DIPHENOLOXIDASES - PRESENCE IN MEMBRANE OF HUMAN ERYTHROCYTE

Diphenoloxidases, [EC 1.14.18.1] enzymes which accelerate the auto-oxidation of epinephrine and Dopa, were previously described in blood platelets. Earlier, these enzymes were identified in different animal species and particularly in human red blood cells. Ghosts of red blood cells were separated; the protease inhibitors diisopropylfluorophosphate (DFP) (10-3 M) and 6-aminocaproic acid 10-2 M in sodium phosphate buffer, 5 .times. 10-3 M (pH 8) were used. These ghosts, totally free of Hb, were first pulverized in liquid N then treated ultrasonically. The supernatant showed the presence of a band of diphenoloxidase activity on starch gel electrophoresis and 2 bands on isoelectrofocusing in polyacrylamide gel pH 5-8 after incubation with 0.02 M Dopa, 0.076 M Tris, 0.005 M citric acid, 0.004 M Mg, pH 8.7 for 2 h at 37.degree. C. These enzymes differed from (Na, K)-ATPase in that they were neither inhibited by DFP (10-1 M) nor by EDTA (10-2 M) but were inhibited by lead acteate 10-2 M. Like (Na, K)-ATPase diphenoloxidases were present at membrane level. The role in vivo of the diphenoloxidases in ATPase activity of red blood cells is discussed.