Evidence for Multiple Molecular Forms of Yeast β-Glucosidase in a Hybrid Yeast

Abstract

A mixture of β-glucosidases from Saccharomyces fragilis (Y-18) and S. dobzhanskii (Y-19) eluted from diethylaminoethyl cellulose in two peaks, whereas the enzyme from a hybrid, S. fragilis × S. dobzhanskii (Y-42), eluted in a single broad peak. The highest Y-42 activity fractions eluted at a sodium chloride molarity which was intermediate to the molarities at which most of the Y-18 and Y-19 activity was eluted. In cellulose polyacetate strips, Y-42 enzyme migrated as a diffuse band which spanned the distances migrated by the enzymes from the parent yeast strains. Antisera against either Y-18 or Y-19 enzyme precipitated 80 to 90% of Y-42 enzyme activity. When Y-42 enzyme was dissociated by heat or urea and reacted with parental antiserum, a concomitant increase in the opposite parental activity was demonstrable in both precipitation and complement-fixation (CF) tests. Urea-dissociated β-glucosidases were resolvable by sucrose-gradient centrifugation into multiple bands displaying specific CF activity. When the enzymes were exposed to 4 m urea for 12 min, particles of approximately 110,000 molecular weight were obtained. By extending the exposure time to 40 min, and incorporating 0.5 m urea in the gradients, smaller particles were detected with molecular weights ranging from 18,000 to 23,000. Attempts to regenerate enzyme activity after dissociation with urea were only moderately successful. Results suggested that a slightly acidic environment favored reassociation, as did the presence of 2-mercaptoethanol. Residual urea also seemed important. It is proposed that the structural genes for both Y-18 and Y-19 enzyme are present in Y-42 cells with either independent or closely interacting regulatory mechanisms. Since synthesis of the two parental-type polypeptides may be unequal, the availability of enzyme subunits for subsequent polymerization in the cell cytoplasm might be equalized at the polysome level. Random association of subunits would produce a binomial distribution of true hybrid enzyme molecules.