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)

Glutathione (GSH) is known to play an important role in protecting cells against oxidative stress. The present study was undertaken to assess the ability of GSH to protect isolated rat liver nuclei against NADPH-induced peroxidation. Nuclei were isolated from rat liver homogenates by discontinuous sucrose gradient centrifugation, and lipid peroxidation was induced by 1.7 mM ADP, 0.11 mM EDTA, 0.1 mM FeCl3, and either 1 mM NADPH or 0.5 mM ascorbate. The amount of lipid peroxidation was determined by measuring the formation of thiobarbituric acid-reactive products and the disappearance of lipid unsaturated fatty acid moieties. The addition of GSH (0.1 to 1.0 mM) produced a concentration-dependent lag period prior to the onset of lipid peroxidation. This GSH-induced lag period was abolished by pretreatment of nuclei with trypsin, thiol modifying reagents, disulfides, or heating nuclei at 60 degrees C for 15 min. Nuclei which were incubated with GSH also catalyzed the conversion of cumene hydroperoxide to cumyl alcohol. Similarly, this activity was also inhibited by thiol modifying reagents, disulfides, and heating nuclei at 60 degrees C for 15 min. The data suggest that a GSH-dependent peroxidase activity is associated with rat liver nuclear membranes which are capable of inhibiting lipid peroxidation.
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PMID:Characterization of glutathione-dependent inhibition of lipid peroxidation of isolated rat liver nuclei. 334 68

Pancreatic secretory trypsin inhibitor (PSTI) has been thought to be only a secretory trypsin inhibitor of human pancreas, but the serum content of immunoreactive PSTI is elevated without pancreatic disease. Using the peroxidase-antiperoxidase method, immunoreactive cells for PSTI were found in human pancreas, stomach, duodenum, appendix, colon and urinary tract of both fetus and adult, adult gall bladder, and fetal lung. PSTI-immunoreactive cells were identified in fetal pancreas at the tenth gestational week, and in extrapancreatic tissues at the sixteenth (gastrointestinal and urinary tract) and twentieth weeks (lung). PSTI-immunoreactive cells of fetal lung were present in neuroepithelial bodies. Strongly positive cells in fetal duodenum were argyrophilic and resembled endocrine cells. Immunohistochemical study was also performed on tissues associated with inflammatory diseases of gastrointestinal tract. The distribution pattern of immunoreactive cells in the stomach varied in accordance with chronic gastritis. Immunoreactive cells were also found in endocrine micro-nests and in a carcinoid tumor associated with fundic gastritis. These results suggest that PSTI may play some physiological role other than secretory trypsin inhibition of the pancreas.
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PMID:Immunohistochemical localization of pancreatic secretory trypsin inhibitor in fetal and adult pancreatic and extrapancreatic tissues. 351 Nov 41

Actin, myosin, and laminin have been localized in retinal vessels of normal rats by fluorescence microscopy. Actin was localized with the fluorescent F-actin binding toxin nitrobenzoxadiazole phallacidin (NBD-Ph). Indirect immunofluorescence was used to localize myosin and laminin. In addition, laminin localization was also performed with the Protein A-horseradish peroxidase (PA-HRP) method. NBD-Ph staining gave strong fluorescence in both retinal capillaries and larger vessels. Anti-myosin fluorescence could also be observed in trypsin digests of the retinal vasculature. Strong fluorescence of PA-HRP reaction product could be detected in the walls of vessels exposed to anti-laminin antibody. Actin distribution in vessels of the RCS rat with inherited retinal degeneration (retinal dystrophic RCS rat) was also studied. After exposure to NBD-Ph, all capillaries showed fluorescence. However, it was more intense in many of the capillaries in the outer retina, which also appeared morphologically abnormal. Electron microscopy of retinal capillaries fixed in 2.5% glutaraldehyde containing 8% tannic acid revealed numerous microfilaments in the pericyte cytoplasm and some in the basal portion of endothelial cells. In pericytes, these microfilaments are in close association with the endothelial side of the cell. Tangential sections through this region indicate that these filaments may be anchored to the membrane at this site.
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PMID:Actin, myosin, and laminin localization in retinal vessels of the rat. 352 82

