A nicked 0-toxin (CO), obtained by limited proteolysis with subtilisin Carlsberg, causes almost no hemolysis while it retains a nearly intact cholesterol binding site below 20 "C. Neither electron microscopic evidence for the formation of arc-and ring-shaped structures on the membrane nor toxin-stimulated influx of extracellular Ca2 + are detected in CB-treated cells below 20°C. Thus, event(s) in the lytic process are responsible for the temperature dependency of hemolysis, which is also supported by the observation that C0 requires higher Arrhenius activation energy for hemolysis than the native toxin. Using CB as a probe due to its high affinity for membrane cholesterol without causing any obvious membrane changes, we demonstrated the possible existence of high-and low-affinity sites for &toxin on sheep erythrocytes. Both binding sites disappear by simultaneous treatment of the cells with sublytic doses of digitonin. Furthermore, C0 binds only to cholesterol among the chloroform/methanolextractable, lipid components of sheep and human erythrocytes but not to the protein components derived from them. These results strongly suggest that cholesterol is an essential component of the both high-and low-affinity sites, and also imply that the modes of existence of cholesterol in the red cell membrane are heterogeneous.Although the importance of cholesterol for the structure and function of biological membranes is recognized [l, 21, interesting problems concerning its organization in membranes, such as its precise orientation in the bilayer and its interaction with other membrane constituents, along with the role of cholesterol in membrane function remain to be answered. Cholesterol functions as a receptor for polyene antibiotics [3, 41, saponins [4], and thiol-activated cytolysins (hemolysins) such as 0-toxin (perfringolysin 0) [5] and cereolysin [6]. These molecules have been used as probes to investigate the distribution of cholesterol in membranes due to their specific interaction with cholesterol.

A nicked toxin whose hemolytic activity is temperature dependent was obtained by limited proteolysis of theta-toxin (Mr 54,000) with subtilisin. The nicked toxin (C theta) is a complex of two fragments: the N-terminal fragment (Mr 15,000) with basic isoelectric point and the C-terminal fragment (Mr 39,000) with the single cysteinyl residue of the toxin whose reduced form is essential for the hemolytic activity. C theta hemolyzes erythrocytes only at temperatures above 25 degrees C, whereas the native toxin hemolyzes them even at 10 degrees C. At temperatures below 25 degrees C, C theta does not hemolyze them although it does bind to membrane cholesterol and although no distinct difference was observed between the secondary structure of C theta and that of native toxin. It was found that C theta binds to the cells only in a reversible manner at low temperature, while the native one binds irreversibly to the cells within 10 min, which explains the cold lability of C theta on hemolysis. The structural basis of the cold lability was discussed through comparison of C theta with another nicked derivative of theta-toxin that was also obtained.

We have purified the translation restoring factor (RF) and the eukaryotic initiation factor 2 (eIF-2) stimulating protein (ESP) to near homogeneity from the postribosomal supernatant and the ribosomal salt wash, respectively, of rabbit reticulocyte lysate. They were isolated in the form of eIF-2 complexes, apparently in a 1:1 ratio. Their virtually identical NaDodSO,/ polyacrylamide gel electrophoretic patterns show, in addition to the eIF-2 a (38,000), f3 (52,000), and y (54,000) bands, peptide bands at approximately 80, 65, 57, 40, and 32 kilodaltons. The apparent Mr ofeither complex is about 450,000, whereas that of free translation restoring factor (RF) is approximately 250,000. At 0.5 mM Mge', both ESP and RF stimulate ternary complex (eIF-2.GTP.Met-tRNA1) formation catalyticallywith unphosphorylated eIF-2. Phosphorylation of the eIF-2 a subunit by preincubation with eIF-2 a kinase and ATP, which virtually blocks eIF-2-ESP interaction, results in only partial blocking of the interaction with RF. This may explain the translation restoring activity of RF.

Perfringolysin O (theta-toxin) is a cholesterol-binding and pore-forming toxin that shares with other thiol-activated cytolysins a highly conserved sequence, ECTGLAWEWWR (residues 430-440), near the C-terminus. To understand the membrane-insertion and pore-forming mechanisms of the toxin, we evaluated the contribution of each Trp to the toxin conformation during its interaction with liposomal membranes. Circular dichroism (CD) spectra of Trp mutant toxins indicated that only Trp436 has a significant effect on the secondary structure, and that Trp436, Trp438, and Trp439 make large contributions to near-UV CD spectra. Quenching the intrinsic Trp fluorescence of the wild-type and mutant toxins with brominated lecithin/cholesterol liposomes revealed that Trp438 and probably Trp436, but not Trp439, contributes to toxin insertion into the liposomal membrane. Near-UV CD spectra of the membrane-associated mutant toxins indicated that both Trp438 and Trp439 are required for the CD peak shift from 292 to 300 nm, a signal related to theta-toxin oligomerization and/or pore formation, suggesting a conformational change around Trp438 and Trp439 in these processes.

We have previously suggested the existence of two distinctive states of cholesterol in erythrocyte and lymphoma cell membranes as revealed by high- and low-affinity binding sites for theta-toxin of Clostridium perfringens [Ohno-Iwashita, Y., Iwamoto, M., Mitsui, K., Ando, S., & Nagai, Y. (1988) Eur. J. Biochem. 176, 95-101; Ohno-Iwashita, Y., Iwamoto, M., Ando, S., Mitsui, K., & Iwashita, S. (1990) Biochim. Biophys. Acta 1023, 441-448]. To understand factor(s) which determine membrane cholesterol heterogeneity, we analyzed toxin binding to large unilamellar liposomes composed of cholesterol and phospholipids (phosphatidylcholine/phosphatidylglycerol = 82:18, mol/mol). Liposomes containing phospholipids with 18-carbon hydrocarbon chains at both positions 1 and 2 of the glycerol have both high- and low-affinity toxin-binding sites with Kd values similar to those of intact erythrocytes, whereas liposomes with hydrocarbon chains containing 16 or fewer carbons at either position 1 or 2 have only low-affinity toxin-binding sites. The cholesterol/phospholipid ratio, in addition to the length of phospholipid hydrocarbon chain, also determines the number of toxin-binding sites, indicating that at least these two factors determine the topology of membrane cholesterol by creating distinctively different affinity sites for the toxin. Since theta-toxin binding detects specific populations of membrane cholesterol that are not detectable by the measurements of susceptibility to cholesterol oxidase and cholesterol desorption from membranes, the toxin could provide a unique probe for studying the organization of cholesterol in membranes.