Formation of Adenosine 5′-Phosphorofluoridate and the Assay of Adenyl Cyclase in Barley Seeds

Abstract

Barley seed (Hordeum vulgare L.) homogenates contain an apparent enzymatic activity which catalyzes the synthesis of adenosine 5'-phosphorofluoridate from magnesium-adenosine 5'-triphosphate and sodium fluoride. Formation of this compound may interfere with some adenyl cyclase assays which use fluoride as a component of the incubation medium. Neither adenyl cyclase activity nor endogenous adenosine 3': 5'-monophosphate was detected in barley seed homogenates or extracts.The mechanism of action of some animal hormones involves an alteration in the intracellular level of adenosine 3': 5'-monophosphate (cyclic AMP) (9,21,22,26). These hormones stimulate the activity of a membrane-bound adenyl cyclase system and thereby increase the enzymatic production of cyclic AMP from ATP. Sodium fluoride markedly stimulates adenyl cyclase activity in broken cell preparations from a variety of animal tissues (21,22), and it is routinely used in the assay of adenyl cyclase (11). A speculative explanation for the mechanism of fluoride action has been proposed which involves the formation of adenosine 5'-phosphorofluoridate as a reactive intermediate (1).Recently, preliminary evidence for the presence of adenyl cyclase (30) and endogenous cyclic AMP (16) in plant tissues has been presented. The synthesis of a cyclic AMP-like compound in plant tissues incubated with "4C-adenine has also been observed (15,18,23). Several reports indicate that the cyclic nucleotide promotes the secretion of some hydrolytic enzymes from embryoless barley seeds (4-6, 17, 19, 24 MATERIALS AND METHODSAdenyl Cyclase Assay. Adenyl cyclase activity was assayed by the method of Ramachandran (20). Tissues were homogenized in 10 volumes of cold 10 mm tris, pH 7.5, containing 0.25 M sucrose. Assays were performed for 15 min at 30 C in the presence or absence of 10 mm sodium fluoride. Washed particles from guinea pig hearts were prepared according to the method of Drummond and Duncan (3). The incubation medium for the adenyl cyclase assay was identical to that described previously (3), except for the concentrations of ATP (6 mM) and magnesium sulfate (9 mM).Cyclic AMP Assays. Endogenous cyclic AMP was assayed by the methods of Wastila et al. (28) and Gilman (7). Barley seed extracts were prepared in accordance with the method of Gilman (7).Apparent Enzymatic Formation of AMP-F. The synthesis of AMP-F was measured by a modification of an assay procedure described by Krishna et al. (11) for the determination of adenyl cyclase activity. Seeds were powdered and homogenized in 10 volumes of cold 10 mm MES buffer (pH 6.0), 1 mM dithiothreitol, and 10% (w/v) sucrose. The mixture was centrifuged at 30,000g for 10 min, and the supernatant fraction was used directly in the assay. The incubation medium consisted of 40 mm MES (pH 6.0), 3.3 mm magnesium sulfate, 10 mM caffeine, 0.1 mm ATP (0.5 fic "4C-ATP-34 mc/mmole), 0.1 M sodium fluoride and 0.18 ml of the enzyme preparation in a final volume of 0.6 ml. After an incubation period of 60 min at 30 C, the solutions ...

Self-Incompatibility Involved in the Level of Acetylcholine and cAMP

“…Cyclic AMP (cAMP) is an essential signaling molecule in both prokaryotes and eukaryotes. 1 The existence of cAMP in higher plants was questioned by some reviewers [2][3][4] in the mid 1970's, so that many workers were discouraged from studying roles in plant biology. However, its presence was confirmed by mass spectrometry 5 and infrared spectrometry 6 in the early 1980's and increasing evidence [7][8][9][10][11][12] now suggests that cAMP makes important contributions in plant cells, as in animals.…”