Rapid purification and N-terminal sequencing of a potato tuber cyclic nucleotide binding phosphatase.

A high affinity cyclic nucleotide binding phosphatase was purified to homogeneity from potato tubers by a rapid procedure involving batchwise elution from carboxymethylcellulose and gel filtration. The phosphatase has a molecular weight of 28,000 as estimated from both SDS-PAGE and gel filtration. The phosphatase binds to Con A-agarose and is eluted by 0.5 M alpha-methylglucoside. The phosphatase catalyses the hydrolysis of nucleoside monophosphates, p-nitrophenylphosphate and O-phospho-L-tyrosine, but not of O-phospho-L-serine or O-phospho-L-threonine. N-terminal sequencing of the phosphatase has revealed significant homology with two similar-size soybean leaf and stem storage glycoproteins.

Properties of a calmodulin-activated Ca2+-dependent protein kinase from wheat germ.

A soluble protein kinase that is largely dependent upon Ca2+ for activity was partially purified from wheat germ. The protein kinase (Mr 90 000) catalyzes the phosphorylation of casein, histones and of endogenous proteins. Calmodulin activates the protein kinase with histone as substrate, half-maximal activation being obtained with 1.4 microM sheep brain calmodulin. The rate of casein phosphorylation is half-maximal at 0.3 microM free Ca2+ and maximal at 2.0 microM free Ca2+. Higher Ca2+ is required for histone phosphorylation, namely 80 microM and 500 microM free Ca2+, respectively, for half-maximal and maximal phosphorylation rates. In addition to Ca2+, millimolar Mg2+ is required for maximal activity of the enzyme; millimolar Mn2+ can substitute for the (Ca2+ + Mg2+) requirement. The Km for ATP is 31 microM; other nucleoside 5'-triphosphates and ADP inhibit phosphoryl transfer from ATP to protein. Serine and threonine residues of casein or histones are phosphorylated by the enzyme. The protein kinase is inhibited by relatively high concentrations of chlorpromazine and fluphenazine. The low free Ca2+ required for activation of the enzyme suggests that this type of Ca2+-dependent protein kinase may be involved in Ca2+-mediated stimulus-response coupling in plants.

Purification and sequencing of napin-like protein small and large chains from Momordica charantia and Ricinus communis seeds and determination of sites phosphorylated by plant Ca(2+)-dependent protein kinase.

The basic protein fraction from seeds of castor bean (Ricinus communis L.) contains 4732 Da and 4603 Da proteins phosphorylated in vitro by plant Ca(2+)-dependent protein kinase (CDPK). These proteins, RS1A and RS1B respectively, were purified by cation-exchange HPLC (SP5PW column) and reverse-phase HPLC (C18 column) and identified as napin-like protein small chains by Edman sequencing and electrospray ionization mass spectrometry (ESMS). The other R. communis 4 kDa small chains (RS2A, RS2B, RS2C and RS2D) are not phosphorylated by CDPK and neither is the corresponding 7332 Da large chain (RL) that forms 1:1 disulfide-linked complexes with RS2(A-D). RS1A/B is one of the best substrates found for plant CDPK (K(m) = 1.8 +/- 0.8 microM). RS2(A-D) (but not RL or RS1A/B) strongly inhibit calmodulin (CaM)-dependent myosin light chain protein kinase (MLCK) (IC50 = 0.25 microM) and inhibit the Ca(2+)-dependent enhancement of dansyl-CaM fluorescence. The basic protein fraction from seeds of bitter melon (Momordica charantia) also contains napin-like proteins that are 1:1 disulfide-linked complexes of a small chain (MS1, MS2, MS3 or MS4) and a large chain (ML). The M. charantia small chains were purified and completely sequenced by Edman degradation and ESMS. M. charantia small chains MS1, MS2, and MS4 (but not MS3) are phosphorylated by CDPK to unit stoichiometry on S21 within the sequence R17SCES21FLR. The R. communis small chain RS1A is phosphorylated on S34 within the sequence R31QSS34SRR. Both of these phosphorylation site motifs are consistent with those found for other plant CDPK substrates.

Inhibition of plant calcium-dependent protein kinases by basic polypeptides.

