Difference in enzymatic activities between soluble and membrane bound choline acetyl transferase from squid head ganglia

Abstract

Choline acetyl transferase (Acetyl CoA: choline O-acetyl transferase, EC 2.3. 1.6, ChAT) catalyzes the synthesis of acetylcholine in the nerve terminal. This enzyme is present in several soluble isozyme forms as well as in a integral membrane protein form. Here the interaction between the soluble enzyme form and membrane is studied. When a system which contains both membrane and enzyme ($S_2$) is exposed to increasing concentration of KCl, there was a decrease in specific enzyme activity while the specific activity of the enzyme in the absence of membrane ($S_3$) was found to be independent of the KCl concentration. From these observation it was concluded that the enzyme binds to the membrane electrostatically and that the specific activity of the enzyme in solution is smaller than that of membrane bound form. That the increase of KCl brings about the release of bound enzyme was verified from the parallel increases in protein concentration and ChAT activity of the supernatant solutions when KCl concentration is increased. The determinations of these quantities were made after the supernatants were separated from the membranes by ultracentrifugation. In the case of $S_2$ fraction, the Km for acetyl CoA obtained in the presence of KCl was larger than the value without the salt. The enzyme activity was also determined in the presence of liposomes of phosphatidyl choline and phosphatidyl serine, CM cellulose and chondroitin sulfate. Increase in enzyme activity was observed only when the enzyme is present together with negatively charged surfaces. Since chondroitin sulfate had no effect, however, it is clear that the negative surface alone is not a sufficient condition for enhancing enzyme activity. A preliminary study on the pH effect has been made.