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)

The proteolytic cleavage of Chlamydia trachomatis LGV-434 surface proteins and resultant effects on infectivity and association with cultured human epithelial (HeLa) cells have been examined. Of several proteases examined, trypsin, chymotrypsin, and thermolysin extensively cleaved the chlamydial major outer membrane protein (MOMP). Two proteases, trypsin and thermolysin, cleaved the MOMP to the extent that monomeric MOMP was not detectable by immunoblotting with monospecific polyclonal antibodies. In the case of thermolysin, not even antigenic fragments were detected. Surprisingly, infectivity toward HeLa cells was not diminished. In addition, the association of intrinsically 14C-radiolabeled elementary bodies (EBs) with HeLa cells or their dissociation by proteinase K was not measurably affected by prior trypsinization of the EBs. Trypsinization of lactoperoxidase surface-iodinated elementary bodies demonstrated that most of the 125I-labeled surface proteins were cleaved. In all cases, however, a number of proteolytic cleavage fragments remained associated with the EB surface after surface proteolysis. When trypsinized EBs were electrophoresed under nonreducing conditions and immunoblotted with either polyclonal or type-specific monoclonal MOMP antibodies, MOMP was found in a large oligomeric form that failed to enter the polyacrylamide stacking gel. Additionally, trypsinized viable EBs bound radioiodinated type-specific MOMP monoclonal antibody as efficiently as did the control nontrypsinized organisms. Taken together, the findings indicate that although the MOMP is highly susceptible to surface proteolysis, the supramolecular structure of the protein on the EB surface is apparently maintained by disulfide interactions. Thus, if surface-exposed chlamydial proteins are involved in the initial interaction of chlamydiae with eucaryotic cells, the functional domains of these proteins which mediate this interaction must be resistant to proteolysis and remain associated with the EB surface.
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PMID:Effect of proteolytic cleavage of surface-exposed proteins on infectivity of Chlamydia trachomatis. 258 Jul 94

Thyroid peroxidase was isolated from porcine thyroids by two methods. Limited trypsin proteolysis was employed to obtain a cleaved enzyme, and affinity chromatography was used to isolate intact thyroid peroxidase. Enzyme isolated by both methods was used in the examination of the heme site of native thyroid peroxidase and its complexes by EPR spectroscopy. Intact thyroid peroxidase showed a homogeneous high-spin EPR signal with axial symmetry, in contrast to the rhombic EPR signal of native lactoperoxidase. Reaction of cyanide or azide ion with native thyroid peroxidase resulted in the loss of the axial EPR signal within several hours. The EPR spectroscopy of the nitrosyl adduct of ferrous thyroid peroxidase exhibited a three-line hyperfine splitting pattern and indicated that the heme-ligand structure of thyroid peroxidase is significantly different from that of lactoperoxidase.
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PMID:Electron paramagnetic resonance spectroscopy of thyroid peroxidase. 283 83

To test a possibility that free band 3 and ankyrin-linked band 3 are exchanged in situ, band 3 was labeled with 125I, using intact red blood cells and lactoperoxidase. The cytoplasmic surface of this labeled band 3 was considered to be intact. When Triton shells were incubated with Triton supernatants prepared from 125I-labeled intact erythrocytes at 37 degrees C in the presence of Mg-ATP under isotonic conditions, the incorporation of free 125I-labeled band 3 to shells was observed. This incorporation was affected by the presence of Triton X-100 in the incubation mixture, and significantly decreased when the content of Triton X-100 was less than 0.04% (v/v). On the other hand, ankyrin-linked 125I-labeled band 3 was released when shells prepared from 125I-labeled intact erythrocytes were incubated with the Triton supernatants at 37 degrees C under the same condition as when free 125I-labeled band 3 incorporation was observed. These results strongly suggest that free and ankyrin-linked band 3 exchanged with each other in the presence of Triton X-100. A water-soluble 43 kDa fragment of band 3 inhibited the incorporation of free 125I-labeled band 3 to the shells and also inhibited the Mg-ATP-dependent shape change of ghosts in the absence of Triton X-100. Both of these inhibitory effects remained, even after 10 min of heat treatment at 100 degrees C, but drastically decreased by treatment with trypsin. Our results strongly suggest that a dynamic exchange of the free band 3 for ankyrin-linked band 3 may occur in intact erythrocytes, and it may even contribute to the shape change of erythrocytes.
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PMID:Dynamic association of band 3 with triton shells in human erythrocyte ghosts. 295 91

