Studies on pig liver mevalonate-5-diphosphate decarboxylase

A procedure in which three sequential enzymes of cholesterol biosynthesis, mevalonate kinase (ATP: (R)-mevalonate 5-phosphotransferase, EC 2.7.1.36), phosphomevalonate kinase (ATP: (R)-5-phosphomevalonate phosphotransferase, EC 2.7.4.2) and mevalonate-5-diphosphate decarboxylase (ATP: (R)-5-diphosphomevalonate carboxy-lyase (dehydrating), EC 4.1.1.33), from pig liver, could be purified in the one operation is described. Mevalonate kinase and phosphomevalonate kinase were utilized for the enzymic synthesis of mevalonate 5-diphosphate (both 1-14C-labelled and unlabelled), the substrate for mevalonate-5-diphosphate decarboxylase, using excess free ATP4-. A radioactive assay for the enzyme, based on the release of 14CO2 from [1-14C]mevalonate-5-diphosphate, was developed. The assay allowed reassessment of the metal and nucleotide specificity of the decarboxylase. ATP could be partially replaced by GTP and ITP, but no activity was observed with CTP, UTP or TTP. Apparent activation of the enzyme by ATP4- was observed as found for mevalonate kinase (C.S. Lee and W.J. O'Sullivan (1983) Biochim. Biophys. Acta 747, 215-224) and phosphomevalonate kinase (C.S. Lee and W.J. O'Sullivan (1985) Biochim. Biophys. Acta 839, 83-89). The presence of 1 mM excess free ATP4-, above that complexed as the substrate MgATP2-, decreased the Km for MgATP2- from 0.45 mM to 0.15 mM. MgADP- was shown to act as a competitive inhibitor with respect to MgATP2-.     

Inhibition of mevalonate 5-diphosphate decarboxylase by fluorinated substrate analogs

Mevalonate 5-diphosphate decarboxylase (MDD) is a peroxisomal enzyme in the cholesterol biosynthetic pathway, which plays an important role in regulating cholesterol biosynthesis. In the present study, rat MDD was cloned and purified to apparent homogeneity. Two fluorinated MDD substrate analogs, P'-geranyl 2-fluoromevalonate 5-diphosphate (4) and 2-fluoromevalonate 5-diphosphate (6), were synthesized, and both were found to be irreversible inhibitors of rat MDD. These two inhibitors were characterized, and mechanisms of the inactivation process were proposed. Kinetic studies indicate both analogs only bind into mevalonate binding-site of MDD. Compound 4 shows competitive inhibition on mevalonate kinase (MVK), and its IC(50) value was determined to be comparable with that of geranyl diphosphate. Further kinetic studies indicate compound 4 only bind into ATP binding-site of MVK. These studies provide an example for a single inhibitor to carry out sequential blocking of two enzymes in cholesterol biosynthesis, which may provide useful information for drug discovery for the purpose of treating cardiovascular disease and cancer or for pest control.     

Functional evaluation of conserved basic residues in human phosphomevalonate kinase

Phosphomevalonate kinase (PMK) catalyzes the cation-dependent reaction of mevalonate 5-phosphate with ATP to form mevalonate 5-diphosphate and ADP, a key step in the mevalonate pathway for isoprenoid/sterol biosynthesis. Animal PMK proteins belong to the nucleoside monophosphate (NMP) kinase family. For many NMP kinases, multiple basic residues contribute to the neutralization of the negatively charged pentacoordinate phosphate reaction intermediate. Loss of basicity can result in catalytically impaired enzymes. On the basis of this precedent, conserved basic residues of human PMK have been mutated, and purified forms of the mutated proteins have been kinetically and biophysically characterized. K48M and R73M mutants exhibit diminished Vmax values in both reaction directions (>1000-fold) with only slight Km perturbations (<10-fold). In both forward and reverse reactions, R110M exhibits a large (>10,000-fold) specific activity diminution. R111M exhibits substantially inflated Km values for mevalonate 5-phosphate and mevalonate 5-diphosphate (60- and 30-fold, respectively) as well as decreases [50-fold (forward) and 85-fold (reverse)] in Vmax. R84M also exhibits inflated Km values (50- and 33-fold for mevalonate 5-phosphate and mevalonate 5-diphosphate, respectively). The Ki values for R111M and R84M product inhibition by mevalonate 5-diphosphate are inflated by 45- and 63-fold; effects are comparable to the 30- and 38-fold inflations in Km for mevalonate 5-diphosphate. R141M exhibits little perturbation in Vmax [14-fold (forward) and 10-fold (reverse)] but has inflated Km values for ATP and ADP (48- and 136-fold, respectively). The Kd of ATP for R141M, determined by changes in tryptophan fluorescence, is inflated 27-fold compared to wt PMK. These data suggest that R110 is important to PMK catalysis, which is also influenced by K48 and R73. R111 and R84 contribute to binding of mevalonate 5-phosphate and R141 to binding of ATP.     

