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 insertion of pig intestinal microvillus aminopeptidase (EC 3.4.11.2) into the membrane was studied by the hydrophobic photolabel [125I]iodonaphthylazide. The aminopeptidase was either labelled in the microvillus membrane and purified, or labelled after detergent solubilization and purification in a buffer containing Triton X-100, and then isolated from the reaction mixture. Of the three subunits A (Mr 162000), B (Mr 123000) and C (Mr 62000) of the aminopeptidase, A and B, but not C contained radioactivity, indicating that both subunit A and B carry anchoring peptide. The radioactivity when released from the subunits by trypsin treatment was connected to low-molecular-weight material.
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PMID:The insertion of pig microvillus aminopeptidase into the membrane as probed by [125I]iodonaphthylazide. 705 91

1. RNase St was inactivated by iodoacetate. The inactivation was most rapid at pH 5.0-7.0. Competitive inhibitors protected RNase St from inactivation by iodoacetate. The protective effect of 2'-GMP was most effective among nucleotides tested. 2. RNase St was inactivated with the concomitant incorporation of one molar equivalent of carboxymethyl group. The carboxymethyl group incorporated into RNase St was liberated by treatment with 0.2 N NaOH or 1 M hydroxylamine. Thus the incorporation of a carboxymethyl group into a carboxyl group was demonstrated. 3. 14C-labeled CM-RNase St was digested successively with alkaline protease and aminopeptidase M. The 14C-labeled amino acid was identified as the carboxymethyl ester of glutamic acid by means of column chromatography. 4. By digestion of reduced carboxymethylated CM-RNase St with trypsin, a peptide containing a 14C-carboxymethyl group was isolated by Dowex AG-50W colum chromatography. alpha-Chymotryptic digestion of the radioactive tryptic peptide, Glu48-Lys65, produced a tetrapeptide containing a 14C-carboxymethyl group, that is, Tyr59-His60-Glu61-Tyr62. Therefore, it was concluded that Glu61 in RNase St was the site of carboxymethylation. 5. When RNase St was inactivated by iodoacetamide at pH 8.0, about 2 histidine residues were modified. The molar ratio of the products of carboxyamidomethylation were 52.3%, 21.7%, 21.0%, and 4.8% for 3-CAM-His, 1,3-di-CAM-His, 1-CAM-His, and di-CM-Lys, respectively. 6. CD spectra of CM-RNase St and CAM-RNase St were practically the same as that of the native RNase St indicating the maintenance of the native conformation during modification. 7. The binding constants of CM-RNase St and CAM-RNase St with 2'-GMP were about 1/150 and 1/38 of that of the native enzyme, respectively.
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PMID:Alkylation of a ribonuclease from Streptomyces erythreus with iodoacetate and iodoacetamide. 728 72

We have developed a method to rapidly identify the antigenic determinant for an antibody using in situ proteolysis of an immobilized antigen-antibody complex followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF). A mouse anti-bombesin monoclonal antibody was immobilized to agarose beads and then the antigen, gastrin-releasing peptide (GRP), was allowed to bind. Direct analysis of the immobilized antigen-antibody complex by MALDI/TOF is demonstrated and allows identification of ca. 1 pmol of the bound GRP. To identify the epitope, the immobilized antigen-antibody complex was subjected to proteolysis with trypsin, chymotrypsin, thermolysin, and aminopeptidase M. Following proteolysis, the part of the antigen in contact with the antibody and protected from proteolysis was identified directly by MALDI/TOF. Subsequently, the epitope was eluted from the immobilized antibody with 0.1 M glycine buffer (pH 2.3), separated by reversed-phase HPLC, and its identity confirmed by MALDI/TOF. Using this approach, the epitope for the anti-bombesin monoclonal antibody was shown to comprise the last 7-8 residues (HWAVGHLM-NH2) of GRP.
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PMID:Epitope mapping of the gastrin-releasing peptide/anti-bombesin monoclonal antibody complex by proteolysis followed by matrix-assisted laser desorption ionization mass spectrometry. 753 May 43

A short treatment of dog renal brush-border membrane vesicles (BBMV) with sodium cholate, followed by dialysis of the detergent, reorients the polarity of H(+)-ATPase in the membrane and exposes its ATP binding sites to the extravesicular space, as previously shown with pig BBMV. In cholate-pretreated vesicles, the H(+)-ATPase remains fully active, but is inserted under the reversed polarity in sealed vesicles. A large spontaneous N-ethylmaleimide-sensitive ATPase activity is thus observed, as well as a steep intravesicular acidification upon external ATP addition, two findings absent in native vesicles. The ability of nitrate plus ATP to dissociate the hydrolytic subunits ot the proton pump in cholate-pretreated vesicles, but not in native vesicles, demonstrates that most of the ATP binding subunits are accessible to ATP following cholate treatment. The sensitivity of the cytoplasmic domain of the H(+)-ATP activity to trypsin also confirms the reorientation of the enzyme in cholate-pretreated vesicles. The H(+)-ATPase and alkaline phosphatase remain largely associated with the membranes after the treatment with cholate, but gamma-glutamyltranspeptidase, aminopeptidase N, and neutral endopeptidase are largely solubilized. Upon dialysis of cholate, all these enzymes are in part reinserted in the membrane according to their original polarity. The reorientation process is however specific for the H(+)-ATPase. Cholate treatment does not increase the formation of inside-out vesicles. Thus the treatment with cholate really reorients the polarity of the H(+)-ATPase in vesicles and allows for study of the proton pumping capacity of vacuolar H(+)-ATPase of proximal tubules.
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PMID:Effect of cholate on H(+)-ATPase and other proteins of dog renal brush-border membrane. 812 55

