Activity of glutamine synthetase (EC 6.3.1.2) in the soluble protein fraction of nodules from 21-day- old lupin plants was measured. On a fresh tissue weight basis, activity was approximately 500 times higher than in the soluble protein fraction from roots of inoculated plants and 300 times higher than in the bacteroid soluble protein fraction. The activity of glutamine synthetase in the bacteroids was approximately 10 times lower than in cultured rhizobia. Glutamine synthetase activity in the nodule cytoplasm was found to increase during nodule development over a time course which followed the induction of leghaemoglobin and nitrogenase. Glutaminase activity, which could be inhibited by L-5-diazo-4-oxo-norvaline, was detected in extracts of bacteroids and cultured rhizobia. The glutamine synthetases present in cultured rhizobia and in the nodule cytoplasm had different electrophoretic mobilities on polyacrylamide gels.
An L-asparaginase cDNA clone, BR4, was isolated from a Lupinus arboreus Sims developing seed expression library by screening with polyclonal antibodies to the seed asparaginase. The cDNA hybridised with an oligonucleotide probe designed from amino acid sequence data and was found on sequencing to be 947 bp in length. Six polypeptide sequences obtained previously could be placed along the longest open reading frame. Computer-aided codon use analysis revealed that the cDNA sequence was consistent with other plant genes in terms of codon use. The cDNA insert was used to analyse asparaginase transcription in various tissues by northern blot analysis. A transcript size of approximately 1.2 kb was detected in L. arboreus seed total and poly(A)+ RNA. The level of this transcript declined from 30 days after anthesis to an undetectable level by day 55. Furthermore, under the high stringency conditions used, the seed asparaginase cDNA did not hybridise with total or poly(A)+ RNA isolated from root tips, suggesting that the asparaginase known to be present in this tissue may be the product of a different gene. Southern analysis suggested the seed asparaginase is a single-copy gene. The plant asparaginase amino acid sequence did not have any significant homology with microbial asparaginases but was 23% identical and 66% similar (allowing for conservative substitutions) to a human glycosylasparaginase.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.