Wheat embryo Ca2+-dependent protein kinase (CDPK) is inhibited by a variety of polypeptides including actin, gramicidin S, melittin, protamine, various histone preparations, histone H4 and by basic amino-acid homopolymers. Melittin (Ki 9 microM) is a non-competitive inhibitor of wheat germ CDPK and also inhibits wheat leaf CDPK and silver beet leaf CDPKs. Protamine inhibits wheat germ CDPK in an apparently competitive fashion (Ki 0.2 microM) and is also a potent, albeit less effective, inhibitor of the leaf CDPKs. Various basic amino-acid homopolymers are also potent, apparently competitive inhibitors of wheat embryo CDPK, namely poly(L-lysine) (IC50 2 nM), poly(L-ornithine) (IC50 3 nM) and poly(L-arginine) (IC50 17 nM) and also inhibit the leaf CDPKs, albeit at higher concentrations. Histone H4 and various calf thymus histone preparations inhibit wheat embryo CDPK in a fashion that is not competitive and calmodulin can substantially reverse such inhibition.

Purification and sequencing of a family of wheat lipid transfer protein homologues phosphorylated by plant calcium-dependent protein kinase.

Four low molecular weight, basic proteins (WBP1A, WBP1B, WBP2 and WBP3) that are substrates for wheat germ Ca(2+)-dependent protein kinase (CDPK) were purified from wheat germ by a procedure involving batchwise cation exchange on carboxymethylcellulose (CM52), acid precipitation, cation exchange HPLC on an SP5PW column and reverse-phase HPLC on a C18 column. While WBP1A, WBP1B and WBP3 are phosphorylated by wheat germ CDPK exclusively on Ser residues, WBP2 is phosphorylated on both Ser and Thr residues. CDPK-catalysed phosphorylation sites on WBP1A and WBP1B were determined. With all four proteins the phosphorylated form comigrates with non-phosphorylated protein (Mr about 9 kDa) on SDS-PAGE. Average molecular masses of reduced WBP1A, WBP1B, WBP2 and WBP3 measured using electrospray ionisation mass spectrometry (ESMS) are 9389 Da, 9274 Da, 9479 Da and 9467 Da, respectively. The complete amino-acid sequences of WBP1A and WBP1B (determined by Edman sequencing and ESMS of proteolytically derived fragments) and N-terminal sequences of WBP2 and WBP3 are highly homologous to each other and to sequences of low molecular weight, basic plant lipid transfer proteins (LTPs).

Purification and characterization of a heat-stable wheat substrate for wheat embryo calcium-dependent protein kinase.

A heat-stable wheat protein (WP) that is a good substrate for wheat embryo Ca(2+)-dependent protein kinase (CDPK) was purified from wheat embryo by a procedure involving batchwise anion exchange chromatography on DEAE-cellulose (DE52), passage through Phenyl-Sepharose CL-4B, heat and acid treatment and anion exchange HPLC on a DEAE-5PW column. WP is phosphorylated by CDPK to a stoichiometry of about 0.8 mol phosphoryl per mol WP. The Km for WP is 3.5 microM. WP is phosphorylated by CDPK on Ser residues. [32P]phosphoWP exactly copurifies on SDS-PAGE with WP (59 kDa). Phosphorylation of WP by CDPK is largely Ca(2+)-dependent. The N-terminal amino acid sequence of WP has homology with bacterial azurins. Evidence for two serine phosphorylation sites was obtained from sequencing of phosphopeptides derived from tryptic and chymotryptic digests of phosphoWP. One major site of phosphorylation is inferred to be on a serine within the sequence KKMASMK. WP is one of the best endogenous protein substrates yet found for wheat embryo CDPK. A 59kDa protein is phosphorylated in vivo in sprouting wheat.

Purification and sequencing of multiple forms of Brassica napus seed napin small chains that are calmodulin antagonists and substrates for plant calcium-dependent protein kinase.

