PHOSPHATASE OF RABBIT POLYMORPHONUCLEAR LEUCOCYTES

Abstract

Rabbit polymorphonuclear leucocytes contain an active phosphatase that readily hydrolyzes disodium phenyl phosphate. The pH activity curve of the enzyme was found to have two maxima, one in the region of pH 10 and the other in the region of pH 5. The alkaline phosphatase was much more active than the acid phosphatase. The concentration of alkaline phosphatase in rabbit white cells was approximately one thousand times that of the enzyme in the serum. Under the conditions of study, the alkaline phosphatase activity was proportional to the concentration of the enzyme. The effect of substrate concentration on the enzyme activity was studied and the Michaelis constant (Ks) determined. An excess of substrate inhibited the enzyme. The course of the reaction was linear with time for the first 60 min.; after 90 min. the activity fell off faster than would be expected if the reaction were of the first order.Magnesium and glycine, in low concentrations, caused an increase in the enzyme activity, whereas zinc, cyanide, borate, phosphate, bile salts, and glycine, in higher concentrations, were inhibitory. Fluoride had no demonstrable effect. Surface-active substances, such as saponin, bile salts, or alkyl sulphate, liberated the enzyme from the cells. Similar results were obtained when α-glycerophosphate or β-glycerophosphate was used as the substrate.The alkaline phosphatase can be considered to belong to Class AI of Folley and Kay (22) and the acid phosphatase to Class AII. The alkaline phosphatase can also be considered to be a Phosphatase II of Cloetens (9).