Karel R. Schubert

Subcellular organization of ureide biogenesis from glycolytic intermediates and ammonium in nitrogen-fixing soybean nodules.

Subcellular organelle fractionation of nitrogen-fixing nodules of soybean (Glycine max (L.) Merr.) indicates that a number of enzymes involved in the assimilation of ammonia into amino acids and purines are located in the proplastids. These include asparagine synthetase (EC 6.3.1.1), phosphoribosyl amidotransferase (EC 2.4.2.14), phosphoglycerate dehydrogenase (EC 1.1.1.95), serine hydroxymethylase (EC 2.1.2.1), and methylene-tetrahydrofolate dehydrogenase (EC 1.5.1.5). Of the two isoenzymes of asparate aminotransferase (EC 2.6.1.1) in the nodule, only one was located in the proplastid fraction. Both glutamate synthase (EC 1.4.1.14) and triosephosphate isomerase (EC 5.3.1.1) were associated at least in part with the proplastids. Glutamine synthetase (EC 6.3.1.2) and xanthine dehydrogenase (EC 1.2.1.37) were found in significant quantities only in the soluble fraction. Phosphoribosylpyrophosphate synthetase (EC 2.7.6.1) was found mostly in the soluble fraction, although small amounts of it were detected in other organelle fractions. These results together with recent organelle fractionation and electron microscopic studies form the basis for a model of the subcellular distribution of ammonium assimilation, amide synthesis and uredie biogenesis in the nodule.

Ureide biogenesis in leguminous plants

Abstract Allantion and allantoic acid are the predominant forms of organic nitrogen produced by N-fixing nodules of some important legume species. Only recently has the significance and biosynthetic origin of these substances in leguminous plants been realized.

Biosynthesis of purines by a proplastid fraction from soybean nodules

A proplastid-containing fraction was rapidly prepared from soybean nodules by a combination of differential and step gradient centrifugation. This fraction was capable of incorporating [U-14C]glycine into purines in the presence of added phosphoribosylpyrophosphate or ribose 5-phosphate, glutamine, aspartate, ATP, bicarbonate, methenyl tetrahydrofolate, MgCl2, and KCl. The primary product was IMP; some inosine was also formed. Soluble and bacteroid fractions from soybean nodules gave considerably lower rates of incorporation. Labeled carbon from both [U-14C]serine and [3-14C]serine was incorporated into purines when tetrahydrofolate and NADP+ were substituted for methenyl tetrahydrofolate. In this case, small amounts of label were also found in AMP and xanthine monophosphate (XMP). Labeled bicarbonate was incorporated into IMP and inosine by the proplastid fraction. Labeled formate, however, was not a competent substrate for purine synthesis, indicating the absence of formyl tetrahydrofolate synthetase activity in this fraction. When labeled IMP was incubated with a proplastid preparation, most of the label appeared in inosine. XMP and xanthosine were also formed if NAD+ or NADP+ was added to the incubation mixture indicating the presence of IMP dehydrogenase activity in the proplastid fraction.

Uricase from soybean root nodules: Purification, properties, and comparison with the enzyme from cowpea☆

A 45-fold purification of uricase (urate:O2 oxidoreductase, EC 1.7.3.3) from soybean root nodules by ammonium sulfate fractionation, gel filtration, and affinity chromatography is described. Electrophoresis on nondenaturing gels using an activity stain or on sodium dodecyl sulfate (SDS) gels demonstrated that the enzyme obtained was nearly homogeneous. The subunit molecular weight of uricase estimated from SDS gels was 32,000 +/- 3000. Gel-filtration studies indicated that the native enzyme is a monomer at pH 7.5 which associates to form a dimer at pH 8.8. Enzyme activity was stabilized by the addition of dithiothreitol. The pH dependence of the enzyme showed an optimum of 9.5. Initial rate kinetics showed Km values of 10 and 31 microM for uric acid and oxygen, respectively, with an intersecting pattern of substrate dependence. Uricase activity was inhibited strongly by xanthine, which was competitive with respect to uric acid (Ki = 10 microM). No significant inhibition was observed in the presence of a variety of amino acids, ammonium, adenine, or allopurinol, in contrast with results reported for the cowpea enzyme. Gel-filtration chromatography and SDS-gel electrophoresis of uricase purified by the same method from cowpea nodules indicated that the native enzyme exists as a monomer of Mr 50,000 at pH 7.5.

Purine biosynthesis and catabolism in soybean root nodules: Incorporation of 14C from 14CO2 into xanthine☆

Abstract Nodulated root systems of soybean plants were exposed to 14CO2 in the presence and absence of allopurinol. After 5 h about one-fifth of the label in the perchloric acid-soluble fraction of the nodules was found to be in xanthine in the allopurinol-treated plants. Control plants contained much lower levels of xanthine, but with similar specific activity. Hypoxanthine was not detected in either control or allopurinol-treated plants, even though it would be expected to accumulate in the latter. Degradation of labeled xanthine from allopurinol-treated plants using xanthine oxidase and uricase resulted in the loss of most of the label. The preferential incorporation and accumulation of 14C from 14CO2 into C6 of xanthine in allopurinol-treated plants is consistent with the involvement of phosphoribosylaminoimidazole carboxylase in the de novo synthesis of purines. The accumulation of xanthine and absence of hypoxanthine in nodules of allopurinol-treated plants confirms earlier observations. In addition, the similar specific activities of 14C in xanthine in allopurinol-treated and control plants indicate that the xanthine which accumulates in allopurinol-treated plants is the product of de novo purine biosynthesis.

Ultrastructural and immunochemical analyses of the distribution of microfilaments in seedlings and plants ofGlycine max

SummaryFilamentous structures were observed when cytoplasmic extracts of various tissues of soybean plants and seedlings were examined by electron microscopy. Three main lines of evidence indicate that these structures represented microfilaments derived from the soybean tissues: a) the diameter of the filaments was estimated to be 6–7 nm; b) the addition of rabbit heavy meromyosin resulted in the decoration of the filaments, yielding characteristic arrow-head patterns; and c) ATP reversed the decoration of the filaments by heavy meromyosin. When the various anatomical parts of soybean plants and seedlings were compared for the presence of microfilaments, the root tips and radicles showed the highest frequency while the petioles and cotyledons yielded no observable filaments. In order to substantiate these findings, a quantitative radioimmunoassay was developed using rabbit antibodies directed against calf thymus actin. These studies demonstrated that the concentration of actin in extracts of the root tip was 15-fold higher than in those of the petiole and leaf. Similar comparisons of various parts of soybean seedlings showed that the radicle was rich in actin. These results suggest that actin filaments are found predominantly in the subterranean parts of plants.