Six napin small (S) subunits and six napin large (L) subunits were resolved from the seeds of kohlrabi (Brassica napus var. rapifera) by a procedure involving extraction, batchwise elution from carboxymethylcellulose (CM52) and reverse-phase HPLC after treatment with guanidine hydrochloride and 2-mercaptoethanol. The precise average molecular masses of the ca. 4.5 kDa small subunits and the ca. 10 kDa large subunits were determined by electrospray ionisation mass spectrometry (ESMS). The amino-acid sequences of six small subunits (S1A, S1B, S2, S3A, S3B and S4) were deduced from the ESMS-based masses of tryptic fragments, Edman sequencing and previously published data. The deduced structures were precisely consistent with this data and with the ESMS-based average molecular masses of these polypeptides. The structures of the small subunits (39-41 residues) are very similar with variations involving single substitutions at or near the N-terminus and 1 to 3 changes within the last 7 amino acids. Particular B. napus small and large chains are phosphorylated by plant Ca2+-dependent protein kinase (CDPK). The best site of phosphorylation on small chains is inferred to be either S34 or S39 of S1B. The napin-containing basic protein fraction from B. napus seeds largely abolishes the Ca2+-dependent fluorescence enhancement of dansyl-calmodulin and also inhibits calmodulin (CaM)-dependent myosin light chain kinase (MLCK). The resolved napin small chains also inhibit MLCK. All of the kohlrabi napin small chains, as well as homologous Brassicaceae small chains, have a central 23 amino-acid sequence that can potentially form an alpha-helix in which all the basic residues are located on one side. This structural element may be involved in the interaction of these proteins with CaM and the biological activity of antifungal proteins of this kind.

Purification and sequencing of multiple forms of Brassica napus seed napin large chains that are calmodulin antagonists and substrates for plant calcium-dependent protein kinase.

Six napin large (L) chains (as well as six napin small chains) were resolved from the seeds of kohlrabi (Brassica napus var. rapifera) by a procedure involving extraction, batchwise elution from carboxymethylcellulose (CM52) and reversed-phase HPLC after treatment with guanidine hydrochloride and 2-mercaptoethanol. The precise average molecular masses of the circa 4.5 kDa small subunits and the circa 10 kDa large subunits were determined by electrospray ionisation mass spectrometry (ESMS). Of six large subunits resolved (L1A), L1B, L1C, L2A, L2B and L2C), the complete amino acid sequences of four (L1A, L2A, L2B and L2C) and the near-complete sequences of two (L1B and L1C) were deduced from the ESMS-based masses of tryptic fragments, Edman sequencing and previously published data. The deduced structures are precisely consistent with this data and with the ESMS-based average molecular masses of these polypeptides. ESMS analysis of unreduced napin extract revealed only seven circa 14.5 kDa complexes, the observed masses being in close agreement with those calculated for 1:1 complexes of particular small and large subunits assuming four disulfides in each napin complex. The structures of the napin large subunits (86-91 residues) are very similar and all amino acid differences observed are confined to only 25 positions. The L2A, L2B AND L2C large chains (but not the L1A, L1B and L1C large chains) are phosphorylated well by plant Ca2+-dependent protein kinase (CDPK). The CDPK-catalyzed phosphorylation site on the large chain L2A is inferred to be S57 within the sequence LQQVIS57RIYQT (the site being S60 within the same sequence in L2B and L2C). The napin-containing basic protein fraction from B. napus seeds largely abolishes the Ca2+-dependent fluorescence enhancement of dansyl-calmodulin and also inhibits calmodulin (CaM)-dependent myosin light chain kinase (MLCK). The resolved napin long chains also inhibit MLCK. Each kohlrabi large chain contains 2 sequences (corresponding to L(10)-Q(20) and Q(51)-L(64) of L1A) which have the potential to form amphipathic alpha-helices. Each large chain also contains a Q-rich 19 amino acid sequence (corresponding to L(30)-Q(48) of L1A) which has the potential to form a '2-sided' alpha-helix with basic residues confined to one side. These structural elements may be involved in the inferred interaction of these proteins with CaM and may be relevant to the biological activity of antifungal proteins of this kind.

Purification and characterization of wheat and pine small basic protein substrates for plant calcium-dependent protein kinase.

A wheat basic protein (WBP) was purified to homogeneity from wheat germ by a protocol involving extraction, centrifugation, batchwise elution from carboxymethylcellulose (CM-52), acidification with trifluoroacetic acid, neutralization and HPLC on a SP5PW cation exchange column. WBP is a 10 kDa protein and is phosphorylated on serine residues by wheat germ Ca(2+)-dependent protein kinase (CDPK). [32P]phosphoWBP exactly comigrates with WBP on SDS-PAGE. WBP does not inhibit either wheat germ CDPK or calmodulin-dependent myosin light chain kinase. Apart from histone H1, WBP is the best endogenous substrate yet found for wheat embryo CDPK. A 12 kDa pine basic protein (PBP) was purified to homogeneity from seeds of stone pine (Pinus pinea L.) by a simple procedure involving batchwise elution from carboxymethylcellulose and cation exchange HPLC. PBP is also a good substrate for CDPK and is phosphorylated on Ser residues. N-terminal sequencing of WBP and PBP revealed that these proteins are homologous to a family of small basic plant proteins having a phospholipid transfer function.

