Molecular Pharmacology, Vol 17, 199-205, Copyright © 1980 by the American Society for Pharmacology and Experimental Therapeutics

Electrophoretic Analysis of ³H-Pargyline-Labeled Monoamine Oxidases A and B from Human and Rat Cells

¹ Department of Human Genetics, Yale University School of Medicine, New Haven, Connecticut 06510

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.

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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.