A radioimmunoassay for (p)ppGpp and its application to Streptomyces hygroscopicus.
Aradioimmunoassay for (p)ppGpp was set up and applied to Streptomyces hygroscopicus. The (p)ppGpp specific antibodies obtained from rabbits immunized with ppGpp-human serum albumin could not distinguish between guanosine-5'-triphosphate-3'-diphosphate (pppGpp) and guanosine-5'-diphosphate-3'-diphosphate (ppGpp) whereas the cross reacitivity against structurally related nucleotides was negligible. Therefore, the antibodies were used for determination of (p)ppGpp, i.e. the sum of pppGpp and ppGpp. The pretreatment of biological samples for quantification of (p)ppGpp included formic acid extraction of cell pool followed by step gradient elution on DEAE-Sephadex A25 (Cl-) column for separation of (p)ppGpp from nucleotides with three or less phosphate groups. The very sensitive radioimmunoassay allowed determination of very low amounts: 1--50 pmoles (p)ppGpp per assay tube. The application of the RIA to S. hygroscopicus revealed a basal level in exponentially growing mycelium of about 2 pmoles (p)ppGpp per mg dry weight. The equilibrated pool size after induction of stringent response with serine hydroxamate was found to be about 30 pmoles (p)ppGpp per mg dry weight.
D002848:Chromatography, DEAE-Cellulose; D006150:Guanine Nucleotides / Q000032:analysis*; D006158:Guanosine Pentaphosphate / Q000032:analysis*; D006159:Guanosine Tetraphosphate / Q000032:analysis*; D010761:Phosphorus Radioisotopes; D011863:Radioimmunoassay; D013302:Streptomyces / Q000032:analysis*; D014316:Tritium
D006150:Guanine Nucleotides; D010761:Phosphorus Radioisotopes; D014316:Tritium; D006159:Guanosine Tetraphosphate; D006158:Guanosine Pentaphosphate