The activation of 14C-labeled estradiol by "true" and "pseudo" peroxidases to form conjugates and other products was compared in four model systems using H2O2, glutathione, Mn2+ or irradiated riboflavin. Albumin was used as acceptor except in the glutathione system. The binding of estradiol to glutathione in the presence of the true peroxidases, lacto- or uterine peroxidase (no H2O2 added), was also examined and the conditions shown to differ from those required with the pseudoperoxidases, microperoxidase or trypsin-digested cytochrome c. The conjugates were purified by chromatography after elution from Amberlite XAD-2 and the relative amounts of these products assessed by autoradiography. The ratio of steroid to glutathione in the main water-soluble metabolite formed with lactoperoxidase was found to be approx 1:1 in a double label experiment with [14C]estradiol and [3H]glutathione. It was also shown, using estradiol labeled with 3H in different positions of the steroid molecule, that lactoperoxidase acts non-specifically in catalyzing the formation of glutathionyl conjugates as indicated by the release of 3H2O. The possible role of peroxidase and glutathione in the metabolism of estrogens and in the formation of artifactual products is discussed.
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PMID:Metabolism of estradiol by true and pseudoperoxidases. 299 58

The incorporation of 125I, using lactoperoxidase, and the subsequent inactivation of beta-galactosidase in the period when incorporation and inactivation were stoichiometric were investigated in detail. The high pressure liquid chromatographic (HPLC) radioactive profiles of the tryptic peptides of samples taken in the stoichiometric period showed that, although two labelled peptides predominated, there were other labelled peptides. The predominating peptides were shown to be the mono- and di-iodinated forms of the peptide containing Tyr-253. This confirmed the result of an earlier study, but quantitation showed that this iodination accounted for only 15-18% of the total. To show that the other labelled peptides in the HPLC profiles were not merely oxidized or partially digested forms of the peptide containing Tyr-253, two experiments were carried out. In one of the experiments, two of the other labelled peptides were isolated and identified as iodinated forms of the peptide containing Tyr-285 (5-7% of the incorporation). In the other experiment, four additional labelled fractions from the HPLC eluate were treated further with trypsin. No further digestion was observed and thus these peptides did not result from incomplete digestion of the sequence containing Tyr-253. Overall, these results show that, although the incorporation of 125I was stoichiometric with inactivation, no single Tyr was responsible for the inactivation as was tentatively suggested previously. The competitive inhibitor isopropyl-beta-D-thiogalactopyranoside (IPTG) was effective in reducing the rates of inactivation of the enzyme and incorporation of 125I, but the same peptides were labelled in the presence of IPTG as in its absence.
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PMID:A detailed examination of the iodination of beta-galactosidase: stoichiometric inactivation by nonspecific iodination. 309 Oct 51

Insulin is able to down-regulate its specific cell surface receptor in cultured human lymphocytes. The effect of vanadate, a known insulinomimetic agent, was examined to determine whether it could mimic insulin to down-regulate the insulin receptor. Exposure of cultured human lymphocytes (IM-9) to vanadate (0-200 microM) resulted in a time- and dose-dependent decrease in cell surface insulin receptors to 60% of control, while insulin (100 nM) down-regulated to 40%. The vanadate effect, in contrast to the rapid effect of insulin, was slow to develop (4-6 h). Surface receptor recovery after 18 h exposure was rapid after vanadate removal (20 min), but it required hours after insulin suggesting the presence of an intracellular (cryptic) pool of receptors after vanadate treatment. Insulin binding to Triton X-100-solubilized whole cells after 18 h treatment revealed that total cell receptors had decreased to 50% of control after insulin but increased to 120 and 189% of control after 100 and 200 microM vanadate, respectively. Furthermore, vanadate inhibited the insulin-mediated loss of total cell receptors from 50 to 28%. Removal of cell surface receptors by trypsin before cell solubilization revealed that 100 microM vanadate increased insulin binding to 321% of control indicating an accumulation of intracellular receptors. Labeling of cell surface proteins with Na125I and lactoperoxidase followed by immunoprecipitation of solubilized receptors with anti-receptor antibody after incubation for various times up to 20 h and quantitation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that, while insulin shortened t1/2 from 7.3 to 5.3 h, vanadate prolonged receptor t1/2 to 14 h. No effect of vanadate was detected on insulin receptor tyrosine kinase activity with up to 4 h incubation at the vanadate concentrations used in this study. Furthermore, human growth hormone surface receptors were similarly down-regulated by vanadate. We conclude that 1) vanadate has an apparent insulin-like effect to down-regulate cell surface insulin receptors in cultured human lymphocytes; 2) in contrast to insulin-induced down-regulation which is associated with receptor degradation vanadate causes an accumulation of intracellular (cryptic) receptors and inhibits insulin receptor degradation; and 3) these effects of vanadate may be exerted on other cell surface receptors.
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PMID:Vanadate down-regulates cell surface insulin and growth hormone receptors and inhibits insulin receptor degradation in cultured human lymphocytes. 328 33

Studies of virus-specific proteins of type D retroviruses by radioimmunoprecipitation (RIP) using various radioactive precursors demonstrated in HEp-2 cells producing these retroviruses the presence of two glycosylated gag-coded polyproteins with molecular weights of 78 K and 90 K. The same polyproteins were found by lactoperoxidase iodination and RIP on the plasma membrane of the cells. Comparative studies of these glycoproteins by the limited proteolysis method showed the sites for trypsin action in them to be located in similar places of protein bases which indicated their structural relationship. It is suggested that glycosylated gag-coded polyproteins of retroviruses (including type D retroviruses) play the role of receptors for certain mitogens the seizure of which stimulates cell proliferation.
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PMID:[Nonstructural glycosylated polyproteins coded for by the gag gene of retrovirus type D expressed on the plasma membrane]. 349 57

