Comparative study of soluble and membrane-bound forms of choline acetyl transferase from squid head ganglia

Abstract

Choline acetyl transferase (EC 2.3.1.6) (ChAT) present in cholinergic nerve endings catalyzes the synthesis of acetyl choline (Ach) from choline and acetylCoA. The membrane-bound form (integral membrane protein) of the choline acetyl transferase was prepared from squid head ganglia. The preparation procedure includes repeated hypo-osmotic disruption and washing by ultracentrifugation in a high ionic strength buffer. It appears that approximately 20\% of the synaptosomal choline acetyl transferase is present as a integral protein, non-ionically bound to membrane. It was found that at high concentrations, the acetylcholine(Ach) inhibits soluble ChAT to a greater degree than the membrane-bound enzyme form. The activities of soluble and membrane-bound ChAT also decrease with increasing calcium concentration but the activity of soluble ChAT is inhibited less than that of membrane-bound ChAT. The activity of soluble ChAT is completely lost at 2.0 mM SDS concentration whereas the membrane-bound ChAT still exhibits about 74\% of the control value at 3.0 mM SDS concentration. Soluble ChAT exhibits a sharp pH optimum near 6.6 in potassium phosphat buffer wheas membrane-bound ChAT shows a broader pH-activity profile. Optimum temperature of both soluble and membrane-bound forms of ChAT is $32-35\,^\circ\!C$ but over the range of $35-56\,^\circ\!C$, the membrane-bound ChAT is more stable than the soluble enzyme. Soluble and membrane-bound forms of ChAT exhibit identical Km values of approximately 0.12 mM for the substrate of acetyl-CoA. These results strongly indicate that two forms of ChAT, one soluble and one membrane-bound, are present in squid head ganglia.