Ecto-Enzymes of the Guinea Pig Polymorphonuclear Leukocyte

Abstract

A suspension of intact guinea pig polymorphonuclear leukocytes hydrolyzed added ATP, AMP, and p-nitrophenyl phosphate under physiologically appropriate conditions. These enzymatic activities were not due to artifacts such as breakage of the cells during the incubation period. It thus seemed possible that the hydrolyses were being catalyzed by ecto-enzymes, i.e. enzymes on the plasma membrane with their active sites facing the external medium. Three types of experiment were designed to test this hypothesis. First, the activities of intact cells were compared to those of homogenates, sonicates, and cells treated with detergent. Disruption of cells resulted in an approximately 2-fold increase in maximal ATPase and p-nitrophenyl phosphatase activities, suggesting that the plasma membrane was acting as a permeability barrier to the substrates involved. Disruption did not increase AMPase activity, leaving open the possibility that an ecto-enzyme is the only protein in polymorphonuclear leukocytes capable of hydrolyzing AMP. Second, the products of ATPase, AMPase, and p-nitrophenyl phosphatase activities of intact cells were localized by using radioactively labeled substrates. The concentration of inorganic phosphate produced by these reactions was 18 to 100 times greater in the extracellular medium than in the intracellular milieu. This suggests that the substrates are cleaved outside the cells, or that they are cleaved inside and the products are transported out. The latter possibility was militated against by the following experiment. Cells were loaded with inorganic [33P]phosphate, then allowed to hydrolyze substrates labeled with 32P. The distributions of the two isotopes were compared. Almost all of the inorganic [32P]phosphate was found outside of the cells, while 90% of the inorganic [33P]phosphate remained inside. Third, the cells were treated with the diazonium salt of sulfanilic acid, a reagent known not to penetrate into intact erythrocytes. This treatment rapidly and dramatically inhibited the intact-cell ATPase, AMPase, and p-nitrophenyl phosphatase, while lactate dehydrogenase, a soluble cytoplasmic enzyme, was unaffected. Control experiments demonstrated that in sonicates lactate dehydrogenase was as susceptible to inhibition by the diazonium salt as were the other three activities.