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 binding of concanavalin A (Con A) to the cell surface of zoospores and cysts of Phytophthora palmivora was studied by radiometry (125I-Con A), ultraviolet microscopy (fluorescein-Con A) and electron microscopy peroxidase-diaminobenzidine technique). Zoospores were found to secrete during the early stages of encystment a Con A-binding material susceptible to trypsin digestion. This glycoprotein is contained in the so-called peripheral vesicles and is probably responsible for the adhesion of the encysting zoospores to solid surfaces.
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PMID:Adhesion of Phytophthora palmivora zoospores: detection and ultrastructural visualization of concanavalin A-receptor sites appearing during encystment. 117 43

Two patients (G2, G3) with iodine organification defect were studied. The first patient (G2), a 25-year-old women with no clinical hypothyroidism, had had her goiter for 10 years; 62% of the thyroidal iodine was released by perchlorate indicating iodine organification defect. The thyroid tissue obtained at thyroidectomy contained a normal concentration of thyroid peroxidase (I2 formation from I-) when tested after solubilization of the enzyme by trypsin and digitonin treatment of the particulate material. 1. The enzymatic activity (G2-TPO) behaved on DEAE cellulose chromatography very differently from those of hog (P-TPO) or another human goiter peroxidase (G1-TPO) (Pommier, et al., J Clin Endocrinol Metab 39: 69, 1974): the molarity of elution was 2M NaCl instead of 0.15 mM. 2. Both P-TPO and G2-TPO catalyzed iodide peroxidation (I- leads to I2) but the Km (iodide) value for G2-TPO was much lower (2.3 x 10(-2) M) when compared with that of P-TPO (3.7 x 10(-3) M) or G1-TPO (3.5 x 10(-3) M). In addition, the optimum pH for this reaction differed markedly (pH 6.1 instead of 7.9). 3. G2-TPO was poorly efficient in catalyzing the oxidation of gaiacol to tetragaiacol. 4. G2-TPO was unable to perform the iodination of non-iodinated goiter thyroglobulin whatever the pH and the iodide concentration. 5. Thyroglobulin from this goiter (G2) was almost not iodinated (0.0014%), i.e., 0.07 atoms iodine/mole thyroglobulin), and its total content in the gland was very low (0.3-4 g/1000 g wet tissue instead of 25 g). A clear discrepancy was thus shown between the euthyroid state of this patient and the total lack of iodinating activity of the isolated peroxidase. The second patient (G3), a 17-year-old man with clinical hypothyroidism, had had his goiter for 5 years. 100% of the thyroidal iodine was released by perchlorate indicating a complete iodine organification defect. The thyroid tissue obtained at thyroidectomy contained no peroxidase activity when tested before and after treatment of the particulate material by trypsin and digitonin and even in the presence of hematin. Thyroglobulin from this goiter, which was almost non-iodinated (0.0014%), was present in normal amounts in the gland (congruent to 25 g/1000 g).
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PMID:Thyroid iodine organification defects: a case with lack of thyroglobulin iodination and a case without any peroxidase activity. 126 32

The purity of horseradish peroxidase isoenzyme C was demonstrated using isoelectric focusing, polyacrylamide gel electrophoresis at two pH values and cellulose acetate electrophoresis at two pH values. The glycopeptides obtained upon trypsin digestion were isolated using the plant lectin, concanavalin A, and were resolved using paper electrophoresis. The carbohydrate content of the native peroxidase was 86% accounted for by the carbohydrate content of the glycopeptides thus suggesting little loss of carbohydrate during glycopeptide isolation and purification. In each of the seven glycopeptides isolated glucosamine was associated with asparagine, thus suggesting the carbohydrate chains are covalently bound to the peptide chain through N-glycosidic linkages. The purity of each glycopeptide was demonstrated by the sequential release of single amino acid residues by Edman degradation. As six glycopeptides had unique amino acid sequences, it was concluded that the carbohydrate prosthetic group was distributed in at least six units along the protein backbone. Five glycopeptides possessed the amino acid sequence about the point of carbohydrate attachment of Asn-X-(Ser, Thr) where X is any amino acid. The size of the carbohydrate units ranged from 1600 to 3000 daltons. The predominant carbohydrate residues in each glycopeptide were mannose and glucosamine with lesser and varying amounts of fucose, xylose, and arabinose. There was no apparent correlation of the carbohydrate composition with the amino acid sequence.
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PMID:The isolation and characterization of the glycopeptides from horseradish peroxidase isoenzyme C. 126 13

