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Metabolism and Enzymology

Aspartate Aminotransferase in Alfalfa Root Nodules ¹

I. Purification and Partial Characterization

U.S. Department of Agriculture/Agricultural Research Service, University of Minnesota, St. Paul, Minnesota 55108, Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, Minnesota 55108

Aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1 [AAT]), a key enzyme in the assimilation of C and N compounds, was purified from the cytosol of alfalfa (Medicago sativa L.) root nodules. Isoforms that increased during nodule development, AAT-2a, AAT-2b, and AAT-2c, were purified greater than 447-fold to apparent homogeneity, and high titer polyclonal antibodies were produced. The native molecular weight of the AAT-2 isoforms was approximately 80 kilodatons with a subunit molecular weight of 40 kilodatons, indicating that the holoenzymes are dimers. The AAT-2 isoforms comprised approximately 0.4% of the total soluble nodule protein. The AAT specific activity was measured in leaf, stem, root, and nodule organs, and zymograms of each were compared. Enzyme activity was 4- to 37-fold greater in effective (nitrogen fixing) nodules than in leaves, stems, and roots. Effective nodule AAT-specific activity was 3- to 8-fold greater than that of plant-controlled ineffective nodules. No differences in K_m were observed between AAT-1 and AAT-2. Antibodies raised against AAT-2 were more selective against AAT-2 than AAT-1. Evidence obtained from zymograms suggests that the expression of alfalfa nodule AAT is controlled at two different gene loci, AAT-1 and AAT-2, resulting in different dimeric isoforms.

¹ Joint contribution from the Minnesota Agriculture Experiment Station (Paper No. 16,053, Scientific Journal Series) and U.S. Department of Agriculture/Agricultural Research Service. The research was supported in part by USDA/Competitive Research Grants office grant 87-CRCR-1-2588.

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