Synthesis and applications of 8-azido photoaffinity analogs of P1,P3-bis(5'-adenosyl)triphosphate and P1,P4-bis(5'-adenosyl)tetraphosphate.
32P-labeled photoaffinity analogs of bis(5'-adenosyl)-tetraphosphate and bis(5'-adenosyl)triphosphate which contain a single photoreactive 8-azidoadenosine group distal to the radiolabel have been synthesized from commercially available components using a combination of chemical and enzymatic procedures including a water-soluble carbodiimide. The method is simple, rapid, and produces yields of high specific activity products of around 60%. The analog of bis(5'-adenosyl)-tetraphosphate is very similar to the parent compound in its inhibition of rat liver adenosine kinase and its efficiency as a substrate for the bis(5'-nucleosidyl)tetraphosphate pyrophosphohydrolase from Artemia embryos. In the latter case, ATP and 8-azidoAMP are the preferred products. As would be expected, this analog is a much more effective photoprobe for both adenosine and adenylate kinases than the corresponding analog of bis(5'-adenosyl)triphosphate. Both compounds have been used to photoaffinity label crude extracts of Artemia, Vero cells, and Clostridium acetobutylicum and preferential specific labeling of different polypeptides by each analog has been shown. In extracts of C. acetobutylicum, the labeling of a polypeptide of Mr 48,500 by the bis(5'-adenosyl)tetraphosphate analog was totally dependent on the presence of Co2+ ions. These compounds should therefore prove valuable both for the active site labeling of purified binding proteins and for the detection and identification of new target proteins for these nucleotides.
Prescott
M
M
Department of Biochemistry, School of Life Sciences, University of Liverpool, United Kingdom.
McLennan
A G
AG
D000241:Adenosine / Q000031:analogs & derivatives*; D000345:Affinity Labels / Q000138:chemical synthesis / Q000378:metabolism; D000818:Animals; D001156:Artemia / Q000032:analysis; D001386:Azides; D001426:Bacterial Proteins / Q000302:isolation & purification; D002352:Carrier Proteins / Q000302:isolation & purification; D003013:Clostridium / Q000032:analysis; D015226:Dinucleoside Phosphates / Q000378:metabolism; D010455:Peptides / Q000302:isolation & purification; D014709:Vero Cells / Q000032:analysis
D000345:Affinity Labels; D001386:Azides; D001426:Bacterial Proteins; D002352:Carrier Proteins; D015226:Dinucleoside Phosphates; D010455:Peptides; C012760:8-azidoadenosine; C020733:diadenosine tetraphosphate; C011440:diadenosine triphosphate; D000241:Adenosine