Interchain disulfide bonds and subunit organization in human serum cholinesterase.

Abstract

Highly purified “usual” human serum cholinesterase was studied. The enzyme is a tetramer with a molecular weight of approximately 340,000. Its four subunits appear to be identical. On 1% sodium dodecyl sulfate (SDS) gel electrophoresis the enzyme had a molecular weight of about 180,000 when dissolved in 1% SDS, but it had a molecular weight of about 90,000 when dissolved in 1% SDS and 40 mM dithiothreitol. This was evidence that the subunits were arranged as a dimer of dimers ((cw)&, with each (a)~ dimer composed of two identical subunits joined by interchain disulfide bonds. Purified “atypical” cholinesterase had the same dimer of dimers structure and the same molecular weight on SDS-gel electrophoresis as usual cholinesterase. Additional structural experiments were conducted with purified usual choline&erase. The number of interchain disulfide bonds was quantitated by selectively reducing these bonds in the absence of a denaturant, followed by alkylation with [l-‘4C]iodoacetate. One SH per subunit was alkylated. As the enzyme contains no free SH groups, the alkylated residues must previously have been components of disulfide bonds. It was shown that the alkylated -SH groups arose from interchain disulfide bonds, for on SDS-gel electrophoresis of the alkylated enzyme, the labeled product all migrated with the subunit molecular weight of 90,000. It was concluded that each dimer contained only one interchain disulfide bond between two subunits. After reduction and alkylation of the interchain disulfide bonds, the enzyme was still a tetramer and was fully active. Therefore, it was concluded that the interchain disulfides were not necessary for either the tetramerit structure or for activity, and that the four subunits were held together by noncovalent bonds. The interchain disulfide bonds stabilized the enzyme as shown by measurements of rates of heat inactivation. Native enzyme had a half-life of 54 min at 55”C, whereas enzyme that had been selectively reduced and [14C]carboxymethylated, had a half-life of 16 min at 55°C. The interchain disulfide bonds were easily reduced and alkylated, suggesting that these bonds are located near the surface of the molecule. In addition to the single interchain disulfide bond, each subunit contains several (three to five) pairs of intrachain disulfide bonds.