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:3.5.1.4 (deaminase)
5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In this study, we demonstrated that benzene and its metabolites, phenol and hydroquinone, were toxic to human burst-forming unit-erythroid (BFU-E) growth, hydroquinone being the most toxic. Phenol (10(-4) M) was also found to have a marked toxicity on stromal cell colony formation. BFU-E binding with human-tumor necrosis factor (rHu-TNF) was linear with the number of BFU-E colonies. Recombinant rHu-TNF suppressed BFU-E growth in a dose-dependent manner and this was reversed with anti-TNF antibody. Binding studies of rHu-TNF for human K562 cells indicated that K562 cells have a binding constant of approximately 1075 per cell. The heme pathway enzymes, uroporphyrinogen deaminase, and heme oxygenase activities were measured in BFU-E cultures exposed to iron, interleukins (1 and 2), and various lymphocyte and macrophage-conditioned media with or without hemin. In most instances, hemin was found to stimulate the heme synthetic pathway in the presence of these agents. Iron and adherent (macrophage) cell conditioned media (CM) were found to stimulate heme oxygenase activity. Macrophage CM was found to suppress erythropoiesis in contrast to phytohemagglutinin-stimulated leukocyte (PHAL)-CM, which enhanced erythroid growth. In addition, porphobilinogen deaminase levels were greater in 14-day cultures containing hemin plus PHAL-CM as compared with hemin alone. These results are discussed with respect to the generation of hematopoietic inhibitory-stimulatory factors by the marrow microenvironment and their effects on heme synthesis and degradation.
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PMID:Microenvironmental cytokines and expression of erythroid heme metabolic enzymes. 331 Dec 13

The colorimetric estimation of amidase activity, using both qualitative and quantitative determinations of ammonia, is widely used for the differentiation of mycobacteria. At present the generally used phenol-hypochlorite method requires heating of the test solution to 90 degrees C for 30 min or to boiling for 5 min. At room temperature at least 2 h are necessary to obtain a full and stable color. Heating is also disadvantageous because it increases the vaporization of toxic phenol vapors and it may lead to the formation of insoluble manganese dioxide, which interacts with the photometric determination. We found that the addition of ketones (preferably acetone) to the catalyst solution (MnSO4) accelerates the reaction in such a manner that heating is not necessary and the full color development can be obtained within 6 min. The proposed method is superior to the conventional ones because (1) the fully developed color can be obtained after 6 minutes without heating; (2) boiling, which increases the volatilization of phenol and creates dangers for the laboratory staff and the equipment, can now be reduced; and (3) the formation of manganese dioxide in the test solution is avoided.
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PMID:An improved method of ammonia determination, applicable to amidases and other ammonia-producing enzyme systems of mycobacteria. 397 82

Previous results [J. F. Kuttesch, Jr. and J. A. Nelson, Cancer Chemother, Pharmac. 8, 221 (1982)] from this laboratory indicate that mechanisms exist for renal secretion of 2'-deoxyadenosine and possibly for reabsorption of adenosine in humans and in mice. Since significant metabolism of these purine nucleosides occurs even in the presence of adenosine deaminase inhibitors, the renal handling of a compound which is not significantly metabolized by the deaminase or by kinases was studied. Unlike 2'-deoxyadenosine itself, the 2'-deoxyadenosine analog, [4-amino-7-(2'-deoxy-beta-D-erythro-pentofuranosyl)-pyrrolo-(2,3-d)pyrimidine; 2'-deoxytubercidin], is not significantly metabolized by mammalian tissues. In mice, the renal plasma clearance of 2'-deoxytubercidin exceeded that of inulin by about 3-fold. Also, mouse kidney slices concentratively accumulated 2'-deoxytubercidin by a saturable and metabolically dependent process. The uptake by mouse kidney slices was inhibited by classical substrates for the organic cation secretory system (tetraethylammonium, choline and N1-methylnicotinamide) but was not markedly inhibited by classical substrates for the organic anion secretory system (p-aminohippurate, phenol red and probenecid). Since 2'-deoxytubercidin inhibited the active, concentrative uptake of [14C]tetraethylammonium, but failed to inhibit the uptake of p-[14C]aminohippurate by mouse kidney slices, it is concluded that 2'-deoxytubercidin may be secreted by the organic cation system. Additional studies are required, however, to unequivocally establish the relationships between 2'-deoxytubercidin, 2'-deoxyadenosine and tetraethylammonium renal secretory mechanisms.
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PMID:Renal transport of 2'-deoxytubercidin in mice. 621 93

