Activation of acetylcholine esterase (ACHE) as a sign for erythrocyte membrane alteration

In vitro activity determination of membrane bound acetylcholine esterase (ACHE) of intact erythrocytes and differently altered RBC forms by means of the ELLMAN method yielded increase in activity in erythrocyte/vesicle mixtures and ghosts. Alteration of the cells by treatment with periodate, neuraminidase and trypsin leads to no, weak or clear decrease in the activity of this enzyme. Old cells also show a clear decrease in activity. When cells were treated with diamide ACHE is at first functionally inhibited, finally, however, it reaches supernormal values of activity. Possible causes for the different behaviour of ACHE in dependence on the degree of alteration of the erythrocytes are discussed. The results of the measurements were compared with the ultrahistochemical ACHE reactions according to KARNOVSKY (thiocholine--copper ferricyanide method) and IWAYAMA (lead thiocholine method). The influence of several components of the KARNOVSKY'S histochemical reaction mixture on ACHE activity was tested. The investigations suggest that disintegration of the erythrocyte membrane is accompanied with activation of the ACHE molecules which are nearly inactive in the intact membrane. This activation contributes to the positive histochemical ACHE test, which is an indicator reaction of membrane disintegration. Nearly intact membranes show a negative reaction. On the basis of these results we could show that physiological enucleation of normoblasts is connected with the formation of disintegrated membrane areas and their discharge from the future reticulocyte membrane.