Localization of enzymes involve in polyphosphoinositide metabolism on the cytoplasmic surface of the human erythrocyte membrane

Abstract

1. 1. Impermeable inside-out and right-side-out vesicles were prepared from membranes of human erythrocytes. During preparation of each kind of impermeable vesicle, permeable vesicles were also obtained. 2. 2. Incubation of vesicles with [γ- 32P]ATP at 37 °C for periods of up to 1 h did not change the topography or the permeability of the vesicles. 3. 3. Vesicles incorporated labeled phosphate from [γ- 32P]ATP into both diphosphoinositide and triphosphoinositide, but impermeable inside-out vesicles incorporated significantly more nuclide than did right-side-out vesicles. 4. 4. Permeable vesicles derived during the preparation of inside-out vesicles were as active as impermeable inside-out vesicles in the incorporation of labeled phosphate into the polyphosphoinositides. However, permeable vesicles derived during the preparation of right-side-out vesicles were not as active. 5. 5. Impermeable right-side-out vesicles, treated with 0.01% saponin, incorporated labeled phosphate into the polyphosphoinositides at a level comparable to that of impermeable inside-out vesicles. 6. 6. These data show that the enzymes involved in metabolism of diphosphoinositide and triphosphoinositide are located on the cytoplasmic surface of the erythrocyte membrane.