Acetylcholinesterase in the human erythron. II. Biochemical assay.
Acetylcholinesterase (AChE) is an integral erythrocyte membrane protein. A role for the enzyme in the developing human erythron is being explored. Assays of AchE by the standard Ellman technique overestimate the amount of enzyme by failing to account for the contribution of hemoglobin to the optical density of the reaction mixture. Furthermore, reliance on substrate selection alone for specificity is unsatisfactory. Incorporation of inhibitors of "true" AchE and of pseudocholinesterase confer greater ability to distinguish one enzyme from the other. In our experience, the inhibitor constant (Kl) for edrophonium, which is highly specific for AChE, is approximately 5 x 10(-5) M against adult human erythrocytes that contain significantly more total cholinesterase activity than do erythrocytes from umbilical cord blood. This consists of both "true" and "pseudo" enzyme, the former predominating and accounting for 0.75-1.65 (mean 1.02, median 0.87) femtomoles of substrate hydrolysed per min per cell in adult blood, with values of 0.15-1.04 (mean 0.71, median 0.73) obtained on cord blood. Moreover, the enzyme activity in neonatal erythrocytes has a rather different inhibitor profile from that of adult cells. AChE was also demonstrated in fresh (ALL) and cultured (K562 and HL60) human leukemic cells, as well as in primitive granulocyte-macrophage and erythroid cells cloned from normal human bone marrow. In the erythroid colonies the enzyme activity was 0-3.76 (mean 1.20, median 0.76) femtomoles per min per cell, apparently the first successful measurement of AChE in such cells.
Barr
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RD
Department of Pediatrics, Faculty of Health Sciences, McMaster University, Hamilton, Ontario, Canada.
Koekebakker
M
M
Lawson
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AA
D000110:Acetylcholinesterase / Q000378:metabolism*; D002999:Clone Cells / Q000201:enzymology; D004912:Erythrocytes / Q000201:enzymology*; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000201:enzymology; D006801:Humans; D007945:Leukemia, Lymphoid / Q000201:enzymology; D013997:Time Factors
D000110:Acetylcholinesterase