Immunoaffinity purification and comparison of allantoinases from soybean root nodules and cotyledons.
Allantoinase (allantoin amidohydrolase, EC 3.5.2.5) catalyzes the conversion of allantoin to allantoic acid in the final step of ureide biogenesis. We have purified allantoinase more than 4000-fold by immunoaffinity chromatography from root nodules and cotyledons of soybean (Glycine max [L] Merr.). We characterized and compared properties of the enzyme from the two sources. Seed and nodule allantoinases had 80% identity in the first 24 amino acid residues of the N terminus. Two-dimensional gel electrophoresis of the purified enzymes showed that multiple forms were present in each. Allantoinases from nodules and cotyledons had very low affinity for allantoin with a Km for allantoin of 17.3 mM in cotyledons and 24.4 mM in nodules. Both had activity in a broad range of pH values from 6.5 to 7.5. In addition, purified allantoinase from both sources was very heat stable. Enzyme activity was stable after 1 h at 70 degrees C, decreased gradually with heating to 85 degrees C, and was lost at 90 to 95 degrees C. Although these studies have revealed some differences between allantoinases in seeds and nodules, the differences were not reflected in key enzyme properties. The immunoaffinity approach enabled purification of allantoinase from soybean root nodules and simplified its purification from cotyledons, thereby allowing characterization and comparison of the enzyme from the two sources.
Bell
J A
JA
Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana 47907-1155, USA.
Webb
M A
MA
D000581:Amidohydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D000906:Antibodies; D002846:Chromatography, Affinity; D018548:Cotyledon / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008969:Molecular Sequence Data; D018517:Plant Roots / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D013025:Glycine max / Q000201:enzymology*; D013816:Thermodynamics
D000906:Antibodies; D000581:Amidohydrolases; C019659:allantoinase