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Metabolism and Enzymology

Aspartate Aminotransferase in Alfalfa Root Nodules ¹

II. Immunological Distinction between Two Forms of the Enzyme

Plant Science Research Unit, U.S. Department of Agriculture, Agricultural Research Service, University of Minnesota, St. Paul, Minnesota 55455, Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, Minnesota 55455

Aspartate aminotransferase (AAT), a key enzyme in the biosynthesis of aspartate and asparagine, occurs as two forms in alfalfa (Medicago sativa L.), AAT-1 and AAT-2. Both forms were purified to near homogeneity, and high titer polyclonal antibodies produced to the native proteins. Alfalfa AAT-1 was purified from root suspension culture cells, while AAT-2 was purified from effective root nodules. Antibodies prepared to AAT-1 and used as probes for western blots readily recognized native and SDS forms of AAT-1 but did not recognize either native or SDS forms of AAT-2. Conversely, antibodies to AAT-2 readily recognized native and SDS forms of AAT-2 but did not recognize AAT-1. Immunotitrations further confirmed the immunological distinction between AAT-1 and AAT-2. AAT-1 antibodies immunotitrated 100% of the in vitro activity of purified AAT-1 but had no effect on AAT-2 in vitro activity. Likewise, AAT-2 antibodies removed 100% of the in vitro activity of purified AAT-2 but did not affect AAT-1 in vitro activity. Sequential titration of total AAT activity from roots and nodules showed that AAT-1 comprised the major form (62%) of AAT in roots, while AAT-2 was the predominant form (90%) in nodules. Last, SDS-PAGE western blots showed that the molecular masses of AAT-1 and AAT-2 were 42 and 40 kilodaltons, respectively. These data indicate that AAT is under the control of at least two distinct genes in alfalfa.

¹ Joint contribution from the U.S. Department of Agriculture-Agricultural Research Service and the Minnesota Agriculture Experiment Station (Paper No. 17,650, Scientific Journal Series). The research was supported in part by USDA-CRGO grant 87-CRCR-1-2588 and NSF-DCB Grant 8905006.

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