ABSTRACT
Two isoenzymic forms of aspartate aminotransferase are present in the plant fraction of developing lupin root nodules. One of these forms, aspartate aminotransferase-P2 (AAT-P2), increases dramatically with the onset of biological nitrogen fixation and is associated with the assimilation of ammonia by the plant in the Rhizobium-legume symbiosis. A day 18 lupin nodule cDNA library in the lambda ZapII vector was immunoscreened with a monoclonal antibody specific for AAT-P2 and yielded two near-full-length 1700 bp clones. These clones were sequenced. Amino acid sequences from three peptides derived from immunopurified AAT-P2 were aligned, and showed 100% homology with the amino acid sequence deduced from the cDNA clones. The DNA sequence showed 50% homology with AAT sequences from a range of animal sources. Conversion of the clones to the phagemid form allowed their expression in Escherichia coli where both exhibited enzyme activity that could be immunoprecipitated with AAT-P2-specific monoclonal antibodies. Western blot analysis revealed protein moieties with molecular masses of 39, 43, 45 and 55 kDa. The 5' end of the clones coded for a hydrophobic leader sequence of about 50 amino acids indicative of a targeting sequence and consistent with the plastid localisation of nodule AAT-P2.
Subject(s)
Aspartate Aminotransferases/genetics , DNA/genetics , Fabaceae/genetics , Isoenzymes/genetics , Plants, Medicinal , Amino Acid Sequence , Animals , Aspartate Aminotransferases/isolation & purification , Base Sequence , Blotting, Western , Cloning, Molecular/methods , DNA/isolation & purification , Fabaceae/enzymology , Fabaceae/physiology , Isoenzymes/isolation & purification , Molecular Sequence Data , Protein Conformation , Rhizobium/physiology , Sequence Homology, Nucleic Acid , SymbiosisABSTRACT
Twenty-one monoclonal antibodies were raised against the aspartate aminotransferase-P(2) isoenzyme from root nodules of Lupinus angustifolius [L.] cv Uniharvest. Induction of this isoenzyme is positively correlated with the onset of N(2) fixation in effective root nodules and is associated with the assimilation of ammonia by the plant in the Rhizobium-legume symbiosis. The monoclonal antibodies produced were all of the IgG class, recognized five different epitopes on the protein, and represented greater than 90% of the available epitopes. These epitopes were not unique to lupin nodule aspartate aminotransferase-P(2) but were shown to be present on the enzyme from tobacco leaves and potato. Four of the epitopes were conformational with a fifth epitope recognized by the appropriate monoclonals in both its native and denatured forms. None of the monoclonal antibodies produced reacted with Rhizobium Iupini NZP2257 extracts. Antibodies against two epitopes showed some cross-reaction with the constitutive aspartate aminotransferase-P(1) isoenzyme also found in lupin root nodules. However, affinity of these monoclonals for AAT-P(1) was three orders of magnitude lower than for AAT-P(2). Monoclonals against the other epitopes appeared to be specific for aspartate aminotransferase-P(2).