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)

p100 is a recently identified 100 kDa protein which shares a putative receptor-binding sequence with the signal transducing G-proteins Gt and Gi. In liver, p100 immunoreactivity is distributed between the cytosolic and the microsomal fractions [Traub, Evans & Sagi-Eisenberg (1990) Biochem. J. 272, 453-458; Udrisar & Rodbell (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6321-6325]. More specifically, we have localized the membrane-associated form of p100 to an endosomal subfraction of rat liver microsomes. In this study we have investigated the nature of the interaction between p100 and microsomal membranes. p100 was located on the cytoplasmic surface of the microsomal vesicles, and could be released by treatment with 0.5 M-NaCl or 0.5 M-Tris/HCl, pH 7.0. However, p100 was not released by non-ionic detergents, such as Triton X-100. Binding of p100 to the membrane was reversible, as both membrane-released and cytosolic p100 could re-bind stripped (Tris-washed) microsomes. Soluble p100 could not, however, bind to untreated microsomes. Binding to stripped microsomes approached saturation and was inhibited by up to 60% by either heat treatment or mild trypsin treatment of the vesicles. This implies that the interaction between p100 and the microsomal vesicles involves the direct binding of p100 to vesicular proteins. This binding was regulated by both adenine and guanine nucleotides. As p100 contains a region similar to the C-terminal decapeptide of alpha i, (the alpha-subunit of Gi) and has a localization that is restricted to an endosomal subfraction, we propose that cytosolic p100 may bind to cytoplasmically exposed domains of internalized receptors. Thus, like the adaptins, p100 may be involved in the process of sorting and receptor trafficking through the endosomal compartment of the cells.
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PMID:Characterization of the interaction between p100, a novel G-protein-related protein, and rat liver endosomes. 174 44

N-linked oligosaccharides devoid of glucose residues are transiently glucosylated directly from UDP-Glc in the endoplasmic reticulum. The reaction products have been identified, depending on the organisms, as protein-linked Glc1Man5-9GlcNAc2. Incubation of right side-sealed vesicles from rat liver with UDP-[14C]Glc, Ca2+ ions and denatured thyroglobulin led to the glucosylation of the macromolecule only when the vesicles had been disrupted previously by sonication or by the addition of detergents to the glucosylation mixture. Similarly, maximal glucosylation of denatured thyroglobulin required disruption of microsomal vesicles isolated from the protozoan Crithidia fasciculata. Treatment of the rat liver vesicles with trypsin led to the inactivation of the UDP-Glc:glycoprotein glucosyltransferase only when proteolysis was performed in the presence of detergents. The glycoprotein glucosylating activity could be solubilized upon sonication of right side-sealed vesicles in an isotonic medium, upon passage of them through a French press or by suspending the vesicles in an hypotonic medium. Moreover, the enzyme appeared in the aqueous phase when the vesicles were submitted to a Triton X-114/water partition. Solubilization was not due to proteolysis of a membrane-bound enzyme. The enzyme could also be solubilized from C. fasciculata microsomal vesicles by procedures not involving membrane disassembly. About 30% of endogenous glycoproteins glucosylated upon incubation of intact rat liver microsomal vesicles with UDP-[14C]GLc could be solubilized by sonication or by suspending the vesicles in 0.1 M Na2CO3. These and previous results show that the UDP-Glc:glycoprotein glucosyltransferase is a soluble protein present in the lumen of the endoplasmic reticulum. In addition, both soluble and membrane-bound glycoproteins may be glucosylated by the glycoprotein glucosylating activity.
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PMID:The UDP-Glc:glycoprotein glucosyltransferase is a soluble protein of the endoplasmic reticulum. 184 Apr 23

