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)

We have used isolated perfused rat livers to examine the intracellular processing of 125I-epidermal growth factor (EGF) and to determine where in the endocytic pathway the hydrolases which degrade EGF are acting. Following uptake of 125I-EGF at 37 or 16 degrees C, subcellular fractions enriched in endosomes and lysosomes were isolated, and their 125I-EGF content was examined by reverse-phase high performance liquid chromatography. Three forms of EGF processed at their carboxyl termini are generated in endosomes. At 37 degrees C, EGF is first processed in early endosomes by a carboxypeptidase B-like protease and is further processed in late endosomes by a trypsin-like protease and then a carboxypeptidase B-like protease. At 16 degrees C, entry of EGF into late endosomes is slowed, and only the first processed form is generated over 60 min. Longer perfusions (180 min) at 16 degrees C result in some processing (7%) by proteases found in late endosomes. EGF-horseradish peroxidase cytochemistry confirmed that the additional processing detected at 180 min correlated with movement of EGF from tubulovesicular to multivesicular endosomes. These results, combined with in vitro incubations of EGF in isolated endosomal and lysosomal fractions, suggest that different proteases are active at selective points in the endocytic pathway and that the full complement of proteases needed for complete degradation of EGF is active only in lysosomes.
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PMID:Sequential processing of epidermal growth factor in early and late endosomes of rat liver. 167 62

The products generated by the reaction of Limulus alpha 2-macroglobulin with trypsin were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Unreacted Limulus alpha 2-macroglobulin had a subunit molecular mass of 185 kDa. Trypsin-reacted samples contained two prominent peptides smaller (85 and 100 kDa) and three peptides larger (200, 250, and 300-350 kDa) than the unreacted subunit. Reaction of methylamine-treated Limulus alpha 2-macroglobulin with trypsin resulted in the same two prominent reaction products smaller than 185 kDa, but all of the reaction products larger than 185 kDa were absent. The covalent binding of biotinylated trypsin with Limulus alpha 2-macroglobulin was detected by probing Western blots with horseradish peroxidase-avidin. Surprisingly, the only reaction products that contained trypsin were bands at 100 and 120 kDa. The staining of these bands with horseradish peroxidase-avidin was weak: most of the biotinylated trypsin that remained associated with alpha 2-macroglobulin during gel filtration chromatography was located at the dye front following reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The reaction products larger than 185 kDa did not contain trypsin. Methylamine-reacted Limulus alpha 2-macroglobulin failed to bind any biotinylated trypsin. In contrast to the reaction of trypsin with Limulus alpha 2-macroglobulin, all high molecular mass bands generated by the reaction of human alpha 2-macroglobulin with biotinylated trypsin stained intensely with horseradish peroxidase-avidin. Thus, Limulus alpha 2-macroglobulin forms thiol ester-dependent, high molecular mass products involving isopeptide bonding between trypsin-generated fragments, without the incorporation of trypsin into the complexes. Most of the alpha 2-macroglobulin-associated trypsin is non-covalently trapped rather than covalently cross-linked.
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PMID:Reaction of proteinases with alpha 2-macroglobulin from the American horseshoe crab, Limulus. 171 64

A monoclonal antibody (Mab), 45J, which reacts with intact fibrinogen, has been employed to demonstrate the interaction of the carboxy terminal regions of the A alpha-chain in non-denatured plasma fibrinogen. The 45J Mab recognizes an epitope in the mid section of the carboxy terminal end of the A alpha chain. The epitope is destroyed by plasmin and trypsin digestion. The 45J Mab and a horseradish peroxidase conjugate of the 45J Mab (45J-HRP) were used in an ELISA to demonstrate that the antibody could recognize two copies of the same epitope on purified fibrinogen or denatured plasma fibrinogen. Fibrinogen in non-denatured plasma could not be detected by this single antibody ELISA. This immunochemical study demonstrates that only one copy of the epitope on the C-terminal protuberance of the A alpha-chain is exposed in non-denatured plasma. However, once the plasma fibrinogen has been denatured, as in the purification process, both copies of the epitope are available for antibody binding. This finding suggests that in plasma there is an intramolecular interaction between the carboxy terminal ends of the fibrinogen A alpha-chains which can be destroyed by denaturation.
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PMID:Immunochemical evidence for intramolecular interaction of the carboxy terminal A alpha-appendages of plasma fibrinogen. 172 3

