PubMed Health⌕ Search

Biomedical subjects

W Nastainczyk

Publications and source records attributed to W Nastainczyk.

60 records · Page 4Linked to original sources
PubMed1977

Kinetic studies on a 4-methoxybenzoate O-demethylase from Pseudomonas putida.

A direct, sensitive and reliable photometric assay procedure for monitoring the activity of non-specific 4-methoxybenzoate O-demethylases of microorganisms is described. The assay is based on the O-demethylation of 3-nitro-4-methoxybenzoate to the yellow-coloured product 3-nitro-4-hydroxybenzoate. Using this assay and by monitoring the oxidation rate of reduced pyridine nucleotides, the kinetic properties of a purified, reconstituted enzyme system composed of 4-methoxybenzoate monooxygenase (O-demethylating) and a reductase from Pseudomonas putida have been investigated. It has been found that the KM value of the monoxygenase of this enzyme system towards different substrates (i.e. tight couplers, uncouplers and partial uncouplers) rises from the extremely low value of 0.07 muM for the tight couplers to about 55 muM for the uncouplers. The effect of possible inhibitors and metal ions on the reconstituted enzyme system was investigated. The inhibition pattern was almost identical to that found for the purified reductase, only batho-phenanthrolinedisulfonate showing a greater inhibition of the reconstituted enzyme system. The affinity of the reductase towards NADH was found to be approximately 200-fold greater than that towards NADPH. Futhermore, the affinity of this reductase to NADH depended on the nature of the electron acceptor. The affinity to NADH was more than 10 times higher when the monooxygenase-substrate complex was used as the electron acceptor, than when cytochrome c or 2,6-dichloroindophenol was used. These differences are discussed on the basis of enzyme-enzyme interactions between the reductase and the monooxygenase.

Benzoates↗
PubMed1976

Binding of a metyrapone spin label to microsomal cytochrome P-450.

Probing of the active site of microsomal cytochrome P-450 was carried out with a spin label derived from 2-methyl-1,2-bis(3-pyridyl)-1-propanone (metyrapone). Its optical binding spectra to cytochrome P-450 resemble the spectra with metyrapone. The electron paramagnetic resonance spectrum of the spin label in microsomes indicates binding with strong immobilization. Since the apparent optical and EPR binding constants agree very well (Ks approximately 2-10(-5) M), and metyrapone is found to displace the spin label, we conclude, that the spin label binds to the active site of cytochrome P-450. Addition of detergents or an increase in temperature mobilizes the bound spin label slightly. The EPR signal accounts for only 60% of the bound spin label due to its dipolar interaction with the low-spin ferric heme of cytochrome P-450. From this finding, the distance between nitroxide and iron is evaluated to be 11 A. This supports the model that one of the pyridine nitrogens of metyrapone is coordinated to the iron of cytochrome P-450. The bound spin label shows virtually no interaction with ferricyanide as if the active site of cytochrome P-450 is a hydrophobic pocket not accessible to ions.

Animals↗
PubMed1976

Binding of thiols to microsomal cytochrome P-450.

Lipophilic thiol compounds interact spectrally with liver microsomes from phenobarbital-pretreated rats by formation of unusual optical difference spectra with peaks at 378, 471, 522 and 593 nm in the oxidized state. The binding kinetics were biphasic. The EPR spectrum of cytochrome P-450 was slightly modified but the magnitude of the low-spin signal was unchanged. n-Octanethiol competitively displaced metyrapone and n-octane from the active site of cytochrome P-450. Other thiols behaved similarly with variations in the magnitude and the affinity of the binding process. Tertiary thiols caused the formation of the high-spin cytochrome P-450 substrate complex, and model studies with myoglobin revealed that steric hindrance prevented the liganding of the tertiary thiol group to the ferric cytochrome P-450. Addition of thiols to dithionite reduced microsomes resulted in relatively small spectral changes with maxima at 449 nm typical for ligand complexes of the ferrous cytochrome. It was concluded that lipophilic thiols can be bound as ligands by at least two species of oxidized cytochrome P-450 which represent, however, not more than about one fifth of the total cytochrome P-450 content in liver microsomes from phenobarbital-pretreated rats.

Animals↗