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A complete nodulin-35 (N-35) cDNA encoding nodule-specific uricase (EC 1.7.3.3) was isolated from a soybean (Glycine max L. var. Prize) nodule cDNA expression library using a previously isolated partial cDNA clone. The N-35 cDNA was expressed in Escherichia coli driven by the lacZ promoter and was found to be functionally active. The uricase activity was detected in the cytoplasmic fraction of E. coli with the same pH optimum and apparent Km values as that in the nodules. Because a stop codon is located 15 base pairs upstream of the N-35 initiation codon, it appears that a fusion protein with LacZ was not made, but reinitiation occurred due to the presence of a putative Shine-Dalgarno sequence in the appropriate region. The size of the N-35 polypeptide made in E. coli is identical to that present in soybean nodules and is able to assemble into a tetrameric holoenzyme with the same molecular weight as the native uricase. Thus, the presence of peroxisomes does not appear to be essential for the proper assembly of the holoenzyme in E. coli. These data also indicate that posttranslational modifications or membrane transport are not essential either for the assembly of N-35 into a holoenzyme or for the activity of uricase.  相似文献   

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Aspartate aminotransferase (AAT) is a key plant enzyme affecting nitrogen and carbon metabolism, particularly in legume root nodules and leaves of C4 species. To ascertain the molecular genetic characteristics and biochemical regulation of AAT, we have isolated a cDNA encoding the nodule-enhanced AAT (AAT-2) of alfalfa (Medicago sativa L.) by screening a root nodule cDNA expression library with antibodies. Complementation of an Escherichia coli AAT mutant with the alfalfa nodule AAT-2 cDNA verified the identity of the clone. The deduced amino acid sequence of alfalfa AAT-2 is 53 and 47% identical to animal mitochondrial and cytosolic AATs, respectively. The deduced molecular mass of AAT-2 is 50,959 daltons, whereas the mass of purified AAT-2 is about 40 kilodaltons as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the protein's N-terminal domain (amino acids 1-59) contains many of the characteristics of plastid-targeting peptides. We postulate that AAT-2 is localized to the plastid. Southern blot analysis suggests that AAT-2 is encoded by a small, multigene family. The expression of AAT-2 mRNA in nodules is severalfold greater than that in either leaves or roots. Northern and western blots showed that expression of AAT activity during effective nodule development is accompanied by a sevenfold increase in AAT-2 mRNA and a comparable increase in enzyme protein. By contrast, plant-controlled ineffective nodules express AAT-2 mRNA at much lower levels and have little to no AAT-2 enzyme protein. Expression of root nodule AAT-2 appears to be regulated by at least two events: the first is independent of nitrogenase activity; the second is associated with nodule effectiveness.  相似文献   

4.
In soybean root nodules the enzyme uricase is expressed concomitantly with nodule development. The initial expression of this protein does not depend on active nitrogen fixation, as demonstrated by analysis of uricase activity in effective and ineffective root nodules. However, the maximal level of uricase activity is determined by the infecting Rhizobium japonicum strain. Sterile root cultures and callus tissue, devoid of the microsymbiont, were incubated at varying oxygen concentrations and analyzed for uricase activity. The specific activity of uricase was increased by lowering the oxygen concentration, with the highest activity obtained around 4−5% oxygen. The increase in uricase activity was due to increased uricase synthesis, as demonstrated by in vivo labelling of callus culture followed by immunoprecipitation with antibodies raised against highly purified nodule uricase.  相似文献   

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Nodulin-35, a protein specific to soybean root nodules, was purified under non-denaturing conditions (DEAE-cellulose followed by Sephacryl S-200 chromatography) to homogeneity. The holoprotein showed uricase (EC 1.7.3.3) activity. Analytical ultracentrifugation under non-denaturing conditions revealed a molecule of 124 kd, S°20W = 8.1; however, under denaturing conditions a value of 33 kd, S°20W = 1.9, was obtained. This indicated that nodulin-35 is the 33-kd subunit of a specific soybean root nodule uricase (uricase II) and that the enzyme contains four similar subunits. The native molecule contains ˜1.0 mol Cu2+ per mol, has an isoelectric point of ˜9.0 and a pH optimum for uricase activity at 9.5. Uricase activity found in young uninfected soybean roots is due to another form of enzyme (uricase I) which is of ˜190 kd, has maximum activity at pH 8.0 and does not contain any subunit corresponding in size to nodulin-35. Uricase I, also present in young infected roots, declines at a time when nodulin-35 appears. Monospecific antibodies prepared against uricase II (nodulin-35) showed no cross-reactivity. Uricase II was localized in the uninfected cells of the nodule tissue. These results are consistent with the concept that a nodule-specific ureide metabolism takes place in peroxisomes of uninfected cells, and suggest the participation of uricase II in this pathway.  相似文献   

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Tissue distribution of uricase (urate oxidase, EC 1.7.3.3) was studied by immunoblotting and RNA slot blot analysis. For immunoblotting, highly specific monoclonal antibodies against rat liver uricase were obtained, and for mRNA detection, a cloned uricase cDNA was used. Among seven tissues studied, uricase was immunologically detected only in the liver. The contents of uricase in other tissues, i.e., brain, thymus, heart, spleen, kidney and lactating mammary gland, were estimated to be less than 2% of that in the liver. Uricase mRNA was also detected only in the liver. The steady-state level of the mRNA in the isolated hepatocytes was relatively constant during the 8-day culture period when compared with those of other mRNAs expressed in the liver, suggesting a unique control mechanism of its expression.  相似文献   

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The effect of lowering oxygen concentration on the expression of nodulin genes in soybean callus tissue devoid of the microsymbiont has been examined. Poly(A)+ RNA was isolated from tissue cultivated in 4% oxygen and in normal atmosphere.Quantitative mRNA hybridization experiments using nodule-specific uricase (Nodulin-35) and sucrose synthase (Nodulin-100) cDNA probes confirmed that the synthesis of the uricase and sucrose synthase is controlled by oxygen at the mRNA level.The steady-state levels of uricase and sucrose synthase mRNA increased significantly (5–6- and 4-fold respectively) when the callus tissue was incubated at reduced oxygen concentration. Concomitant with the increase in mRNA level a 6-fold increase in specific activity of sucrose synthase was observed.Two messengers representing poly-ubiquitin precursors also responded to lowering the oxygen concentration. The increase was about 5-fold at 4% oxygen. No expression at atmospheric oxygen or in response to low oxygen was observed when using cDNA probes for other nodulin genes such as leghemoglobin c3, nodulin-22 and nodulin-44.  相似文献   

10.
Uricase (nodulin-35) cDNA, LjUr, was isolated from nodules of a model legume, Lotus japonicus. LjUr expression was most abundant in nodules, although it was detected in nonsymbiotic tissues as well, particularly in roots. Expression in nodules was detected in uninfected cells, nodule parenchyma, and, more intensely, in vascular bundles. Phylogenetic analysis of uricase sequences from various legumes indicated that uricases of amide- and ureide-transporting legumes form two distinct clades. LjUr is in the cluster of amide-transport legumes even though L. japonicus bears determinate nodules.  相似文献   

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