Differences from normal in microsomal antigen (M-Ag) may be involved in the development of autoimmune thyroid disease. We compared the M-Ag in Graves' thyroid immunologically and biochemically to that in normal thyroid. The concentration of M-Ag, measured with an enzyme-linked immunosorbent assay, was significantly greater in the Graves' microsomes than in normal microsomes. Binding of a patient's microsomal antibody to Graves' microsomes was completely inhibited when the serum was first incubated with normal thyroid microsomes. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and Western blotting were done with a monoclonal antibody to denatured M-Ag. In both Graves' and normal thyroids, M-Ag existed as 107-, 101-, and 95-kDa peptides. After incubation with V8 protease, the residual antigenic peptide had a molecular weight of less than 60,000 and after incubation with trypsin, 95- and 87-kDa peptides and several smaller antigenic peptides were found. There were no significant differences in the pattern of normal and Graves' microsomes after digestion. Two-dimensional gel electrophoresis of Graves' microsomes showed that the isoelectric point for the 107-kDa peptide was at pH 7.2; that for the 101-kDa peptide was at pH 6.2, and that for the 95-kDa peptide was at 6.5. These values were not different from those observed for normal microsomes. These results indicate that M-Ag in Graves' thyroid does not differ from that in normal thyroid, and that microsomal antibodies in autoimmune thyroid disease probably do no arise from differences in the antigen.
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PMID:Thyroid microsomal antigen in Graves' thyroid is not different from that in normal thyroid. 184 66

Synthetic mRNAs were produced using either the complete coding sequence of a human preproendothelin-1 cDNA clone or a truncated form in which the portion encoding the first 17 amino acids, representing a putative signal peptide for insertion into the endoplasmic reticulum, was replaced with a methionine codon. The mRNAs were translated in vitro in the presence or in the absence of microsomal membranes. Protection from trypsin digestion demonstrated that the full-length polypeptide, but not the truncated form, could be inserted into the membranes. Sequence analysis revealed that membrane insertion is accompanied by removal of the first 17 amino acids. These results indicate that the first 17 amino acids of human preproendothelin-1 are a functional signal peptide which allows the protein to enter the secretory pathway.
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PMID:The signal peptide of human preproendothelin-1. 186 85

Procathepsins L and D, the proenzyme forms of two lysosomal proteases, are shown to bind to mouse fibroblast microsomal membranes at acidic pH. The propeptide of procathepsin L is necessary for membrane association because the mature forms of this lysosomal protein did not bind to the membranes. Both proenzymes were eluted from the membranes by increasing either the pH or the ionic strength of the buffer, so they are peripheral proteins that interact ionically with the membranes. The proenzymes were not eluted from the membranes with 50 mM mannose or 10 mM mannose 6-phosphate, which suggests that carbohydrate does not mediate membrane binding. Membrane binding is probably a specific, protein-mediated interaction since treatment of the microsomes with trypsin reduced by half the amount of procathepsin L which bound to the membranes, and binding of procathepsin L to the membranes was saturable. One or more "lysosomal proenzyme receptors" capable of binding to lysosomal proenzymes at acidic pH could complement the mannose 6-phosphate receptor system in prelysosomes and sort certain lysosomal proenzymes to lysosomes.
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PMID:Procathepsins L and D are membrane-bound in acidic microsomal vesicles. 186 63

One of the arginine esteropeptidases in human submaxillary gland was purified from microsomal membranes. The enzyme is inactive in membranes and requires trypsin treatment for its full activation. The trypsin-activated enzyme was purified to homogeneity. Its molecular weight was determined to be 94,000 by SDS-polyacrylamide gel electrophoresis. Among various substrates examined, the obtained enzyme exhibited high specific activities toward Tos-Arg-OMe (esterolysis) and D-Ile-Pro-Arg-pNA (amidolysis). The enzyme was inhibited by some serine proteinase inhibitors, whereas inhibitors of other types of proteinases did not affect or only scarcely affected it. The enzyme appears to be distinct from other arginine esteropeptidases previously described.
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PMID:A new latent arginine esteropeptidase from human submaxillary gland. 187 81

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

Monoclonal antibodies (mAbs) against the soluble form (S-COMT) of catechol-O-methyltransferase (COMT, EC 2.1.1.6) were produced using a purified preparation of the enzyme from pig liver as antigen. The selected monoclonal antibodies recognized the enzyme with different capacities. One of them (Co60-1B/7) showed a significant cross reaction with S-COMT from rat and human liver. A protein band of 23 kDa was recognized by the mAbs on Western blots of the soluble fraction of pig liver. The mAbs were also able to recognize the membrane-bound form of the enzyme, which was found to be mainly localized in the microsomal fraction of pig and rat liver as well as of the human hepatoma cell line Hep G2. The protein bands detected in microsomes had a molecular mass of 26 kDa in pig and rat liver and displayed a slightly higher molecular mass (29 kDa) in the Hep G2 cell line. A single step method for the immunoaffinity purification of pig liver S-COMT was developed by using a Sepharose 4B column to which the mAb Co54-5F/8 was covalently coupled. Acid elution conditions were optimized to obtain the enzyme in active form with a good yield. SDS-PAGE analysis of the purified preparation revealed a single protein band with a molecular mass of 23 kDa with 154-fold enrichment in enzyme activity over the starting material. Since the N-terminus was blocked, purified enzyme preparations were cleaved with trypsin. Two fragments of 22 and 33 amino acids in length could be sequenced by Edman degradation.
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PMID:Immunoaffinity purification and partial amino acid sequence analysis of catechol-O-methyltransferase from pig liver. 193 84

