Localization of the Alkaline Phosphatase and 5'-Nucleotidase Activities of the Diatom Phaeodactylum tricornutum

Summary: Intact cells of phosphate-depleted Phaeodactylum tricornutum possessed non-specific alkaline phosphatase (EC 3.1.3.1; PMEase) and 5′-nucleotidase (EC 3.1.3.5; 5′-NDase) activities; there was also an extracellular PMEase. The optimum pH for the cell-bound PMEase was > 10.3, and for the 5′-NDase was 9–9.5. The extracellular PMEase had an optimum pH > 10.3 and accounted for > 30% of the total PMEase activity at this pH; there was no extracellular 5′-NDase activity. The activities of these enzymes increased during phosphate-deprivation, but the rate of AMP hydrolysis (by the action of both PMEase and 5′-NDase) always exceeded that of p-nitrophenylphosphate at the physiological pH (for a marine organism) of 8–8.5. By the use of differential centrifugation after cell disruption in a French pressure cell, a highly purified fraction of cell walls was prepared. This fraction was virtually devoid of membranous material as viewed by electron microscopy, and exhibited PMEase, but no 5′-NDase activity. By using a different centrifugation procedure after disruption by shaking with glass beads, a microsomal fraction (pelleted by forces of 14000–156000 g) was prepared. This fraction was free of cell wall fragments as viewed by electron microscopy, and exhibited 5′-NDase activity but no PMEase activity. It is concluded that the PMEase was associated with cell walls, whilst the membrane-bound 5′-NDase which sedimented as vesicles in the microsomal fraction was associated with plasma membranes.