Terence J. Scallen

Sterol carrier and lipid transfer proteins.

The discovery of the sterol carrier and lipid transfer proteins was largely a result of the findings that cells contained cytosolic factors which were required either for the microsomal synthesis of cholesterol or which could accelerate the transfer or exchange of phospholipids between membrane preparations. There are two sterol carrier proteins present in rat liver cytosol. Sterol carrier protein 1 (SCP1) (Mr 47 000) participates in the microsomal conversion of squalene to lanosterol, and sterol carrier protein 2 (SCP2) (Mr 13 500) participates in the microsomal conversion of lanosterol to cholesterol. In addition SCP2 also markedly stimulates the esterification of cholesterol by rat liver microsomes, as well as the conversion of cholesterol to 7 alpha-hydroxycholesterol - the major regulatory step in bile acid formation. Also, SCP2 is required for the intracellular transfer of cholesterol from adrenal cytoplasmic lipid inclusion droplets to mitochondria for steroid hormone production, as well as cholesterol transfer from the outer to the inner mitochondrial membrane. SCP2 is identical to the non-specific phospholipid exchange protein. While SCP2 is capable of phospholipid exchange between artificial donors/acceptors, e.g. liposomes and microsomes, it does not enhance the release of lipids other than unesterified cholesterol from natural donors/acceptors, e.g. adrenal lipid inclusion droplets, and will not enhance exchange of labeled phosphatidylcholine between lipid droplets and mitochondria. Careful comparison of SCP2 and fatty acid binding protein (FABP) using six different assay procedures demonstrates separate and distinct physiological functions for each protein, with SCP2 participating in reactions involving sterols and FABP participating in reactions involving fatty acid binding and/or transport. Furthermore, there is no overlap in substrate specificities, i.e. FABP does not possess sterol carrier protein activity and SCP2 does not specifically bind or transport fatty acid. The results described in the present review support the concept that intracellular lipid transfer is a highly specific process, far more substrate-specific than suggested by the earlier studies conducted using liposomal techniques.

Sterol carrier protein2 (SCP2)-mediated transfer of cholesterol to mitochondrial inner membranes.

Rats treated with cycloheximide accumulate cholesterol in the adrenal mitochondria. This cholesterol is largely associated with the mitochondrial outer membrane. The addition of homogeneous sterol carrier protein2 (SCP2) to these mitochondria stimulated pregnenolone production to a greater extent than with mitochondria from untreated rats. Adrenal mitochondria from cycloheximide-treated rats were incubated in the presence of aminoglutethimide and cycloheximide to prevent further utilization of cholesterol for pregnenolone synthesis. Outer and inner membrane fractions of these mitochondria incubated with and without 0.75 microM SCP2, were prepared following hypotonic disruption. Incubations of mitochondria in the presence of SCP2 caused a marked shift in cholesterol from outer to inner membrane fractions. The findings provide direct evidence for a role of SCP2 or a similar peptide in modulating transfer of cholesterol to the inner membrane site of cholesterol side chain cleavage.

Sterol Carrier and Lipid Transfer Proteins

Publisher Summary The discovery of the lipid exchange, transfer, or carrier proteins is a result of the findings that cells contained cytosolic factors that were required for microsomal synthesis of cholesterol or could accelerate the transfer or exchange of phospholipids between membrane preparations. Most of the lipid exchange transfer or carrier activities have been characterized as cytosolic factors. These activities might be concentrated in cell particulate fractions, such as microsomes, and might be released to varying extents by tissue homogenization and/or treatment of the “cytosolic” fraction. The phospholipid exchange and transfer activities have been determined by the exchange or transfer of labeled phospholipids between microsomes and mitochondria or liposomes. These more direct assays involving aspects of cholesterol metabolism suggest the possibility that there is greater biological specificity for sterol carrier protein 2 (SCP 2) than is implied by the term non-specific lipid transfer protein. Another protein with apparent biological specificity is fatty acid-binding protein (FABP). Three approaches have been employed to determine fatty acid-binding protein (FABP) activities: Sephadex G-50 chromatography of liver cytosol following addition of [ 14 C] oleate; use of hydrophobic Lipidex 1000 to separate FABP-bound from unbound fatty acids; and immunoprecipitation technique.

