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.
Choline acetyltransferase (Acetyl CoA : Choline O-acetyl transferase, EC 2.3.1.6, ChAT)는 신경말단에서 neurotransmitter 인 acetylcholine 을 합성하는 enzyme 이다. 이 enzyme 은 integral membrane protein form 뿐만 아니라 여러가지 soluble isozyme forms이 존재한다.
본 연구에서는 soluble enzyme form 과 membrane의 상호작용에 대하여 연구했다. Membrane 과 enzyme이 동시에 존재하는 계에서 KCl의 농도를 증가시켰더니, membrane 이 존재하지 않을때 똑같이 KCl 의 농도를 증가 시켜도 enzyme 의 specific activity 변화가 없는데, enzyme 의 specific activity 가 줄었다. 위의 실험적 사실로 부터 이 enzyme 이 membrane 에 electrostatically bound 를 이루며, 이때 soluble form 때 보다 specific enzyme activity 가 증가한다는 결론을 얻었다.
KCl 농도의 증가로 ionic strength 가 증가하여 이 enzyme 이 membrane 에서 분리되어 soluble form 으로 되었는지를 알기 위해 ultracentrifugation 을 하여 얻은 supernatant 의 protein 양과 enzyme 의 total activity 를 조사했다. Membrane 과 enzyme 이 동시에 존재하는 계에서 KCI salt 를 넣지 않은 쪽의 Km 값이 salt를 첨가한 쪽의 Km 값의 절반 정도였다. Phosphatidyl choline, phosphatidylserine 의 artificial vesicles, CM cellulose, chondriotine sulfate 로 처리한 enzyme 의 activity 를 조사했다.
그 결과 negative charge surface 가 존재할때 enzyme activity 의 증가현상이 나타났다. 그러나 chondriotin sulfate 는 아무런 영향이 없었다. 이는 enzyme activity 를 증가 시키는데는 negative surface 만으로는 충분하지 않음을 나타낸 것이다.
Ionic strength 대신 pH 변화에 의한 영향에 대해서도 예비 조사를 하였다.
