AMPHIPHILIC FORMS OF BUTYRYLCHOLINESTERASE IN MUCOSAL CELLS OF RAT INTESTINE

The properties of a cholinesterase from mucosal cells of rat intestine have been characterized. The enzyme was identified as butyrylcholinesterase because it was more sensitive to iso-OMPA (IC50 = 1.0 X 10(-6) M) than to BW284C51 (IC50 = 5.5 x 10(-5) M) and was not inhibited by substrate excess. It displayed a higher affinity for acetylthiocholine than for butyrylthiocholine. A major molecular form was observed sedimenting at 5.9 S. Two other minor molecular forms were identified as a hydrophilic tetramer (G4, sedimenting at 10.5 S) and a monomer (G1, sedimenting at 4.3 S). The 5.9S component was referred to as "G" form (G for globular) and not "G2" as usual dimers for the following reasons: (i) the G form was unaffected by the reducing agents, beta-mercaptoethanol and dithiothreitol, which converted disulfide-linked dimers of acetylcholinesterase into monomers, (ii) the G form was shifted from 5.9 to 3.4 S when the sucrose gradient contained Triton X-100. This value of 3.4 S (in Triton X-100) appeared too low for a typical G2 form. The shift in the S value was partly reversible: the 3.4 S form resedimented at 5.2 S in the absence of detergent. The behavior of the G form in sucrose gradients indicated that it was amphiphilic. This was confirmed in nondenaturing electrophoreses and also by quantitative binding of the G form to octyl-Sepharose. The hydrophobic domain of the G form was not a glycolipid, as shown by its insensitivity to Bacillus thuringiensis phosphatidylinositol-specific phospholipase C and its nonaggregating properties in the absence of nondenaturing detergent. Proteinase K failed to remove the hydrophobic domain(s). In contrast, bromelain rapidly generated active peptides significantly less amphiphilic than the native form. Indirect estimation of G and G1 molecular weights from Stokes radii obtained from HPLC and sedimentation coefficients gave respectively 126 000 and 72 000, which were compatible with those of dimers and monomers. The G1 form was also amphiphilic but was more readily eluted from octyl-Sepharose column than the G form. Taken together these data suggested that the G form was a dimer of amphiphilic catalytic subunits not linked by a disulfide bond and with a special type of hydrophobicity. Depending on their type of hydrophobic domain, amphiphilic cholinesterase dimers (G2 forms) have been referred to as class I (glycolipid anchor) or class II (hydrophobic sequence of the catalytic peptide) by Bon et al. [Bon et al. (1988) J. Neurochem. 51, 776-785, 786-7941. The major molecular form of rat mucosal cells is tentatively characterized here as the first example in mammals of very rare amphiphilic butyrylcholinesterase dimers of class II.