Phenomenological association of alterations of immune system function at the time of puberty (e.g. involution of the chicken bursa of Fabricius) has led to postulation that the humoral immune system may negatively affect the hypothalamo-adenohypophyseal-gonadal axis of the neonate. Presently, we examined the effect of an acidic aqueous bursa of Fabricius extract, derived from prepubescent chickens, on in vitro basal and LH-stimulated progesterone biosynthesis by isolated ovarian granulosa cells of the largest preovulatory chicken follicles (F1 and F2). Crude extracts of < 5kDa and > 3kDa inhibited LH-stimulated progesterone secretion (P < 0.05). The bioactive component was observed to be heat labile and is sensitive to the endopeptidases chymotrypsin, trypsin and papain. The peptide is not sensitive to the exopeptidase, aminopeptidase M. Partial purification by reversed phase HPLC resulted in a fraction capable of inhibiting in vitro steroidogenesis. This fraction suppressed LH-stimulated progesterone biosynthesis to approximately basal levels (79% suppression). Following removal of the peptide, granulosa cells were capable of LH-stimulated progesterone biosynthesis similar to control cells. Bursal extract significantly inhibited cAMP analog-stimulated progesterone biosynthesis. These data indicate that the anti-steroidogenic peptide derived from the chicken bursa of Fabricius is a single heat labile, amino terminally blocked peptide with bioactivity independent of the gonadotropin receptor of the granulosa cell.
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PMID:Detection and partial characterization of an anti-steroidogenic peptide from the humoral immune system of the chicken. 845 Jul 12

The serum level of placental leucine aminopeptidase (P-LAP) increases during pregnancy. P-LAP degrades several peptide hormones such as oxytocin and vasopresin, suggesting a role in maintaining homeostasis during pregnancy. In the study reported here, we have isolated a cDNA clone with 4084 base pairs encoding P-LAP from a human placental cDNA library. The amino acid sequence deduced from the cDNA contained all of the sequences of the peptide fragments obtained by digestion of the purified protein with trypsin. The predicted P-LAP contains the HEXXH consensus sequence of zinc metallopeptidases, indicating that the enzyme belongs to this family, which includes aminopeptidase N and aminopeptidase A. The deduced sequence also contains a hydrophobic region near the N terminus, suggesting that the enzyme is a type II integral membrane protein. Northern blot analysis revealed that P-LAP was expressed in several tissues, some of which expressed two forms of mRNAs. These results suggest that the enzyme is synthesized as an integral membrane protein and is released into blood under some physiological conditions.
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PMID:Human placental leucine aminopeptidase/oxytocinase. A new member of type II membrane-spanning zinc metallopeptidase family. 855 Jun 19

The present study sought to determine the expression of alpha- and beta-tryptase in in vitro differentiated human cord blood derived mast cells. We also analysed the glycosaminoglycan composition and the phenotype of the cells. The major protease in human mast cells is tryptase, and cDNAs for two different human tryptases have been characterized, the so-called alpha- and beta-tryptase. By reverse transcriptase-polymerase chain reaction (RT-PCR) we could show that stem cell factor (SCF)-dependent cord blood derived mast cells express both alpha- and beta-tryptase. Furthermore, the cells were stained with a monoclonal antibody (mAb) against tryptase, and the tryptase was enzymatically active cleaving the substrate Z-Gly-Pro-Arg- methoxy-2- naphthylamide (MNA). The majority of the cord blood derived mast cells could also be stained with mAbs against chymase, cathepsin G and CD68. They also expressed Kit/SCFR (CD117), CD13, CD29 and CD45 on the cell surface. The proteoglycan-derived polysaccharide composition of the cells was estimated to be 25-35% of heparin origin and 65-75% of chondroitin sulphate origin. Hence, the cord blood derived mast cells exhibit a phenotype in common with the so-called MCTC type of human mast cells.
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PMID:Stem cell factor-dependent human cord blood derived mast cells express alpha- and beta-tryptase, heparin and chondroitin sulphate. 869 Apr 66