Glycopeptide-containing fractions in HPLC peptide maps can be detected by a simple application of the microtiter plate-bound streptavidin-biotinylated glycopeptide-lectin method (M.-C. Shao, 1992, Anal. Biochem., 205, 77-82). To illustrate this application, the glycoproteins, ovalbumin and asialofetuin, reduced and S-alkylated with vinylpyridine, were digested with trypsin-L-1-p-tosylamino-2-phenylethylchloromethyl ketone and the tryptic peptides were fractionated by reverse-phase HPLC, monitoring for absorbance at 230 nm. Aliquots of the HPLC fractions (typically 0.2-0.5% of the total volume) were biotinylated and complexed with streptavidin in the wells of a microtiter plate, allowing the streptavidin-glycopeptide complex to adhere to the plate. Suitable lectins, such as concanavalin A, Datura stramonium agglutinin, and peanut agglutinin, all of which had been coupled to horse radish peroxidase, were added, and after thorough washing, only the wells containing streptavidin-bound glycopeptides retained the complementary lectin and gave a positive peroxidase reaction. Less than 1 pmol of glycopeptide can be detected. The demonstration that the glycopeptide detection could be inhibited either by addition of an excess of the appropriate sugar inhibitor to the different lectins or by digestion of the biotinylated glycopeptides with N-glycosidase F or O-glycosidase shows that the glycopeptide-lectin interaction is the basis for the reaction.
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PMID:Method for the detection of glycopeptides at the picomole level in HPLC peptide maps. 128 90

The horseradish peroxidase labeled affinity purified mice anti-UEA (Urea soluble egg antigen of Schistosoma japonicum) antibody was used in enzyme-linked immunoelectrotransfer blot (EITB) to monitor the changes after UEA being processed by M phi. The immune responsive peptides were detected in the culture supernatant and homogenate of M phi pulsed with UEA in vitro (M phi+). After processing by M phi the high molecular weight UEA was cleaved into low molecular weight peptides, as shown, by the reactive bands. They markedly differed from that native UEA or trypsin-digested UEA. The bands of M phi supernatant and homogenate showed similarity with certain quantitative differences. According to the result described above, we considered: 1. UEA could be processed into smaller pieces by M phi, the style of processing is cleavage. 2. The processed peptides might be released to extracellular environment.
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PMID:[Preliminary study on the role of macrophage in processing and presenting antigens of Schistosoma japonicum]. 130 72

We have assigned the biosynthetic processing steps of cathepsin D to intracellular compartments which are involved in its transport to lysosomes in HepG2 cells. Cathepsin D was synthesized as a 51-kDa proenzyme. After formation of 51-55-kDa intermediates due to processing of N-linked oligosaccharides, procathepsin D was proteolytically processed to an intermediate 44-kDa and the mature 31-kDa enzyme. The intersection of the biosynthetic pathway of cathepsin D with the endocytic pathway was labeled with horseradish peroxidase and monitored biochemically by 3,3'-diaminobenzidine cytochemistry. Horseradish peroxidase was used either as a fluid-phase marker to label the entire endocytic pathway or conjugated to transferrin (Tf) to label endosomes only. Directly after biosynthesis cathepsin D was accessible neither to horseradish peroxidase nor Tf-horseradish peroxidase. Newly synthesized 51-55-kDa species of cathepsin D present in the trans-Golgi reticulum were accessible to both horseradish peroxidase and Tf-horseradish peroxidase. The accessibility of trans-Golgi reticulum to both endocytosed horseradish peroxidase and Tf-horseradish peroxidase was monitored by colocalization with a secretory protein, alpha 1anti-trypsin. The proteolytic processing of 51-55-kDa to 44-kDa cathepsin D occurred in compartments which were fully accessible to fluid-phase horseradish peroxidase. Tf-horseradish peroxidase had access to only 20% of 44-kDa cathepsin D while it had no access to 31-kDa cathepsin D. In contrast, the 31-kDa species was completely accessible to fluid-phase horseradish peroxidase. We conclude that proteolytic processing of 51-55-kDa to 44-kDa cathepsin D occurs in endosomes, whereas the processing of 44-31-kDa cathepsin D takes place in lysosomes.
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PMID:Identification of subcellular compartments involved in biosynthetic processing of cathepsin D. 132 3

Treatment of prostaglandin endoperoxide (PGH) synthase apoprotein with a 100- or 1000-fold excess of N-acetylimidazole (NAI) led to time-dependent inactivation of both cyclooxygenase and peroxide activities. Reconstitution of apoprotein with heme prior to incubation with NAI substantially protected the enzyme from inactivation. Pretreatment of the protein with either acetylsalicylic acid (aspirin) or (+/-)-2-fluoro-alpha-methyl-4-biphenylacetic acid (flurbiprofen), which inhibit cyclooxygenase activity, did not alter the time course of peroxidase inactivation by NAI. Treatment of NAI-inactivated apoPGH synthase with hydroxylamine led to substantial regeneration of both cyclooxygenase and peroxidase activities. Quantitation of radioactivity following incubation of PGH synthase with [3H-acetyl]NAI indicated incorporation of 1.7 +/- 0.9 acetyl groups/70-kDa subunit. Cleavage of acetylated protein with trypsin under nondenaturing conditions followed by high-performance liquid chromatography analysis demonstrated that most of the radioactivity was incorporated into the 33-kDa fragment although significant radioactivity was also detectable in the 38-kDa fragment. Chymotryptic peptide mapping of acetylated protein revealed numerous potential sites of acetylation distributed in widely divergent regions of the protein. No apparent differences were observed between the chymotryptic maps of apo- and holoenzyme, suggesting that the adduct responsible for loss of catalytic activity is unstable to the chromatographic conditions. The different biochemical properties of PGH synthase acetylated by NAI or aspirin suggest that a major determinant of the specificity of aspirin for Ser530 is binding of the salicylate moiety to this region of the PGH synthase protein.
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PMID:Acetylation of prostaglandin endoperoxide synthase by N-acetylimidazole: comparison to acetylation by aspirin. 139 Jul 34

