The release of extracellular enzymes from yeast by "osmotic shock".

Abstract

A procedure for subjecting cells of Saccharomyces chevalieri to “osmotic shock” is described. Exponentially growing yeast cells are first suspended in a concentrated solution of mannitol plus EDTA, 2-mercaptoethanol and Tris. After this high osmotic treatment, the cells collected by centri-fugation are suddenly resuspended in a solution of MgCl2 of low osmotic strength. This treatment causes the rapid and specific release of extracellular enzymes from yeast cells, namely: invertase, a Co2+ activated 5′-nucleotidase, acid phosphatase and alkaline pyrophosphatase. Also, the capacity to accumulate l-proline is greatly diminished. At the same time, some material absorbing at 260 nm and 7% of the cell wall protein are released into the shock medium. Alkaline pyrophosphatase and 5′-nucleotidase are described for the first time as extracellular enzymes for yeast. Intracellular yeast enzymes such as alcohol dehydrogenase, alkaline phosphatase and cytochrome-c oxidase remain within the cell after “osmotic shock”. The viability of yeast cells in 80% after being subjected to the “osmotic shock” procedure. Examination of yeast cells in the electron microscope shows that a hairy-like structure in the external portion of the cell wall is practically lost after the osmotic shock treatment. No other difference between shocked and unshocked cells can be visualized.