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)

A marked decrease in activity of ornithine decarboxylase in thymus and spleen occurs soon after treatment of rats with a glucocorticoid. In the present study, evidence was obtained that extracts of these tissues prepared 5 h after administration of dexamethasone, when the enzyme activity is very low, contain an inhibitor of ornithine decarboxylase. The inhibitor is also present at 12 h after treatment and, in lesser amount, at 2.5 h, but was not evident at 24 h. The inhibitory activity was destroyed by treatment with heat or with trypsin, and was not lost on dialysis of the extract. Preliminary experiments indicate that the Mr of the inhibitor is greater than 50 000, which differentiates it from antizyme, an inhibitor of ornithine decarboxylase found in several other cell types. The inhibitor seems to act by a non-catalytic and non-competitive mechanism. The inhibition is dependent on the amount of inhibitor and does not change with time. Since inhibition is not changed by dialysis of the inhibitory extract, its activity apparently does not require small-Mr substances. This differentiates it from inhibitors which inactivate ornithine decarboxylase by covalent modification, such as the polyamine-dependent protein kinase or transglutaminase. The formation of this inhibitor is an early event in lymphoid tissues in response to dexamethasone and may be important in causing the inhibition of cell division which precedes the destruction of lymphocytes.
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PMID:An inhibitor of ornithine decarboxylase in the thymus and spleen of dexamethasone-treated rats. 397 59

Lytic infection of CV1 cells with herpes simplex virus type 2 does not stimulate ornithine decarboxylase activity and there is no correlation between polyamines and a growth-stimulating factor (GSF) which is present in the supernatant of the cultures. The factor was partially purified by gel filtration on Sephadex G-50 and polyacrylamide gel electrophoresis. Gel filtration as well as dialysis through membranes with different pore diameters indicated a molecular weight between 3,500 and 10,000 daltons. GSF is not extracted from aqueous solutions by diethyl ether. The activity is partially sensitive to trypsin digestion and is completely destroyed by periodate oxidation. These results suggest that GSF is a glycoprotein or a glycopeptide whose mitogenic activity resides mainly in the sugar moiety.
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PMID:Isolation and characterization of the growth-stimulating factor in supernatants of herpes simplex virus type 2 infected cells. 628 45

The mode of interaction of deoxycholate (DOC) or lithocholate (LC) with F344 rat colon was examined by measurements of uptake, 31P nuclear magnetic resonance (NMR) spectroscopy and observation of morphological changes. DOC as well as LC was taken up by the colon in a nonsaturable manner with respect to concentration and time, up to 30 min. None of several metabolic inhibitors reduced the uptake of the bile acids, nor did pretreatment of colon segments with chloroform-methanol (2:1, (v/v), heat or trypsin. Further, the bile acids were not transported by the colon against concentration gradients, and they were bound to both the mucosa and serosa equally. From these findings, it is concluded that the bile acids are transported in a passive manner, and no specific receptor for them is contained in colonic mucosa. The uptake of the bile acids by the colon varied with temperature and was related to the fluidity of the colonic membranes. The extent of uptake of dehydrocholate and taurocholate, which do not induce ornithine decarboxylase (ODC) activity, was almost the same as that of LC. The 31P NMR spectra of the colonic mucosal cells indicated that the proportion of the bilayer structure is increased by 0.5 mM DOC. Among a variety of bile acids examined, the extent of membrane alteration was in parallel with the extent of ODC induction. Treatment of the colonic mucosa with 0.5 mM DOC caused marked degeneration of the surface but not the deeper layers of the mucosa. Thus, physiological concentrations of bile acids influence the membrane organization of the colonic mucosa in a nonspecific manner that is possibly related to the tumor-promoting activity.
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PMID:Binding of bile acids with rat colon and resultant perturbation of membrane organization as studied by uptake measurement and 31P nuclear magnetic resonance spectroscopy. 650 Feb 37

The effect of repeated oral administration of prostaglandin analogue (dmPGE2) on intestinal macromolecular transport and digestive enzymes development were investigated in the suckling rats. By the administration of dmPGE2 for 7 days, precocious induction of maltase activity, depression of amylase activity and enhancement of trypsin activity in the pancreas occurred. Absorption of bovine IgG was dose dependently depressed by dmPGE2 treatments. The intestinal cessation was also observed in the adrenalectomized pups, but was not influenced by difluoromethyl ornithine administration. These results suggest that oral administration of PGE2 induces precocious maturation of the small intestine and exocrine pancreas and that the intestinal cessation is not directly related to ornithine decarboxylase activity in the suckling rats.
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PMID:Precocious cessation of intestinal macromolecular transport and digestive enzymes development by prostaglandin E2 in suckling rats. 752 52

