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)

Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is a granule-associated enzyme that catalyzes the production of alpha-amidated peptides from their glycine-extended precursors, a posttranslational modification often required for full biological activity. PAM activity in crude homogenates of bovine neurointermediate pituitary has an acidic pH optimum for the peptide substrate alpha-N-Ac-Tyr-Val-Gly. During purification, the pH optimum shifts, so that purified bovine (b)PAM exhibits an alkaline pH optimum for this substrate with virtually no activity below pH 6.5. A factor that restores the ability of purified bPAM to produce alpha-amidated products at pH 6 was identified. In rat anterior pituitary this factor (denoted SPAM for stimulator of PAM activity) was a soluble protein with a mol wt of 44 K by gel filtration; its stimulatory activity could be reduced or eliminated by trypsin digestion or boiling. SPAM stimulated PAM activity at acidic pH by increasing the apparent Vmax and decreasing the apparent Michaelis-Menten constant (Km) for the peptide substrate. Like PAM, SPAM activity is localized to the secretory granule. Levels of SPAM activity in various rat tissues correlated closely with levels of PAM activity, with the greatest amount of SPAM activity in atrium, anterior pituitary, and neurointermediate pituitary. The distribution of PAM and SPAM between soluble and membrane fractions also correlated closely. In AtT-20 cell lines transfected with a complementary DNA (cDNA) encoding the full-length bPAM precursor, both SPAM and PAM activities were increased compared to wild type cells; both activities were decreased in a cell line expressing an antisense rat (r)PAM mRNA. In marked contrast, an AtT-20 cell line transfected with a cDNA encoding a truncated, soluble form of bPAM had elevated levels of PAM activity, but levels of SPAM activity were not increased compared to wild-type cells. These results suggest that SPAM activity is closely linked to the expression of full-length PAM. The interaction of PAM and SPAM may represent a site for regulation of the synthesis of bioactive peptides, particularly at low intragranular pH.
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PMID:pH-dependent stimulation of peptidylglycine alpha-amidating monooxygenase activity by a granule-associated factor. 224 28

A unique monoclonal antibody was obtained by immunizing mice with complement-inactivated fetal bovine serum (FBS). This antibody, named SI-1, stained epidermal basal cells of humans, pig, guinea pig, and rat by an indirect immunofluorescence technique after pretreatment of cryostat sections with alkali buffer (pH 9.6). After dissociating pig epidermal cells by trypsin, the SI-1 antibody stained exclusively and strongly one type of uniquely shaped cells. They were small and hanging-bell or columnar in shape with one convoluted side on the base, consisting of less than 2.8% of the dissociated epidermal cell population. The antigen contained in FBS was partially purified by affinity chromatography using the SI-1 antibody. The affinity-purified antigen inhibited the spreading of PAM cells, a spontaneously transformed murine keratinocyte line, in serum-free medium in a dose-dependent manner at concentrations of 10(-5) to 10 ng/ml. The antigen also inhibited the spreading of trypsinized pig epidermal cells in the range of 10(-2) to 10(3) ng/ml in the presence of 0.05% FBS. Although there have been a few reports indicating that serum inhibited both spreading and attachment, a specific factor in serum has not been purified before. This is, to our knowledge, the first presentation of a cell-spreading inhibitor contained in serum.
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PMID:A cell-spreading inhibitor exists in serum and in epidermal basal cells. 242 17

31P Nuclear Magnetic Resonance (NMR) studies were performed on mono- and diisopropylphosphoryl derivatives of alpha-chymotrypsin, trypsin, and subtilisin. Questions addressed included the pKa of the active center Asp...His...Ser triad in both species. While the pKa in the diisopropylphosphoryl derivatives is near 7.4 (found in this and other laboratories earlier) and reflects a nearly normal imidazolium titration curve, the apparent pKa in the monoisopropylphosphoryl enzymes (obtained by "aging" of the diisopropylphosphoryl derivatives and monitored by 31P NMR) is between 9.7 and 11.4 depending on the protease. This latter "titration" of the 31P NMR signal is reversible and presumably reflects the interaction of the imidazolium positive charge with the monoanionic phosphodiester. Of the two tetrahedral intermediates, the properties of the monoisopropylphosphoryl enzyme are probably more representative of the tetrahedral oxyanionic intermediate invoked during peptide hydrolysis. The same NMR technique was used to determine the action of PAM (pyridine-2-aldoxime methiodide, a known "antidote" for acetylcholinesterase inactivated by diisopropylfluorophosphate), on the inactivated enzymes. It was clear that the "antidote" could reverse the diisopropylphosphorylation but was ineffective on the monoisopropylphosphoryl ("aged") enzyme. 11B NMR studies were performed on phenylboronic (PBA) acid and 3,5-bis-trifluoromethylphenylboronic acid in the absence and presence of chymotrypsin and subtilisin. At 22 degrees C the former, but not the latter, compound was in fast exchange between the free and enzyme bound states. The relaxation parameters could be calculated for the bound PBA in chymotrypsin and the fluorinated analogue in subtilisin and clearly indicated that the boron nucleus was tetrahedral in the active centers, a good analogue for the tetrahedral oxyanionic intermediate.
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PMID:Multinuclear magnetic resonance studies on serine protease transition state analogues. 276 49

