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The multiple actions of sterol carrier protein-2 (SCP-2) in intracellular lipid circulation and metabolism originate from its gene and protein structure. The SCP-x/pro-SCP-2 gene is a fusion gene with separate initiation sites coding for 15-kDa pro-SCP-2 (no enzyme activity) and 58-kDa SCP-x (a 3-ketoacyl CoA thiolase). Both proteins share identical cDNA and amino acid sequences for 13-kDa SCP-2 at their C-termini. Cellular 13-kDa SCP-2 derives from complete, posttranslational cleavage of the 15-kDa pro-SCP-2 and from partial posttranslational cleavage of 58-kDa SCP-x. Putative physiological functions of SCP-2 have been proposed on the basis of enhancement of intermembrane lipid transfer (e.g., cholesterol, phospholipid) and activation of enzymes involved in fatty acyl CoA transacylation (cholesterol esters, phosphatidic acid) in vitro, in transfected cells, and in genetically manipulated animals. At least four important SCP-2 structural domains have been identified and related to specific functions. First, the 46-kDa N-terminal presequence present in 58-kDa SCP-x is a 3-ketoacyl-CoA thiolase specific for branched-chain acyl CoAs. Second, the N-terminal 20 amino acid presequence in 15-kDa pro-SCP-2 dramatically modulates the secondary and tertiary structure of SCP-2 as well as potentiating its intracellular targeting coded by the C-terminal peroxisomal targeting sequence. Third, the N-terminal 32 amino acids form an amphipathic a-helical region, one face of which represents a membrane-binding domain. Positively charged amino acid residues in one face of the amphipathic helices allow SCP-2 to bind to membrane surfaces containing anionic phospholipids. Fourth, the hydrophobic faces of the N-terminal amphipathic a helices along with beta strands 4, 5, and helix D form a ligand-binding cavity able to accommodate multiple types of lipids (e. g., fatty acids, fatty acyl CoAs, cholesterol, phospholipids, isoprenoids). Two-dimensional 1H-15N heteronuclear single quantum coherence spectra of both apo-SCP-2 and of the 1:1 oleate-SCP-2 complex, obtained at pH 6.7, demonstrated the homogenous formation of holo-SCP-2. While comparison of the apo- and holoprotein amide fingerprints revealed about 60% of the resonances remaining essentially unchanged, 12 assigned amide residues underwent significant chemical-shift changes upon oleic acid binding. These residues were localized in three regions: the juncture of helices A and B, the mid-section of the beta sheet, and the interface formed by the region of beta strands 4, 5, and helix D. Circular dichroism also showed that these chemical-shift changes, upon oleic acid binding, did not alter the secondary structure of SCP-2. The nuclear magnetic resonance chemical shift difference data, along with mapping of the nearby hydrophobic residues, showed the oleic acid-binding site to be comprised of a pocket created by the face of the beta sheet, helices A and B on one end, and residues associated with beta strands 4, 5, and helix D at the other end of the binding cavity. Furthermore, the ...

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CHEMISTRY: MONEY ET AL. 901discussed with respect to the possibilities of reducing or augmenting antigenicity, modifying protein properties, and permanently attaching drugs to receptor sites. Potential applications to the treatment of cancer, organ transplantation, production of antibodies to viruses, treatment of sickle-cell anemia, myasthenia gravis, phenylketonuria, and mental illness have been mentioned. The usefulness of protein transformation in the investigation of receptor sites has been pointed out.The author would like to thank Nechama S. Kosower of the Department of Medicine, Albert Einstein College of Medicine, Yeshiva University, for many useful and critical discussions.* Supported in part through grant GP-251 from the National Science Foundation.f Alfred P. Sloan fellow, 1960-1964. 1 Kosower, E. M., these PROCEEDINGS, 51, 1141PROCEEDINGS, 51, (1964. 2Hunter, M. J., and M. L. Ludwig, J. Am. Chem. Soc., 84, 3491 (1962 March 3, 1966 Numerous theories regarding the biosynthesis of the Yohimbe, Strychnos, and Cinchona alkaloids have been propounded. '-5 In each of these hypotheses, condensation of tryptophan (or tryptamine) with various C9-Cjo units is invoked to rationalize the diversity of structure found in these bases, conveniently summarized in the pattern (Fig. 1, I). Incorporation experiments6 have demonstrated that administration of tryptophan-2-C14 to Rauwolfia serpentina plants leads to ajmaline-5-C" (II). However, in spite of numerous feeding experiments,7 8 the origin of the Cg-Cio unit remains obscure. Thus, radioactive tyrosine, alanine, and mevalonic acid when fed to R. serpentina were not incorporated7 to any significant extent into ajmaline. These results seemed to invalidate those hypotheses requiring intervention and subsequent fission ----) of the units III, IV, and V which,

Although sterol carrier protein-2 (SCP-2) stimulates sterol transfer in vitro, almost nothing is known regarding the identity of the putative cholesterol binding site. Although great advances have been made in our understanding of vascular lipid transport via serum lipoproteins, much less is known regarding intracellular trafficking pathways of lipids. While spontaneous desorption and intracellular diffusion of lipids occur, they do not account for (i) the asymmetric distribution of cellular lipids, (ii) the synthesis of cholesterol in and targeted efflux from the relatively cholesterol poor endoplasmic reticulum, (iii) the lysosomal release and intracellular targeting of cholesterol, fatty acids, and glycerides, or (iv) high density lipoprotein-mediated reverse cholesterol transport. Cellular lipids are transferred and targeted within the cell via vesicular pathways, cytosolic proteins, and possibly via as yet undefined interactions between cytosolic proteins and vesicular pathways.In addition to vesicular pathways, intracellular cholesterol is believed to be transferred and targeted within the cell via proteins such as SCP-2 1 (reviewed in Refs. 1-5), caveolin (reviewed in (Refs. 4 and 6 -11), or steroidogenic acute regulatory protein (12). SCP-2 is primarily a soluble protein, caveolin exists in both membrane-bound and soluble homo-and heterocomplex forms, and steroidogenic acute regulatory protein traffics from the endoplasmic reticulum to the inner mitochondrial membrane by mechanism(s) not yet understood.A variety of studies show positive correlation between SCP-2 expression and intracellular cholesterol transfer in vivo: biliary cholesterol secretion (13-15), lung surfactant formation (16), intestinal cholesterol absorption (17, 18), macrophage foam cell formation (19), diabetes (20), and cholesterol oxidation (21-23). Likewise, studies with intact transfected cells overexpressing SCP-2 confirm a role for SCP-2 in intracellular sterol trafficking (24 -30). Despite these observations, very little is known regarding the mechanism whereby SCP-2 transfers cholesterol between membranes. It has been suggested that interaction of SCP-2 with cholesterol (31-34) is essential for SCP-2-mediated intermembrane sterol transfer in vitro (35). Unfortunately, clear demonstration of cholesterol binding or saturation binding of other sterols to SCP-2 has been difficult to achieve.In addition to its involvement in intermembrane cholesterol transfer, recent reports that SCP-2 also interacts with long chain fatty acids (LCFA) and long chain fatty acyl-CoAs (LCFA-CoA) suggest additional role(s) for this protein in intracellular LCFA and/or LCFA-CoA trafficking. The pathway(s) whereby LCFA and their CoA derivatives traffic and are targeted within the cell are as yet undefined (reviewed in Refs. 36 and 37). Increasing evidence shows that the intracellular fatty acid binding proteins function in intracellular trafficking by enhancing LCFA uptake, intracellular diffusion, or metabolic targeting (reviewed in Refs. 36 -39). For over...