PLANT PHYSIOLOGY , Vol 107, Issue 2 435-441, Copyright © 1995 by American Society of Plant Biologists
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BIOCHEMISTRY AND ENZYMOLOGY |
Immunoaffinity Purification and Comparison of Allantoinases from Soybean Root Nodules and Cotyledons
J. A. Bell and M. A. Webb
Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana 47907-1155
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[deg]C, decreased
gradually with heating to 85[deg]C, and was lost at 90 to 95[deg]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.
