The Synthesis of Phosphatidic Acid and Protein-bound Phosphorylserine in Salt Gland Homogenates.

Kinetic data obtained from studies of the avian salt gland have shown that a fraction of phosphatidic acid is formed which undergoes continuous turnover of its phosphate group during stimulated secretory activity and which disappears when this activity stops; the turnover of phosphatidic acid (determined with radioactive phosphate) appears, therefore, to be associated in some way with the stimulated active transport of Na+ in the salt gland (1). One explanation for these observations is thab the phosphorylation of diglyceride by adenosine triphosphate to form phosphatidic acid, followed by its dephosphorylation to re-form diglyceride, might be part of the mechanism whereby ATP is utilized for ion transport. The presence of enzymes (diglyceride kinase and phosphatidic acid phosphatase) which catalyze these two reactions has been demonstrated in the membrane fraction of salt gland and other tissues (2-5). In recent years much work in the transport field has been devoted to studies of the (Naf + K+)-dependent, ouabaininhibitable adenosine triphosphatase activity, which was first shown by Skou (6) in a microsome fraction from crab nerve, and which has since been found in preparations from many other tissues. Because this (Na+ + K+)-dependent ATPase activity has many of the same properties as Na+-transporting systems, it has been suggested that this ATPase activity is closely linked to the process of Na+ transport. The experiments reported here were designed to test whether a turnover of phosphatidic acid, such as occurs during Naf pumping in the intact salt gland cell, might also take place during the (Naf + K+)-dependent ATPase activity which occurs in salt gland homogenates (7). No component of phosphatidic acid that showed kinetic behavior which would support the participation of the phosphatidic acid cycle in the (Na+ + K+)dependent ATPase activity in cell-free preparations could be found. These results could be interpreted to mean that the rapid turnover of a fraction of the phosphatidic acid in the intact cells of the salt gland cannot be accounted for by allocating to this phosphatidic acid a role as an intermediate in the utilization of ATP for Na+ transport. However, the possibility that the steady state level of the phosphorylated intermediate may be so low as to be undetectable in the cell-free preparations cannot be excluded. Results from experiments in which phosphatidic acid was made radioactive in the intact cell before homogenization are compatible with this possibility. Heald (8) and Judah et al. (9-12) have put forward the sug-