Inhibition of eukaryote signal-regulated protein kinases by plant-derived catechin-related compounds.

The cladodes of Phyllocladus trichomanoides, the bark of Pseudotsuga menziesii and the heartwood of Acacia melanoxylon contain catechin derivatives that are potent inhibitors of rat brain protein kinase C. Most of these compounds are also inhibitors of bovine heart cyclic AMP-dependent protein kinase catalytic subunit and wheat Ca(2+)-dependent protein kinase. However, these compounds are either not inhibitors or are relatively poor inhibitors of avian myosin light chain kinase. The most potent protein kinase inhibitors are the procyanidin dimer, trimer and tetramer from the bark of Pseudotsuga menziesii. These compounds are among the most potent plant-derived protein kinase inhibitors yet found.

Selective inhibition of cyclic AMP-dependent protein kinase by amphiphilic triterpenoids and related compounds.

A set of plant- and animal-derived amphiphilic triterpenoids have been shown to be potent and selective inhibitors of the catalytic subunit of rat liver cyclic AMP-dependent protein kinase (cAK). Thus plant-derived 18 alpha- and 18 beta-glycyrrhetinic acid, ursolic acid, oleanolic acid and betulin and animal-derived lithocholic acid, 5-cholenic acid and lithocholic acid methyl ester are inhibitors of cAK with IC50 values (concentrations for 50% inhibition) in the range 4-20 microM. These compounds are ineffective or relatively ineffective as inhibitors of various other eukaryote signal-regulated protein kinases namely wheat embryo Ca(2+)-dependent protein kinase (CDPK), avian calmodulin-dependent myosin light chain kinase (MLCK) and rat brain Ca(2+)- and phospholipid-dependent protein kinase C (PKC). These naturally occurring triterpenoids have a common structural motif involving polar residues located at opposite ends of an otherwise non-polar triterpenoid nucleus. A variety of triterpenoids not possessing this structural motif are relatively inactive as inhibitors of cAK and of CDPK, PKC and MLCK. The terpenoid amphiphilic compound crocetin is also a potent and relatively selective inhibitor of cAK (IC50 value for cAK 3.0 microM). 12-Hydroxystearic acid and 10-hydroxydecanoic acid do not inhibit CDPK, PKC or MLCK but are selective inhibitors of cAK (IC50 values 127 and 138 microM, respectively), consistent with a simple model for amphiphile inhibition of cAK involving two polar groups separated by a non-polar region. However, laurylgallate and 15-pentadecanolide are also potent and selective inhibitors of cAK (IC50 values 1.5 and 20 microM, respectively) although the structures of both of these compounds involve a large non-polar portion associated with only one polar region. Crocetin and the plant-derived amphiphilic triterpenoids described here are the most potent non-aromatic plant-derived inhibitors of cAK yet found.

Inhibition of cyclic AMP-dependent protein kinase by curcumin.

Curcumin [diferuloylmethane; 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione], a major bioactive secondary metabolite found in the rhizomes of turmeric (Curcuma longa), is an inhibitor of Ca(2+)- and phospholipid-dependent protein kinase C (PKC) and of the catalytic subunit (cAK) of cyclic AMP-dependent protein kinase (IC50 values 15 and 4.8 microM, respectively). Curcumin inhibits plant Ca(2+)-dependent protein kinase (CDPK) (IC50 41 microM), but does not inhibit myosin light chain kinase or a high affinity 3',5'-cyclic AMP-binding phosphatase. Curcumin inhibits cAK, PKC and CDPK in a fashion that is competitive with respect to both ATP and the synthetic peptide substrate employed. The IC50 values for inhibition of cAK by curcumin are very similar when measured with kemptide (LRRASLG) (in the presence or absence of ovalbumin) or with casein or histone III-S as substrates. However, the presence of bovine serum albumin (0.8 mg ml-1) largely overcomes inhibition of cAK by curcumin.

Inhibition of wheat embryo calcium-dependent protein kinase by acridines and azaacridines.