The present work concerns our studies to search for factor(s) which may influence the hemostatic process in or around metastasis of tumours. We studied the platelet aggregating property of a methyl cholanthrene induced experimental tumour. Platelet aggregating material was found to be different from the known aggregating agents like thrombin, ADP, collagen, thromboxane A2 and trypsin. It depends on a critical level of calcium for its action. PAM is a high molecular weight substance which contains sialic acid. It is trypsin and plasmin insensitive. The activity of this substance is not being destroyed by phospholipase-C. Metabolic study indicates that PAM acts by mitochondrial energy metabolic pathway of the platelets.
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PMID:A new platelet aggregating material (PAM) in an experimentally induced rat fibrosarcoma. 674 May 52

Proteins encompassing the two catalytic domains (monooxygenase and lyase) and the COOH-terminal domain of rat peptidylglycine alpha-amidating monooxygenase (rPAM)3 were purified from recombinant Escherichia coli overexpressing each domain and used to raise domain-specific polyclonal antibodies. Four alternatively spliced forms of PAM RNA (PAM-1, -2, -3, and -4) were transcribed in vitro and used to synthesize PAM proteins in a cell-free translation system. The orientation of the proteins in microsomal membrane vesicles was analyzed using trypsin protection assays and immunoprecipitation with the domain-specific antibodies. Only one of the two potential N-glycosylation sites (Asn765-Phe-Ser) in PAM-1 was efficiently utilized by microsomal membranes. PAM-1 and PAM-2 were shown to be type Ia membrane proteins with their two catalytic domains residing within microsomal vesicles and their COOH-terminal domains exposed to the cytosol. In contrast, PAM-3 and PAM-4 were shown to be soluble proteins contained entirely within vesicles. Thus, the COOH-terminal domain underwent topological switching between the cytosolic (PAM-1 and -2) and luminal (PAM-3) compartments as a function of alternative splicing of exons Ba/Bb. Computer analyses of the PAM protein sequence correlated the exons encoding PAM-1 with a model for the structural and functional domains of the PAM protein. The dual topologies of the PAM proteins confer an important means of functional regulation to this secretory granule associated neuropeptide processing enzyme.
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PMID:Topological switching of the COOH-terminal domain of peptidylglycine alpha-amidating monooxygenase by alternative RNA splicing. 768 Jan 92

1. The effect of 2,3-butanedione monoxime (BDM), a 'chemical phosphatase', on Na(+)/Ca(2+) exchange current (I(NCX)) was investigated using the whole-cell voltage-clamp technique in single guinea-pig cardiac ventricular myocytes and in CCL39 fibroblast cells expressing canine NCX1. 2. I(NCX) was identified as a current sensitive to KB-R7943, a relatively selective NCX inhibitor, at 140 mM Na(+) and 2 mM Ca(2+) in the external solution and 20 mM Na(+) and 433 nM free Ca(2+) in the pipette solution. 3. In guinea-pig ventricular cells, BDM inhibited I(NCX) in a concentration-dependent manner. The IC(50) value was 2.4 mM with a Hill coefficients of 1. The average time for 50% inhibition by 10 mM BDM was 124+/-31 s (n=5). 4. The effect of BDM was not affected by 1 microM okadaic acid in the pipette solution, indicating that the inhibition was not via activation of okadaic acid-sensitive protein phosphatases. 5. Intracellular trypsin treatment via the pipette solution significantly suppressed the inhibitory effect of BDM, implicating an intracellular site of action of BDM. 6. PAM (pralidoxime), another oxime compound, also inhibited I(NCX) in a manner similar to BDM. 7. Isoprenaline at 50 microM and phorbol 12-myristate 13-acetate (PMA) at 8 microM did not reverse the inhibition of I(NCX) by BDM. 8. BDM inhibited I(NCX) in CCL39 cells expressing NCX1 and in its mutant in which its three major phosphorylatable serine residues were replaced with alanines. 9. We conclude that BDM inhibits I(NCX) but the mechanism of inhibition is not by dephosphorylation of the Na(+)/Ca(2+) exchanger as a 'chemical phosphatase'.
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PMID:Inhibitory effect of 2,3-butanedione monoxime (BDM) on Na(+)/Ca(2+) exchange current in guinea-pig cardiac ventricular myocytes. 1125 Aug 83

