Solubilization of frog brain and retina cholinesterase and studies of different molecular forms

Abstract

1. 1. The cholinesterase (ChE) of frog brain and retina could be easily solubilized. About 10% of the brain and 20% of the retina ChE were found to be soluble in 0.05 M phosphate buffer. After treatment with 0.5% (v/v) Triton X-100, about 30% of the total ChE activity of the brain and only 10% for retina was left particle bound. NaCl by itself did not solublize ChE. Use of higher NaCl concentrations in combination with Triton X-100 as well as higher detergent concentrations alone seemed to cause an inhibiting effect of the solubilized ChE from retina. 2. 2. The solubilized ChE from brain as well as retina were electrofocused as one main activity peak, corresponding to isoelectric points of pH 6.1 and 6.0, respectively. A second molecular form at pH 5.9 was distinguishable for the brain, but not for retina ChE. 3. 3. Sucrose gradient centrifugation indicated that the ChE solubilized from the brain and retina consists of two molecular forms exhibiting S values of 5.1 ± 0.24, 10.9 ± 0.33 and 6.1 ± 0.30, 10.9 ± 0.43, respectively. After solubilization by higher Triton X-100 concentrations the soluble extracts from brain and retina seemed to contain the activity of these forms in different proportions. 4. 4. Polyacrylamide gel electrophoresis separated three molecular forms of the brain ChE. One of these forms was found to have a molecular weight of 394,000 ± 20,000. The others were found to have an identical molecular weight of 550,000 ± 10,000. Two molecular forms exhibiting molecular weights of 292,000 ± 10,000 and 470,000 ± 10,000, could be separated for retina. The high as well as the low molecular weight form of brain and retina seemed to differ with respect to charge. 5. 5. The results found clearly indicate that the molecular forms of the frog ChE exhibit a high degree of lability. 6. 6. Substrate specificity, substrate inhibition, K m values and inhibition rates were examined without finding any significant differences between ChE solubilized from the brain and retina of the frog ( Rana temporaria).