The inhibition of wheat Ca(2+)-dependent protein kinase (CDPK) by substituted acridines and substituted 5-, 6-, 7- and 8-azaacridines (5-AA, 6-AA, 7-AA and 8-AA) was examined. Of a total of 71 substituted acridines and azaacridines examined, only 20 have IC50 values for wheat CDPK of less than 200 microM. Of these, effective compounds all have neutral or basic 4-substituents, except for 2,7-dibromo-4-carboxy-5-AAO (IC50 73 microM), the carboxymethyl ester of which is a much better inhibitor (IC50 20 microM). There is a large aza position effect so that various 4-substituted azaacridines can be either very active or very poor inhibitors depending upon the azaacridine nucleus substituted. One of the most potent inhibitors found is the 8-AA 4-N-(2-dimethylaminoethyl)carboxamide (4-P) derivative (IC50 1.5 microM), but the corresponding 4-substituted acridine analogue is a very poor inhibitor. Other potent inhibitors found include 1-nitro-4-P-8-AA (IC50 4 microM) and 7-bromo-4-methyl-5-AA (IC50 0.7 microM). These potent and relatively specific CDPK inhibitors may be useful in obtaining evidence for CDPK involvement in plant cell responses to specific signals.

Inhibition of wheat embryo calcium-dependent protein kinase and other kinases by mangostin and gamma-mangostin.

The hull of the fruit of the mangosteen tree (Garcinia mangostana) contains four inhibitors of plant Ca(2+)-dependent protein kinase. Two of these inhibitors have been purified and identified as the xanthones 1,3,6-trihydroxy-7-methoxy-2,8-bis(3-methyl-2-butenyl)-9H- xanthen-9-one (mangostin) and 1,3,6,7-tetrahydroxy-2,8-bis(3-methyl-2-butenyl)- 9H-xanthen-9-one (gamma-mangostin). Both xanthones also inhibit avian myosin light chain kinase and rat liver cyclic AMP-dependent protein kinase. This is the first report of inhibition of plant and animal second messenger-regulated protein kinases by plant-derived xanthones.

Inhibition of signal-regulated protein kinases by plant-derived hydrolysable tannins.

A variety of hydrolysable tannins purified from Phyllanthus amarus are potent inhibitors of rat liver cyclic AMP-dependent protein kinase catalytic subunit (cAK) with IC50 values (concentrations for 50% inhibition) in the range 0.2-1.7 microM. The three most effective compounds of this series of hydrolysable tannins have five phenolic substituents. These three compounds are also the most effective inhibitors of wheat embryo Ca(2+)-dependent protein kinase (CDPK), rat brain Ca(2+)- and phospholipid-dependent protein kinase C (PKC) and Ca(2+)-calmodulin-dependent myosin light chain kinase (MLCK). The order of sensitivity for protein kinase inhibition by the hydrolysable tannins studied is cAK > CDPK > PKC > MLCK. Thus the IC50 values for protein kinase inhibition by the most potent compound are 0.2 microM (for cAK), 1.8 microM (for CDPK), 26 microM (for PKC) and 56 microM (for MLCK) when protein kinase affinity is measured using synthetic peptide substrates. These hydrolysable tannin inhibitors found are the most specific and potent plant-derived inhibitors of cAK yet found.

Differential inhibition of eukaryote protein kinases by condensed tannins.

Condensed tannins, isolated from a variety of plant sources, were characterized according to the constituent flavans, being based on procyanidin and/or prodelphinidin and having a cis or trans stereochemistry at positions 2 and 3. All the tannin preparations are potent inhibitors of rat liver cyclic AMP-dependent protein kinase catalytic subunit (cAK) with IC50 values (concentrations for 50% inhibition) ranging from 0.009 to 0.2 microM. The tannin preparations are very good inhibitors of rat brain Ca(2+)- and phospholipid-dependent protein kinase C (PKC) (IC50 values in the range 0.3-7 microM), wheat embryo Ca(2+)-dependent protein kinase (CDPK) (IC50 values in the range 0.8-7 microM) and of calmodulin (CaM)-dependent myosin light chain kinase (MLCK) (IC50 values in the range 7-24 microM). One of the most effective preparations, that from the leaves of Ribes nigrum, has IC50 values with respect to cAK, PKC, CDPK and MLCK of 0.009, 0.6, 2.0 and 16 microM, respectively. In general, the order with respect to sensitivity to inhibition by these condensed tannins is cAK > PKC > CDPK > MLCK. The Ribes nigrum preparation is a competitive inhibitor of cAK with respect to both ATP and synthetic peptide substrate. These condensed tannin preparations are the most potent plant-derived inhibitors of cAK yet found.