A polyclonal antibody was raised against trypsin purified from the midgut of blood-fed Aedes aegypti. Using this antibody and our modification of the peroxidase-antiperoxidase immunocytochemical reaction, strong activity was found in the lumen of the midgut at the light-microscopical level. The activity was localized mainly in the posterior part of the distensible, abdominal midgut, along the periphery of the blood bolus and within the peritrophic membrane. Immunoreactivity appeared 8 h after the blood meal and was most prominent around 24 h, coinciding with our previous spectrophotometric determinations of trypsin. At the electron-microscopical level, secretory granules, immunocytochemically labelled with anti-trypsin antibody and protein A-colloidal gold, were first detected about 12 h after the blood meal. At 18 h, the secretory pathway could be followed immunocytochemically from the formation of granules in the Golgi complex until their release by exocytosis in the midgut lumen. By 24 h, there was a reduction in secretory granules, and large lysosomes appeared. The process of secretion described for this mosquito is comparable to similar events in vertebrate secretory systems and the presence of an intracellular trypsinogen is suggested.
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PMID:Mosquito trypsin: immunocytochemical localization in the midgut of blood-fed Aedes aegypti (L.). 352 50

Avidin-biotin-peroxidase complex immunohistochemistry was used on formalin fixed, paraffin embedded, trypsin digested, skin biopsies to detect immunoglobulin deposition in dogs with autoimmune skin disease. Immunostaining by the avidin-biotin-peroxidase complex technique revealed intercellular and/or basement membrane immunoglobulin deposits in 27 of 28 dogs considered to have autoimmune skin disease by clinical and histological evaluation and in six of 19 dogs considered to have autoimmune skin disease by clinical evaluation but without histological confirmation. Similar immunostaining was not evident in five biopsies of normal skin or in biopsies from four dogs with noninflammatory dermatoses, but was present in biopsies from one of ten dogs considered by clinical and histological criteria to have an inflammatory dermatosis other than autoimmune skin disease. Detection of immunoglobulin deposits in skin biopsies by avidin-biotin-peroxidase complex immunohistochemistry offers numerous advantages over conventional immunofluorescence methods including the opportunity to precisely compare histological and immunological findings.
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PMID:Avidin-biotin-peroxidase complex immunohistochemistry to detect immunoglobulin in formalin fixed skin biopsies in canine autoimmune skin disease. 355 68

The interaction between thyroid microsomal autoantibodies and thyroid microsomal antigen/thyroid peroxidase (TPO) has been studied using both intact antigen preparations and their water-soluble trypsin fragments. In an analysis of sera from 30 patients with Graves' or Hashimoto's diseases, microsomal antibodies showed similar reactivity towards trypsin fragments (with TPO activity) and intact detergent (sodium deoxycholate, DOC)-solubilized human microsomal antigen preparations (r = 0.96). This raised the possibility that both the peroxidase-active site and the major autoantigenic site(s) of microsomal antigen were present on the same trypsin fragments. Studies with porcine TPO showed that only a few sera contained microsomal antibodies which cross-reacted strongly with the porcine preparations. Further analysis was carried out by immunoprecipitation of 125I-labelled microsomal antigen followed by SDS-PAGE and autoradiography. These studies suggest that intact human microsomal antigen (a single-chain protein with Mr = 110,000) contains an intrachain loop of amino acids formed by a disulphide bridge. Trypsin treatment cleaves the antigen close to its transmembrane section and releases a water-soluble fragment (Mr = 100,000), containing the intact disulphide-linked loop of amino acids. Further trypsin action causes cleavage of the peptide bonds within the loop in some preparations. Consequently, three major water-soluble trypsin fragments (Mr = 100,000, 73,000 and 68,000) are formed all of which contain an intact disulphide bridge and have microsomal antibody binding activities. The integrity of the disulphide bridge in intact antigen/TPO preparations and their trypsin fragments is essential for autoantibody binding activity.
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PMID:Structure-activity analysis of microsomal antigen/thyroid peroxidase. 366 90

