Search PubMed⌕ Search

Biomedical subjects

A Stier

Publications and source records attributed to A Stier.

At least 37 records · Page 2Linked to original sources
PubMed1988

Resonance Raman study of the cytochrome P-450 LM2-halothane intermediate complex.

Resonance Raman (RR) and absorption spectroscopic studies of purified rabbit liver cytochromes P-450 show that the form 2 isomer (LM2) but not the form 4 isomer (LM4) forms a long-lived complex with halothane after dithionite reduction, absorbing light at 470 nm, in which ferric 6-coordinated heme iron in the low-spin configuration is liganded to 2-chloro-1,1-difluoroethylene. The RR data exclude the possibility that the CF3CHCl- carbanion is a ligand and are consistent with the involvement of an active-site pocket in the cytochrome P-450 polypeptide.

Animals↗
PubMed1988

Surface enhanced resonance Raman study of phenobarbital-induced rabbit liver cytochrome P-450 LM2.

Surface enhanced resonance Raman (SERR) spectroscopy has been used to study the vibrational spectra of the heme of purified rabbit liver cytochrome P-450 LM2 which was adsorbed on colloidal silver suspensions or on a silver electrode. Bases on a comparison with the resonance Raman (RR) spectra of the 'solute' species the high sensitivity of the SERR technique is demonstrated. Two different features were chosen in order to determine the structural and functional integrity of the adsorbed P-450. Both, substrate-induced spin state changes on the oxidized P-450 and the effect of the thiolate ligand on the oxidation state marker band v4 in the reduced P-450 could be observed in the SERR spectra of the adsorbed as well as in the RR spectra of the dissolved enzyme. These findings indicate that the protein structure near the substrate binding site and the coordination by thiolate are not affected by the interaction with the metal surface. Both structural elements are crucial for the function of P-450. Thus the elementary processes of the enzymatic action of P-450 can be investigated by this highly sensitive version of RR spectroscopy.

Adsorption↗
PubMed1988

Heterogeneity of biotransformation of fluoranthene in perifused liver cells.

Pathways of biotransformation of the carcinogenic polycyclic hydrocarbon fluoranthene in individual isolated perifused liver cells were delineated using noninvasive microfluorescence spectroscopic techniques. An example of heterogeneity of coupling between phase I and phase II enzymes detected in a population of beta-naphthoflavone-induced rat liver cells demonstrates the usefulness of the experimental model of perifused liver cells.

Animals↗
PubMed1988

A perfusion chamber for high-resolution light microscopy of cultured cells.

A perfusion chamber suitable for general microscopic work on upright and inverted microscopes with cultured cells is described. The device features a minimal internal volume and large field of view within small outer dimensions. In particular the chamber body is very flat to accommodate large-aperture condenser and objective lenses. It is constructed of innocuous, sterilizable materials, incorporates permanent seals for the windows, is robust and simple to use.

Cells, Cultured↗
PubMed1985

The site of cyclic AMP-dependent protein kinase catalyzed phosphorylation of cytochrome P-450 LM2.

The phenobarbital-inducible form of cytochrome P-450 purified from rabbit liver microsomes is phosphorylated by cAMP-dependent protein kinase at a single site, the serine residue in position 128 of the amino acid sequence. The serine is located in a characteristic recognition sequence for cAMP-dependent protein kinase and is part of a primary structure which is conserved during evolution, present also in phenobarbital-inducible rat cytochrome and cytochrome P-450 CAM from Pseudomonas putida. The contribution of these findings to our understanding of the structure and membrane topology of cytochrome P-450 LM2 and its turnover regulated by phosphorylation is discussed.

Amino Acid Sequence↗
PubMed1985

Conversion of hepatic microsomal cytochrome P-450 to P-420 upon phosphorylation by cyclic AMP dependent protein kinase.

Cytochrome P-450, purified from liver microsomes of phenobarbital-induced rabbits, was phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase. Upon phosphorylation P-450 was found to be converted to its denatured form, P-420, as verified spectroscopically from the CO-bound form of the reduced cytochrome. The conversion was dependent on both kinase and ATP. Thus, cyclic AMP may regulate the biotransformation system through the control of the degradation rate of microsomal P-450 in vivo.

Adenosine Triphosphate↗
PubMed1985

Anoxia influences the lateral diffusion of a lipid probe in the plasma membrane of isolated cardiac myocytes.

Using the technique of fluorescence photobleaching recovery we have measured the characteristics of lateral diffusion of oleylaminofluorescein (OAF) in the plasma membrane of isolated rat cardiac myocytes under normoxic and anoxic conditions. The normoxic pattern is one of slow diffusion and low recovery (D = 1.8 +/- 0.3 X 10(-10)cm2/s, r = 0.44 +/- 0.059), while under anoxic conditions faster diffusion and higher recovery (D = 2.5 +/- 0.4 X 10(-9)cm2/s, r = 0.58 +/- 0.063) are observed, the change proceeding via an intermediate stage with a yet faster diffusing species (D greater than 2.5 X 10(-9)cm2/s). The process is reversible. We hypothesize that under normoxic conditions the lateral diffusion of OAF is hindered by the division of the cell membrane into a patchwork of more or less isolated domains by lateral and longitudinal barriers of spectrin (7, 13) which are rearranged under anoxic conditions to another pattern which permits the label greater, but still not unrestricted, freedom of movement.

Amines↗
PubMed1985

Effects of nitrogen mustard on potassium transport systems and membrane structure of Ehrlich ascites tumor cells.