We have recently improved the automation of an in-gel digestion system, DigestPro 96, using in situ alkylation of proteins with acrylamide, conducted during one-dimensional (ID) SDS-PAGE. The improved method included the processes of destaining, dehydration, trypsin digestion, and extraction but excluded the reduction and alkylation steps following staining of proteins with CBB. The extracted peptide mixtures were directly loaded onto a micro C18 LC column of the mass spectrometer. The resultant spectra were processed with "Mascot" search engine to estimate the sequence coverage of the bovine serum albumin (BSA). The original method, designed for Laemmli ID SDS gel applications, consisted of reduction and post-alkylation with iodoacetamide, which produced carboxyamidemethyl (CAM; -S-CH2CONH2) derivatives. The original method also included a desalting step essential for mass spectrometry, especially matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We compared the original and improved methods using BSA (3 pmol loaded to the gel, one third of digested peptide mixture injected into LC-MS). The original method yielded both CAM and propionicamide (PAM;-S-CH2CH2CONH2) derivatives. The source of PAM derivatives is the unpolymerized acrylamide formed during electrophoresis. The sequence coverage of CAM derivatives of BSA by the original method was 10% with desalting and 19% without desalting. The sequence coverage of PAM derivative by the improved method was 32%. Our results clearly show the advantage of our improved automated in-gel digestion method for in situ PAM alkylated protein with respect to peptide recovery, compared with the original method with CAM post-alkylation.
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PMID:Improvement of automatic in-gel digestion by in situ alkylation of proteins. 1367 49

Human keratinocytes are known to express the protease-activated receptors, PAR-1 and PAR-2. Activation of PAR-1 results in increased proliferation, whereas PAR-2 activation results in decreased keratinocyte proliferation. Trypsin activates PAR-1 and in higher concentrations, PAR-2. The aim of this study was to evaluate the overall effect of trypsin on keratinocyte proliferation in a mouse in vivo and in vitro model. Daily topical application of 0.3-300 pmol trypsin/cm2 on hairless mouse skin induced dose-dependent epidermal hyperproliferation as determined by an increase in 5-bromo-2'-deoxyuridine incorporation of up to eight-fold in basal keratinocytes and an up to three-fold increase in keratinocyte layers. This was accompanied by an increased transepidermal water loss. These effects of trypsin were abolished by the addition of the trypsin inhibitor n-p-tosyl-l-lysine-chloromethyl ketone. Histological analysis revealed acanthosis, hypergranulosis, and spongiosis in the epidermis as well as vasodilatation and an inflammatory infiltrate in the upper dermis. In the murine keratinocyte cell line PAM-212 activation of PAR-1 with specific activating peptides resulted in a calcium influx and an increase of proliferation, whereas activation of PAR-2 caused a diminished proliferation. Incubation with trypsin, PAR-1-, and PAR-2-activating peptides induced cytokine-induced neutrophil chemoattractant (KC) mRNA expression as a marker for inflammation in PAM-212 in a dose-dependent manner. In conclusion, our results suggest that trypsin induces in vivo epidermal proliferation and inflammation. Proliferation seems not to be signaled by PAR activation, but PAR-2-induced KC chemokine expression may contribute in part to trypsin-induced inflammation.
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PMID:Trypsin induces epidermal proliferation and inflammation in murine skin. 1508 39

We have previously demonstrated that pregnant ovine endometrium expresses the gastrin-releasing peptide (GRP) gene at a high level following conceptus implantation. Here we report the isolation, characterization and biological activity of ovine GRP 1-46, the primary product of this gene in the pregnant endometrium. Full thickness 125-140-day pregnant sheep uterus (term is 145 day) was homogenized in 80% acetonitrile/2% trifluoroacetic acid (1:7 ACN/TFA), concentrated on reverse-phase C18 cartridges and chromatographed successively on gel filtration (Sephadex G-50) and reverse-phase HPLC (C18 muBondapak). Purification was monitored by RIA. Purified GRP peptide was analysed by mass spectrometry giving a major mass ion at 4963 which corresponds exactly to GRP 1-46. Other mass ions from pro-GRP did not contain a biologically active N-terminus or antigenic determinant. Proteolytic cleavage of pro-GRP to give rise to GRP(1-46) would require preferential cleavage at the Glu-Glu bond by a Glu-C2-like enzyme, rather than the trypsin-like and C-terminal amidation enzymes (PAM) that produce GRP(18-27) and GRP(1-27) in other tissues. GRP 1-46 was synthesized and receptor binding and biological activity tested on a range of rodent and human cell lines that express GRP-related receptors GRPR, NMBR and BRS3. GRP 1-46 bound GRPR and NMBR with low affinity, and mobilized inositol phosphate in cell lines expressing the GRPR and NMBR, but not BRS-3. This study describes a new processed product of the GRP gene, GRP 1-46, which is highly expressed in the pregnant sheep endometrium and which acts as a weak agonist at the GRPR and NMBR.
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PMID:Isolation, identification and biological activity of gastrin-releasing peptide 1-46 (oGRP 1-46), the primary GRP gene-derived peptide product of the pregnant ovine endometrium. 1994 25