Additions of micromolar concentrations of hematin to washed rat pulmonary microsomal preparations resulted in marked (5-7-fold) increases in the NADPH-dependent generation of phenolic metabolites of benzo[a]pyrene (BaP). 9-Hydroxy-BaP was identified as the major reaction product. Additions of pulmonary cytosolic fractions to microsomes produced no measurable effect but cytosol and hematin added together elicited 25-30-fold increases in total phenolic products. Cytosolic fractions from other tissues, including rat kidneys and perfused rat livers, were also highly effective in enhancing the hematin-mediated increases in monooxygenase activity. However, cytosol from human placental tissues was only minimally effective when either pulmonary or placental microsomes were utilized as enzyme source. Superoxide dismutase and catalase (alone or in combination) had no measurable effect on hematin-mediated increases. Horseradish peroxidase effectively inhibited the hematin-dependent reactions but hematin-independent reactions were inhibited with equal effectiveness. Carbon monoxide profoundly inhibited all hematin-mediated increases in metabolite formation. The activating cytosolic component was non-dialyzable, inactivated by trypsin and heat, and eluted in the void volume from Sephadex G-150 columns. This suggested that the cytosolic factor(s) responsible for the increased hematin-dependent oxidation was a protein(s) with a high molecular weight or perhaps an aggregate or oligomer of proteinaceous material. HPLC profiles indicated a major effect on the generation of phenolics; quinones were also increased but only minimal increases in diols were observed. Results were consistent with the hypothesis that hematin-mediated increases in pulmonary monooxygenase activity result from an increased association of a small pool of pulmonary P-450-apoprotein(s) with the hematin prosthetic group to result in increased levels of an unidentified holocytochrome(s) with a relatively high substrate turnover number. The current data suggest a quaternary interaction among P-450 apoprotein(s), heme prosthetic group, reaction products (particularly 3-hydroxy-BaP) and a cytosolic protein(s). We postulate that the mechanism of action of the cytosolic factor is to facilitate the interaction of hematin with the apocytochrome.
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PMID:Cytosolic activation of hematin-dependent microsomal monooxygenase activity in the lung. 370 23

Specific radioimmunoassays for the 7-S domain of type IV collagen and the fragment P1 of laminin were used to quantify these basement membrane proteins in human kidney cortex at different ages and in some patients with diabetes mellitus. The antigens were solubilized by treating the tissue samples with the proteolytic enzymes collagenase, trypsin and pepsin. Total collagen content (as indicated by hydroxyproline concentration) increased with age, and the proportion of the collagen that could be solubilized by any enzyme treatment decreased. The type IV collagen concentration increased significantly with age, whereas the laminin concentration tended to decrease. In the one case of a type I diabetic the amounts of both antigens exceeded those in the age matched controls. In four type II diabetics the results were comparable with those for other aged cases. The distribution of the proteins was studied using the peroxidase-antiperoxidase method. The staining intensity and thickness of both antigens increased with age in the mesangium and Bowmans capsules, the change in type IV collagen staining being more evident. In diabetic patients these changes were more pronounced and other basement membranes appeared thicker in the stainings. These results indicate that basement membrane material accumulates in the kidney cortex during aging and that an alteration takes place in the composition of the basement membranes, the proportion of type IV collagen increasing and that of laminin decreasing.
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PMID:Effect of age and diabetes on type IV collagen and laminin in human kidney cortex. 378 96

