Collapsin, a member of the newly recognized semaphorin family, contributes to axonal pathfinding during neural development by inhibiting growth cone extension. The mechanism of collapsin action is poorly understood. Here we use a Xenopus laevis oocyte expression system to identify molecules involved in collapsin signalling, because several experiments have raised the possibility that heterotrimeric GTP-binding proteins might participate in these events. A collapsin response mediator protein of relative molecular mass (M(r)) 62K (CRMP-62) required for collapsin-induced inward currents in X. laevis oocytes is isolated. CRMP-62 shares homology with UNC-33, a nematode neuronal protein required for appropriately directed axonal extension. CRMP-62 is localized exclusively in the developing chick nervous system. Introduction of anti-CRMP-62 antibodies into dorsal root ganglion neurons blocks collapsin-induced growth cone collapse. CRMP-62 appears to be an intracellular component of a signalling cascade initiated by an unidentified transmembrane collapsin-binding protein.

AB STRACTThe terminal enzyme of the NADHdependent stearyl coenzyme A desaturase system has been isolated from rat liver microsomes. This desaturase is a single polypeptide of 53,000 daltons containing 62% nonpolar amino-acid residues and one atom of non-heme iron. The purified protein forms high molecular weight aggregates that can be dispersed by detergent procedures.Desaturase activity requires NADH, stearyl coenzyme A, oxygen, lipid, and the three enzymes, cytochrome b5 reductase (EC 1.6.2.2), cytochrome b5, and desaturase. Cytoehrome b5 is the direct electron donor to the desaturase, which appears to utilize the iron in the oxidationreduction sequence during desaturation of stearyl coenzyme A.The characterization of the liver microsomal stearyl coenzyme A desaturase system by workers in several laboratories (1-11), using spectral methods and partially purified components from hen and rat liver microsomes has shown that the oxygen-and NADH-dependent formation of oleyl coenzyme A involves cytochrome b5 reductase (EC 1.6.2.2; NADH : ferricytochrome b5 oxidoreductase), cytochrome b5, and a terminal, cyanidesensitive oxidase, the desaturase. The present report describes the isolation of the terminal component, the desaturase, from liver of rats induced for this enzyme. A sequence of extractions of contaminating proteins from microsomes, followed by solubilization of the desaturase by detergents and subsequent purification, results in a protein preparation that appears to be homogeneous by several criteria. It contains one molecule of non-heme iron, which appears to participate in stearyl coenzyme A desaturation in the presence of oxygen and reduced cytochrome b5. METHODS AND MATERIALSSodium deoxycholate (Matheson, Coleman and Bell) was purified as described (12). Sephadex G-75 was obtained from Pharmacia, and Whatman DEAE-cellulose (DE-52), from Reeve Angel. Triton X-100 was the purified product of Packard Instrument Co. The ['4C]deoxycholate was obtained from Mallinckrodt Chemical Works, the NADH, stearyl coenzyme A, and bathophenanthroline sulfonate were products of Sigma Chemical Co., and [14C]stearyl coenzyme A was a product of New England Nuclear. The egg lecithin was obtained from Supelco in a chloroform solution. We prepared clear liposomes by removing solvent in a nitrogen stream, adding 0.02 M Tris-acetate buffer, pH 8.1 (Tris buffer), to yield a suspension of 12.5 mg/ml, sonicating under nitrogen at 00 for 15-20 min at 40 mW, and centrifuging the slightly turbid suspension for 15 min at 100,000 X g to yield the clear liposome suspension, which was stored under nitrogen at 00 and used for no longer than 10 days. Abbreviation: Tris buffer, Tris-acetate buffer (pH 8.1). 4565Cytochrome b5 was prepared from either calf or rabbit liver microsomes by the detergent extraction procedure that yields the complete form of this heme protein (13). The proteins from the two species were indistinguishable as electron donors for the desaturase. The soluble catalytic fragment of cytochrome b5 was prepared by tryptic dige...

A species of cytochrome b 5 with a monomer molecular weight of 16,700 has been isolated from rabbit-liver microsomes by a procedure that uses detergents and avoids the use of any proteolytic or lipolytic enzymes. This detergent-extracted cytochrome b 5 is larger than the trypsin- or lipase-extracted enzyme, and appears to contain an extremely hydrophobic appendage of 40 amino acids, probably at the N-terminus. The hydrophobic character of the extra amino acid sequence leads to aggregation in the absence of detergents, and may be of considerable importance in the binding of the enzyme to microsomes. It is suggested that the hydrophilic portion of the cytochrome molecule, which bears the heme and is enzymatically functional, is oriented toward the surface of the membrane where it readily reacts with nonmicrosomal proteins, while the hydrophobic “tail” anchors the heme protein tightly to the membrane.

Two methods are reported for the formation of large, uniform-sized phospholipid vesicles. (5) or ether injection (6). Vesicles may be formed with phospholipid mixtures, purified phospholipids, or synthetic phospholipids. The methods of preparation and the properties of such vesicles have recently been reviewed (1, 7). Although multilamellar liposomes are easy to prepare, they have been criticized as a model system for the study of membrane phenomena (1). Thus, the kinetics of transport processes are more complex due to multiple diffusion barriers and it is not easy to obtain a uniform-sized population of vesicles. These disadvantages are overcome by the use of small, unilamellar vesicles; however, the surface of these vesicles has a high degree of curvature which affects many of the physical properties of the phospholipid in the vesicles (8, 9). One would then expect the large, unilamellar vesicles to resemble most closely biological membranes. It was previously shown (4) that treatment of 1 mol of egg lecithin with 2 mol of bile salt followed by gel filtration to remove the bile salt results in the formation of a uniform population of single-bilayer vesicles having an average diameter of 300 A. We now report that the treatment of 1 mol of egg lecithin with 0.5 mol of bile salt results in the formation of a uniform population of single-bilayer vesicles having an average diameter of 1000 A. These preparations, which have a large internal volume and are free of multilamellar structures, may be useful in cases in which previous techniques (5, 6) cannot be applied.MATERIALS AND METHODS Egg phosphatidylcholine (10), sodium deoxycholate (11), cytochrome b5 heme peptide (12), the catalytic fragment of NADH-cytochrome b5 reductase (13) (pH 5) or 1% ammonium molybdate (pH 5). One drop of liposomes (30-100 ,uM phospholipid) was placed on a Formvar-coated grid and, after 30 sec, one drop of staining solution was added; 30 sec later, the solution was drained off with filter paper and the grid was allowed to air dry. The grids were examined in a Hitachi llE electron microscope operated at 75 kV. Vesicle diameters were measured on several electron micrographs covering different areas on a grid and then averaged from at least 400 measurements.Method I: Formation of Large Vesicles from Small, Preformed Vesicles. Small, uniform-sized egg lecithin vesicles were prepared as described (11). Any solute to be entrapped was added to the vesicle solution at this point and the vesicles were adjusted to 20 mM phospholipid. The solution was then warmed to 25°C and an aliquot of 250 mM sodium deoxycholate was rapidly added and mixed to give a final mixture containing a ratio of deoxycholate to phospholipid of 1:2. The large vesicles began to form almost immediately, as indicated by. the increase in the light scattering of the solution, a change from nearly clear for the small vesicles to a transparent opalescence for the large vesicles. Vesicle formation was complete within 5-10 min at 250C (vesicles could also be formed at 40C...

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