Limits...
Radiolabeling of lipo-chitooligosaccharides using the NodH sulfotransferase: a two-step enzymatic procedure.

Gressent F, Cullimore JV, Ranjeva R, Bono JJ - BMC Biochem. (2004)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: 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

ABSTRACT

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.

Show MeSH

Related in: MedlinePlus

Time course of PAPS formation. The reaction mixture containing 26 μM Na2SO4 (10 μCi of labeled sulfate) was incubatedat 37°C in 20 mM Tris-HCl buffer pH 8.5, 40 mM MgCl2, 4 mM ATP. The reaction was started by addition of 30 μl of yeast extract, either (A) without GTP (■) or (B) in the presence of 10 mM GTP (●).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC404373&req=5

Figure 1: Time course of PAPS formation. The reaction mixture containing 26 μM Na2SO4 (10 μCi of labeled sulfate) was incubatedat 37°C in 20 mM Tris-HCl buffer pH 8.5, 40 mM MgCl2, 4 mM ATP. The reaction was started by addition of 30 μl of yeast extract, either (A) without GTP (■) or (B) in the presence of 10 mM GTP (●).

Mentions: A procedure that allows the formation of [35S]PAPS using Na2 35SO4 and Mg-ATP as substrates and yeast extract as a source of enzymes has been previously described [8]. Under the set experimental conditions (4 mM ATP, 40 mM MgCl2, 30 μl yeast extract in 20 mM Tris buffer pH 8.5) we have found that PAPS synthesis is transient with about 13% of the initial sulfate being incorporated by 30 min but then with it being degraded by 120 min (Fig. 1A). If the yeast extract was concentrated two-fold by ultrafiltration, the initial velocity of the reaction increased, and the same maximum was reached within 15 min, but the product was degraded twice as fast (data not show). These data indicate that the PAPS loss was not due to chemical or physical degradation, but that it was enzymatically degraded. Additions to the reaction mixture of 50 I.U of inorganic pyrophosphatase or 100 I.U of pyruvate kinase and phosphoenolpyruvate (which catalyses the conversion of ADP into ATP), in order to eliminate inorganic pyrophosphate (PPi) or ADP which are potent inhibitors of ATP sulfurylase and APS kinase respectively [9], were unable to improve the final yield. Moreover, preincubation of the yeast extract at 37°C during 30 min before addition of ATP, Mg2+ and labeled inorganic sulfate did not change the time course of the reaction, showing that the PAPS synthesis enzymes were not heat inactivated or inhibited by chemicals contaminating the yeast extract. In contrast, if the preincubation was done in the presence of ATP and Mg2+, the subsequent addition of sulfate did not lead to the formation of PAPS. This observation suggests that the yield of PAPS might be improved by altering the composition of the reaction mixture.


Radiolabeling of lipo-chitooligosaccharides using the NodH sulfotransferase: a two-step enzymatic procedure.

Gressent F, Cullimore JV, Ranjeva R, Bono JJ - BMC Biochem. (2004)

Time course of PAPS formation. The reaction mixture containing 26 μM Na2SO4 (10 μCi of labeled sulfate) was incubatedat 37°C in 20 mM Tris-HCl buffer pH 8.5, 40 mM MgCl2, 4 mM ATP. The reaction was started by addition of 30 μl of yeast extract, either (A) without GTP (■) or (B) in the presence of 10 mM GTP (●).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC404373&req=5

Figure 1: Time course of PAPS formation. The reaction mixture containing 26 μM Na2SO4 (10 μCi of labeled sulfate) was incubatedat 37°C in 20 mM Tris-HCl buffer pH 8.5, 40 mM MgCl2, 4 mM ATP. The reaction was started by addition of 30 μl of yeast extract, either (A) without GTP (■) or (B) in the presence of 10 mM GTP (●).
Mentions: A procedure that allows the formation of [35S]PAPS using Na2 35SO4 and Mg-ATP as substrates and yeast extract as a source of enzymes has been previously described [8]. Under the set experimental conditions (4 mM ATP, 40 mM MgCl2, 30 μl yeast extract in 20 mM Tris buffer pH 8.5) we have found that PAPS synthesis is transient with about 13% of the initial sulfate being incorporated by 30 min but then with it being degraded by 120 min (Fig. 1A). If the yeast extract was concentrated two-fold by ultrafiltration, the initial velocity of the reaction increased, and the same maximum was reached within 15 min, but the product was degraded twice as fast (data not show). These data indicate that the PAPS loss was not due to chemical or physical degradation, but that it was enzymatically degraded. Additions to the reaction mixture of 50 I.U of inorganic pyrophosphatase or 100 I.U of pyruvate kinase and phosphoenolpyruvate (which catalyses the conversion of ADP into ATP), in order to eliminate inorganic pyrophosphate (PPi) or ADP which are potent inhibitors of ATP sulfurylase and APS kinase respectively [9], were unable to improve the final yield. Moreover, preincubation of the yeast extract at 37°C during 30 min before addition of ATP, Mg2+ and labeled inorganic sulfate did not change the time course of the reaction, showing that the PAPS synthesis enzymes were not heat inactivated or inhibited by chemicals contaminating the yeast extract. In contrast, if the preincubation was done in the presence of ATP and Mg2+, the subsequent addition of sulfate did not lead to the formation of PAPS. This observation suggests that the yield of PAPS might be improved by altering the composition of the reaction mixture.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: 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

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus