Heterogeneity and Partial Purification of Human Ery throcy te Membrane Acetylcholinesterase

Abstract

1) Triton X-100 solubilised human erythrocyte acetylcholinesterase (E 0 ), when subjected to chromatography on Sephadex G-200, showed one enzyme activity peak (Eg) and a number of protein peaks (Pg). The same sample could be separated into several subfractions of enzyme activity on DEAE-cellulose by gradient elution with increasing sodium chloride. When the gel filtered enzyme peak (Eg) alone was rechromatographed on an ion-exchange column under identical conditions, it showed only one enzyme peak. But when Eg in combination with protein peaks (Pg) without enzyme activity is rechromatographed as a physical mixture on a DEAE-cellulose column under the same conditions, the preparation could again be resolved into at least two fractions with enzyme activity.2) Disc electrophoresis of fractions from DEAEcellulose chromatography separated multiple bands which differ significantly from those produced by electrophoresis of E 0 . This suggests that E 0 had undergone some form of conformational modification during the ion-exchange chromatography. However, this modification of E 0 was avoided, if it was subjected to Sephadex G-200 chromatography before the DEAE-cellulose step.3) A three-step technique (fractionation on Sephadex G-200, DEAE-cellulose chromatography and electrofocusing) has been performed for the purification of erythrocyte acetylcholinesterase. An enzyme preaparation of high purity with a specific activity of 81 U/mg of protein was obtained. Heterogenität und partielle Reinigung der Acetylcholinesterase aus menschlichen ErythrozytenmembranenZusammenfassung: 1) Menschliche Erythrozyten-Acetylcholinesterase (E 0 ), in Triton X-100 gelöst, zeigt nach der Chromatographie an Sephadex G-200 einen Aktivitätsgipfel (Eg), aber verschiedene Proteingipfel (Pg). Nach Gradientenelution mit steigender NaCl-Konzentration an DEAE-Cellulose erhält man jedoch mehrere Fraktionen mit Enzymaktivität. Wird aber Eg unter gleichen Bedingungen an einer lonenaustauschefsäule rechromatographiert, wird nur ein einheitlicher Enzymaktivitätsgipfel erhalten. Mischt man Eg und Pg und rechromatographiert dann an einer DEAE-Cellulose-Säule, so läßt sich eine Auftrennung in mindestens zwei Fraktionen mit Enzymaktivität beobachten.