We have determined and analyzed the nucleic acid sequence of a 14,855-bp region that contains the complete gene cluster encoding the 4-hydroxyphenylacetic acid (4-HPA) degradative pathway of Escherichia coli W (ATCC 11105). This catabolic pathway is composed by 11 genes, i.e., 8 enzyme-encoding genes distributed in two putative operons, hpaBC (4-HPA hydroxylase operon) and hpaGEDFHI (meta-cleavage operon); 2 regulatory genes, hpaR and hpaA; and the gene, hpaX, that encodes a protein related to the superfamily of transmembrane facilitators and appears to be cotranscribed with hpaA. Although comparisons with other aromatic catabolic pathways revealed interesting similarities, some of the genes did not present any similarity to their corresponding counterparts in other pathways, suggesting different evolutionary origins. The cluster is flanked by two genes homologous to the estA (carbon starvation protein) and tsr (serine chemoreceptor) genes of E. coli K-12. A detailed genetic analysis of this region has provided a singular example of how E. coli becomes adapted to novel nutritional sources by the recruitment of a catabolic cassette. Furthermore, the presence of the pac gene in the proximity of the 4-HPA cluster suggests that the penicillin G acylase was a recent acquisition to improve the ability of E. coli W to metabolize a wider range of substrates, enhancing its catabolic versatility. Five repetitive extragenic palindromic sequences that might be involved in transcriptional regulation were found within the cluster. The complete 4-HPA cluster was cloned in plasmid and transposon cloning vectors that were used to engineer E. coli K-12 strains able to grow on 4-HPA. We report here also the in vitro design of new biodegradative capabilities through the construction of a transposable cassette containing the wide substrate range 4-HPA hydroxylase, in order to expand the ortho-cleavage pathway of Pseudomonas putida KT2442 and allow the new recombinant strain to use phenol as the only carbon source.
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PMID:Molecular characterization of the 4-hydroxyphenylacetate catabolic pathway of Escherichia coli W: engineering a mobile aromatic degradative cluster. 855 Apr 3

Horseradish peroxidase isoenzyme C (HRP) contains eight N-linked glycans composed of Man, Xyl, Fuc, and GlcNAc. These glycans were resistant to enzymatic hydrolysis by endoglycosidases peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase F, endo-beta-N-acetyl-glucosaminidase H, and endo-beta-N-acetylglucosaminidase F under conditions where ovalbumin was deglycosylated. However, using anhydrous trifluoromethanesulfonic acid (TFMS) in the presence of 90 mM phenol for 5 min at--10 degrees C, all carbohydrate except GlcNAc was removed. Sixty percent of deglycosylated HRP was active after this TFMS treatment. Benzhydroxamic acid affinity chromatography separated active and inactive deglycosylated HRP. TFMS treatment, however, introduced negative charges in all inactive HRP and in about 90% of the active deglycosylated HRP. The nature of this modification has not been identified. After ion-exchange chromatography, homogeneous and fully active deglycosylated HRP, showing the original pI of 9, electronic absorption spectrum, and enzyme kinetics, was obtained, In this purified product no amino acid modifications were detected by amino acid analysis, partial sequencing, and mass spectrometry of tryptic peptides. The deglycosylated product showed greatly reduced solubility in salt solution compared to that of authentic HRP.
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PMID:Mild chemical deglycosylation of horseradish peroxidase yields a fully active, homogeneous enzyme. 857 87

