The modulation of cytochrome P450 activities by their membrane lipid environment

Abstract

Cytochrome P450 (GYP) are a family of membrane-bound enzymes which form a component of the mixed function oxidase (MFO) system and are involved in the metabolism of many endogenous and exogenous compounds and, as such, play key roles in many physiological, pharmacological and toxicological processes. Due to the diversity of their substrates, it is essential to gain an understanding of how their activities can be modulated. The membrane lipid environment of CYP isoforms has proved to be an essential component for their optimal activities and consequently investigations into this aspect of modulation are fundamental to many areas of research.
The work described in this thesis investigates 3 methods for the modulation of hepatic microsomal membrane lipids in order to relate 3 components of the lipid bilayer to the associated GYP isoform activities. The effect of incubating post-mitochondrial fractions with a range of phospholipase A2 (PLA2) concentrations at 37 °G on 4 CYP-catalysed reactions
during microsomal fraction preparation was examined. The size of the membrane free fatty acid (FFA) pool was found to increase substantially following PLA2 treatment, and the main hydrolysis product was revealed to be arachidonate. Although the bulk fluidity of the membrane was unchanged at specific PLA2 concentrations, differential sensitivities of the GYP isoforms were observed at these concentrations. By assaying the activity of the NADPH-P450 reductase component of the MFO independently, in addition to using an oxygen surrogate to by-pass the reductase enzyme, it was concluded that it was
specifically the GYP proteins that were susceptible to the membrane modulation which consequently lead to the observed decreases in the rates of reaction. The subsequent removal of the FFA pool with bovine serum albumin (BSA) was found not to restore the GYP isoform activities. It has been suggested that the inhibitory effect of FFAs may be due to conformational changes in the lipid environment of the isozymes. Also, hydrolysis of membrane phospholipids by PLA2 activity results in the formation of FFAs and Iysophospholipids, both of which can affect substrate partitioning within a membrane. Thus, the remaining lysophospholipids following BSA treatment may have contributed to the decreased activities of the GYP isoforms.
A range of PLA2 concentrations at the lower incubation temperatures of 10 °G and 20 °C were used to retailor microsomal lipids in order to investigate the possibility that the GYP activities were associated with specific lipid domains. As with the 37°G incubation, the main hydrolysis product within the FFA pool was found to be arachidonate. Although arachidonate release was not significantly affected at any of the incubation temperatures, the activities of individual GYP isoforms were significantly decreased with increases in incubation temperature at specific PLA2 concentrations. Kinetic studies suggested that PLA 2 treatment of the microsomal membrane modulated CYP activity through the hydrolysis of associated lipid domains, and not by competitive inhibition by the endogenously released arachidonate.
In an attempt to elucidate it microsomal membrane fatty acid saturation affected the activities of associated CYP isoforms, a preliminary study was conducted in which membrane lipid species were chemically modified by the use of catalytic hydrogenation. Membrane fractions were successfully hydrogenated, particularly long-chain fatty acids such asarachidonate. The activities of those CYP isoforms investigated were modulated to varying degrees upon hydrogenation of the membrane environment. It was concluded that the use of such catalysts should provide a suitable system for investigations fatty acid unsaturation and CYP activities.
Finally, as phosphatidylcholine (PC) has been shown to be important for certain CYP isoform activities, a preliminary study was carried out to determine the effect of the biosynthetic pathway for PC synthesis on mouse hepatic MFO activity. Using microsomal fractions obtained from a transgenic strain in which one of the pathways for PC synthesis was absent, it was found that the PC fatty acid profile was changed compared to that of the wildype fractions. Additionally, the activities of certain CYP isoforms were affected by the biosynthetic origin of PC synthesis. This sensitivity of specific isozymes to the PC biosynthetic pathway may have also been sex-dependent.
The work described in this thesis suggests several strategies which could be exploited further to refine and improve experimental studies of CYP isoforms. The results obtained support the concept that the membrane composition and
environment of CYP isoforms differentially affects their activities. Due to the complexity of these remarkable biochemical systems, further studies are clearly required to define the membrane properties involved in the modulation of CYP
activity.