Immunological, isoelectric, hydrophobic and molecular weight differences between soluble and ionically membrane-bound fractions of choline-o-acetyltransferase prepared from mouse and rat brain.
Choline-O-acetyltransferase (EC 2.3.1.6; ChAT) was prepared from synaptosomal fractions (P(2)) of mouse and rat brain in the presence of proteolytic inhibitors by the method of Gray and Whittaker (1962) as modified by (Salehmoghaddam and Collier, 1976). The P(2) fraction was hypo-osmotically shocked with glass distilled water and centrifuged to separate the cytoplasmic (S(3)) and vesicle-bound (P(3)) fractions. Fraction S(3) was saved for ChAT assay and compared with the ChAT fraction eluted from the P(3) by salt at a pH 7.4 or by detergent (Benishin and Carroll, 1983). These three fractions of ChAT were then compared by molecular weights, isoelectric points, immunoblotting with monoclonal or polyclonal antibodies and hydrophobicity. The results show that the S(3) fraction of ChAT has a molecular weight of 66 K(d), whereas the ionically-bound fraction of ChAT has a molecular weight of 73-78 K(d). SDS-PAGE of these two ChAT fractions followed by immunoblotting revealed the presence of two immunoreactive bands at 28-29 K(d) and 50-51 K(d) for the ionically bound ChAT fraction. Conversely, none of these antibodies immunostained any protein bands for the S(3) ChAT fraction even though one monoclonal antibody had been prepared against this ChAT fraction and the S(3) ChAT fraction had a similar specific activity prior to SDS-PAGE as did the salt solubilized ChAT fraction. However, anti-ChAT monoclonal antibody MB16 binds the native S(3) ChAT fraction in the co-precipitation assay. The S(3) fraction of ChAT had only one isoelectric point at pH 7.8, whereas the ionically bound and detergent soluble ChAT fractions had two isoelectric points at pH 8.1-8.15 and 7.45-7.5. The S(3) ChAT fraction also differed in hydrophobicity from the other two ChAT fractions. These differences between the S(3) and salt soluble ChAT fractions were not obviated by addition of Triton X-100 and thus could not be attributed to the association of lipids with either of the fractions. We conclude that the water soluble fraction of ChAT in central nerve terminals differs in its physical properties and its subcellular location from that which ionically binds to membranes.
Badamchian
M
M
Department of Pharmacology, Texax Tech University Health Sciences Center, Lubbock, TX 79430, U.S.A.
Morrow
K J
KJ
Carroll
P T
PT