Monospecific polyclonal rabbit antibodies to a purified form of haem oxygenase of chick liver, showing sequence similarity to mammalian haem oxygenase-1, were raised and used to study characteristics of the oxygenase. The antibodies inhibited activity of the purified oxygenase, but not other enzyme components (NADPH:cytochrome reductase and biliverdin reductase) of the standard assay mixture of haem oxygenase. In addition, the antibodies inhibited activity of haem oxygenase in microsomes (microsomal fractions) from Cd(2+)-treated chick liver, spleen, testis and brain. Western (immuno-) blots of microsomal proteins of selected organs from chick, rat and man, and homogenates of chick-embryo liver-cell cultures, probed with the antibodies, showed a major protein with a molecular mass of 33-34 kDa and a lower-molecular-mass protein (28-29 kDa) of variable intensity. Studies with trypsin and selected proteinase inhibitors established that the smaller peptide was a proteolytic product of the larger. Treatment of chick-embryo liver-cell cultures with CdCl2, a potent inducer of haem oxygenase, increased the degree of proteinase-mediated cleavage of the 33 kDa protein to the lower-molecular-mass form. These results indicate that, under at least some conditions, such cultures should be homogenized in the presence of trypsin inhibitor to prevent proteolytic degradation of the enzyme and allow maximal expression of haem oxygenase activity. The antibodies also reacted with haem oxygenase from spleen, testis and brain of both chicks and rats, and the spleen of humans. A method for quantifying the amount of haem oxygenase protein was developed with use of slot-blots and laser densitometry; linearity was observed from 0 to 5 ng of haem oxygenase protein per slot, and the method was applied to sonicated cultured chick-embryo liver cells treated with Cd2+ (0.3 mM) or iron plus glutethimide. In both cases, increases in enzyme activity were of similar magnitude to increases in amounts of enzyme protein. Approximate amounts of haem oxygenase protein in microsomes of several organs from intact animals could also be estimated by the use of slot-blot-laser densitometry, and the amounts measured were increased by the addition of purified haem oxygenase to the microsomal preparations. Results of these studies indicated that haem oxygenase-1 could be detected in microsomes from all chick or rat organs studied, including testis and brain.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Immunochemical studies of haem oxygenase. Preparation and characterization of antibodies to chick liver haem oxygenase and their use in detecting and quantifying amounts of haem oxygenase protein. 195 81

The subcellular distribution of enkephalin (EK) precursor proteins was investigated to clarify the intracellular site of biosynthesis of EK in rat dental pulp tissue. The contents of met-EK-like peptides in nuclear, microsomal, and supernatant fractions of the pulp tissue were markedly increased after sequential digestion with trypsin and carboxypeptidase B, indicating the enrichment of the precursors in these fractions. Sephadex G-100 gel filtration showed a common peak of the precursor proteins in the homogenate and its microsomal and supernatant fractions, and the molecular weight was determined to be about 58,000 by SDS polyacrylamide gel electrophoresis. Both the partially purified precursor protein from the supernatant fraction and N alpha-benzoyl-DL-arginine-beta-naphthylamide (BANA) were used as substrates for a lysosomal enzyme separated by Sephadex G-75 gel filtration. The major peak of EK-producing activity of the enzyme was identical with that of BANA-degrading activity of the enzyme. These results demonstrate the EK-producing activity of lysosomal proteinase, and also indicate the usefulness of the two substances as substrates for the enzyme.
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PMID:Subcellular distribution of enkephalin precursor proteins in rat dental pulp and the usefulness as a substrate for enkephalin-producing enzymes. 199 Feb 37


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