Use of monoclonal antibodies for the purification and characterization of the threonine-sensitive isozyme of maize homoserine dehydrogenase.
Monoclonal antibodies, highly specific for the threonine-sensitive isozyme of maize homoserine dehydrogenase, have been prepared and utilized to purify the enzyme to homogeneity. The results of one- and two-dimensional polyacrylamide gel electrophoresis under denaturing conditions indicate that the enzyme is composed of subunits of identical molecular weight. Apparent microheterogeneity of the subunits was observed during isoelectric focusing, but peptide maps generated by partial cleavage with three different chemical reagents did not reveal any differences among the proteins separated by isoelectric focusing. It is concluded that the subunits of the active dimeric and tetrameric configurations of the maize enzyme are identical or very similar. Evidence is presented which indicates that the enzyme purified by immunoaffinity chromatography retains all of the properties of freshly isolated enzyme, including the ability to undergo several ligand-induced slow transitions among four unique states and complex kinetic responses to physiological substrates. Two monoclonal antibodies are shown to interact differently with the purified enzyme. One, MC-11, reacts with all enzyme molecules, while the other, MC-3, is able to resolve two antigenically distinct subpopulations. These populations are present in approximately equal amounts in etiolated shoots and leaves of light-grown seedlings. However, the results of kinetic and hysteretic studies indicate that they are functionally indistinguishable. The antibodies appear to recognize a structural difference between the enzyme populations which does not result in detectable alterations in their catalytic or regulatory properties.
Krishnaswamy
S
S
Bryan
J K
JK
D000429:Alcohol Oxidoreductases / Q000302:isolation & purification*; D000911:Antibodies, Monoclonal; D002021:Buffers; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006715:Homoserine Dehydrogenase / Q000302:isolation & purification*; D007120:Immunochemistry; D007527:Isoenzymes / Q000302:isolation & purification*; D007700:Kinetics; D010446:Peptide Fragments / Q000032:analysis; D013912:Threonine / Q000494:pharmacology; D003313:Zea mays / Q000201:enzymology*
D000911:Antibodies, Monoclonal; D002021:Buffers; D007527:Isoenzymes; D010446:Peptide Fragments; D013912:Threonine; D000429:Alcohol Oxidoreductases; D006715:Homoserine Dehydrogenase