Previously, we purified and characterized a pro-phenol-oxidase (pro-PO) of 79 kDa from coleopteran insect, Holotrichia diomphalia larvae [Kwon et al. (1997) Mol. Cells 7, 90-97]. Here, we describe the identification of two pro-PO-activating factors (PPAF), named PPAF-I and PPAF-II, directly involved in the activation of the isolated pro-PO. When pro-PO was incubated with either PPAF-I or PPAF-II, no phenol oxidase activity was observed. However, incubation of pro-PO with both PPAF-I and PPAF-II specifically exhibited phenol oxidase activity. The purified PPAF-I with a molecular mass of 33 kDa on SDS/PAGE had characteristics of a serine protease. It exhibited amidase activity against fluorogenic peptide substrates, tert-butoxycarbonyl-phenylalanyl-seryl-arginyl-4-methylcoumaryl-7-amide being the best among the substrates examined. The activity was completely inhibited by 0.02 mM p-nitrophenyl-p'-guanidinobenzoate HCl and diisopropylflurophosphate. The NH2-terminal sequence of PPAF-I had significant sequence similarity to those of serine proteases. On the other hand, the purified PPAF-II had a molecular mass of 40 kDa on SDS/PAGE and 400 kDa determined by gel filtration, indicating an oligomeric protein. The NH2-terminal sequence of PPAF-II showed no similarity to known proteins. PPAF-II exhibited no amidase activity against the fluorogenic substrates. Reconstitution experiments and immunoblotting analysis using affinity-purified antibody against pro-PO demonstrated that PPAF-I first cleaves the intact pro-PO to an intermediate of 76 kDa with no phenol oxidase activity, and then, PPAF-I converts the intermediate to the active phenol oxidase of 60 kDa in the presence of PPAF-II. These results indicate that the activation of pro-PO system in hemolymph of H. diomphalia larvae is accomplished by at least two activating factors, a serine protease and a protein cofactor.
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PMID:In vitro activation of pro-phenol-oxidase by two kinds of pro-phenol-oxidase-activating factors isolated from hemolymph of coleopteran, Holotrichia diomphalia larvae. 965 93

Peptide-N4-(N-acetyl-beta-d-glucosaminyl asparagine amidase) from Aspergillus tubigensis (PNGase At) was expressed in baculovirus-infected insect cells. The recombinant PNGase At was secreted and purified to homogeneity with a yield of 9.5 mg per liter of infected cell medium. Recombinant PNGase At migrated upon SDS-PAGE as a single-chain protein with a molecular mass of 78 kDa. This contrasts with the native Aspergillus enzyme which is "nicked" and migrates as two subunits each with a molecular weight about 43 kDa. Quantitation of total sugar by phenol-sulfuric acid suggests that the enzyme expressed in baculovirus-infected insect cells was substituted with 8-10 chains of carbohydrate of which 75% was released by Endoglycosidase F1. ESI-MS analysis of the oligosaccharides released from the recombinant PNGase At revealed similarity in the number of glycosylated residues but a significant difference in their composition, when compared to the carbohydrates of the native PNGase At. Despite differences in the primary structure and in the composition of glycan residues, the recombinant enzyme had the same specific activity as the native enzyme.
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PMID:Overexpression of PNGase at from baculovirus-infected insect cells. 979 Aug 95

