Analysis of 3',5'-cAMP and adenylyl cyclase activity in higher plants using polyclonal chicken egg yolk antibodies.
Polyclonal antibodies were raised in chicken against an adenosine 3',5'-monophosphate-diphtheria toxoid antigen construct. The antibodies obtained show selectivity and high affinity toward 3',5'-cyclic nucleotides while exhibiting negligible affinity for 2',3'-cyclic nucleotides and other related adenine compounds. This paper reports on the development of an immunoaffinity purification procedure allowing both adenosine 3':5'-monophosphate (3',5'-cAMP) and adenylyl cyclase activity measurement in plant tissue samples. Basically, the technique consists of sequential purification of samples on solid-phase columns, the newly developed immunoaffinity columns, and quantitative analysis in ion-suppression HPLC coupled to photo diode array detection. The described method results in a drastic reduction of processing time compared to existing procedures and combines high yields (70-80%) and thorough purification, hence significantly increasing the sensitivity of quantification of 3',5'-cAMP content in higher plant material. Used in adenylyl cyclase activity measurement it also allows for a routine positive identification of the newly formed compound, 3',5'-cAMP, a feature generally lacking in existing adenylyl cyclase assays.
Roef
L
L
Department of Biology, University of Antwerp, Wilrijk, Belgium.
Witters
E
E
Gadeyne
J
J
Marcussen
J
J
Newton
R P
RP
Van Onckelen
H A
HA
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D002645:Chickens; D002846:Chromatography, Affinity / Q000379:methods; D000242:Cyclic AMP / Q000032:analysis*; D004530:Egg Yolk / Q000276:immunology*; D010944:Plants / Q000201:enzymology / Q000378:metabolism*; D051381:Rats; D014407:Tumor Cells, Cultured
D000906:Antibodies; D000242:Cyclic AMP; D000262:Adenylyl Cyclases