The Structure of the Yeast Cell Wall

The autolytic release of fl-fructofuranosidase (EC 3.2.1.26) and acid phosphatase (EC 3.1.3.2) from the cell wall of yeast was studied under different organic solvents and with a variety of pII and temperature conditions. /3Fructofuranoeidase (but not acid phosphatase) was found to be a convenient marker for studying changes in the wall structure. The ethyl acetate-treated cell was characterized in terms of changes in cell volume, dry weight, total nitrogen, and phospholipid; and it was used as a model of wall structure. The P-fructofuranosidase content is little affected by ethyl acetate treatment, but a Z-day exposure completely inactivates the autolytic enzyme system. Approximately 50% of the marker can then be enzymatically released by incubation with a cell-free extract from fresh yeast. I f ethyl acetate-treated cells are previously treated with dithiothreitol, 100% of the marker now becomes accessible and is liberated by cell-free extract. The effect of sulfhydryls was shown to be on a wall component. The cell-free extract was partially purified by heat treatment, ammonium sulfate precipitation, and dialysis with full retention of autolytic activity. Diethylaminoethyl-cellulose chromatography was used to resolve the extract into two active fractions. Fraction I is a /3(1 ---f 6) glucanase, while Fraction II is a /3(1 + 3) glucanase; both appear to be endohydrolases in their action pattern. Fractions I and II will account for, at most, 77% release of marker from ethyl acetate-treated cells. A third (or more) fraction(s) was clearly indicated but not identified. In living yeast cells the autolytic enzyme system is located internal to the protoplasmic membrane and, during tolueneinduced autolysis, is retained by the cell at least until 20% of the marker has been liberated.