By Ehrlich ascites tumor cells 86Rb+ has been shown to be a suitable tracer for K+-transport. Sixty percent of the total 86Rb-uptake into these cells is ouabain-inhibitable, 30% is sensitive to furosemide and 10% enters the cells by ouabain and furosemide-insensitive systems. N-Mustard inhibits both the ouabain-sensitive and the furosemide-inhibitable systems. The uptake which is resistant to both inhibitors is not affected by the alkylating drug. At N-mustard concentrations below 10 microM, the reduction of the Rb-uptake is predominantly due to the inhibition of the furosemide-sensitive transport. Higher concentrations are required before a significant inhibition of the ouabain-sensitive transport can be observed. The dose response curve of the furosemide-sensitive transport--not, however, of the ouabain inhibitable pump--corresponds to the dose response curve for the antiproliferative activity of N-mustard. The recovery of the furosemide-sensitive transport after a single exposure to N-mustard is relatively slow and--in contrast to the repair of DNA cross-links--is characterized by an initial 4-hr lag period. Furosemide alone does not interfere with cell multiplication. The inhibition of the transport system alone does, therefore, not explain the antitumor activity of N-mustard. The effect is discussed as a marker for membrane lesions after exposure to alkylating agents. In order to investigate the influence of N-mustard on membrane structure, membranes were labelled with diiodofluoresceiniodoacetamide. Anisotropy curves obtained from time-dependent depolarization of delayed fluorescence indicated a mustard induced immobilization of membrane constituents. Lateral diffusion of lipophilic probes was determined by following the quenching of fluorescence of pyrene by cetylpyridinium. The latter studies yielded no evidence for a change in membrane lipid fluidity. The data are interpreted as the results of cross-links of membrane proteins by the bifunctional alkylating agent.

Animals↗
PubMed1984

Phosphorylation of rabbit liver cytochrome P-450 LM2 and its effect on monooxygenase activity.

The phosphorylation of rabbit liver microsomal cytochrome P-450 LM2 by catalytic subunit of cyclic AMP-dependent protein kinase (W. Pyerin et al. (1983) Carcinogenesis 4, 573) has now been studied in detail with purified soluble form of cytochrome P-450 as well as with the purified protein incorporated into model membranes. The apparent Km values for P-450 of the phosphorylation reaction in all experimental systems were in a range of 2-8 microM, while the Vmax values were dependent on the state of P-450. Upon phosphorylation, the reconstituted enzyme activities with benzphetamine (N-demethylation) and 7-ethoxycoumarin (O-deethylation) as substrates were reduced to 30-40% of control.

Animals↗
PubMed1982

Spin-trapping studies on the free-radical products formed by metabolic activation of carbon tetrachloride in rat liver microsomal fractions isolated hepatocytes and in vivo in the rat.

1. The metabolic activation of carbon tetrachloride to free-radical intermediates is an important step in the sequence of disturbances leading to the acute liver injury produced by this toxic agent. Electron-spin-resonance (e.s.r.) spin-trapping techniques were used to characterize the free-radical species involved. 2. Spin trapping was applied to the activation of carbon tetrachloride by liver microsomal fractions in the presence of NADPH, and by isolated intact rat hepatocytes. The results obtained with the spin trap N-benzylidene-2-methylpropylamine N-oxide ('phenyl t-butyl nitrone') (PBN) and [13C]carbon tetrachloride provide unequivocal evidence for the formation and trapping of the trichloromethyl free radical in these systems. 3. With the spin trap 2-methyl-2-nitrosopropane, however, the major free-radical species trapped are unsaturated lipid radicals produced by the initiating reaction of lipid peroxidation. 4. Although pulse radiolysis and other evidence support the very rapid formation of the trichloromethyl peroxy radical from the trichloromethyl radical and oxygen, no clear evidence for the trapping of the peroxy radical was obtainable. 5. The effects of a number of free-radical scavengers and metabolic inhibitors on the formation of the PBN-trichloromethyl radical adduct were studied, as were the influences of changing the concentration of PBN and incubation time. 6. High concentrations of the spin traps used were found to have significant effects on cytochrome P-450-mediated reactions; this requires caution in interpreting results of experiments done in the presence of PBN at concentrations greater than 50 mM.

Animals↗
PubMed1982

Modification of liver microsomal lipids by halothane metabolites; a multi nuclear NMR spectroscopic study.

N-trifluoroacetyl phosphatidyl ethanolamine was found as an intermediate of biotransformation of halothane after intense in vivo exposure of non-induced and phenobarbital induced rabbits. The metabolite has been identified in detergent treated liver microsomes by 31P-NMR-spectroscopy and in microsomal lipid extracts by 19F-NMR- and 1H-NMR-spectroscopy as well as by thin layer chromatography. The same exposure changed phospholipid composition of liver microsomal membranes: most prominent was a substantial decrease of the ratio of phosphatidyl ethanolamines to phosphatidylcholines.

Animals↗
PubMed1982

Cytochrome P-450 rotamers control mixed-function oxygenation in reconstituted membranes. Rotational diffusion studied by delayed fluorescence depolarization.

1. Rotational diffusion of purified rabbit-liver microsomal cytochrome P-450 LM2 in reconstituted lipid-vesicle membranes was investigated by measurement of time-dependent polarized emission of delayed fluorescence. 2. Cytochrome P-450 labelled with diiodofluorescein iodoacetamide exhibited strict uniaxial rotation about the normal to the membrane. 3. Benzphetamine retards rotation, while reduction of the cytochrome P-450 substrate-complex accelerates rotation. 4. A model is proposed in which cytochrome P-450 forms disc-shaped rotamers immersed in the bilayer membrane to a depth which is varied by substrate-induced and redox state-dependent conformational changes. The model describes a regulating mechanism of the electron transfer-controlling mixed-function oxygenation.

Animals↗