Resolution and Properties of Two High Affinity Cyclic Adenosine 3′:5′-monophosphate-Binding Proteins from Wheat Germ

Abstract

A high affinity cAMP-binding protein (cABP II) was purified to homogeneity from wheat germ. The apparent molecular weight of cABP II, as determined from gel exclusion chromatography, is 5.2 x 10' (at low ionic strength) and 2.8 x 10' (at high ionic strength). One polypeptide subunit (molecular weight, 80,000) was resolved by polyacrylamide gel electrophoresis of cABP II under subunit dissociating conditions. The purification protocol employed resolves cABP II from a distinct, less acidic cAMPbinding protein (cABP I). The Kd values for cAMP are about 10' molar and 10-molar for cABP II and cABP I, respectively. The cAMP-binding sites of cABP I and cABP II have a marked adenine-analog specificity, binding adenine, adenosine, adenine-derived nucleosides and nucleotides and a variety of adenine derivatves having cytokinin activity. While cABP II is phosphorylated in reactions catalyzed by endogenous protein kinases, there is no evidence for modulation of these cABP II-protein kinase interactions by cAMP.Cyclic AMP has major amplifying regulatory functions in prokaryotes (24) and eukaryotes (12) but, despite considerable investigation (1,20,32), there is still no direct evidence for an explicit regulatory function for cAMP in higher plants (1). Reports and speculative interpretations of physiological responses of plants to exogenous cAMP have been criticized (20,35), as has much of the evidence for the existence of cAMP in higher plants (1)(2)(3)20). Adenylate cyclase has yet to be convincingly demonstrated in cellfree extracts from higher plants (18), and there is, as yet, no evidence for the presence in higher plants of cAMP-dependent protein kinases (1, 35). Nevertheless, possible key elements of a cAMP regulatory system have been demonstrated in higher plants, including the presence of low concentrations of cAMP of nonmicrobiological origin (3, 4), net synthesis and secretion of cAMP (3), and the presence of phosphodiesterases that catalyze the hydrolysis of cAMP (5,7,9,21). Further, albeit circumstantial, evidence for a cAMP regulatory system in higher plants derives from the multiplicity of high affinity cyclic nucleotide-binding proteins in higher plants (8,15,16,19,(25)(26)(27)30). 5'-Nucleotidases that are competitively inhibited by cAMP and cGMP2 are present in wheat seedlings (26,27) and potato tubers (25). Cyclic GMP at p.M concentrations promotes GTP binding to wheat embryo elongation factor 1 and promotes polypeptide synthesis in the cell-free translation system from wheat embryo (19 activity has been resolved in Phaseolus (8), wheat embryo (30), and artichoke rhizomes (15,16). Protein kinases present in partially purified preparations of cAMP-binding proteins from artichoke rhizome and wheat embryo were not activated by cAMP (15,30). The present paper describes the resolution and properties of two cAMP-binding proteins from wheat germ and the interaction of one of these proteins with endogenous protein kinase. MATERIALS AND METHODS (NH4)2SO4 PrecipitationAssay for cAMP Binding. The (NH4)2SO4-pre...

Purification and Properties of a High Specific Activity Protein Kinase from Wheat Germ

“…Casein, phosvitin, and cABPII are phosphorylated by the purified casein kinase, which has absolute dependence on added protein substrate for activity (Table II). Neither BSA nor a preparation of calf thymus histones are phosphorylated by the casein kinase (Table II) 32P-1abeled casein from reactions catalyzed by casein kinase was hydrolyzed in 6 N HCl at 100°C for 1 to 4 h, and the hydrolysates were subjected to high-voltage electrophoresis as described previously (15). Peaks of radioactivity corresponding to phosphothreonine (6% of total) and to phosphoserine (17%) were found, in addition to lower mobility material near the point of application (37%) and a higher mobility anodic peak corresponding to Pi (24% of total).…”

“…However, the latter enzyme, unlike CBP kinase (16) and the casein kinase, is insensitive to N-ethylmaleimide (19). We have previously reported the phosphorylation of casein and cABPII by three wheat germ protein kinase fractions (15). However, these fractions were very impure (specific activities, 0.2-3.0 nmol/min-mg) and were isolated in low yield (2 nmol/min.…”

“…The only endogenous substrate for the wheat germ casein kinase that we have purified to date is the cyclic AMP-binding protein cABPII (15). The relatively low rates of phosphorylation of cABPII catalyzed by the purified casein kinase cannot be due to contaminating CBP kinase-for which enzyme cABPII is a good substrate (16)-since casein kinase preparations phosphorylating cABPII had no detectable activity with CBP as substrate (Table II).…”

“…CM-Sepharose CL-6B was obtained from Pharmacia Fine Chemicals AB. cABPII was purified to homogeneity as described previously (15). BSA, dephosphorylated casein (partially hydrolyzed and dephosphorylated for protein kinase studies), calf thymus histones (Sigma type II-A), wheat seed a-amylase inhibitor, and wheat germ lectin were obtained from Sigma Chemical Co. CBP and CBP kinase were purified, and casein was coupled to cyanogenbromide-activated Sepharose 4B as described in the accompanying paper (16).…”

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

“…Convincing data have been obtained, which are related to the main components of the scrutinized system: cAMP, adenylate cyclase, phosphodiesterase, cAMP-binding proteins (Brown et al, 1980;Polya and Bowman, 1981;Phedenko et al, 1983;Yavorskaya and Kalinin, 1984;Tarchevsky, 2001), nucleotide-gated channels (Martinez-Atienza et al, 2007). The concentration of the endogenous cAMP is an indicator of the functional activity for this signaling.…”