Determination of cAMP in plant cells by a modified enzyme immunoassay method

Abstract

Presently, there is no doubt about the functioning of the adenylate cyclase signaling system in plants, but the role of this system in various physiological-biochemical processes has been investigated insufficiently. Cyclic adenosine monophosphate (cAMP), the key component produced by adenylate cyclase, whose concentrations in plant cells vary rather widely, is the indicator of functional activity for this signaling way. In the latter case, in the process of determination of concentrations of this messenger, one encounters difficulties related to insufficient sensitivity of the methods most frequently applied. In this connection, the proposed mechanism is a modification of the method of the enzyme immunoassay (EIA), which is based on immediate measurement of cAMP concentrations in the sample with the use of antibodies. This modification allows us to determine the concentrations of cAMP with the precision of 5 pM, which exceeds the sensitivity of other methods by approximately 10 times. The specificity of the assay has been confirmed by other two independent tests--the capillary electrophoresis and the nuclear magnetic resonance (NMR). It has also been compared to the data obtained with the use of the commercial kit from Sigma-Aldrich. The modification has been tested on such plant objects as in vitro potato plants, and suspension cells of potato and Arabidopsis.

The cyclic nucleotide cGMP is involved in plant hormone signalling and alters phosphorylation of Arabidopsis thaliana root proteins

“…In all the above studies, cGMP levels were measured with immunoassays (e.g. Lomovatskaya et al , 2011), a method that has intrinsic disadvantages (Isner and Maathuis, 2011). The current data suggest that, when measured in real time at the single-cell level, ABA perception increases cGMP levels.…”

Cyclic Nucleotides and Nucleotide Cyclases in Plant Stress Responses

“…Cyclic AMP formation is catalyzed by adenylate cyclases (E.C.4.6.1.1) that convert ATP to cAMP and pyrophosphate (Helmreich et al, 1976), while the enzyme cAMP PDE (E.C.3.1.4.17) hydrolyzes 3′,5′-cAMP to 5′-AMP (Robison & Sutherland, 1971). Endogenously applied cAMP can be used as an indicator of functional activity for adenylate cyclase signaling system (Lomovatskaya et al, 2011). The presence and localization of AC activity was first experimentally demonstrated in meristems of young pea hypocotyls (Hilton & Nesius, 1978) making use of adenylyl-imidodiphosphate as a substrate specific for the enzyme and lead nitrate as a precipitating product that can be visualized with electron microscopy and quantitated in situ.…”

“…The radioimmune assay is based on the primary binding of standard antigen with antibodies followed by unlabeled antigen that competitively replace the radioactively labeled antigen, while the bioluminescent method permits the monitoring of cAMP levels in the living system, however, the use of the latter requires fluorophores with different spectrum properties than chlorophyll and phenols. A recent improved enzyme immunoassay method for determining cAMP concentration in plant tissue was proposed (Lomovatskaya et al, 2011) and allows for the detection of cAMP down to 5 pM, and this is about 10 times more sensitive than previously reported methods. Enzymes that hydrolyze cAMP -cAMP PDEs -were first reported in pea seedlings (Lieberman & Kunishi, 1969), but apparently, such an activity was not detected in barley (Vandepeute et al, 1973).…”

A Modified Enzyme Immunoassay Method for Determination of cAMP in Plant Cells

“…It has been determined in the comparative experiments conducted earlier (Lomovatskaya et al, 2011) that the sensitivity of modified method is about 5 pM, which is eight to ten times more precise than that obtained using the acetylated version of EIA from Sigma-Aldrich and two times better than that from GE Healthcare. Furthermore, the EIAs from SigmaAldrich and GE Healthcare require the use of compounds, which possess lachrymator and corrosive effects; the prices of the reagents used for our modified method are some three times cheaper than those using the method from Sigma-Aldrich.…”

“…The concentration of cAMP in plant samples (plants of potato in vitro) was determined by capillary electrophoresis. We also used this method to compare the value of concentration to the value obtained by EIA (Lomovatskaya et al, 2011).…”