Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.64 (MPP)
 1,876 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

(3-si,4-re)-2,5-Dihydroxyacetanilide epoxidase (DHAE I), a key enzyme in the biosynthesis of the epoxysemiquinone antibiotic LL-C10037 alpha by Streptomyces LL-C10037 [Gould, S.J., & Shen, B. (1991) J. Am. Chem. Soc. 113, 684-686], and (3-re,4-si)-2,5-dihydroxyacetanilide epoxidase (DHAE II) isolated from Streptomyces MPP 3051--which yields the (3R,4S)-epoxyquinone mirror image product of DHAE I--are described. DHAE I was purified 640-fold. Gel permeation chromatography indicated an Mr of 117,000 +/- 10,000; SDS-PAGE gave a major band of 22,300 daltons, indicating that DHAE I is either a pentamer or hexamer in solution. The enzyme had a pH optimum of 6.5, a Km of 8.4 +/- 0.5 microM, and a Vmax of 3.7 +/- 0.2 mumol min-1 mg-1. DHAE II was purified 1489-fold. The enzyme was shown to be a dimer of Mr 33,000 +/- 2000, with 16,000-dalton subunits, with a pH optimum of 5.5 and a Km of 7.2 +/- 0.4 microM. Both enzymes required only O2 and substrate; flavin and nicotinamide coenzymes had little or no effect. Neither catalase nor EDTA affected the activity of either enzyme, but complete inhibition of both was obtained with 1,10-phenanthroline. The activity of the purified DHAE I could be enhanced, but only by Mn2+ (relative V = 246 at 0.04 mM), Ni2+ (relative V = 266 at 0.2 mM), or Co2+ (relative = 498 at 0.2 mM). Reconstitution from a DHAE I apoenzyme, generated by treatment with 1,10-phenanthroline followed by Sephadex G-25 chromatography, occurred only by addition of one of these three metals.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Opposite facial specificity for two hydroquinone epoxidases: (3-si,4-re)-2,5-dihydroxyacetanilide epoxidase from Streptomyces LL-C10037 and (3-re,4-si)-2,5-dihydroxyacetanilide epoxidase from Streptomyces MPP 3051. 189 11

Domains important for the activity of the heterodimeric mitochondrial processing peptidase (MPP) were investigated, by inserting one alanine residue at ten positions along the polypeptide chain of the beta-subunit (beta-MPP). An alanine residue inserted after Glu70, Ser114, Lys215 and Ser314 respectively, abolished the cleavage activity of MPP. When the alpha-subunit (alpha-MPP) was co-expressed with N-terminal hexa-histidine tagged beta-MPP, alpha-MPP was co-eluted from a nickel-derivatized affinity resin, with a 1:1 stochiometry, both with wild-type beta-MPP and with the mutants with alanine inserted after Ser114 and Ser314. The mutants with alanine inserted after Glu70 and Lys215 did not associate with alpha-MPP. The mutagenesis studies indicate that: (1) the whole HXXEHX76H region of beta-MPP is important for the proper conformation of the active site of MPP and may also be in contact with alpha-MPP; (2) the non-conserved central region surrounding Lys215 is involved in the interaction with alpha-MPP; and (3) the carboxy-terminal region of beta-MPP surrounding Ser314 is also of importance for the catalysis. Cross-linking studies indicated that purified alpha-MPP bound a precursor protein in the absence of any beta-MPP. Furthermore, the interaction of MPP and its subunits with a peptide substrate, as analyzed by surface plasmon resonance, showed that alpha-MPP bound a peptide substrate as efficiently as MPP. The data suggest that the alpha-subunit is responsible for the binding of mitochondrial presequences prior their presentation to the catalytic site of MPP.
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PMID:Functional cooperation of the mitochondrial processing peptidase subunits. 929 49

In hospitals a large variety of substances are in use for medical purposes such as diagnostics and research. After application, diagnostic agents, disinfectants and excreted non-metabolized pharmaceuticals by patients, reach the wastewater. This form of elimination may generate risks for aquatic organisms. The aim of this study was to present: (i) the steps of an ecological risk assessment and management framework related to hospital effluents evacuating into wastewater treatment plant (WWTP) without preliminary treatment; and (ii) the results of its application on wastewater from an infectious and tropical diseases department of a hospital of a large city in southeastern France. The characterization of effects has been made under two assumptions, which were related to: (a) the effects of hospital wastewater on biological treatment process of WWTP, particularly on the community of organisms in charge of the biological decomposition of the organic matter; (b) the effects on aquatic organisms. COD and BOD5 have been measured for studying global organic pollution. Assessment of halogenated organic compounds was made using halogenated organic compounds absorbable on activated carbon (AOX) concentrations. Heavy metals (arsenic, cadmium, chrome, copper, mercury, nickel, lead and zinc) were measured. Low most probable number (MPP) for faecal coliforms has been considered as an indirect detection of antibiotics and disinfectants presence. For toxicity assessment, bioluminescence assay using Vibrio fischeri photobacteria, 72-h EC50 algae growth Pseudokirchneriella subcapitata and 24-h EC50 on Daphnia magna were used. The scenario allows to a semi-quantitative risk characterization. It needs to be improved on some aspects, particularly those linked to: long term toxicity assessment on target organisms (bioaccumulation of pollutants, genotoxicity, etc.); ecotoxicological interactions between pharmaceuticals, disinfectants used both in diagnostics and in cleaning of surfaces, and detergents used in cleaning of surfaces; the interactions into the sewage network, between the hospital effluents and the aquatic ecosystem.
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PMID:Ecotoxicological risk assessment of hospital wastewater: a proposed framework for raw effluents discharging into urban sewer network. 1562 48

The rat organic cation transporter rOCT1 with six histidine residues added to the C-terminus was expressed in Sf9 insect cells, and expression of organic cation transport was demonstrated. To purify rOCT1 protein, Sf9 cells were lysed with 1% (w/v) CHAPS [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate], centrifuged, and subjected to sequential affinity chromatography using lentil-lectin Sepharose and nickel(II)-charged nitrilotriacetic acid-agarose. This procedure yielded approximately 70 microg of purified rOCT1 protein from 10 standard culture plates. Using a freeze-thaw procedure, purified rOCT1 was reconstituted into proteoliposomes formed from phosphatidylcholine, phosphatidylserine, and cholesterol. Proteoliposomes exhibited uptake of [3H]-1-Methyl-4-phenylpyridinium ([3H]MPP) that was inhibited by quinine and stimulated by an inside-negative membrane potential. MPP uptake was saturable with an apparent K(m) of 30 +/- 17 microM. MPP uptake (0.1 microM) was inhibited by tetraethylammonium, tetrabutylammonium, and tetrapentylammonium with IC50 values of 197 +/- 11, 19 +/- 1, and 1.8 +/- 0.03 microM, respectively. With membrane potential clamped to 0 mV using valinomycin in the presence of 100 mM potassium on both sides of the membrane, uptake of 0.1 microM MPP was trans stimulated 3-fold by 2.5 mM intracellular choline, and efflux of 0.1 microM MPP was trans stimulated 4-fold by 9.5 mM extracellular choline. The data show that rOCT1 is capable and sufficient to mediate transport of organic cations. The observed trans stimulation under voltage-clamp conditions shows that rOCT1 operates as a transporter rather than a channel. Purification and reconstitution of functional active rOCT1 protein is an important step toward the biophysical characterization and crystallization.
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PMID:Purification and functional reconstitution of the rat organic cation transporter OCT1. 1614 24