Purification of Allantoinase from Soybean Seeds and Production and Characterization of Anti-Allantoinase Antibodies

Abstract

Allantoinase catalyzes the hydrolysis of allantoin to allantoic acid, a reaction important in both biogenesis and degradation of ureides. Ureide production in cotyledons of germinating soybean (Glycine max 1.) seeds has not been studied extensively but may be important in mobilizing nitrogen reserves. Allantoinase was purified approximately 2500-fold from a crude extrad of soybean seeds by differential centrifugation, heat treatment, ammonium sulfate fractionation, ethanol fractionation, and fast protein liquid chromatography (Pharmacia) with Mono-Q and Superose columns. The purified enzyme had a subunit size of 30 kD. Polyclonal antibodies produced against the purified protein titrated allantoinase activity in a crude extract of seed proteins. Antibodies recognized the 30-kD band in western blot analysis of crude seed extracts, indicating that they were specific for allantoinase.

Immunoaffinity Purification and Comparison of Allantoinases from Soybean Root Nodules and Cotyledons

“…The nodule sequence had 80% identity (Fig. 5) with the N-terminal sequence of seed allantoinase (Webb and Lindell, 1993). Neither sequence shared any significant similarity with other sequences in computer data banks, including sequences for allantoinase in yeast (Buckholz and Cooper, 1991).…”

“…The polyclonal antibodies used in these studies were made against native allantoinase purified from soybean seeds, as detailed by Webb and Lindell (1993).…”

“…Second-dimension slab gels were prepared according to Laemmli (1970). For western blot analysis, proteins were electroblotted onto nitrocellulose and probed with anti-allantoinase antibodies as described previously (Webb and Lindell, 1993) …”

“…In soybean (Glycine max L.) and some other legumes, ureide metabolism is induced by the onset of rhizobial infection and nodulation (Fujihara et al, 1977;Matsumoto et al, 1977aMatsumoto et al, , 1977bTajima et al, 1977), and root nodules assimilate recently fixed nitrogen into the ureides allantoin and allantoic acid for export to the shoot (Pate, 1973;Matsumoto et al, 1977a;Herridge et al, 1978). Soybean seedlings also produce ureides in the absence of infection with rhizobia (Matsumoto et al, 1977a;Polayes and Schubert, 1984), and ureide production in the cotyledons may contribute to the export of nitrogen reserves to support seedling growth (Webb and Lindell, 1993). Ureides arise from de novo purine synthesis in nodules (Atkins et al, 1980;Boland and Schubert, 1982), whereas in cotyledons of germinating seedlings they may arise from both de novo purine synthesis and purine salvage (Polayes and Schubert, 1984).…”

“…Previous studies of allantoinase in crude preparations (Franke et al, 1965;Thomas et al, 1983;Rao et al, 1988;Reddy et al, 1989) have indicated that there may be tissue-specific isoforms. Webb and Lindell (1993) recently described the purification of allantoinase from soybean seeds. Allantoinase from soybean nodules was previously resistant to purification (Webb, 1988;M.A.…”