Effect of filipin on the permeability of red beet and potato tuber discs.

The discovery of an acylated steryl glucoside in plant tissue in 1964 (13) raised the number of known forms of sterols in plants to four. These are the free sterols, sterol esters, steryl glycosides, and acylated steryl glycosides. Kiribuchi et al. (12) analyzed the sterol content of soybean and found that sterol ester was present in very small amount and that the sterol was mostly in the form of steryl glucoside and acylated steryl glycoside. The most reliable quantitative analyses of the different forms of sterols in plant tissue are those of Galliard (4, 5). He has found that 9.1% of the total lipid of potato tubers is sterol in the four forms mentioned above: 0.6% free sterol, 0.1% sterol esters, 2.0%o steryl glucoside, and 6.4% acylated steryl glucoside (4). In the case of postclimacteric apples 29.2% of the total lipid was sterol derivatives: 20.1% free sterol, 2.1% sterol esters, 5.4% steryl glucoside, and 1.6% acylated steryl glucoside (5). Several recent papers have examined the biosynthesis of steryl glucoside and acylated steryl glucoside (3, 9, 10, 14); however, there has been no indication of the physiological function of these sterols and derivatives. Grunwald (8) has made an approach to this problem by studying the effect of added sterols on the efflux of betacyanin from red beet discs. The polyene antibiotic filipin, isolated from Streptomyces filipinensis, has been shown to antagonize fungal spore germination and growth (6, 7). A tentative structure has been proposed for filipin showing it to be a 35 C pentaene (1); however, it now appears that filipin is a complex of at least four components (15). The effect on growth can be prevented and reversed by addition of cholesterol to the medium (6, 7). Work with model membrane systems of lipid monolayers and bilayers has shown that the artificial membranes are affected by filipin only when cholesterol has been previously incorportited into the membrane (2, 11). Mycoplasma laidlawii is susceptible to filipin only if the growth conditions have permitted the organism to incorporate cholesterol into the membrane (16). The inhibition of yeast growth by filipin can be antagonized by sitosterol, ergosterol, and stigmasterol as well as cholesterol (6, 7). These observations indicate that filipin may be used as a diagnostic test for sterol-containing membranes. It therefore occurred to us to test the effect of filipin on higher plant tissues. Cores of plant material were cut with a cork borer 7.5 mm in diameter. Discs approximately 1 mm thick were cut free-hand from this core. The discs were washed in tap water before use. Aging of the discs for 24 hr in tap water did not change the response to filipin. The reaction mixtures consisted of 8.0 ml of