Biosynthetic preparation of the NO-glucosiduronic acid of N-acetyl-N-phenylhydroxylamine

Kato, Keitaro; Ide, Hiroyuki; Hirohata, Itsuyo; Fishman, William H.

doi:10.1042/bj1030647

Cited by 12 publications

(9 citation statements)

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“…In a preliminary investigation of the flavin content, a sample of the most highly purified NADH dehydrogenase was extracted by the method of Kato et al [24]. On examination of the thin-layer plate under ultraviolet light, a fluorescent spot was observed with an R, value corresponding to FAD.…”

Section: Is-fold Purification Was Achieved With a 4 % Yield From Washedmentioning

confidence: 99%

Purification and characterization of the rotenone-insensitive NADH dehydrogenase of mitochondria from Arum maculatum

Cook

Cammack

1984

Eur J Biochem

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The non-ionic detergent lauryl dimethylamine N-oxide (LDAO) has been used to extract the NADH dehydrogenases of Arum /naculatun? mitochondria. Affinity chromatography on S'ADP-Sepharose 4B was used to separate the rotenone-sensitive (complex I) NADH dehydrogenase from thc rotcnone-insensitive NADH dchydrogenase. An 1 8-fold purification of the rolenonc-insensitive NADH dehydrogenase was achicvcd. The enzyme is specific for NADH with optimal activity around pH7.2. The apparent K, Tor NADH is 28pM, with dichloroindophcnol as acceptor at pH 7.2. The rotenone-insensitive NADH dehydrogenase appears to be a flavoprotein and no iron-sulphur centres were detected by electron spin resonance spectroscopy.One of the major differences between animal mitochondria and the mitochondria isolated from plant tissues is the ability of the latter to oxidize externally added NADH [I]. The cxogenous NADH oxidase activity is insensitive to thc complcx I inhibitors rotenone and piericidin.All plant mitochondria investigated have been found to oxidize externally added NADH to some extent. An exception is red beetroot (Beta idgaris L.) mitochondria [2] where the activity was only developed when beetroot slices were aged in aerated CaSO, solution.One of the most striking examples of rotenone-insensitive exogenous NADH oxidation occurs in the thermogenic spadix of aroids such as Arum twarulatum, where NADH oxidation rates may reach 7000 nmol min-' mg-[3].The enzyme responsible for this activity is an NADH dehydrogenase which is located on the outer surface of the inner mitochondrial membrane [l]. The dehydrogenase transfers eleclrons from exogenous NADH to the ubiquinone pool [4]. Electron transfer through cytochrome c oxidase appears to be coupled to two sites of phosphorylation [5 ~ 81.Studies on the kinetic properties of the rotenone-insensitive external NADH dehydrogenase are hindered by the prcscncc of an NADH dehydrogenase located on the outer membrane [I] and by the Complex I NADH dehydrogenase. There is also cvidence for a rotenone-resistant NADH dehydrogenase activity on the inner surface of the inner plant mitochondria1 membrane [9]. The elucidation of the kinetic, structural and thermodynamic properties of the respiratory-linkcd cxternal NADH dehydrogenase await its isolation from these contaminating activities.In this publication we describe the separation of the rotenone-insensitive NADH dchydrogenase and the rolenonc-sensitive complex I NADH dehydrogenase activity in A. rmculatum mitochondria by affinity chromatography on S'ADP-Sepharose 4B. Some properties of the rotenoneinsensitive NADH dehydrogenase are described. MATERIALS AND METHODS Preyxirution oj' Arum mitochopidrial rnenihraiiesA m n i nzaculutum inflorescences were collected from the wild and stored at 6 'C until used. Mitochondria were prepared as previously described [lo] and frozen at 77K until used.For the subsequent stages, the buffer was 10mM Mops, pH 7.2. Mitochondria1 membranes were prepared by diluting thawed 4 . niaculatum mitochondria tenfold with buff...

