Reproducible and rapid methods for the isolation and assay of a-factor, a yeast mating hormone.
The Saccharomyces cerevisiae mating hormone a-factor has been difficult to isolate reproducibly in sufficient yields by methods using ion-exchange chromatography, probably because of its pronounced hydrophobicity. In this work, a hydrophobic adsorbent (Amberlite XAD-2), in an insoluble bead from, was used to isolate larger (up to sixfold greater than previous reports) and quite reproducible (12% standard deviation) quantities of a-factor by adsorption from cell-free filtrates of a cultures. Moreover, when the beads were added to the cultures at the time of inoculation, sixfold greater yields were obtained than when a-factor was adsorbed to the beads from cell-free filtrates. a-Factor was readily eluted from the beads with 1-propanol. The same adsorbent could also be used in the partial purification of the less hydrophobic alpha-factor. Adsorption of both hormones by Amberlite XAD-2 gave a degree of purification comparable to that obtained by the first steps of previously published methods while providing larger yields of hormones. The present procedure is shorter, simpler, and, for a-factor, more reproducible. The activities of both hormones were quantitated by using an assay in which the size distribution of cells in the population was monitored after the addition of hormone of the opposite mating type. The extent of increase in cell size which accompanies hormone treatment is a function of the hormone concentration. To ensure solubilization of a-factor in the aqueous bioassay system, samples were diluted into bovine serum albumin solutions and sonicated before assaying. The resulting assay is most sensitive at hormone concentrations between 0.05 and 2 U/ml, can reliably detect as little as 0.16 ng of hormone, gave results reproducible within 16%, and is convenient for a large number (>100) of samples.
D000327:Adsorption; D001681:Biological Assay; D005656:Fungal Proteins / Q000032:analysis / Q000302:isolation & purification* / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000032:analysis*
D005656:Fungal Proteins