Isolation and characterization of acetylcholinesterase and other particulate proteins in the hemolymph of Aplysia californica

Abstract

Hemolymph of the marine mollusc, Aplysia californica, contains four large particles: acetylcholinesterase, hemocyanin, a hemagglutinin, and a structure tentatively identified as erythrocurorin. We purified the acetylcholinesterase 20-fold by differential centrifugation and filtration through a column of 4% agarose. The freshly isolated esterase complex was found to have a sedimentation coefficient of 69, but the negatively stained enzyme lacked a definite structure in the electron microscope, and appeared as irregular aggregates of a 60 A subunit. The complex was unstable below pH 5 or during storage at 7 degrees. Under these conditions, enzymatic activity remained essentially unchanged. Treatment of the purified enzyme with trichloroacetic acid, organic solvents, and sodium dodecyl sulfate broke the complex down into two major subunits with molecular weights of about 70,000. Exposure of the enzyme to [3H]diisopropylfluorophosphate resulted in the labeling of one of these subunits. Although similar in specificity, the cholinesterase of the blood differed from the enzyme in Aplysia nervous tissue, which is associated with membrane. Treatment with sodium deoxycholate activated the membrane-associated enzyme but inhibited slightly that of the hemolymph; tyrocidine inhibited the hemolymph enzyme but not the enzyme of nervous tissue; and mild digestion with trypsin released the membrane-bound enzyme in an active, soluble form, but inactivated the enzyme of hemolymph. The other particulates of Aplysia hemolymph were partially characterized. Aplysia hemocyanin was similar in structure to other molluscan hemocyanins. When negatively stained, the unit particle appeared to be a disc with a diameter of 280 A and a width of 45 A. These discs were stacked to form long cylindrical arrays. The purified hemocyanin was found to contain 0.26% copper (dry weight). Using differential centrifugation and gel filtration we also obtained a 9-fold purification of Aplysia hemagglutinin. This particle was 120 A in diameter with a dark staining central core of 40 A consisting of 6 subunits. The particle tentatively identified as erythrocurorin appeared as a structure 200 A in diameter consisting of 5 V-shaped subunits.