Molecular Forms of Yeast Invertase

Abstract

The molecular forms of yeast invertase have been studied. It is shown that by gel filtration on Sephadex G‐200 it is possible to demonstrate the presence not only of a light, carbohydrate‐free, invertase, and a heavy invertase containing 50% carbohydrate, but also of a continuous spectrum of molecular forms that probably represent the sequential addition of mannose to the light form during the secretion process, which culminates in the formation on the heavy enzyme that is found outside the cytoplasmic membrane. The elution volume –void volume ratio in Sephadex G‐200 varies from 1.75 of the light to 1.05 of the heavy invertase. The separation of invertase has also been achieved by ion‐exchange chromatography and by isoelectric focusing and is facilitated by removal of the heavy form by ammonium sulphate precipitation. During the protoplasting process the removal of the cell wall is accompanied by the loss of most of the heavy form. The intermediate forms are exclusively detected inside the protoplast, together with the light invertase and a small amount of heavy invertase. The effect of 2‐deoxy‐d‐glucose and cycloheximide on the biosynthesis and distribution of molecular forms of yeast invertase has also been studied. In the presence of 10 mM glucose Saccharomyces 303–67 repressed cells readily synthesize invertase during the two‐hour incubation period. Upon the addition of 2‐deoxy‐d‐glucose, at a concentration of 75 μg/ml, the observed inhibition in the cells is 60%, but if the activity is measured after breaking the cells, only a 31% inhibition is found, revealing an accumulation of invertase inside the protoplast. 2‐Deoxy‐d‐glucose originates a pile‐up of the light and intermediate forms at the expense of the formation of the heavy enzyme, showing that the inhibition of the glycosilation and, therefore, the secretion process, has taken place. In he absence of de novo invertase synthesis originated by cycloheximide, the glycosilation process still takes place as indicated by the accumulation of the heavy form at the expense of the light, carbohydrate‐free, enzyme. Copyright © 1975, Wiley Blackwell. All rights reserved