Product identification and kinetic studies of nucleotidyl cyclase activity in isolated chloroplasts by quantitative fast-atom bombardment mass spectrometry

Abstract

While the natural occurrence of adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate in higher plants is now well established, the existence in plants of specific nucleotidyl cyclase activities functioning to synthesize these two cyclic nucleotides has been the topic of recent controversy. Here positive-ion fast-atom bombardment mass spectrometry is used to unequivocally identify the products of the putative nucleotidyl cyclase activities in a membrane fraction from isolated spinach chloroplasts, and quantitative mass spectrometry is used in a study of enzymic reaction kinetics to determine the relevant kinetic parameters necessary for the elucidation of the binding interactions between the substrates and the two enzymes. Copyright # 1999 John Wiley & Sons, Ltd. Received 12 March 1999; Revised 27 March 1999; Accepted 30 March 1999 In animals cyclic nucleotides play a vital role in biochemical signal transduction and the regulation of cellular metabolism. Adenosine 3',5'-cyclic monophosphate (cyclic AMP) was first discovered as the mediator of glucagon action in glycogen metabolism in the mammalian liver, 1 this observation leading to the second messenger hypothesis, a concept describing the role of cyclic AMP as the intracellular regulator whose synthesis is altered in response to a wide range of extracellular hormones and neurotransmitters. This change in intracellular cyclic AMP in turn elicits changes in the activity of enzymes and proteins within the cell. 2 The change in cyclic AMP synthesis from adenosine 5'-triphosphate (ATP) is the result of a change in activity of adenylyl cyclase, a multicomponent enzyme located in the cell membrane able to respond to neurotransmitters and hormones binding to receptors on the cell surface. 3A second cyclic nucleotide, guanosine 3',5'-cyclic monophosphate (cyclic GMP), performs a similar but more restricted second messenger role, mediating the actions of a diverse set of primary messengers including natriuretic peptides, light and nitric oxide, 4-6 with the synthesis of cyclic GMP from guanosine 5'-triphosphate (GTP) catalyzed by a second nucleotidyl cyclase, guanylyl cyclase.

Cytoenzymological analysis of adenylyl cyclase activity and 3′:5′‐cAMP immunolocalization in chloroplasts of Nicotiana tabacum

“…Although these effects were not studied with respect to cAMP metabolism, it is striking that cAMP fluctuations in lower plants have been associated with photosynthetic activity (Segovia et al ., 2001; Gordillo et al ., 2004), chlorophyll synthesis (Berchtold & Bachofen, 1977), blue light signalling (Iseki et al ., 2002) and red light signalling (Okamoto et al ., 2004). By contrast, biochemical evidence has been acquired for the presence of phosphodiesterase (Brown et al ., 1980) and adenylyl cyclase (Newton et al ., 1999b; Witters et al ., 2004) in chloroplasts of higher plants. Together with the cytoenzymological data and immunocytochemical data presented here, chloroplasts can be regarded as ultimate candidates for further investigation in order to assign to them a physiological functional cAMP metabolic activity.…”

Product identification and adenylyl cyclase activity in chloroplasts of Nicotiana tabacum

“…When coupled to capillary liquid chromatography, cyclic nucleotides can be quantified on‐line at the low‐femtomole detection level 13. In addition to the well‐established evidence of the natural occurrence of cyclic nucleotides in higher plants there is growing evidence for the presence of the metabolic enzymes: adenylyl cyclase (AC) and cyclic nucleotide phosphodiesterase, as well,14 and references therein,15 and various physiological functions of cyclic nucleotides have been summarised in a recent review by Trewavas et al 16. Adenylyl cyclase activity has been demonstrated in higher plants by the use of histochemical and biochemical methods.…”