ANALYSIS OF 3',5'-CAMP AND ADENYLYL-CYCLASE ACTIVITY IN HIGHER-PLANTSUSING POLYCLONAL CHICKEN EGG-YOLK ANTIBODIES

Polyclonal antibodies were raised in chicken against an adenosine 3',5 '-monophosphate-diphtheria toroid antigen construct, The antibodies ob tained show selectivity and high affinity toward 3',5'-cyclic nucleoti des while exhibiting negligible affinity for 2',3'-cyclic nucleotides and other related adenine compounds. This paper reports on the develop ment of an immunoaffinity purification procedure allowing both adenosi ne 3':5'-monophosphate (3',5'-cAMP) and adenylyl cyclase activity meas urement in plant tissue samples. Basically, the technique consists of sequential purification of samples on solid-phase columns, the newly d eveloped immunoaffinity columns, and quantitative analysis in ion-supp ression HPLC coupled to photo diode array detection. The described met hod results in a drastic reduction of processing time compared to exis ting procedures and combines high yields (70-80%) and thorough purific ation, hence significantly increasing the sensitivity of quantificatio n of 3',5'-cAMP content in higher plant material. Used in adenylyl cyc lase activity measurement it also allows for a routine positive identi fication of the newly formed compound, 3',5'-cAMP, a feature generally lacking in existing adenylyl cyclase assays. (C) 1996 Academic Press, Inc.