ENZYMATIC RADIOLABELING TO A HIGH SPECIFIC ACTIVITY OF LEGUME LIPO-OLIGOSACCHARIDIC NODULATION FACTORS FROM RHIZOBIUM-MELILOTI

被引:27
作者
BOURDINEAUD, JP
BONO, JJ
RANJEVA, R
CULLIMORE, JV
机构
[1] CNRS,INRA,BIOL MOLEC RELAT PLANTES MICROORGANISMES LAB,F-31326 CASTANET TOLOSAN,FRANCE
[2] UNIV TOULOUSE 3,CTR RECH BIOL & PHYSIOL VEGETALE,CNRS,URA 1457,F-31162 TOULOUSE,FRANCE
关键词
D O I
10.1042/bj3060259
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
In this paper we describe the two-step coupled S-35-radiolabelling of the lipo-oligosaccharidic nodulation (Nod) factors of the bacterium Rhizobium meliloti to a specific radioactivity of 800 Ci/mmol. These radiolabelled Nod factors bind to a particulate fraction from roots of the bacterium's symbiotic host, Medicago truncatula, with an equilibrium dissociation constant (K-D) of 117 nM, similar to that observed with a synthetic tritiated ligand. The first step of the S-35-labelling involves the synthesis of 3'-phosphoadenosine 5'-phospho[S-35]sulphate ([S-35]PAPS) from ATP and [S-35]sulphate using yeast enzymes. The second step exploits the sulphotransferase activity of the R. meliloti NodH protein, which has been expressed in Escherichia coli, to transfer the labelled sulphate group from PAPS to non-sulphated Nod factors. This enzyme was found to be active in E. coli cultured at 18 degrees C but not 37 degrees C. NodH could also transfer the sulphate group from PAPS to a model substrate, tetra-N-acetyl chitotetraose, with apparent K-m values of 56 and 70 mu M respectively, and exhibited an apparent K-m value for non-sulphated Nod factors of 28 mu M. Coupling the two steps of the radiolabelling resulted in an efficiency of S-35 incorporation from inorganic sulphate to the Nod factors of similar to 10%. These labelled factors will be a valuable tool in the search for high-affinity receptors for the lipo-oligosaccharidic nodulation factors.
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页码:259 / 264
页数:6
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