Directed evolution of N-carbamyl-D-amino acid amidohydrolase from Agrobacterium tumefaciens NRRL B11291 was attempted in order to simultaneously improve oxidative and thermal stability. A mutant library was generated by DNA shuffling, and positive clones with improved oxidative and thermal stability were screened on the basis of the activity staining method on a solid agar plate containing pH indicator (phenol red) and substrate (N-carbamyl-D-p-hydroxyphenylglycine). Two rounds of directed evolution resulted in the best mutant 2S3 with a significantly improved stability. Oxidative stability of the evolved enzyme 2S3 was about 18-fold higher than that of the wild type, and it also showed an 8-fold increased thermostability. The K(m) value of 2S3 was comparable to that of wild-type enzyme, but k(cat) was slightly decreased. DNA sequence analysis revealed that six amino acid residues (Q23L, V40A, H58Y, G75S, M184L, and T262A) were substituted in 2S3. From the mutational analysis, four mutations (Q23L, H58Y, M184L, and T262A) were found to lead to an improvement of both oxidative and thermal stability. Of them, T262A had the most significant effect, and V40A and G75S only increased the oxidative stability.
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PMID:Directed evolution of N-carbamyl-D-amino acid amidohydrolase for simultaneous improvement of oxidative and thermal stability. 1205 52

The pyridoxal 5'-phosphate-dependent enzymes have been evolved to catalyze diverse substrates and to cause the reaction to vary. 1-Aminocyclopropane-1-carboxylate deaminase catalyzes the cyclopropane ring-opening reaction followed by deamination specifically. Since it was discovered in 1978, the enzyme has been widely investigated from the mechanistic and physiological viewpoints because the substrate is a precursor of the plant hormone ethylene and the enzymatic reaction includes a cyclopropane ring-opening. We have previously reported the crystal structure of the native enzyme. Here we report the crystal structures of the two reaction intermediates created by the mutagenesis complexed with the substrate. The substrate was validated in the active site of two forms: 1). covalent-bonded external aldimine with the coenzyme in the K51T form and 2). the non-covalent interaction around the coenzyme in the Y295F form. The orientations of the substrate in both structures were quite different form each other. In concert with other site-specific mutation experiments, this experiment revealed the ingenious and unique strategies that are used to achieve the specific activity. The substrate incorporated into the active site is reactivated by a two-phenol charge relay system to lead to the formation of a Schiff base with the coenzyme. The catalytic Lys51 residue may play a novel role to abstract the methylene proton from the substrate in cooperation with other factors, the carboxylate group of the substrate and the electron-adjusting apparatuses of the coenzyme.
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PMID:Reaction intermediate structures of 1-aminocyclopropane-1-carboxylate deaminase: insight into PLP-dependent cyclopropane ring-opening reaction. 1288 62

The gram-positive bacterium Staphylococcus aureus is a major pathogen responsible for a variety of diseases ranging from minor skin infections to life-threatening conditions such as sepsis. Cell wall-associated and secreted proteins (e.g., protein A, hemolysins, and phenol-soluble modulin) and cell wall components (e.g., peptidoglycan and alanylated lipoteichoic acid) have been shown to be inflammatory, and these staphylococcal components may contribute to sepsis. On the host side, many host factors have been implicated in the innate detection of staphylococcal components. One class of pattern recognition molecules, Toll-like receptor 2, has been shown to function as the transmembrane component involved in the detection of staphylococcal lipoteichoic acid and phenol-soluble modulin and is involved in the synthesis of inflammatory cytokines by monocytes/macrophages in response to these components. Nod2 (nucleotide-binding oligomerization domain 2) is the intracellular sensor for muramyl dipeptide, the minimal bioactive structure of peptidoglycan, and it may contribute to the innate immune defense against S. aureus. The staphylococcal virulence factor protein A was recently shown to interact directly with tumor necrosis factor receptor 1 in airway epithelium and to reproduce the effects of tumor necrosis factor alpha. Finally, peptidoglycan recognition protein L is an amidase that inactivates the proinflammatory activities of peptidoglycan. However, peptidoglycan recognition protein L probably plays a minor role in the innate immune response to S. aureus. Thus, several innate immunity receptors may be implicated in host defense against S. aureus.
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PMID:Recognition of Staphylococcus aureus by the innate immune system. 1602 Jun 88


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