Attempts to Demonstrate Acetylcholinesterase Activity in Blood and Bone-Marrow Cells by a Modified Thiocholine Technique

Abstract

The thiocholine method of Koelle (1951) was used in attempts to demonstrate acetylcholinesterase activity in artificial enzyme-gelatin mixtures and in suspensions and smears of isolated platelet, ghosts, erythrocytes, and megakaryocytes of different species. When the last processing step with ammonium sulphide was omitted, the following observations were made: After a short time of incubation, small granular or rod-like deposits were found at the presumed sites of AChE activity. These deposits apparently grew into large crystals during prolonged incubation. Such crystals were demonstrated at the surface of platelet ghosts and erythrocytes of species bearing AChE activity, and further in megakaryocytes of some species presumably bearing this activity. The crystals were provisionally assumed to be a Cu thiocholine salt representing sites of AChE activity. If an enzyme inhibitor was added or a substrate used which is not split by AChE no crystals appeared.

Studies on the Histogenesis of Blood Platelets

“…On enzymatic hydrolysis of AThCh, the liberated thioeholine reacts with copper salt to give a relatively insoluble product, presumed to be copper thioeholine (CuThCh) (7). Under the phase microscope the pre sumed CuThCh precipitate appears as deposits of needleshaped crystals indicating AChE activity (5). No crystals appear if an enzyme inhibitor -physostigmincis added to the incubation medium (5).…”

“…Under the phase microscope the pre sumed CuThCh precipitate appears as deposits of needleshaped crystals indicating AChE activity (5). No crystals appear if an enzyme inhibitor -physostigmincis added to the incubation medium (5).…”

“…A modified thioeholine method was used in the present experiments to demonstrate the AChE activity in platelets and bone marrow cells (5). The cells were incubated in a medium containing copper glycinate and acetylthiocholine (AThCh) and, in separate experiments, in a medium containing copper glycinate and butyrylthiocholine (BuThCh).…”

Cellular characterization of thrombocytes in Xenopus laevis with specific monoclonal antibodies

“…1A). Acetylcholinesterase staining, which detects MKs in mice, rats, and cats [23,33], was used to differentially stain spindle and mature thrombocytes among other blood cells in X. laevis (Fig. 1B).…”

The Cytological Localization of Intracellular Neuronal Acetylcholinesterase

“…When the former crystals are in aqueous suspension, or in smear preparations, treatment with (NH4)2S can cause their dissolution and replacement by amorphous CuS, but the two sites may not be identical (12,33). This modification does appear to have definite advantages when the method is used for isolated intact blood cells (12) and neurons (38). Under the circumstances of the present study, the sites of localization of AChE appeared identical with and without (NH4)~S development, and the latter step permitted more convenient observation with the ordinary light microscope.…”