observed in these reactions most likely reflects the accessibility of the reactive cystine or methionine side chains present in the three related proteinases, and hence a difference in the compactness of their protein structures.
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PMID:Properties of a subtilisin-like proteinase from a psychrotrophic Vibrio species comparison with proteinase K and aqualysin I. 1010 4

The comet assay is a widely used biomonitoring tool for DNA damage. The most commonly used cells in human studies are lymphocytes. There is an urgent need to find an alternative target human cell that can be collected from normal subjects with minimal invasion. There are some reports of buccal cells, collected easily from the inside of the mouth, being used in studies of DNA damage and repair, and these were of interest. However, our preliminary studies following the published protocol showed that buccal cells sustained massive damage and disintegrated at the high pH [O. Ostling, K.J. Johanson. Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem. Biophys. Res. Commun. 123 (1984) 291-298] used, but that at lower pH were extremely resistant to lysis, an essential step in the comet assay. Therefore, the aims of this study were to develop a protocol than enabled buccal cell lysis and DNA damage testing in the comet assay, and to use the model to evaluate the potential use of the buccal cell model in human biomonitoring and nutritional study. Specifically, we aimed to investigate intra- and inter-individual differences in buccal cell DNA damage (as strand breaks), the effect of in vitro exposure to both a standard oxidant challenge and antioxidant treatment, as well as in situ exposure to an antioxidant-rich beverage and supplementation-related effects using a carotenoid-rich food. Successful lysis was achieved using 0.25% trypsin for 30 min followed by proteinase K (1mg/ml) treatment for 60 min. When this procedure was performed on cells pre-embedded in agarose on a microscope slide, followed by electrophoresis (in 0.01 M NaOH, 1mM EDTA, pH 9.1, 18 min at 12 V), a satisfactory comet image was obtained, though inter-individual variation was quite wide. Pre-lysis exposure of cells to a standard oxidant challenge (induced by H2O2) increased DNA strand breaks in a dose related manner, and incubation of cells in Trolox (a water soluble Vitamin E analogue) conferred significant protection (P<0.05) against subsequent oxidant challenge. Exposure of buccal cell in situ (i.e. in the mouth) to antioxidant-rich green tea led to an acute decrease in basal DNA strand breaks. In a controlled human intervention trial, buccal cells from 14 subjects after 28 days' supplementation with a carotenoid-rich berry (Fructus barbarum L.) showed a small but statistically significant (P<0.05) decrease in DNA strand breaks. These data indicate that this buccal cell comet assay is a feasible and potentially useful alternative tool to the usual lymphocyte model in human biomonitoring and nutritional work.
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PMID:A buccal cell model comet assay: development and evaluation for human biomonitoring and nutritional studies. 1608 24


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