Solubilization of chicken brain cholinesterase, separation and characterization of molecular forms

Abstract

1. The cholinesterase (ChE) of chicken brain cortex could be easily solubilized. About 10% was found to be soluble in 0.05 M phosphate buffer. After treatment of 0.25% (v/v) Triton X-100 alone, about 20% and in combination with 1 M NaCl not more than 5% of the total activity was left particle bound. NaCl by itself did not solubilize ChE. 2. Two isozymes of ChE could be separated by polyacrylamide gel electrophoresis, isoelectric focusing and sucrose gradient centrifugation. 3. The S values (and approximate molecular weights) of the two forms separated by sucrose gradient centrifugation were estimated to be 7.4 ± 0.44 (133,000 ± 12,000) and 11.2 ± 0.67 (247,000 ± 23,000). The molecular weights of these forms determined by electrophoresis in different polyacrylamide gel concentrations were found to be about twice as large, namely 223,000 ± 18,000 and 450,000 ± 22,000. 4. Substrate specificity, substrate inhibition, K m values and inhibition rates were examined without finding any significant differences between the forms of ChE separated by sucrose gradient centrifugation and isoelectric focusing. 5. The results were found to be in general accord with the suggestion that the multiple forms of brain ChE may be related to the aggregation of a single low molecular weight species.