Radiolabeling of lipo-chitooligosaccharides using the NodH sulfotransferase: a two-step enzymatic procedure.
BACKGROUND
The NodH sulfotransferase from Sinorhizobium meliloti has been used to radiolabel lipochitooligosaccharidic (LCO) Nod factor signals with 35S from inorganic sulfate in a two-step enzymatic procedure. The first step involved the production of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), a sulphate donor, using enzymes contained in a yeast extract, and the second step used the NodH enzyme. However with this established procedure, only a low incorporation of the initial inorganic sulfate into the Nod factors was obtained (about 7% after purification of the labeled compounds). The aim of this work was to optimize the radiolabelling of Nod factors with 35S.


RESULTS
The limiting step has been shown to be the sulfation of ATP and its subsequent conversion into PAPS (first step), the sulfate donor for the NodH sulfotransferase activity (second step). By the addition of GTP to the reaction mixture and by manipulating the [ATP]/[Mg2+] ratio the yield of PAPS has been increased from 13% to 80%. Using the radiolabeled PAPS we have shown that the efficiency of sulfate transfer to LCOs, by the recombinant S. meliloti NodH sulfotransferase is strongly influenced by the length of the oligosaccharide chain. Variations in the substitutions on the non-reducing sugar, including the structure of the fatty acyl chain, had little effect and Nod factors from the heterologous bacterium Rhizobium tropici could be sulfated by NodH from S. meliloti.


CONCLUSIONS
By characterizing the two steps we have optimized the procedure to radiolabel biologically-important, lipo-chitooligosaccharide (LCO) Nod factors to a specific radioactivity of about 800 Ci x mmol(-1) with an incorporation of 60% of the initial inorganic sulfate. The two-step sulfation procedure may be used to radiolabel a variety of related LCO molecules.
Gressent
Frédéric
F
Signaux et Messages Cellulaires chez les Végétaux, UMR CNRS-UPS 5546, Pôle de Biotechnologie Végétale, 24 chemin de Borde Rouge, BP 17 Auzeville, 31326 Castanet-Tolosan, France. gressent@jouy.inra.fr
Cullimore
Julie V
JV
Ranjeva
Raoul
R
Bono
Jean-Jacques
JJ
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001426:Bacterial Proteins / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D007553:Isotope Labeling / Q000379:methods*; D007700:Kinetics; D008070:Lipopolysaccharides / Q000737:chemistry* / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D010724:Phosphoadenosine Phosphosulfate / Q000096:biosynthesis; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000378:metabolism*; D013462:Sulfur Radioisotopes
D001426:Bacterial Proteins; D008070:Lipopolysaccharides; D013462:Sulfur Radioisotopes; C023023:lipid-linked oligosaccharides; D010724:Phosphoadenosine Phosphosulfate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; C092976:NodH protein, Sinorhizobium meliloti; D015238:Sulfotransferases; D008274:Magnesium