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Section: Is-fold Purification Was Achieved With a 4 % Yield From Washedmentioning

confidence: 99%

Purification and characterization of the rotenone-insensitive NADH dehydrogenase of mitochondria from Arum maculatum

Cook

Cammack

1984

Eur J Biochem

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“…The molecular extinction coefficient of N-acetyl-N-phenylhydroxylamine in CHC13 at 256mu is 7-86 x 105 cm./mole/l. Kato et al (1967) have demonstrated the purity of this isolated compound with respect to elemental analysis and the identification of the infrared spectrum of its derivative by its correspondence with that of the synthetic compound. Instability in alkali was characteristic of the sodium salt and of the derivative.…”

Section: Methodsmentioning

confidence: 85%

“…The isolation, identification and proof of structure of the sodium salt of (N-acetyl-N-phenylhydroxylamine ,B-D-glucosid)uronic acid has been reported by Kato, Ide, Hirohata & Fishman (1967). We now describe the experiments that characterize the action of /-glucuronidase on this conjugate.…”

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confidence: 85%

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Behaviour of the NO-β-d-glucosiduronic acid of N-acetyl-N-phenylhydroxylamine as a substrate for β-glucuronidase

Ide

Green

Kato

et al. 1968

Biochemical Journal

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1. Biosynthetic sodium (N-acetyl-N-phenylhydroxylamine NO-beta-d-glucosid)-uronate is hydrolysed completely by purified mouse urinary beta-glucuronidase into the products N-acetyl-N-phenylhydroxylamine and glucuronic acid. The hydrolysis is inhibited by saccharo-(1-->4)-lactone. These results not only confirm the identity and purity of the substrate but also establish it as a substrate for beta-glucuronidase. 2. Mammalian and bacterial beta-glucuronidase preparations hydrolysed the substrate at a rate one-fifth of that for (phenolphthalein beta-d-glucosid)uronic acid under the optimum conditions of hydrolysis for each source. 3. The pH optimum is 4.1 and the Michaelis constant, K(m), is 3.3x10(-4)m with purified mouse urinary beta-glucuronidase as the enzyme source acting on the NO-beta-d-glucosiduronic acid. The aglycone after extraction into chloroform was quantitatively determined spectrophotometrically at its absorption maximum (256mmu). 4. The hydrolysis was studied as a function of time and temperature. 5. From a consideration of the chemical and enzymic properties of this NO-beta-d-glucosiduronic acid it is possible to suggest its catabolism in vivo.

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“…Recently, the genes that code for toxin A and toxin B have been cloned and sequenced [11][12][13]. Molecular analysis of C. difficile isolates using the polymerase chain reaction (PCR) has demonstrated that toxic strains possess the toxin A [14][15][16] and toxin B genes [14], while non-toxic strains do not possess the toxin determinants. Therefore, conducting PCR with primers specific for toxin A or toxin B can differentiate between toxin and non-toxic strains; however, all toxic strains exhibit the same PCR pattern when primers derived from the toxin-gene sequences are used.…”

Section: Introductionmentioning

confidence: 99%

Typing of toxic strains of Clostridium difficile using DNA fingerprints generated with arbitrary polymerase chain reaction primers

McMillin

1992

FEMS Microbiology Letters

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Clostridium difficile is the causative agent for pseudomembranous colitis in humans. Toxic strains of C. difficile produce two toxins, toxin A and toxin B. A reliable and definitive method of typing the toxic strains of C. difficile is needed since nosocomial cross infection is a primary concern in hospitals and other health care facilities. A method for typing toxic strains of Clostridium difficile using arbitrary polymerase chain reaction (PCR) primers is presented in this study. The C. difficile strains were initially characterized for the toxin A genetic determinant using specific PCR primers which differentiate toxin positive from toxin negative strains. These toxic strains were then PCR typed using six arbitrary primers which generated DNA patterns that were unique for all toxic strains examined. The use of this typing scheme in clinical applications is discussed.

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Biosynthetic preparation of the NO-glucosiduronic acid of N-acetyl-N-phenylhydroxylamine

Cited by 12 publications

References 5 publications

Purification and characterization of the rotenone-insensitive NADH dehydrogenase of mitochondria from Arum maculatum

Purification and characterization of the rotenone-insensitive NADH dehydrogenase of mitochondria from Arum maculatum

Behaviour of the NO-β-d-glucosiduronic acid of N-acetyl-N-phenylhydroxylamine as a substrate for β-glucuronidase

Typing of toxic strains of Clostridium difficile using DNA fingerprints generated with arbitrary polymerase chain reaction primers

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