ABSTRACT
The effects of chlorpromazine on the mobility of cytochrome P-450 and the fluidity of lipid membranes have been investigated in bovine adrenocortical submitochondrial particles (SMP). Rotational diffusion of the cytochrome was measured by observing the decay of absorption anisotropy, ra(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. Analysis of ra(t) was based on a 'rotation-about-membrane-normal' model. The anisotropy decayed within 2 ms to a time independent value r3. The presence of chlorpromazine decreased the mobile population of cytochrome P-450 from 28 to 23%. The rotational relaxation time phi a of the mobile population (approximately 1100 microseconds) was, however, not significantly changed by chlorpromazine. The lipid fluidity was examined by observing time-resolved fluorescence anisotropy, rf(t), of 1,6-diphenyl 1,3,5-hexatriene (DPH). The anisotropy rf(t) decayed within 70 ns to a time independent value r infinity. The motion of DPH was analyzed based on a 'wobbling-in-cone' model. The presence of chlorpromazine decreased the cone angle from 42 degrees to 39 degrees, while the rotational relaxation time phi f (approximately 2 ns) was not significantly changed by the presence of chlorpromazine. These results demonstrate that chlorpromazine decreased the mobility of not only lipids but also membrane proteins.
Subject(s)
Antipsychotic Agents/pharmacology , Chlorpromazine/pharmacology , Cytochrome P-450 Enzyme System/metabolism , Intracellular Membranes/drug effects , Membrane Lipids/metabolism , Phospholipids/metabolism , Adrenal Cortex/drug effects , Adrenal Cortex/metabolism , Animals , Cattle , Diffusion , Intracellular Membranes/metabolism , Kinetics , Mitochondria/drug effects , Mitochondria/metabolism , RotationABSTRACT
Highly purified cytochrome P450scc from bovine adrenal cortex mitochondria was inserted in artificial phospholipid membranes prepared from phosphatidylcholine to study the main principles of its membrane organization in the model system. Topology of the cytochrome P450scc polypeptide chain in proteoliposomes was studied by limited proteolysis with trypsin or chymotrypsin followed by immunochemical identification of the products of proteolysis products of the membrane-bound heme protein. It is shown that limited proteolysis of cytochrome P450scc in proteoliposomes results in a significant decrease of Vmax for the reaction of cholesterol hydroxylation to pregnenolone in the reconstituted system in the presence of exogenously added adrenodoxin-reductase and adrenodoxin. However, after proteolytic modification of cytochrome P450scc with trypsin and chymotrypsin the affinity of the heme protein to adrenodoxin is increased. Different models of membrane organization as well as functional specificity of cytochrome P450scc in artificial membranes are discussed.
