Abstract
Abstract The heat-stable factor required for fatty acid, hydrocarbon, and drug hydroxylation in a reconstituted liver microsomal enzyme system containing hemoprotein P-450 has been identified as phosphatidylcholine. Synthetically prepared dioleoylglyceryl-3-phosphorylcholine was fully active when substituted for the microsomal factor, as judged by the rate of laurate, hexane, octane, ethylmorphine, or benzphetamine hydroxylation in the presence of TPNH, oxygen, hemoprotein P-450, and TPNH-hemoprotein P-450 reductase. Various acyl derivatives of glyceryl-3-phosphorylcholine showed, at their optimal concentrations, the following increasing order of activity with benzphetamine as the substrate: distearoyl; a mixture of 1-monopalmitoyl and 1-monostearoyl; dipalmitoyl; dioleoyl; and a mixture of dilauroyl and monolauroyl. Electron transfer from TPNH to hemoprotein P-450, as determined from the carbon monoxide reduced difference spectrum under anaerobic conditions, was completely dependent upon the presence of microsomal lipid. Stopped flow measurements showed that the rate of electron transfer was biphasic in the presence of the lipid, with a rapid phase largely completed in less than 1 sec and having a first order rate constant of about 100 min-1, followed by a slow phase having a first order rate constant of about 6 min-1, but only the slow rate could be detected in the absence of the lipid. Under similar conditions, but with air as the gas phase, the turnover number (moles of benzphetamine hydroxylated per mole of hemoprotein P-450) was 22 min-1. These results show that the lipid is essential for the enzymatic reduction of hemoprotein P-450 and that only the rapid phase of the reaction is adequate to support the observed rate of substrate hydroxylation.
Highlights
Under similar conditions, but with air as the gas phase, the turnover number was 22 min-l
Vol 245, 1\Jo. 18 reaction was found to be completed mostly in less than 1 see, and the first order rate constants for the rapid and slow phases of hemoprotein P-450 reduction in the complete system containing Fraction B were determined to be about 100 mine1 and 6 min-r, respectively, whereas in the absence of the lipid only the slow rate could be detected. In these and other stopped flow experiments at intermediate lipid concentrations the rapid phase of hemoprotein P-450 reduction was at least as rapid as the hydroxylation reaction, which has a turnover number of 22 min+
These results clearly show that the presence of the lipid is necessary for the enzymatic reduction of hemoprotein P-450
Summary
The heat-stable factor required for fatty acid, hydrocarbon, and drug hydroxylation in a reconstituted liver microsomal enzyme system containing hemoprotein P-450 has been identified as phosphatidylcholine. Hemoprotein P-450 is known to play a central role in the hydroxylation of a variety of substrates in liver microsomes This pigment was obtained from rabbit liver microsomes by Lu and Coon [1, 2] in a solubilized form capable of catalyzing fatty acid w-hydroxylation in the presence of TPNH, oxygen, and two additional microsomal components: hemoprotein P-450 reductase (previously referred to as TPNH-cytochrome c reductase) and a heat-stable fraction called Fraction 13. The hemoprotein P-450 and reductase fractions, after concentration by adsorption onto alumina Cy gel and elution with phosphate buffer [3], were more completely and more consistently dependent upon the lipid component for hydroxylation activity than when prepared without the sonic disruption step.
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