Abstract
3H-Pargyline binds specifically to both A and B forms of monoamine oxidase (MAO) under appropriate conditions. In this study the properties of 3H-pargyline-labeled MAO from rat and human cells were analyzed in three electrophoretic systems: sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, isoelectric focusing, and nonequilibrium pH gradient gel electrophoresis. Four types of samples were labeled with 3H-pargyline: crude mitochondrial preparations from rat hepatoma (with A and B activity), rat glioma (with A activity) and human fibroblasts (with predominantly A activity); and particulate fractions from human platelets (with B activity). Autoradiography of SDS-polyacrylamide gels of all four samples showed a single protein band labeled, with molecular weight of 57,000 ± 3000. Autoradiography of isoelectric focusing and nonequilibrium pH gradient gels revealed a single protein band with basic pI. Two-dimensional gels indicated an identity between this basic protein and the 57,000-dalton protein band. Quantitative analysis of the amount of label recovered in the specifically labeled protein band showed that binding of 3H-pargyline to the A form was more labile than to the B form under the conditions used for solubilization and electrophoresis in nonequilibrium pH gradient gels. These results show that A and B forms of MAO from rat and human cells have similar molecular weight and net charge, but differ in their interaction with pargyline, and suggests they represent distinct enzyme entities.
ACKNOWLEDGMENTS We thank Richard M. Cawthon for valuable discussions; Carmela M. Castiglione for technical help; Dr. Gerry Degnen for advice and encouragement in setting up two-dimensional gel electrophoresis; Dr. Uta Francke for supplying us with fibroblast line LN BUR; and Ms. Regina Gambardella for skilled preparation of this manuscript.
- Copyright © 1980 by The American Society for Pharmacology and Experimental Therapeutics
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