Prostaglandin endoperoxide (PGH) synthase has a single iron protoporphyrin IX which is required for both the cyclooxygenase and peroxidase activities of the enzyme. At room temperature, the heme iron is coordinated at the axial position by an imidazole, and about 20% of the heme iron is coordinated at the distal position by an imidazole. We have used site-directed mutagenesis to investigate which histidine residues are involved in PGH synthase catalysis and heme binding. Individual mutant cDNAs for ovine PGH synthases were prepared with amino acid substitutions at each of 13 conserved histidines. cos-1 cells were transfected with each of these cDNAs, and the cyclooxygenase and peroxidase activities of the resulting microsomal PGH synthases were measured. Mutant PGH synthases in which His-207, His-309, or His-388 was replaced with either glutamine or alanine lacked both activities. Gln-386 and Ala-386 PGH synthase mutants exhibited cyclooxygenase but not peroxidase activities. Other mutants exhibited both activities at varying levels. Because binding of heme renders native PGh synthase resistant to cleavage by trypsin, we examined the effects of heme on the relative sensitivities of native, Ala-204, Ala-207, Ala-309, Ala-386, and Ala-388 mutant PGH synthases to trypsin as a measure of the heme-protein interaction. The Ala-309 PGh synthase mutant was notably hypersensitive to tryptic cleavage, even in the presence of exogenous heme; in contrast, the native enzyme and the other alanine mutants exhibited similar, lower sensitivities toward trypsin and, except for the Ala-386 mutant, were partially protected from trypsin cleavage by heme. Preincubation of the native and each of the alanine mutant PGH synthases, including the Ala-309 mutant, with indomethacin protected the proteins from trypsin cleavage. Thus, all the mutant proteins retain sufficient three-dimensional structure to bind cyclooxygenase inhibitors. Our results suggest that His-309 is one of the heme ligands, probably the axial ligand, of PGH synthase. Two other histidines, His-207 and His-388, are essential for both PGH synthase activities suggesting that either His-207 or His-388 can serve as the distal heme ligand; however, the trypsin cleavage measurements imply that neither His-207 nor His-388 is required for heme binding. This is consistent with the fact that only 20% of the distal coordination position of the heme iron of PGH synthase is occupied by an imidazole side chain.
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PMID:Essential histidines of prostaglandin endoperoxide synthase. His-309 is involved in heme binding. 190 Oct 57

Unambiguous identification of lymphocytes is sometimes difficult because of weak immunostaining of the cell membrane immunoglobulins. A simple method of intensifying the diaminobenzidine (DAB) peroxidase reaction was therefore devised. Paraffin wax sections of formalin fixed tonsils and lymphomas were digested with trypsin and immunostained for kappa and lambda light immunoglobulin chains and CD3 antigen by various peroxidase linked detection systems. After reaction with hydrogen peroxide and DAB the sections were immersed in methenamine silver solution at 60 degrees C for three to seven minutes. The light brown stain on the cell membranes of the mantle zone lymphocytes became dark brown and the stronger stain of the plasma cells became black. Mantle zone B lymphocytes and CD3 positive T lymphocytes were precisely outlined even at low magnification and the lymphomas were easily classified as monoclonal or polyclonal. At high magnification, staining was clearer than with the immunogold-silver stain. Cryostat and paraffin wax sections of other tissues immunostained for various antigens showed similar intensification. Silver methenamine provides an easy means of increasing the sensitivity and visual impact of an immunoperoxidase/DAB reaction in any preparation.
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PMID:Silver enhancement of polymerised diaminobenzidine: increased sensitivity for immunoperoxidase staining. 191 4

