Search

Due to Triton X-114 fractionation of synaptosomes isolated from the electric organ of the fish Torpedo, the existence of a hydrophilic and an amphiphilic form of the enzyme choline-O-acetyltransferase (ChAT) was revealed. Amphiphilic ChAT which represents about 10% of total enzyme activity in synaptosomes, reached 40% of ChAT activity measured in preparations of synaptosomal plasma membranes (SPM) which were washed with solutions of increasing ionic strength. ChAT activity bound to washed SPM could be partially solubilized using proteinase K but not phospholipase C. No ChAT solubilization occurred by treating intact synaptosomes with proteinase K. Water/Triton X-114 partition coefficients of hydrophilic and amphiphilic ChAT were found to be 6.5 and 0.17, respectively. Sedimentation coefficients determined by centrifugation in linear density gradients of sucrose containing Triton X-100, were 4.2S and 4.4S for amphiphilic and hydrophilic ChAT, respectively. On the other hand, removal of Triton X-114 from the detergent phase containing amphiphilic ChAT activity led to enzyme aggregation. Finally, amphiphilic ChAT was slightly more acidic (pH 6.6) than was hydrophilic enzyme (6.8-7.0). We conclude that in Torpedo synaptosomes two forms of ChAT activity, a soluble and a membrane-bound form, are indeed present which differ in their hydrophobicity. The soluble form is hydrophilic. The membrane-bound form is amphiphilic and it aggregates upon removal of detergent. These are two characteristics of integral membrane proteins. Membrane-bound ChAT is most probably intracellularly oriented and not bound to membrane through a 'receptor' protein.

A monoclonal antibody raised against cholinergic synaptosomal plasma membranes isolated from Torpedo electric organ, inhibited completely amphiphilic and hydrophilic choline acetyltransferase (ChAT) activities extracted and separated by using Triton X-114 phase partition of synaptosomes. We tested whether ChAT inhibition was direct or not. We found that the antibody was inhibiting ChAT in preparations of very low purity as well as ChAT that was immunoprecipitated by using a non-inhibitory anti-ChAT polyclonal antibody. Also, inclusion of acetylcoenzyme A at 20 times its Km during incubation of ChAT and antibody, completely prevented ChAT inhibition. This same concentration of the ChAT substrate could significantly but not completely dissociate the complex enzyme-antibody. These results spoke in favour of a direct inhibition of ChAT; the antibody most probably binds to an epitope that may be located at or near the acetylcoenzyme A binding site. The inhibitory effect on hydrophilic and amphiphilic ChAT was dependent on the antibody concentration, but amphiphilic activity required higher concentrations to be affected to the same extent as hydrophilic activity. This was found not only with Torpedo, but also with rat and human ChAT activities. Thus, the antibody appears to be able to distinguish the two forms of ChAT activity.