The yeast Saccharomyces cerevisiae has been studied during cultivation with a naturally occurring antimycotic azasterol. At very low concentrations (1.0 to 10.0 ng/ml), where growth retardation is not observed, an unusual sterol, ergosta-8,14-dien-3,8-ol, accumulates in high concentrations. Upon removal of the azasterol from the culture, the 8,14-diene is converted to ergosterol. Much smaller amounts of another 8,14-sterol, but with an additional unsaturation, have also been observed. Total sterol accumulation is higher in cultures containing subinhibitory levels of the antimycotic agent than the amounts of normal sterol accumulation in control cultures. With between 10 and 100 ng of azasterol per ml a transitory cessation ofgrowth is observed from which the culture is able to recover. At much higher concentrations growth inhibition and even cell lysis results. Competitive inhibition of sterol 24(28)methylene reductase is demonstrated.Antibiotic agents specifically effective against fungi are rare in nature. A family of these agents has recently been isolated from Geotrichum flavo-bruneum (5), and the most abundant of the compounds was identified as 15-aza-24-methylene-D-homocholestadiene (6) ( Fig. 1). These azasterols inhibit fungal growth at very low concentrations but are virtually without effect on bacteria. We became interested in these compounds when we recognized that they are structural analogues of yeast sterols, especially fecosterol. In a previous paper we showed in vitro inhibition offecosterol formation by the antimycotic compound (3).Inhibition of growth and lysis of fungi has been demonstrated with the sterol-binding polyene antibiotics. The MATERIALS AND METHODSSaccharomyces cerevisiae strain 3701B was used in this study. Cultures were grown in broth containing 1% tryptone, 0.5% yeast extract, and 2% carbon source. All growth experiments were conducted at 28°C with shaking. Growth inhibition studies were conducted in broth with either 2% glucose or 2% ethanol. A logarithmic culture to be used as inoculum was harvested by centrifugation, and the cells were resuspended to 1/20 their original volume of broth. Tubes (15 by 150 mm) were prepared with 10 ml of broth and the appropriate azasterol concentration. The optical density of each culture tube was determined before inoculation, and sufficient cells were added to increase the density by 20 Klett units; changes in culture density were monitored with a Klett-Summerson photoelectric colorimeter.Sterol extraction, thin-layer chromatographic separation and purification of sterols, and coupled gas chromatography-mass spectroscopy have been described previously (4). Gas chromatography was performed in a Varian 2740 gas chromatograph with an associated CDS 111 data processor. Ultraviolet absorption spectroscopy was performed on a Cary model 11 recording spectrophotometer.Accumulation oflabeled sterols was accomplished in broth containing L-[methyl-"C]methionine (0.1 ,uCi/ml, 2 ,uM) and the azasterol (75 ng/ml, 0.18 IzM) Cultures (100 ml) were incubat...
15-Aza-25-methylene-D-homocholesta-8,14-dien-3beta-ol, an antimycotic agent, at a concentration of 75 ng/ml inhibited ergosterol biosynthetis in Saccharomyces cerevisiae strain 3701B resulting in the accumulation of an unusual sterol. Experimental data presented indicate that this sterol is ergosta-8,14-dien-3beta-ol. The accumulation of the compound is supportive of current models of biosynthetic pathways for sterols in yeast and is consistent with inhibition by the azasterol of the delta14 sterol reductase.
A naturally occurring azasterol has been shown to inhibit sterol transmethylation in both in vitro and in vivo in the yeast Saccharomyces cerevisiae. The inhibition was competitive, with a calculated dissociation constant of 43 muM. The compound prevented the accumulation of ergosterol in aerobically adapting cells. Cultures forced to gain energy by respiration were found to be much more sensitive to growth inhibition by the azasterol than those cells fermenting glucose. The growth inhibition is reversible at low concentrations of the azasterol.
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