Homocholine and short-chain N-alkyl choline analogues as substrates for Torpedo choline acetyltransferase.
The kinetic parameters, Km and Vmax, for the acetylation of choline and several close analogues were determined by using (a) purified choline acetyltransferase and (b) a hypotonically lysed synaptosomal extract prepared from the electric organ of Torpedo marmorata. Whereas the Km for choline was similar in both cases (0.51 and 0.42 mM), the crude enzyme showed a three- to fivefold greater affinity for its analogues than the purified enzyme, the activity decreasing rapidly with increased N-alkyl substitution. Homocholine was a poor substrate, but was clearly acetylated by both preparations. The effect of salt on analogue acetylation by the crude enzyme was studied by increasing NaCl concentration from zero to 150 mM. There was an increase in both Km and Vmax for all substrates: choline, N,N,N-dimethylmonothylaminoethanol, -monomethyldiethylaminoethanol and -dimethylmonobutylaminoethanol showed the greatest changes, whilst N,N,N-triethylaminoethanol and -dimethylmonopropylaminoethanol and homocholine were much less affected However, in all cases, the kinetic parameter Vmax/Km remained unchanged. The maximal velocities of the different substrates varied more under conditions of high than of low salt. Sodium chloride up to 300 mM had no effect on the amount of enzyme which was bound to membranes in the synaptosomal extract. It is concluded that choline acetyltransferase has a high degree of substrate specificity, which is slightly altered by purification. The effects of salt cannot be explained as a consequence of nonspecific ionic association with membranes.
D000818:Animals; D002794:Choline / Q000031:analogs & derivatives* / Q000378:metabolism; D002795:Choline O-Acetyltransferase / Q000378:metabolism*; D005260:Female; D007700:Kinetics; D009994:Osmolar Concentration; D012965:Sodium Chloride / Q000494:pharmacology; D013570:Synaptic Membranes / Q000378:metabolism; D014101:Torpedo / Q000378:metabolism*
C007954:homocholine; D012965:Sodium Chloride; D002795:Choline O-Acetyltransferase; D002794:Choline