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Purification and characterization of 4-hydroxybenzoate 3-hydroxylase from a Klebsiella pneumoniae mutant strain (1995)

Suárez, Mónica ; Martín, M. ; Ferrer, Estrella ; [et al.]

Springer

Archives of microbiology 164 (1995), S. 70-77

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ISSN: 1432-072X

Keywords: Key words 4-Hydroxybenzoate ; Monooxygenase ; Hydroxylase ; Klebsiella pneumoniae

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract Unlike the parent wild-type strain, the Klebsiella pneumoniae mutant strain MAO4 has a 4-HBA+ phenotype. The capacity of this mutant to take up and metabolize 4-hydroxybenzoate (4-HBA) relies on the expression of a permease and an NADPH-linked monooxygenase (4-HBA-3-hydroxylase). Both enzymes are normally expressed at basal levels, and only the presence of 4-HBA in the media enhances their activities. Strikingly, when the Acinetobacter calcoaceticus pobA gene encoding 4-hydroxybenzoate-3-hydroxylase was expressed in hydroxybenzoate K. pneumoniae wild-type, the bacteria were unable to grow on 4-HBA, suggesting that the main difference between the wild-type and the mutant strain is the capability of the latter to take up 4-HBA. 4-HBA-3-hydroxylase was purified to homogeneity by affinity, gel-filtration, and anion-exchange chromatography. The native enzyme, which appeared to be a dimer of identical subunits, had an apparent molecular mass of 80 kDa and a pI of 4.6. Steady-state kinetics were analyzed; the initial velocity patterns were consistent with a concerted substitution mechanism. The purified enzyme had 362 amino acid residues, and a tyrosine seemed to be involved in substrate activation.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/s002030050237

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Ticlopidine augments luminol-dependent chemiluminescence in human neutrophils (1995)

Bednar, Martin M. ; Dooley, Richard H. ; Tapanes, Ricardo ; [et al.]

New York : Wiley-Blackwell

Journal of Bioluminescence and Chemiluminescence 10 (1995), S. 85-89

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ISSN: 0884-3996

Keywords: Human neutrophils ; ticlopidine ; oxygen-free radicals ; chemiluminescence ; luminol ; Chemistry ; Biochemistry and Biotechnology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Chemistry and Pharmacology

Notes: Neutrophils contribute to the pathophysiology of various ischaemic states. Since many agents thought to be antiplatelet have also been shown to affect neutrophil function, it was of interest to examine the effect of ticlopidine (250 mg, p.o., b.i.d. for three doses), an antiplatelet agent, on fMLP (formyl-methionyl-leucyl-phenylalanine) stimulated neutrophil aggregation and luminol-dependent chemiluminescence in whole blood. Neutrophil aggregation did not significantly change from baseline values during ticlopidine administration. However, luminol-dependent chemiluminescence, an index of respiratory burst metabolism, was noted to be markedly increased during ticlopidine administration. Two hours following the final dose of ticlopidine, the chemiluminescent response (mean ± SEM, n = 5) was significantly increased from 6.27 ± 1.88 to 12.66 ± 2.19 units (p 〈 0.05). A return to baseline (6.68 ± 2.24 units) five days following the administration of ticlopidine was noted. It is concluded from this study that the acute oral administration of ticlopidine may affect neutrophil function as demonstrated by the significant increase in stimulated luminol-dependent chemiluminescence.

Additional Material: 1 Ill.