Histidine 57 of the catalytic triad of trypsin was replaced with alanine to determine whether the resulting variant would be capable of substrate-assisted catalysis [Carter, P., & Wells, J. A. (1987) Science 237, 394-9]. A 2.5-fold increase in kcat/Km was observed on tri- or tetrapeptide substrates containing p-nitroanilide leaving groups and histidine at P2. In contrast, hydrolysis of peptide substrates extending from P6 to P6' is improved 70-300-fold by histidine in the P2 or P1' position. This preference creates new protease specificities for sequences HR decreases, R decreases H, HK decreases, and K decreases H. The ability of histidine from either the P2 or the P1' position of substrate to participate in catalysis emphasizes the considerable variability of proteolytically active orientations which can be assumed by the catalytic triad. Trypsin H57A is able to hydrolyze fully folded ornithine decarboxylase with complete specificity at a site containing the sequence HRH. Trypsin H57A was compared to enteropeptidase in its ability to cleave a propeptide from trypsinogen. Trypsin H57A cleaved the propeptide of a variant trypsinogen containing an introduced FPVDDDHR cleavage site only 100-fold slower than enteropeptidase cleaved trypsinogen. The selective cleavage of folded proteins suggests that trypsin H57A can be used for specific peptide and protein cleavage. The extension of substrate-assisted catalysis to the chymotrypsin family of proteolytic enzymes indicates that it may be possible to apply this strategy to a wide range of serine proteases and thereby develop various unique specificities for peptide and protein hydrolysis.
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PMID:Trypsin specificity increased through substrate-assisted catalysis. 754 82

Trypanosoma brucei ornithine decarboxylase was reconstituted by coexpression of two polypeptides corresponding to residues 1-305 and residues 306-425 in Escherichia coli. The two peptides were coexpressed, at wild-type levels, from a single transcriptional unit that was separated by a 15-nucleotide untranslated region containing a ribosome binding site. The fragmented enzyme was purified and analyzed. The N- and C-terminal peptides are tightly associated into a fully active tetramer which has the same molecular weight as the native dimer. The kinetic constants (Km and kcat) measured for the decarboxylation of ornithine are identical to those obtained for the wild-type enzyme. These results suggest that the enzyme is organized into two structural domains, with a domain boundary in the region of amino acid 305. In contrast, the individual N- and C-terminal peptides are expressed primarily as inclusion bodies. Small quantities of soluble N-terminal peptide could be purified. This truncated protein is capable of inhibiting the wild-type enzyme, suggesting that it is folded into a native-like structure. Limited proteolysis with trypsin or chymotrypsin identifies a likely surface loop at amino acids 160-170, present in both the mouse and T. brucei enzyme, which positions one or more functionally important active site residues (e.g., Lys169). Kinetic analysis of a chimeric enzyme composed of T. brucei and mouse ornithine decarboxylase suggests that the substrate carboxylate binding determinant is located between residues 1 and 170.
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PMID:Domain organization and a protease-sensitive loop in eukaryotic ornithine decarboxylase. 757 30

In the following investigation, we have studied the role of polyamines in the regulation of atrial natriuretic peptide (ANP) using ventricular cardiocytes which in culture synthesize and secrete ANP. Polyamines are cellular cations ubiquitous in eukaryotes, and ANP is a hormone synthesized and secreted by the cardiac atria in adult animal. The cardiocytes were isolated from neonatal rat hearts by enzymatic dissociation using trypsin and collagenase. The functional role of polyamines in regulation of ANP was assessed by exposing the cardiocytes to difluoromethylornithine (DFMO) which is an inhibitor of ornithine decarboxylase, an initial rate-limiting enzyme in the biosynthesis of polyamines. The results showed that DFMO reduced the levels of putrescine (diamine) and spermidine (triamine) in cultured cardiocytes, and it decreased the levels of ANP in media and cellular extracts of cardiocytes as a function of its dose. An addition of putrescine (100 microM) restored within 5-15 min the levels of ANP in media of both control and polyamine-depleted cardiocytes. These results suggest that polyamines are one of the cellular factors involved in regulation of ANP secretion in cultured cardiocytes.
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PMID:Polyamine regulation of atrial natriuretic peptide in cultured cardiocytes. 797 38

