Cawthon R M, Breakefield X O
Biochem Pharmacol. 1983 Feb 1;32(3):441-8. doi: 10.1016/0006-2952(83)90521-x.
[3H]Pargyline-labeled polypeptides associated with the A and B types of monoamine oxidase (MAO) activity in two rat cell lines were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). [3H]Pargyline was bound to MAO A and B in a crude mitochondrial fraction from rat hepatoma cell line MH1C1 and to MAO A in a mitochondrial fraction from rat glioma line C6. Specific radiolabeling of proteins associated with type A or B activity in the hepatoma samples was achieved by incubation with selective B or A inhibitors, respectively, prior to [3H]pargyline binding. Following [3H]pargyline binding, the samples were solubilized by heating in sodium dodecyl sulfate (SDS) in the presence of a reducing agent. SDS-PAGE of [3H]pargyline bound samples revealed a radiolabeled protein band of apparent molecular weight (mol. wt) 63,000 daltons associated exclusively with MAO A activity and a band of apparent mol. wt 60,000 associated exclusively with MAO B activity. Furthermore, when SDS-solubilized, [3H]pargyline-labeled MAO A and B proteins from these cell lines were subjected to limited proteolysis and one-dimensional peptide mapping in SDS gels, different patterns of [3H]pargyline-labeled peptides were obtained. These findings indicate that the A and B forms of MAO activity are associated with enzyme molecules of different primary covalent structures determined by different gene loci.
通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）比较了两种大鼠细胞系中与A、B型单胺氧化酶（MAO）活性相关的[3H]帕吉林标记的多肽。[3H]帕吉林与大鼠肝癌细胞系MH1C1粗线粒体组分中的MAO A和B结合，与大鼠胶质瘤细胞系C6线粒体组分中的MAO A结合。在[3H]帕吉林结合之前，分别用选择性B或A抑制剂孵育肝癌样品，实现与A或B型活性相关蛋白质的特异性放射性标记。[3H]帕吉林结合后，样品在还原剂存在下于十二烷基硫酸钠（SDS）中加热溶解。[3H]帕吉林结合样品的SDS-PAGE显示，一条表观分子量（mol. wt）为63,000道尔顿的放射性标记蛋白带仅与MAO A活性相关，一条表观mol. wt为60,000的带仅与MAO B活性相关。此外，当这些细胞系中经SDS溶解的[3H]帕吉林标记的MAO A和B蛋白进行有限蛋白酶解并在SDS凝胶中进行一维肽图谱分析时，获得了不同的[3H]帕吉林标记肽模式。这些发现表明，MAO活性的A和B形式与由不同基因位点决定的不同一级共价结构的酶分子相关。
