Properties and regulation of a phosphatase of Euglena gracilis, biosynthesis and inactivation

Abstract

Under conditions of limiting orthophosphate concentration in cells of Euglena gracilis, Z strain, there is an increase in phosphatase activity with an optimum at pH 6.4. Derepression begins when phosphate concentration is below 0.5 mM. Maximal values of activity are reached after 75 hours of culture. The increase in phosphatase activity is prevented by hadacidin, cycloheximide and p-fluorophenylalanin indicating that the increased activity is related to enzyme synthesis. The derepressed phosphatase of Euglena is stimulated by EDTA and inhibited by orthophosphate; Ki= 5 × 10−5 M. In contrast to alkaline phosphatase of Escherichia coli the derepressed phosphatase of Euglena is extremely thermolabile. When, after a period of phosphate starvation, phosphate is replenished in a derepressed culture, phosphatase activity drops rapidly to the level of activity characteristic of repressed cells. The disappearance of activity after phosphate addition cannot be attributed exclusively to a repressive mechanism. The rate of decline in the specific activity of phosphatase is much more rapid than can be accounted for by dilution through new protein synthesis. If the synthesis of phosphatase is interrupted by cycloheximide or by p-fluorophenylalanine the level of activity remains constant for at least 48 hours. This rules out the possibility that the phosphatase is an enzyme which undergoes rapid metabolic turnover and that the observed disappearance of activity is merely the expression of general instability of this enzyme following the repression of its synthesis. The disappearance is due to an inactivation process promoted by addition of phosphate. This inactivation process was found to be partly prevented by cycloheximide and is reversibly interrupted by chilling the cells to 20. p-Fluorophenylalanine is without any effect on the rate of inactivation. Prevention of inactivation by cycloheximide is not due to inhibition of phosphate uptake. Inactivation of phosphatase is an irreversible process and new increase of phosphatase activity in inactivated cells is dependent on de novo protein synthesis rather than on a reactivation of the inactivated enzyme molecule.