This study examines the interaction of titin and myosin. In order to analyze the domains of myosin contributing to the binding for titin, we conducted a solid phase binding assay. Different portions of myosin (heavy chains, light chains and myosin fragments) were coated on the microtiter wells and reacted with biotinylated titin. Then the binding of biotinylated titin to these polypeptides was detected by using the avidinbiotin-peroxidase method. The results demonstrated that light meromyosin and subfragment 1 were the major domains of myosin interacting with titin. Titin fragments obtained by trypsin digestion were allowed to react with myosin in an affinity column, and the bound fragments were isolated by an acidic elution. Immunoblot analysis of myosin-bound titin fragments revealed that an A-band domain of titin was responsible for the binding of myosin. In addition, biotinylated titin labelled the outer A-bands and Z-bands in intact myofibrils, thus confirming the in situ binding of titin to myosin.
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PMID:Studies on the interaction between titin and myosin. 150 52

The posterior stomach was isolated from each male Donryu rat and separated into two parts: the tunicae mucosa and submucosa and the tunicae muscularis and serosa. Active and inactive tissue kallikrein were measured using sandwich type enzyme linked immunosorbent assay (s-ELISA) and H-Pro-Phe-Arg-MCA to complement each other. Inactive tissue kallikrein was determined (1) by measuring total tissue kallikrein and active tissue kallikrein in trypsin-treated samples and trypsin-nontreated ones, respectively; and (2) by subtracting active tissue kallikrein from total tissue kallikrein. Although tissue kallikrein was not demonstrable in the tunicae muscularis and serosa, inactive tissue kallikrein in the tunicae mucosa and submucosa reached 79.6% with s-ELISA and 99.1% with H-Pro-Phe-Arg-MCA. Water-immersion stress significantly decreased total tissue kallikrein at Stage IV of ulcers compared with the control value (p less than 0.001 in both measuring methods). Immunohistochemical staining was made using the avidin-biotin-horseradish peroxidase complex method. Tissue kallikrein was proved to be diffusely present as the inactive type within the epithelial cells of the pits in the gastric mucosa of the normal rats. With the progress of ulcers, however, it disappeared from the cells and appeared in the intercellular space. At Stage IV, it began to disappear even from the intercellular space. Based on the previously proposed process of tissue kallikrein release into blood in man, a possible interpretation of the above findings is that inactive tissue kallikrein may serve to maintain the gastric mucosa in a normal state; and that it may be transformed into the active type with ulceration and eliminated in a form of complex with some protease inhibitor in the course of aggravation.
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PMID:[Bio- and histochemical changes of tissue kallikrein in the rat stomach after water immersion-induced gastric ulcer]. 159 73

Vitelline coats (VCs) of Phallusia mammillata were isolated and purified following homogenization of live eggs in order to investigate the molecular basis of sperm-egg recognition. Clean VCs were partly solubilized by sonication in H2O and the soluble fraction (SFVC), derived from the outer surface of VCs, was used for further characterization. Electrophoretic analyses of radioiodinated VCs revealed that SFVC consists of two major glycoprotein components with apparent average Mr's of 450,000 and 180,000, respectively. The 450,000 Mr component is composed of several charge isomers, whereas the 180,000 Mr component is supposed to consist of two oligomers, both with acidic pI, held together by a disulfide linkage(s). Each of the two components possesses WGA-binding sites as shown by transblotting followed by WGA-peroxidase treatment. The amino acid composition of SFVC was determined after acid hydrolysis and its carbohydrate composition was analyzed and quantified by GLC. GlcNAc and GalNAc were found to predominate with 86% by weight of total sugar content and fucose, mannose, and glucose accounted for the remaining 14%. The susceptibility of SFVC to enzymatic (N-glycosidase F) and chemical (TFMS) deglycosylation as well as to protease (trypsin and chymotrypsin) digestion was investigated. Furthermore, sperm receptor activity of SFVC was tested in a fertilization assay. The fertilization rate decreased in a concentration-dependent manner when sperm were preincubated with SFVC. Additionally, sperm treated with SFVC showed binding for FITC-WGA or WGA-gold at the apical portion of the sperm head. Therefore, we strongly assume that one or both of the identified glycoprotein macromolecules of SFVC are involved in sperm-egg recognition.
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PMID:Glycoprotein constituents of the vitelline coat of Phallusia mammillata (Ascidiacea) with fertilization inhibiting activity. 166 Apr 20


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