Studies on the Biological Properties of Polyene Antibiotics (1972)

Abstract The polyene antibiotic filipin has been shown previously to mediate changes in membrane permeability in natural and artificial biological membranes. The concept has emerged that the interaction of filipin is dependent upon the presence of sterol, preferably cholesterol. The present report provides strong evidence that filipin can interact with sterol in a stereochemically and stoichiometrically defined manner to produce a filipin-sterol complex or adduct. Filipin (F) has a characteristic ultraviolet absorption spectrum with 4 maxima. The ratio of absorbance of Peak 3, λ = 320 nm, to Peak 1, λ = 356 nm, is 0.7 in organic solvents and 0.8 in aqueous solution. Addition of cholesterol (C) causes a striking spectral change in only the aqueous solutions of filipin so that, although the absorption maxima do not shift, the ratio of absorbance of Peak 3 to Peak 1 becomes approximately 2.5. The formation of the filipin complex, as defined by the spectral change, is specific for stereols. No significant effect on either the formation or breakdown of the filipin-cholesterol complex (F-C) was evident by addition of bovine serum albumin, sucrose, galactose, lecithin, cetyl alcohol, 4 m NaCl, or 4 m urea, or variation of pH from 2 to 9. Addition of the nonionic detergent, Triton X-100, or organic solvents miscible with H2O, broke the F-C complex, whereas heating to 80–90° and cooling caused a reversible breaking and reforming of the F-C complex. The spectral change also occurs when filipin is added to preparations of egg lecithin-cholesterol liposomes (84:16 to 50:50 mole %). A study of the sterol structural requirements necessary for optimal interaction indicates that with the free steroid in H2O, the interaction is primarily hydrophobic, with the presence of a cholestane ring structure and Δ22 double bond producing the most favorable interaction. But in the liposomal system there is an additional requirement for a 3β-OH on the steroid nucleus. The same structural requirements are also necessary for optimal interaction of filipin with mixed lecithin-sterol monolayers. A quantitative evaluation of the spectral change mediated by cholesterol indicated that the filipin interacted identically with both the free and liposomally bound sterol from 10-6 to 10-4 m antibiotic, such that a defined number of cholesterols were combined with 1 filipin molecule. The spectral change was also evident in red cell ghost membranes and in cells and membranes obtained from Mycoplasma laidlawii grown in the presence of cholesterol. No spectral change was evident in cells or membranes obtained from M. laidlawii grown in the absence of a sterol or in the presence of epi-(3α-hydroxyl)-cholesterol. Analysis of a suspension of synthetic lecithin (L), lecithin-cholesterol (L-C), or L-C-filipin dispersions (L-C-F) by differential scanning calorimetry indicates that addition of C to L (L-C) abolishes the sharp phase transition characteristic of the conversion of lecithin from the crystalline (Lα) to liquidcrystalline (Lβ) form. Addition of F to L (L-F) has no effect on the lecithin phase transition; but addition of F to L-C (L-C-F) causes the phase transition of the lecithin to quantitatively reappear. These results suggest that filipin can interact stoichiometrically with cholesterol and that in membrane systems this interaction is capable of altering some of the physical properties of the membrane, thus providing a basis for filipin-induced changes in membrane permeability.