LAMA-84, a human leucocytic cell line, which upon establishment was described as having megakaryocytic, erythroid and granulocytic characteristics, was analysed for expression of various differentiation markers. In addition to some of the previously described phenotypic characteristics, this cell line was found to express mRNA for several proteins characteristic for basophilic leucocytes and mast cells. The authors show that LAMA-84 cells express mRNA for the mast cell tryptase, the proteoglycan core protein, carboxypeptidase A and the alpha and beta chains of the high affinity IgE receptor (Fc epsilon RI). The authors examined the potential of LAMA-84 to differentiate in serum-free medium or after DMSO or PMA treatment. Depending on the inducing factor, surface expression of the Fc epsilon RI alpha-chain was increased from 20% to 35-50% of the cells and mRNA levels for tryptase were increased in serum-free medium and after DMSO treatment. LAMA-84 was found to express CD13, CDw17, CD29, CD33, CD40, CD45 and CD117. Furthermore, mRNA for the eosinophil/basophil markers Charcot-Leyden crystal (CLC) protein and the major basic protein (MBP), as well as the erythrocyte differentiation marker alpha-globin, was detected. However, the authors observed only trace amounts of mRNA for another erythroid differentiation marker (glycophorin), trace amounts of the megakaryocytic marker GPIIIa, and no detectable level of GPIb alpha. By comparing the expression pattern of a panel of differentiation markers in LAMA-84, and a second human cell line (KU812) expressing a basophil phenotype, it is evident that these cell lines, which presently are the only two cell lines identified with basophilic characteristics, share a large number of phenotypic characteristics.
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PMID:Characterization of a human basophil-like cell line (LAMA-84). 869 92

Aminopeptidase A (glutamyl aminopeptidase; EC 3.4.11.7) has been cloned from porcine brain and kidney cortex cDNA libraries and the complete primary sequence of the enzyme deduced. This predicts a type II integral membrane protein of 942 amino acids with 14 potential N-linked glycosylation sites and a His-Glu-Xaa-Xaa-His zinc binding motif. Aminopeptidase A was purified from porcine kidney cortex by a combination of anion exchange and hydrophobic interaction chromatographies following its release from the membrane by trypsin. The purified protein migrated as three major polypeptides on SDS-polyacrylamide gel electrophoresis of M(r) 147,000, 107,000, and 45,000. N-Terminal sequencing revealed that both the Mr 147,000 and 107,000 polypeptides had the same N-terminal sequence resulting from cleavage of aminopeptidase A by trypsin at the Lys-42-Asp-43 bond just outside the membrane-spanning hydrophobic region. Immunoelectrophoretic blot analysis following electrophoresis under nonreducing conditions revealed that the trypsin-cleaved form of the enzyme no longer migrated as a disulfide-linked dimer, placing the interchain disulfide link N-terminal to Lys-42. N-Terminal sequencing of the M(r) 45,000 polypeptide in the purified preparation of aminopeptidase A revealed that it resulted from cleavage at the Asn-602-Gly-603 bond by an endogenous protease. This posttranslational proteolytic cleavage occurred in porcine kidney cortex microvillar membranes but not in porcine intestinal microvillar membranes. Incubation of purified porcine kidney aminopeptidase N (membrane alanyl aminopeptidase; EC 3.4.11.2) with trypsin resulted in a similar fragmentation pattern to that observed in aminopeptidase A, suggesting that these and other members of the type II membrane-spanning zinc aminopeptidase family may have two distinct domains: an N-terminal domain, containing the zinc binding site and residues identified as being involved in catalysis, and a C-terminal domain of unknown function, that are separated by a protease-susceptible region.
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PMID:Proteolytic fragmentation reveals the oligomeric and domain structure of porcine aminopeptidase A. 906 31

We have been developing a novel bioadhesive drug-carrier matrix that protects embedded therapeutic peptides and proteins from degradation by the most abundant intestinal proteases. Increasing amounts of the Bowman-Birk inhibitor (BBI) were thereby covalently linked to chitosan-EDTA. The bioadhesive properties of the resulting polymer-BBI conjugates and their inhibitory effect toward trypsin (EC 3.4.21.4), chymotrypsin (EC 3.4.21.1), elastase (3.4.21.36), carboxypeptidase A (EC 3.4.17.1), and aminopeptidase N (EC 3.4.11.2) were evaluated in vitro. Whereas unmodified chitosan-EDTA exhibited under our experimental conditions an adhesive strength of 54.4 +/- 7.7 mN, it was determined to be 21.0 +/- 3.8 mN for the comparably most adhesive polymer-BBI conjugate (mean +/- SD; n = 5). All polymer-BBI conjugates showed a strong inhibitory activity toward the serine proteases trypsin and chymotrypsin. However, the protective effect toward elastase was markedly lower. Due to the high binding affinity of chitosan-EDTA toward zinc, which represents an essential cofactor for carboxypeptidase A and aminopeptidase N, all polymer-BBI conjugates displayed additionally a strong protective effect toward these exopeptidases. The novel bioadhesive polymer-BBI conjugates described in this study seem to be very useful drug-carrier matrixes in overcoming the enzymatic barrier to orally administered peptide and protein drugs.
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PMID:Intestinal peptide and protein delivery: novel bioadhesive drug-carrier matrix shielding from enzymatic attack. 954 94


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