Evidence for sterol carrier protein2-like activity in hepatic, adrenal and ovarian cytosol.

Purified sterol carrier protein2 (SCP2) from rat liver stimulated utilization of endogenous cholesterol for pregnenolone synthesis by adrenal mitochondria. Cytosolic preparations of rat liver, adrenal and luteinized ovary were also stimulatory in mitochondrial pregnenolone synthesis to different extents. Treatment of all preparations with rabbit anti-rat SCP2 IgG neutralized the stimulatory effects, and immunoprecipitated proteins gave similar patterns on SDS-gradient polyacrylamide gel electrophoresis. Treatment with rabbit pre-immune IgG had no effect on these parameters. Thus, proteins which are immunochemically compatible with hepatic SCP2 appear to be present in steroidogenic tissues and may play a role in control of mitochondrial cholesterol side chain cleavage activity.

Purification of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Abstract This paper describes a rapid purification procedure for 3-hydroxy-3-methylglutaryl coenzyme A reductase, the major regulatory enzyme in hepatic cholesterol biosynthesis. A freeze-thaw technique is used for solubilizing the enzyme from rat liver microsomal membranes. No detergents or other stringent conditions are required. The purification procedure employs Blue Dextran-Sepharose-4B affinity chromatography, and purification can be carried out from microsomal membranes to purified enzyme in 8 to 10 hours. The purified enzyme has a specific activity of 517 nmoles/min/mg protein, and it is 975-fold purified with respect to the original microsomal membrane suspension. SDS polyacrylamide gel electrophoresis of the purified enzyme shows only trace impurities; the subunit molecular weight for the enzyme measured by this technique is 47,000.

The effect of chronic luteinizing hormone treatment on adult rat Leydig cells

We investigated the chronic effects of luteinizing hormone (LH) treatment on adult rat Leydig cell structure and function. Two groups of sexually mature male Sprague-Dawley rats were used; controls and rats implanted subdermally with LH-filled Alzet miniosmotic pumps (delivers 24 micrograms of LH per day). After 2 weeks of LH treatment, testes of these rats were fixed by 2.5% glutaraldehyde in cacodylate buffer and processed and embedded in epon-araldite for light and electron microscopy and electron microscopic immunocytochemistry. Using light microscopic stereology, Leydig cell volume density, number of Leydig cells per testis, and the average volume of a Leydig cell were determined. Additionally, the organelle volumes per Leydig cells were quantified by electron microscopic stereology. Sterol carrier protein-2 (SCP2) and catalase in Leydig cells were immunolocalized via the Protein A gold method. Isolated and purified Leydig cells were used to determine the LH-stimulated (100 ng/ml) testosterone secretory capacity per Leydig cell in vitro and to compare the SCP2 and catalase content in equal numbers of Leydig cells using immunoblot analysis. After 2 weeks of LH-treatment, Leydig cell number per testis and the average volume showed a two-fold increase. All organelles tested, except the lipid droplets, were significantly (P < 0.05) increased two-fold in volume per Leydig cell. Testosterone secretory capacity per Leydig cell was increased approximately six-fold in the LH-treated group. Immunolabeling studies showed that the intraperoxisomal SCP2 content was significantly greater (P < 0.05) and the catalase content was significantly lower (P < 0.05) in LH-treated rats compared to to controls. Immunoblots showed that the total SCP2 content per cell is greater and the catalase content per cell is similar in Leydig cells of LH-treated rats compared to controls. In summary, chronic LH treatment produced hyperplasia, hypertrophy and increased testosterone secretory capacity in leydig cells of adult rats. However, the increase in the testosterone secretory capacity per Leydig cell exceeds the degree of Leydig cell hypertrophy, which cannot be explained by a generalized increase in volumes of all Leydig cell organelles in the LH-treated rats. These results also suggested that chronic LH treatment induces differential synthesis of peroxisomal proteins, i.e. an increase in SCP2 synthesis and no change in catalase synthesis. This resulted in peroxisomes rich in SCP2 and lower in catalase. Significance of these effects in relation to the increased steroidogenic capacity of Leydig cells remains to be determined.