The coupling of iodotyrosine (coupling reaction) is one of the least studied in the formation of thyroid hormone, particularly in human thyroid diseases. This paper describes a method of measuring iodotyrosine coupling catalyzed by human thyroid peroxidase (TPO) in vitro. There were two important requirements to demonstrate the coupling reaction: 1) thyroglobulin with a low thyroid hormone content, and 2) partially purified TPO. Thyroglobulin with low thyroid hormone content was obtained from Grave's and follicular adenoma tissues after propylthiouracil (PTU) therapy and L-T4 therapy, respectively. TPO was prepared from Graves' thyroid by solubilizing the 100,000 X g pellet of thyroid homogenate with sodium deoxycholate and trypsin, followed by Sephacryl S-300 gel filtration. Before the coupling reaction, thyroglobulin was iodinated with chloramine-T and potassium iodide, followed by dialysis. The coupling reaction was carried out by incubating newly iodinated thyroglobulin with TPO, diiodotyrosine, a coupling stimulator, and a H2O2-generating system (glucose and glucose oxidase) for 20 min at 37 C. After thyroglobulin was digested with Pronase, the thyroid hormone content of the thyroid digest was measured by RIA. Coupling activity was measured by the amount of newly formed T3 (nanograms of T3 per mg thyroglobulin). The time course of coupling reaction showed a progressive increase in coupling activity up to 30 min, and the reaction was temperature and pH dependent, with a pH optimum of 7.0. Coupling activity in the presence of H2O2 and TPO was 43 +/- 5.0 ng T3/mg thyroglobulin (mean +/- SD of triplicate samples), and addition of diiodotyrosine to the H2O2-TPO system caused a nearly 3-fold increase in coupling activity. This method has potential utilization for measurement of peroxidase coupling activity, since there was a linear relationship between the measured coupling activity and the amount of added TPO when the TPO concentration was over 3 micrograms/300 microliter. Methimazole (MMI) and PTU had similar potencies in inhibiting the TPO-catalyzed coupling reaction, whereas MMI was distinctly more potent than PTU as an inhibitor of TPO-mediated iodination in vitro. The different potencies of MMI in the two reactions suggest that different inhibitory mechanisms may be involved in iodination and coupling. The reducing agent, sodium metabisulfite, was also found to be a more potent inhibitor of the TPO-mediated coupling reaction than of the TPO-mediated iodination reaction. The method of iodotyrosine coupling described here may be useful to investigate the coupling step of thyroid hormone formation in human thyroid diseases.
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PMID:Coupling of iodotyrosine catalyzed by human thyroid peroxidase in vitro. 383 97

The location of lectin binding sites and of anionic components was studied in the embryonic rat cerebral cortex after the formation of the cortical plate at embryonic day 18. The cortical layers advanced in differentiation, i.e. the sub-plate region and the marginal zone, showed a predominant staining with peroxidase conjugates of wheat germ agglutinin (WGA), peanut agglutinin (PNA), and after immunocytochemical detection of PNA binding sites. This pattern was obtained also with the colloidal iron hydroxide staining method. In contrast to this, the binding of concanavalin A and of succinylated WGA did not reveal a prevalent staining of the sub-plate region and the marginal zone. The further histochemical analysis of the substances responsible for the selective staining of these layers was performed by lipid extractions and by enzymatic treatment of the tissue sections with trypsin, hyaluronidase or neuraminidase prior to the binding of lectins or colloidal iron. The results obtained indicated high concentrations of sialylated galactosylglycoproteins in coexistence with glycosaminoglycans. Electron microscopy was performed with peroxidase conjugates of WGA and PNA. Binding sites of both of the lectins in the sub-plate region and in the marginal zone were located mainly at cell surfaces of the different cellular structures. The most intensive binding of WGA and PNA was detected at the surface membranes and at intracellular material of amoeboid microglial cells and astrocyte-like cell processes. It can be concluded that in distinct brain areas during early differentiation specific glycoproteins in coexistence with glycosaminoglycans are situated at, or associated with cell surfaces in high concentrations. The identical histochemical features previously described in mesenchymal tissues suggest that these glycoconjugates might be related to common morphogenetic processes in which non-neuronal cells of brain and body are specifically involved.
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PMID:Lectin binding sites and anionic components related to differentiation in the prenatal rat cerebral cortex. 384 46


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