Determination of 5-AMP in the presence of excess 3'(2')-AMP with the aid of antibodies raised against n6-carboxymethyl-5'-AMP conjugates. Use for the quantitation of pyridine nucleotides and of protein-bound ADP-ribose.
5'-AMP antigens were synthesized by conjugation of N6-carboxymethyl-5'-AMP (Cm65'-AMP) to native or methylated serum albumin. Injection of the antigens resulted in antibodies with high affinity and specificity for 5'-AMP in all animals, thus allowing discrimination against 3'(2')-AMP even when present at 10(4)-10(5) times higher concentrations. This specificity was comparable to that of anti 5'-AMP antibodies raised against Cm65'-AMP serum albumin antigens formed in situ from Cm6ADP-ribose serum albumin conjugates by intracellular or pericellular phosphodiesterases. The hapten in the Cm65'-AMP-methylated serum albumin conjugate appeared to be bound almost exclusively via the N6-position. Due to the free exposure of the 5'-phosphate group in this antigen, the resulting antibodies discriminated 5'-AMP derivatives substituted at the phosphate group more efficiently than derivatives with modifications in the adenine ring. It also led to the concomitant formation of adenosine-specific antibodies due presumably to dephosphorylation of the antigen by phosphatases present in the recipient animals. The conjugates formed from Cm65'-AMP and native serum albumin, which appeared to be linked to a large extent via carboxyl groups of the protein and hydroxyl groups of the ribose, recognized modifications in the adenine ring much better than substitutions at the phosphate group. However, in spite of these relatively small differences in specificity, all three types of antibodies could be used successfully to quantitate by radioimmunoassay protein-bound ADP-ribose in adult rat liver and NAD+-NADH in Ehrlich ascites tumor cells as shown by the excellent agreement of the values obtained with the three antisera.
D000246:Adenosine Diphosphate Ribose / Q000032:analysis; D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000276:immunology*; D000818:Animals; D000917:Antibody Formation; D009243:NAD / Q000032:analysis; D011817:Rabbits; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013379:Substrate Specificity
D009243:NAD; D000246:Adenosine Diphosphate Ribose; D012710:Serum Albumin, Bovine; D000249:Adenosine Monophosphate; C029741:N(6)-carboxymethyl-5'-AMP