Accumulation of Squalene during Hepatic Cholesterol Synthesis in vitro

Abstract

The synthesis in vitro of cholesterol from dl‐[2‐14C]mevalonic acid by the rat liver 18000 ×g supernatant was studied. It was shown that in the presence of air and necessary cofactors, the 18000 ×g supernatant incorporated dl‐[2‐14C]mevalonic acid mainly into cholesterol plus cholesterol esters while a small percentage of the radioactivity was associated with squalene, squalene oxide, lanosterol and some unidentified products. However, when serum (rat or human) was added to the incubation mixture, the incorporation of dl‐[2‐14C]mevalonic acid into cholesterol was decreased, with a concomitant accumulation of radioactive squalene. There was a stoichiometric relationship between the concentration of serum in the incubation mixture and the decrease in cholesterol synthesis or the accumulation of squalene.Pretreatment of the serum by dialysis, heat, or reaction with p‐chloromercuribezoate (PCMB), and N‐ethylmaleimide, all failed to annul the squalene accumulating effect of serum.Fractionation of rat serum showed that an α1‐globulin possessed the squalene accumulating activity. Examination of Cohn fractions of human serum showed that the squalene accumulating activity was present in Fraction III‐0, Fraction IV‐1 and Fraction IV‐4. Delipidization of Fraction IV‐1 led to an increase in the squalene accumulating activity of this fraction.Purification of delipidized Fraction IV‐1 by a combination of ammonium sulphate precipitation, ion‐exchange chromatography and perchloric acid precipitation gave a fraction, active in accumulating squalene, with the mobility of an α1‐globulin (α1‐protein). Ultracentrifugal analysis showed that the α‐protein was composed of a major component whose sedimentation velocity was 2.1 Svedberg units and a minor component of higher sedimentation velocity. Incubation of α1‐protein with [14C]cholesterol led to the binding of these lipids to the protein. Comparison of α1‐protein with an apo‐protein of α1‐liporprotein obtained from Dr. A. Scanu (Chicago) showed that both had similar physical, chemical and biological properties, indicating that α1‐protein, the squalene accumulating factor of delipidized Fraction IV‐1, is the apo‐protein of α1‐lipoprotein.The present evidence indicates that the squalene accumulating factor of serum acts by binding squalene.