Previous immunochemical investigations have demonstrated various opioid peptides in the pancreas. However, controversies exist related to the cellular localization of these peptides in the endocrine pancreas. Therefore, the guinea pig endocrine pancreas was immunohistochemically investigated for the presence of opioid peptides derived from pro-dynorphin, pro-enkephalin or pro-opiomelano-cortin. Immunoreactivities were demonstrated on serial semithin sections by the peroxidase anti-peroxidase technique. In routinely immunostained sections, immunoreactivities for dynorphin A and alpha-neo-endorphin were localized in pancreatic enterochromaffin cells, but not in islet cells. Immunoreactivity for Met-enkephalin was confined exclusively to B-cells and was localized only in some secretory granules. However, pre-treatment of semi-thin sections with trypsin and carboxypeptidase B led to a marked increase of Met-enkephalin immunoreactivity in B-cells. In addition, immunoreactivities for Met-enkephalin-Arg-Gly-Leu and bovine adrenal medulla dodecapeptide could be demonstrated in B- and A-cells, and beta-endorphin immunoreactivity was localized in A-cells. In no case, however, were immunoreactivities detected for bovine adrenal medulla docosapeptide, peptide F, corticotropin, melanotropin or dynorphin 1-32. The immunohistochemical findings indicate that opioids of different peptide families are present in the guinea pig endocrine pancreas. Since several opioid peptides of the corresponding pro-hormones could be demonstrated in the reference organs but not in the pancreas, it is concluded that the biosynthetic pathways of the respective precursors are different from those in the adrenal medulla or in the pituitary.
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PMID:Immunohistochemistry of opioid peptides in the guinea pig endocrine pancreas. 197 Sep 50

Dopaminergic neurons of the A 10 cell group in the rat ventral tegmental area (VTA) exhibit electrical and dye coupling. Also, the activity of these neurons at least partially reflects their content of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. We examined the ultrastructural localization of TH to determine the morphological features of dopaminergic neurons in the VTA and the relationships between their TH immunoreactivity content and afferent input. Antiserum against the trypsin-treated form of TH was localized using peroxidase-antiperoxidase (PAP) and immunoautoradiographic methods. Immunoreactivity was detected in perikarya, dendrites, and terminals. The perikarya contained the usual organelles, as well as cilia, lamellar bodies, and subsurface cisterns. Qualitative evaluation of peroxidase reaction product and quantitative analysis of the number of silver grains/unit area revealed varying amounts of TH immunoreactivity in nuclei and cytoplasm. Lightly or intensely labeled nuclei were not necessarily associated with corresponding cytoplasmic labeling density. However, cytoplasmic labeling directly corresponded to the relative frequencies of neuronal appositions and synaptic input. Those neurons with less dense cytoplasmic PAP product received fewer synaptic contacts and were less frequently in apposition to other TH-labeled soma and dendrites than neurons displaying relatively more dense cytoplasmic PAP product. Analysis of single sections revealed that 67% (n = 71) of all TH-labeled somata and 15% (n = 2431) of all TH-labeled dendrites were in apposition to other TH-labeled soma or dendrites. TH-labeled terminals were rarely detected and contained relatively low levels of immunoreactivity. The majority of labeled terminals (n = 29/46) formed synapses with labeled soma and dendrites. Unlabeled terminals (n = 2424) in contact with TH-labeled dendrites appeared to form predominantly symmetric synapses. Ten percent (n = 248) of the unlabeled terminals dually synapsed onto adjacent immunoreactive dendrites, perikarya, or dendrite and perikaryon. We conclude that in the rat VTA, (1) detected TH immunoreactivity in cytoplasm, but not nucleus, corresponds to the level of feedback principally from nondopaminergic afferents; (2) dendrodendritic as well as axodendritic synapses between TH-immunoreactive neurons may mediate dopaminergic autoinhibition; and (3) gap junction-like appositions between neurons and convergent inputs from unlabeled terminals onto TH-immunoreactive profiles provide an anatomical substrate whereby cellular activities might be coordinated under certain conditions.
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PMID:Ultrastructural localization of tyrosine hydroxylase in the rat ventral tegmental area: relationship between immunolabeling density and neuronal associations. 197 39