Ornithine decarboxylase (ODC) is the initial inducible enzyme in the polyamine biosynthetic pathway. In the transformed macrophage-derived RAW264 cell line, ODC was overproduced and existed in both unphosphorylated and phosphorylated forms. To date, the only protein kinase known to phosphorylate mammalian ODC is casein kinase II (CKII). ODC was phosphorylated in vitro by CKII and subjected to exhaustive sequential proteolysis with trypsin and V8 protease. Two-dimensional peptide mapping showed only a single phosphopeptide; two-dimensional phosphoamino acid analysis of the phosphopeptide revealed only 32P-labeled serine. ODC was metabolically radiolabeled with 32Pi in RAW264 cells and also subjected to proteolysis, two-dimensional peptide mapping, and phosphoamino acid analysis. Two phosphopeptides were generated from the metabolically radiolabeled ODC, including one that migrated similarly to the peptide phosphorylated by CKII in vitro. Each of the in situ radiolabeled ODC peptides contained both 32P-labeled serine and threonine residues. Thus, in RAW264 cells, ODC is phosphorylated on at least one serine residue in addition to that phosphorylated by CKII and on at least two threonine residues. Phosphorylated ODC had an increased stability to intracellular proteolysis compared with unphosphorylated ODC, their half-lives being 49.2 +/- 3.78 and 23.9 +/- 2.6 min (p = 0.001), respectively. The phosphorylated and unphosphorylated forms of ODC were independently purified to homogeneity. Kinetic analysis revealed that the catalytic efficiency of the phosphorylated form of ODC was 50% greater than that of the unphosphorylated form; the unphosphorylated ODC had a Vmax of 20.54 +/- 1.65 micromol/min/mg, whereas the phosphorylated form had a Vmax of 30.61 +/- 2.6 micromol/min/mg (p = 0.005). Phosphorylation of ODC by CKII has no effect on enzyme activity. Taken together, these findings demonstrate that regulation of ODC activity is governed by as yet unidentified protein kinases.
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PMID:Multisite phosphorylation of ornithine decarboxylase in transformed macrophages results in increased intracellular enzyme stability and catalytic efficiency. 879 74

Development of eggs after a blood meal in the yellow fever mosquito Aedes aegypti involves hormonal changes, synthesis of nucleic acids, activation of the digestive enzyme trypsin, and production of the yolk protein vitellogenin. Polyamines have been implicated in growth processes and were here examined for possible involvement during egg development. The data suggest that polyamines are important for normal vitellogenesis in the mosquito. Polyamine levels and activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase, key enzymes in the polyamine pathway, were determined in the fat body for two days after a blood meal. During the time that the macromolecules required for vitellogenesis were being synthesized, polyamine levels increased as did the activities of their rate-limiting enzymes. Administration of suicide inhibitors of ornithine decarboxylase, alpha-difluoromethylornithine (DFMO) and alpha-monofluoromethyldehydroornithine methylester (MDME), limited increased polyamine levels and disrupted macromolecular syntheses, particularly during the first twenty-four hours after blood feeding. Specific metabolic processes reduced by DFMO included trypsin activity, and production of RNA, DNA and vitellogenin. MDME had differential effects on transcription of some mRNA species made after an oogenic meal. The level of actin mRNA was not affected by inhibiting polyamine synthesis, but the mRNA levels of vitellogenin, trypsin, and the vitelline membrane protein were decreased. Adding polyamines to a meal containing DFMO or MDME partially reversed the effects of these inhibitors. Increases in spermidine and spermine were associated with these reversals.
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PMID:Polyamines, and effects from reducing their synthesis during egg development in the yellow fever mosquito, Aedes aegypti. 1081 34

Some trypanosomatids, such as Crithidia deanei, are endosymbiont-containing species. Aposymbiotic strains are obtained after antibiotic treatment, revealing interesting aspects of this symbiotic association. Ornithine decarboxylase (ODC) promotes polyamine biosynthesis and contributes to cell proliferation. Here, we show that ODC activity is higher in endosymbiont-bearing trypanosomatids than in aposymbiotic cells, but isolated endosymbionts did not display this enzyme activity. Intriguingly, expressed levels of ODC were similar in both strains, suggesting that ODC is positively modulated in endosymbiont-bearing cells. When the aposymbiotic strain was grown in conditioned medium, obtained after cultivation of the endosymbiont-bearing strain, cellular proliferation as well as ODC activity and localization were similar to that observed in the endosymbiont-containing trypanosomatids. Furthermore, dialyzed-heated medium and trypsin treatment reduced ODC activity of the aposymbiont strain. Taken together, these data indicate that the endosymbiont can enhance the protozoan ODC activity by providing factors of protein nature, which increase the host polyamine metabolism.
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PMID:An endosymbiont positively modulates ornithine decarboxylase in host trypanosomatids. 1654 31


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