Inhibition of oxidative phosphorylation by organotin thiocarbamates.

A series of triphenyl-, tricyclohexyl- and tribenzyltin compounds have been synthesized and examined as inhibitors of mitochondrial oxidative phosphorylation. All compounds tested inhibit oxidative phosphorylation linked to succinate oxidation by potato tuber mitochondria. All of the organotin compounds inhibit ADP-stimulated O2 uptake linked to succinate oxidation with concentrations for 50% inhibition in the range 2-50 microM. This inhibition is not due to inhibition of electron transport from succinate to O2 per se: none of the organotin compounds at 50 microM substantially inhibit the rate of succinate oxidation in the presence of 2,4-dinitrophenol. Representative organotin compounds at 0.5-50 microM do not act as uncouplers of succinate oxidation. It is concluded that the organotin compounds act as energy transfer inhibitors to inhibit oxidative phosphorylation in potato tuber mitochondria. A similar mode of action of representative organotin compounds was found with rat liver mitochondria. These organotin compounds inhibit a hydrophobic Ca2+-dependent plant protein kinase in the absence but not in the presence of thiols.

Inhibition of eukaryote protein kinases and of a cyclic nucleotide-binding phosphatase by prenylated xanthones.

A series of prenylated xanthones are variously potent inhibitors of the catalytic subunit (cAK) of rat liver cyclic AMP-dependent protein kinase (PKA), rat brain Ca2+ and phospholipid-dependent protein kinase C (PKC), chicken gizzard myosin light chain kinase (MLCK), wheat embryo Ca2+-dependent protein kinase (CDPK) and potato tuber cyclic nucleotide-binding phosphatase (Pase). The prenylated xanthones examined are mostly derivatives of alpha-mangostin in which the 3-hydroxyl and 6-hydroxyl are variously substituted with groups R or R', respectively, or derivatives of 3-isomangostin (mangostanol) in which the 9-hydroxyl is substituted with groups R' or the prenyl side chain is modified. The most potent inhibitors of cAK have non-protonatable and relatively small R' and R groups. Conversely, the most potent inhibitors of PKC and MLCK have bulkier and basic R' groups. Some prenylated xanthones are also potent inhibitors of CDPK. PKC and cAK are competitively inhibited by particular prenylated xanthones whereas the compounds that are the most potent inhibitors of MLCK and CDPK are non-competitive inhibitors. Prenylated xanthones having relatively small and non-protonatable R' and R groups inhibit a high-affinity cyclic nucleotide binding Pase in a non-competitive fashion.

Regulation of a plant 5'(3')-ribonucleotide phosphohydrolase by cyclic nucleotides and pyrimidine, purine, and cytokinin ribosides.

A highly specific 5'(3')-ribonucleotide phosphohydrolase has been purified extensively from wheat seedling leaves. While catalyzing the hydrolysis of a wide variety of phosphomonoesters, the enzyme has K(m) values for adenosine 5'-monophosphate and adenosine 3'-monophosphate in the micromolar range and appears highly specific for 5'- and 3'-ribonucleoside monophosphates as substrates at pH 5.0. The cyclic nucleotides adenosine 3':5'-cyclic monophosphate, guanosine 3':5'-cyclic monophosphate, and 8-bromoadenosine 3':5'-cyclic monophosphate, as well as 8-bromoadenosine 5'-monophosphate, are powerful competitive inhibitors of the enzyme; the apparent K(i) values for these nucleotides are 3.4 muM, 1.6 muM, 26 muM, and 9.1 muM, respectively. The enzyme is inhibited noncompetitively by a variety of pyrimidine and purine (including cytokinin) ribosides and 2'-deoxyribosides. Since the cyclic nucleotide competitive inhibitors are also active growth promoters of higher plants, and, furthermore, since elevation of cyclic AMP levels appears to be a consequence of some phytohormone treatments that promote plant growth, it is suggested that negative control of this 5'(3')-ribonucleotide phosphohydrolase may be a significant component of growth regulation through the maximizing of the levels of nucleotide precursors for RNA and DNA synthesis.