Identification and quantitation of adenosine-3?:5?-cyclic monophosphate in plants using gas chromatography-mass spectrometry and high-performance liquid chromatography

Abstract

Direct evidence has been obtained for the presence of adenosine-3':5'-cyclic monophosphate (cAMP) in tobacco (Nicotiana tabacum L.) callus tissue cultures, bean (Phaseolus vulgaris L.) seedlings and immature kernels of sweet corn (Zea mays L.) through the use of a highly specific and sensitive gas chromatography-mass spectrometric assay. Levels of endogenous cAMP ranged from 70 to 126 pmol/g fresh weight. Corresponding levels of cAMP determined for the same samples using radioimmunoassay were consistently three to four times higher. Contrary to previous reports for citrus plants, measurable levels of cAMP could not be detected in young lemon leaves within the limits of detection of the mass-spectrometric assay method. In the case of tobacco callus tissue, the coumarin glucoside, scopolin, which was present in large amounts and showed similar chromatographic behaviour to cAMP, interferred strongly with the mass-spectrometric measurements of cAMP in inadequately purified extracts. The use of high-performance liquid chromatography, in addition to standard chromatographic purification methods, produced highly purified plant extracts for quantitation of cAMP and also provided a method for the separation of cAMP from its 2':3'-isomer.

Determination of cyclic 3′,5′‐adenosine monophosphate in plant tissues by high performance liquid chromatography

“…These results are comparable with those reported for bean and corn tissues (Janistyn, 1986;Newton and Brown, 1986). However, the cAMP content in tissues of tobacco callus (Johnson et al, 1981) appeared to be lower due to the soft conditions of material fixation.…”

“…Plant extracts contain a large number of unidentified substances whose behaviour is similar to that of cAMP in different chromatographic systems. Moreover, the lack of specificity of existing methods for quantitative determination of CAMP in plant tissues does not permit accurate information about cAMP content of higher plants to be obtained (Johnson et al, 1981). This certainly applies to the isotope method of Gilman (1970), which is based on competitive interaction of exogenous labelled cAMP ( [8-3H]-cAMP) with unlabelled cyclophosphate of analysed samples in binding to specific CAMP-binding proteins.…”

“…Similarly the results obtained by radioimmunoassays (RIA) may also be subject to ambiguity. In some cases they appear to be over-rated (Johnson et al, 1981), whilst in others they are seemingly undervalued (Spiteri et af., 1989). The development of new methods based on the use of sensitive detection systems which enable the determination of cAMP independent of the source of its extraction is required to obtain reliable and reproducible results.…”

“…In contrast to methods using an electron-capture detector as in gas chromatography (GC), HPLC does not require the formation of volatile derivatives of the analysed substance and is a highly efficient chromatographic process. This enables the collection of separate fractions of analysed components and hence permits further identification by other methods such as mass spectrometry (MS) (Johnson, 1981;Newton et al, 1980) and, in particular, fast atom bombardment MS (Newton et al, 1989).…”

“…Some publications deal with cAMP analysis by HPLC in cells of animals (Miyatake et al, 1987) or plants (Janistyn, 1986), but the technique has been used mainly for additional purification of plant extracts. Other methods are used for the quantitative estimation of cAMP level (such as combined G U M S ) which prolong the analytical process and cause substantial losses of the substance (Johnson et al, 1981).…”