Single Low Molecular Weight Research Articles

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

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.

Micromethod for quantitation of adenosine deaminase activity in cells from human peripheral blood

Adenosine deaminase (ADA) converts adenosine to inosine. The enzyme can be assayed simply and conveniently by utilizing [ 14C]adenosine as substrate and separating the [ 14C]inosine from the reaction mixture by chromatography on short strips of DE-81 paper. The method is particularly useful in assay and characterization of ADA in small numbers of cells. Lymphocytes, granulocytes, and erythrocytes were isolated from 10 ml samples of human peripheral blood, and ADA activity was measured in each cell type. Extracts of erythrocytes typically contain specific activities of ADA ranging from 1.5 to 2.7 nmoles [ 14C]inosine produced/10 8 cells/min, while lymphocyte and granulocyte extracts contain 50 to 100 times as much ADA activity. The radiometric assay can be used to obtain quantitative information about the behavior of ADA during velocity sedimentation and acrylamide gel electrophoresis. Lymphocytes have both a low and high molecular weight species of ADA. Granulocyte and erythrocyte extracts have a single low molecular weight species. Isoelectric focusing of extracts from the three cell types reveals one major enzyme activity peak with a pI of about 5. These methods should prove useful in studies of the association of quantitative and qualitative variation in ADA activity with human immunodeficiency diseases.