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Lipid peroxidation decreases the rotational mobility of cytochrome P-450 in rat liver microsomes

1985; Elsevier BV; Volume: 817; Issue: 2 Linguagem: Inglês

10.1016/0005-2736(85)90023-9

1879-2642

Josef Gut, Suguru Kawato, Richard J. Cherry, Kaspar H. Winterhalter, Christoph Richter,

Analytical Chemistry and Chromatography

Phenobarbital-induced rat liver microsomes were subjected to NADPH- and Fe2+-catalyzed lipid peroxidation. The formation of approx. 95 nmol malondialdehyde/mg protein during 18 min peroxidation at 37°C was observed. Membrane rigidity measured by means of the steady-state fluorescence anisotropy rs of diphenylhexatriene increased in parallel with the malondialdehyde formation. Both the amount of malondialdehyde and rs remained constant thereafter during incubation of the peroxidized membranes for 2 h. The aminopyrine demethylase activity decreased by about 60% upon lipid peroxidation for 18 min, whereas no significant loss of benzphetamine demethylase activity within the same time range was observed. A time-dependent formation of protein complexes of high molecular weight, comprising most of the microsomal polypeptides, upon lipid peroxidation was observed in SDS-polyacrylamide gel electrophoresis. The effect of microsomal lipid peroxidation on protein-protein interactions was examined by measuring the rotational mobility of intact cytochrome P-450. Rotational diffusion was measured by observing the decay of flash-induced absorption anisotropy r(t) of the P-450 · CO complex. Analysis was based on a ‘rotation-about-membrane normal’ model with the equation r(t) = r1exp(−tφ1) + r2exp(−tφ2). In control microsomes, two classes (rapid and slow) of rotating populations of cytochrome P-450 were observed with φ1 ≈ 150 μs, fraction r1(r1 + r2) ≈ 40% and φ2 ≈ 2 ms, fraction r2(r1 + r2) ≈ 60%. A relatively small decrease in the rotational mobility of P-450 was observed by a 18-min lipid peroxidation, while a subsequent incubation of peroxidized microsomes for 2 h at 37°C resulted in a dramatic immobilization of P-450 by the increase of both r2(r1 + r2) ≈ 75% and φ2 ≈ 10–25 ms. The decrease in the P-450 mobility during 18-min lipid peroxidation would be due to the rigidification of the lipid bilayer. However, because the lipid fluidity remained unchanged thereafter, the significant immobilization of P-450 by the subsequent 2-h incubation is deduced to be due to formation of protein aggregates.