Abstract
Abstract A requirement for the 105,000 x g supernatant of rat liver (S105) is demonstrated for five different reactions in cholesterol biosynthesis. This is best delinated using a buffer-washed acetone powder of liver microsomes. Incubation of S105 alone with each substrate yields chemically unchanged substrate. Evidence that S105 contains a protein responsible for this requirement includes the observations that it is nondialyzable, heat-labile, and destroyed by trypsin. Chromatography of S105 on Sephadex G-75 yields a protein peak which travels at the void volume and has associated with it both endogenous cholesterol and the ability to stimulate sterol synthesis as assayed in the conversion of Δ7-cholestenol to Δ5,7-cholestadienol. In addition, virtually all of the cholesterol present in the 105,000 x g supernatant of liver is bound to protein. These results plus other observations support the following hypothesis. S105 contains a noncatalytic carrier protein (STEROL CARRIER PROTEIN) which originates from the endoplasmic reticulum, binds the substrate, and makes the substrate reactive to the sterol-synthesizing enzymes present in the acetone powder of liver microsomes. The participation of Sterol Carrier Protein (SCP) may be of key importance in the understanding of the enzymatic synthesis of cholesterol.
Highlights
We proposed for the first time that the 105,000 x g supernatant of rat liver contains a noncatalytic carrier protein which originates from the endoplasmic reticulum, binds the substrate, and makes the substrate reactive to the sterol-synthesizing enzymes present in the acetone powder [4,5]
Flask 1 shows that the acetone powder by itself achieved 34% conversion to sterol
See “Materials and Methods” for buffer washing procedure of acetone powder; each incubation flask contained TPNH and DPN which were dissolved in 1 ml of buffer; final concentration of TPNH, 1.2 X lWa M; DPN, 3 X Wa M in an incubation volume of 5 ml
Summary
Preparation of Substrates-Squalene-3H (I) was prepared as previously described [2] ; the specific activity was at least 10,000 cpm per fig (counting efficiency 30%) [2]. Squalene-2, 3-oxide-3H (II) was prepared biosynthetically from nn-mevalonic acid lactone-2-3H. Details of this preparation will be described elsewhere, the high resolution mass spectrum of biologically synthesized squalene-2,3-oxide-3H was essentially identical with the mass spectrum of chemically synthesized squalene-2,3-oxide; specific activity, 6,000 cpm per pg. A5sQholestadienol (VIb) were purchased from Mann; (VIb) was recrystallized prior to use. A5*24-Cholestadienol-26-r4C (VIIa) was purchased from New England Nuclear; specific activity, 57 Ci per mole. Reagents-Reagents were prepared as previously described [2]
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