Lipid peroxidation-cytochrome P450 interactions. Use of linoleic acid hydroperoxide in the characterization of the spin-state of membrane-bound P450.
1. A procedure (linolenic acid hydroperoxide (LAHP) deletion method) is described in which LAHP is added to the reference cuvette of a pair of spectrally balanced cuvettes containing hepatic microsomes to produce a composite high spin (HS)-low spin (LS)-spectrum of P450. 2. The LAHP deletion method was used to determine the spin state of P450 in rat hepatic microsomes with and without the addition of type I compounds. 3. Advantage was taken of the temperature dependency of the spin state of P450 to determine the overall enthalpic and entropic changes for the spin equilibrium to generate computer-derived spectra of HS and LS forms of P450, and to construct a nomogram that allows direct estimation of the percentage of HS and LS spin forms of P450 in intact microsomes at temperatures compatible with biochemical functions. 4. The h.p.l.c. deletion method was used to demonstrate that HS-P450 comprised 57% of the P450 in hepatic microsomes; addition of type I substrates to these microsomes raised the level of HS-P450 to 97%. 5. The percentage of HS-P450 generated by the addition of type I compounds to microsomes declined with increasing deletions of P450 until at the extrapolated 100% level of deletion there was no HS-P450 above that of the original 57% observed in the absence of added compounds. This can be explained if LAHP destroys part of the LS-P450 while altering the remaining LS-P450 such that it retains its LS spectral characteristics but loses its capacity to form HS P450 when type I substrates are added. 6. These studies support the concept that about 50% of hepatic microsomal P450 is functionally in the HS state due to binding with high affinity endogenous substrates or other membrane components; the remaining P450 is LS-P450 that can bind to exogenous substrates to form HS-P450. 7. Applications of the LAHP deletion method for assessment of catalytic properties of membrane-bound P450 at ambient temperatures are discussed.
Department of Pharmacology, University of Minnesota School of Medicine, Minneapolis 55455.
M M
MM
Iba
J E
JE
Gander
G J
GJ
Mannering
D000818:Animals; D003198:Computer Simulation; D003510:Cyclohexanes / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008041:Linoleic Acids; D015227:Lipid Peroxidation / Q000502:physiology*; D008054:Lipid Peroxides; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry; D008566:Membranes / Q000201:enzymology; D008862:Microsomes, Liver / Q000201:enzymology; D051381:Rats; D011919:Rats, Inbred Strains; D013057:Spectrum Analysis / Q000379:methods; D013696:Temperature; D013816:Thermodynamics
D003510:Cyclohexanes; D008041:Linoleic Acids; D008054:Lipid Peroxides; D008565:Membrane Proteins; C008285:linoleic acid hydroperoxide; D003577:Cytochrome P-450 Enzyme System