Aspartate aminotransferase from Panicum maximum Jacq. var. trichoglume Eyles, a C4 plant: purification, molecular properties, and preparation of antibody.
Extracts of the leaf tissue of Panicum maximum Jacq. var. trichoglume Eyles (a phosphoenolpyruvate carboxykinase type of C4 plant) were examined and at least two isoforms of aspartate aminotransferase (EC 2.6.1.1), with different electrophoretic mobilities, were detected. The predominant isoform was purified to homogeneity from mesophyll cells. The purification procedure included fractionation with ammonium sulfate followed by chromatography on diethylaminoethyl-cellulose, Sephacryl S-300, and hydroxyapatite. The purified enzyme had specific activities of 182 and 165 mumol/min/mg protein, measured in terms of the synthesis of oxaloacetate and aspartate, respectively, at pH 8.0. The enzyme, with an apparent molecular size of 100 kDa, appears to be a dimer of a single polypeptide with a molecular size of 42 kDa. Mono specific polyclonal antibodies were raised against the 42-kDa polypeptide. Only a single stained band was detected in extracts of whole leaves by immunoblot analysis with this antibody after two-dimensional polyacrylamide electrophoresis. Furthermore, no difference in mobility was observed between the enzymes extracted from mesophyll and bundle sheath cells on native polyacrylamide gels. These findings are discussed in relation to the other isoform in the leaves of this species.
Numazawa
T
T
Department of Agricultural Chemistry, School of Agriculture, Nagoya University, Japan.
Yamada
S
S
Hase
T
T
Sugiyama
T
T
D000906:Antibodies / Q000032:analysis*; D001219:Aspartate Aminotransferases / Q000276:immunology / Q000302:isolation & purification*; D002848:Chromatography, DEAE-Cellulose; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007527:Isoenzymes / Q000276:immunology / Q000302:isolation & purification*; D007700:Kinetics; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000032:analysis; D010944:Plants / Q000201:enzymology*
D000906:Antibodies; D007527:Isoenzymes; D001219:Aspartate Aminotransferases; D010729:Phosphoenolpyruvate Carboxykinase (GTP)