The fundamental biology of how stable cell-cell bonds develop between activated macrophages and tumor cells, although essential to lysis of the neoplastic targets, remains poorly understood. To investigate whether this phenomenon could be pharmacologically manipulated, we analyzed the effect of phorbol diesters on tumor cell binding by macrophages. Activated murine peritoneal macrophages, treated in vitro with as little as 1 ng/ml of phorbol myristate acetate (PMA), bound significantly more tumor cells than did untreated macrophages. The effect was induced rapidly by PMA (i.e., maximum enhancement was seen within 15 min) and resulted in an average approximately twofold increase in the number of targets bound. The interaction between PMA-treated activated macrophages and tumor cells was completed much more rapidly than by untreated macrophages. The enhanced binding was seen only in macrophages treated with biologically active phorbol esters. Only the selective interaction between activated macrophages and tumor cells was affected (i.e., PMA treatment had no effect on nonselective interactions between activated macrophages and non-neoplastic targets or between nonactivated macrophages and any type of target). Pretreatment of activated macrophages with PMA apparently altered the requirements for microfilaments and microtubules in establishing binding, because cytochalasin B and colchicine, which inhibited control binding, as well as phagocytosis, had no effect on PMA-enhanced binding. PMA treatment did not alter energy requirements for binding, however, because low temperature (4 degrees C) or inhibitors of glycolysis and oxidative phosphorylation blocked both control and PMA-enhanced binding. The enhancement of binding apparently was not due to large quantities of secreted oxygen metabolites but did correlate closely with increased spreading and surface area of the macrophages. PMA treatment resulted in enhanced expression of trypsin-sensitive tumor-cell binding sites on the macrophage surface. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of macrophage membrane proteins labeled with 125I by the lactoperoxidase method revealed at least four trypsin-sensitive cell surface proteins that were re-expressed after PMA treatment. The data suggest that rearrangement and/or induced expression of surface binding sites may be an important step in the binding of tumor cells and indicate that PMA is a useful pharmacologic probe in dissecting the establishment of such binding into discrete steps.
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PMID:Enhancement of selective tumor cell binding by activated murine macrophages in response to phorbol myristate acetate. 351 13

Using the monoclonal antibody LICR-LON-Fib75.1 coupled to Sepharose as an affinity chromatography column, a membrane glycoprotein with an apparent molecular weight of 18,000 on sodium dodecyl sulfate-polyacrylamide gels has been purified from human red blood cells. The purified protein contained 25% carbohydrate by weight, the predominant sugars being galactose, mannose, and glucosamine. Amino acid analysis indicated that the protein was relatively rich in aspartate, glutamate, valine, and leucine and had a low proline and methionine content. The molecule could be removed from intact red blood cells by trypsin and could be labeled with iodine by lactoperoxidase-catalyzed cell surface iodination of red blood cells. The protein could also be labeled using the lipidsoluble photoactivatable reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl) diazirine) and partitioned into the lower phase of the phase-separable detergent Triton X-114. During size-exclusion chromatography in different detergents alterations were observed in the apparent molecular weight of the protein. These results suggest that this Fib75.1-binding protein is an external red blood cell membrane glycoprotein which is capable of binding detergent. Proteins with a similar molecular weight have also been isolated from two human tumor cell lines by immunoprecipitation with this monoclonal antibody.
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PMID:Purification of a human plasma membrane glycoprotein from human red blood cells by affinity chromatography using a monoclonal antibody. 396 59

In this study we have used (phorbol-12-O-tetradecanoylphorbol 13-acetate) and its biologically inactive analogue, 4 alpha-phorbol 12,13-didecanoate), to investigate platelet protein phosphorylation with special emphasis on the properties of a membrane protein-cytoskeleton (transmembrane) complex during platelet activation. Our data indicate that phorbol-12-O-tetradecanoylphorbol 13-acetate (but not 4 alpha-phorbol 12,13-didecanoate) induces both a specific platelet shape change and the preferential phosphorylation of a 180-kDa protein (presumably due to the activation of protein kinase C on the cytoplasmic side of the membrane). Further analysis reveals that the 180-kDa protein can be iodinated by lactoperoxidase and is sensitive to trypsin treatment, indicating exposure of this protein on the outer cell surface. The 180-kDa protein has also been found to contain wheat germ agglutinin-binding sites. All evidence indicates that the 180-kDa polypeptide is a transmembrane glycoprotein and, most importantly, that this protein is found to be preferentially accumulated into a specific membrane-cytoskeleton complex during activation via phorbol-12-O-tetradecanoylphorbol 13-acetate treatment. We believe that the observed phosphorylation of this protein may be closely related to the formation of a complex between several membrane proteins and the cytoskeleton during the initial stages of platelet activation.
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PMID:Phorbol ester-induced phosphorylation of a transmembrane glycoprotein (GP 180) in human blood platelets. 404 78


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