Sterol carrier protein hypothesis: Requirement for three substrate-specific soluble proteins in liver cholesterol biosynthesis

The 105,000 x g supernatant (S105) of liver is required for the conversion of squalene to cholesterol by microsomal membranes. Substantial controversy has existed concerning the properties of what was originally considered to be a single sterol carrier protein present in S105 and required for this conversion. We have now resolved this controversy by the discovery that S105 contains several sterol carrier proteins. Based upon experiments with three substrates, three substrate-specific soluble proteins (with different properties) have been identified which operate at distinct points in microsomal cholesterol synthesis. These proteins are provisionally designated sterol carrier protein1 (SCP1), sterol carrier protein2 (SCP2), and sterol carrier protein3 (SCP3). SCP1 is required for the microsomal conversion of squalene to lanosterol, SCP2 for the microsomal conversion of 4,4-dimethyl-Δ8-cholesterol to C27-sterols, and SCP3 for the microsomal conversion of 7-dehydrocholesterol to cholesterol. Available evidence is consistent with the proposal that a given sterol carrier protein is a soluble constituent of a single microsomal enzyme or enzyme complex, and that it participates both as a carrier for the water-insoluble substrate and as an essential enzyme constituent facilitating catalysis. It may well be that enzymatic transformations of water-insoluble substrates require both microsomal membranes and substrate-specific soluble proteins. This requirement could be a common biological mechanism for water-insoluble substrates.

Solubilization of 3-hydroxy-3-methylglutaryl coenzyme A reductase from lyophilized rat liver microsomes: Lack of evidence for cold lability in this soluble enzyme preparation

Summary This paper describes a simple and reliable method for the solubilization of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase by extracting lyophilized microsomes with buffer. This procedure is suitable for the preparation of large quantities of soluble enzyme and does not require the use of organic solvents, detergents or other stringent conditions which might alter the properties of the enzyme. A test of cold lability in 3-hydroxy-3-methylglutaryl coenzyme A reductase solutions is described. No substantial cold lability was observed. These results indicate that the sensitivity to cold reported by other investigators probably is not an inherent property of the enzyme.

Conversion of diglyceride to triglyceride by rat liver microsomes: A requirement for the 105,000 × g supernatant☆

Abstract This paper describes a requirement for the 105,000 × g supernatant of rat liver for the synthesis of triglyceride from diglyceride and palmityl coenzyme A by rat liver microsomes. ATP and magnesium chloride are also required. The incorporation of both [1- 14 C]-palmityl coenzyme A and [1- 14 C]-diolein into triglyceride has been observed. The 105,000 × g supernatant has no enzymatic activity for this reaction when incubated in the absence of microsomes. The supernatant contains a soluble, essential protein which is nondialyzable, heat sensitive, and destroyed by trypsin. Net synthesis of triglyceride has been demonstrated by chemical analysis.

Characterization of native sterol carrier protein

Recently we made the proposal and presented evidence that a noncatalytic carrier protein (sterol carrier protein or SCP) is involved in the conversion of squalene into cholesterol by liver microsomes [l-7]. This was based on observations made with an acetone powder of rat liver microsomes which required the 105,000 g supematant (SIos) of rat liver for this conversion [8,91. We now report the following accomplishments: a) the purification of native SCP from unheated Slos of rat liver; b) when purified SCP is added to an inactive buffer-washed microsomal preparation from rat liver, it is capable of reconstituting the entire biosynthetic sequence from squalene to cholesterol; c) SCP binds not only water-insoluble precursors of cholesterol, but also lipid components of lipoprotein, i.e., cholesterol, cholesterol ester, phospholipid and tryglyceride; d) the amino acid composition of native SCP closely resembles the amino acid composition of the protein moiety of serum low density lipoprotein (LDL). These findings are compatible with the novel hypothesis that SCP simultaneously serves both as a carrier for the enzymatic synthesis of cholesterol and as the protein component of LDL, specifying LDL assembly. Thus the enzymatic synthesis by the liver of lipid components (e.g., cholesterol) of LDL may coincide with LDL assembly.