Osmotic regulation: physiological significance of proteolytic and nonproteolytic activation of isofloridoside-phosphate synthase.

When cells of Poterioochromonas malhamensis Peterfi are exposed to media of increased osmotic strength, both the internal pool of isofloridoside, and activity in homogenates of isofloridoside-phosphate synthase increase, proportional to the degree of osmotic stress. During the first few minutes of exposure of cells to higher osmolalities, an early relatively small increase in enzyme activity was observed. At the same time a progressive activation of the enzyme in homogenates was noted, providing bovine serum albumin had been omitted from the homogenizing buffer. This in vitro activation was also proportional to the degree of prior osmotic stress, was more pronounced in the presence of fluoride, and was inhibited strongly by adding bovine serum albumin or other proteins. Since earlier work had demonstrated activation of the synthase by adding exogenous proteases, it is likely that this in vitro activation was due to protease activity in the homogenate. The presumed protease must have acquired activity in the cells in response to osmotic stress, and is likely to be responsible for the observed in vivo activation of this biosynthetic enzyme.Between 60 and 90 minutes after increasing the medium osmolarity the isofloridoside pool in cells approached a high steady-state level. About this time it was observed that isofloridoside-phosphate synthase activity passed transiently through a much higher level than before, and a higher molecular weight form of the active enzyme could be observed on gel filtration chromatography.