An enzyme-linked immunosorbent assay (ELISA) has been evaluated for copro-diagnosis of giardiasis with anti-trophozoite antibody to capture specific Giardia lamblia stool antigen (GLSA), which was then detected by specific antibody conjugated with horseradish peroxidase. GLSA was demonstrated in stool eluates from all the 24 confirmed cases of giardiasis. None of the stool eluates from apparently healthy subjects or from patients carrying intestinal parasites other than G. lamblia had GLSA. Of the 25 microscopy-negative clinically suspected cases of giardiasis, 17 (68%) patients had GLSA in their stool eluates; these patients responded to anti-giardial therapy. The specific antigen was isolated and affinity-purified by the use of specific antibody; it had a Mr of 66 Kda, and its immunoreactivity was lost after treatment with heat or trypsin but unaltered by metaperiodate. ELISA seems to be a sensitive and specific method for copro-diagnosis of giardiasis, especially in highly suspected cases.
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PMID:Enzyme-linked immunosorbent assay for copro-diagnosis of giardiasis and characterisation of a specific Giardia lamblia antigen in stools. 203 May 2

The activity of some glycosidases, trypsin-like proteinases, peroxidase, inhibitors of beta-glucuronidase and trypsin-like proteinases, as well as the amount of thiocyanates were studied in mixed saliva (MS), dental deposit (DD) and gums (G) of patients with inflammation of the periodontium. In periodontitis the activity of beta-glucuronidase increases fourfold and that of beta-galactosidase doubles in the G; the activity of beta-glucuronidase and its inhibitors increases, the activity of proteinases diminishes, and the antitryptic activity increases in MS, the activity of peroxidase and the amount of thiocyanates change in this case. Along with the peroxidase-H2O2-thiocyanates system, the inhibitors of beta-glucuronidase and trypsin-like proteinases possess properties of unspecific protection, preventing destruction of the periodontal tissues by glycosides and proteinases of microbial and animal origin.
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PMID:[Enzymatic protective systems of saliva in inflammation of the periodontium]. 205 29

The binding and internalization of endogenous growth hormone in Chang hepatoma cells were localized on the cell surface and in the Golgi-endoplasmic reticulum-lysosome (GERL) area by various indirect immunocytochemical labeling techniques, namely, peroxidase or colloidal gold conjugated to secondary antibody, and avidin-biotin complex methods. Rabbit antiserum and monoclonal antibodies raised against HPLC-purified porcine growth hormone were used in this study. In fixed material, antigen-antibody complexes were found to be homogeneously distributed along the cell membrane. Control groups showed negative binding on the cell surface. Trypsin treatment before immunolabeling removed antibody binding completely, but hyaluronidase was ineffective. Pretreatment of lectins did not block the recognition of primary antibody to antigen molecules on cell surface. Internalization of the antigen-antibody peroxidase or gold complexes was demonstrated in the cells, which were immunolabeled at 4 degrees C, and then reincubated for 0-30 min at 37 degrees C before fixation. After reincubation, the internalized ligand complexes were found in vesicles near the cell surface or in the GERL area near the Golgi apparatus which, however, did not label for peroxidase. These findings suggest that the trypsin-sensitive growth hormone, specifically bound and internalized into Chang hepatoma cells, is localized in the GERL instead of the Golgi apparatus and might be involved in the mechanism of tumor cell growth.
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PMID:Immunocytochemical demonstration of the binding and internalization of growth hormone in GERL of Chang hepatoma cells. 207 35


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