Specificity of antisera prepared against pure bovine MAO-B

Antisera have been prepared against purified bovine MAO-B that appear to react selectively with MAO-B and not MAO-A. Rabbit and mouse antisera indirectly immune precipitated [¹²⁵I]bovine MAO-B using inactivated Staphylococcus aureus cells, and binding of antibodies to bovine and rat MAO-B did not inhibit enzyme activity. Two continuous rat cell lines, hepatoma line MH₁C₁ and glioma line C6, were used to elucidate the specificity of the antisera. MH₁C₁ cells, which express both MAO-A and MAO-B, showed immune-specific staining with rabbit antiserum, and staining was blocked with pure MAO-B. Further, MAO-B activity and [³H]pargyline-labeled MAO molecules could be immune precipitated from solubilized mitochondrial preparations of MH₁C₁ cells; and immune fixation of mitochondrial proteins following SDS polyacrylamide gel electrophoresis (SDS-PAGE) revealed staining of the MAO-B, but not of the MAO-A, flavin-containing subunit. In contrast, no immune-specific immunocytochemical staining was observed in C6 cells, which have only MAO-A activity; no MAO-A activity or [³H]pargyline-labeled MAO could be immune precipitated from solubilized mitochondrial preparations of these cells, and no stained bands were observed for mitochondrial proteins resolved by SDS-PAGE and processed for immune fixation. Further support for the selectivity of this antiserum for MAO-B comes from immunocytochemical staining of rat tissues which express varying amounts of MAO-A and MAo-B activities. Hypothalamus and liver, with high levels of MAO-A and MAO-B activities showed a large number of immunoreactive cells, whereas spleen, heart and superior cervical ganglia, with high MAO-A and low MAO-B activities showed only a few or no stained cells. Catecholamine neurons in the substantia nigra, thought to contain MAO-A, did not show immune-specific staining. Skeletal muscle cells with low MAO-A and MAO-B activities did not stain. These studies provide additional evidence that MAO-A and MAO-B are distinct molecules, differentially expressed in different cell types.