Role of membrane-bound 5''-nucleotidase in nucleotide uptake by the moderate halophile Vibrio costicola

Intact cells of Vibrio costicola hydrolyzed ATP, ADP, and AMP. The membrane-bound 5'-nucleotidase (C. Bengis-Garber and D. J. Kushner, J. Bacteriol. 146:24-32, 1981) was solely responsible for these activities, as shown by experiments with anti-5'-nucleotidase serum and with the ATP analog, adenosine 5'-(beta gamma-imido)-diphosphate. Fresh cell suspensions rapidly accumulated 8-14C-labeled adenine 5'-nucleotides and adenosine. The uptake of ATP, ADP, and AMP (but not the adenosine uptake) was inhibited by adenosine 5'-(beta gamma-imido)-diphosphate similarly to the inhibition of the 5'-nucleotidase. Furthermore, the uptake of nucleotides had Mg2+ requirements similar to those of the 5'-nucleotidase. The uptake of ATP was competitively inhibited by unlabeled adenosine and vice versa; inhibition of the adenosine uptake by ATP occurred only in the presence of Mg2+. These experiments indicated that nucleotides were dephosphorylated to adenosine before uptake. The hydrolysis of [alpha-32P]ATP as well as the uptake of free adenosine followed Michaelis-Menten kinetics. The kinetics of uptake of ATP, ADP, and AMP also each appeared to be a saturable carrier-mediated transport. The kinetic properties of the uptake of ATP were compared with those of the ATP hydrolysis and the uptake of adenosine. It was concluded that the adenosine moiety of ATP was taken up via a specific adenosine transport system after dephosphorylation by the 5'-nucleotidase.     

Pyruvate Kinase of Streptococcus lactis

The kinetic properties of pyruvate kinase (ATP:pyruvate-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis have been investigated. Positive homotropic kinetics were observed with phosphoenolpyruvate and adenosine 5′-diphosphate, resulting in a sigmoid relationship between reaction velocity and substrate concentrations. This relationship was abolished with an excess of the heterotropic effector fructose-1,6-diphosphate, giving a typical Michaelis-Menten relationship. Increasing the concentration of fructose-1,6-diphosphate increased the apparent V_max values and decreased the K_m values for both substrates. Catalysis by pyruvate kinase proceeded optimally at pH 6.9 to 7.5 and was markedly inhibited by inorganic phosphate and sulfate ions. Under certain conditions adenosine 5′-triphosphate also caused inhibition. The K_m values for phosphoenolpyruvate and adenosine 5′-diphosphate in the presence of 2 mM fructose-1,6-diphosphate were 0.17 mM and 1 mM, respectively. The concentration of fructose-1,6-diphosphate giving one-half maximal velocity with 2 mM phosphoenolpyruvate and 5 mM adenosine 5′-diphosphate was 0.07 mM. The intracellular concentrations of these metabolites (0.8 mM phosphoenolpyruvate, 2.4 mM adenosine 5′-diphosphate, and 18 mM fructose-1,6-diphosphate) suggest that the pyruvate kinase in S. lactis approaches maximal activity in exponentially growing cells. The role of pyruvate kinase in the regulation of the glycolytic pathway in lactic streptococci is discussed.     

Improved procedures for the synthesis of phosphomevalonate and for the assay and purification of pig liver phosphomevalonate kinase

An improved procedure for the synthesis of phosphomevalonate using excess free ATP4-, and phenyl agarose to remove contaminating nucleotides, is described. A high-voltage electrophoresis assay, which separates phosphomevalonate from mevalonate 5-diphosphate at pH 3.5, was developed for the assay of phosphomevalonate kinase (ATP:5-phosphomevalonate phosphotransferase, EC 2.7.4.2). High-voltage electrophoresis, at pH 5, could also be used for the separation of mevalonate 5-diphosphate from isopentenyl diphosphate. An alternative method for the purification of phosphomevalonate kinase from pig liver was also developed. The high-voltage electrophoresis assay was used to reassess the metal ion and nucleotide specificity of the pig liver phosphomevalonate kinase. ATP could be partially replaced by ITP and GTP and poorly by CTP and UTP. Apparent activation of the enzyme by free ATP4- was observed as found for mevalonate kinase (C.S. Lee and W.J. O'Sullivan (1983) Biochim. Biophys. Acta 747, 215-224).     

Evidence for the Calvin cycle and hexose monophosphate pathway in Thiobacillus ferrooxidans

The enzymes of the Calvin reductive pentose phosphate cycle and the hexose monophosphate pathway have been demonstrated in cell-free extracts of Thiobacillus ferrooxidans. This, together with analyses of the products of CO(2) fixation in cell-free systems, suggests that these pathways are operative in whole cells of this microorganism. Nevertheless, the amount of CO(2) fixed in these cell-free systems was limited by the type and amount of compound added as substrate. The inability of cell extracts to regenerate pentose phosphates and to perpetuate the cyclic fixation of CO(2) is partially attributable to low activity of triose phosphate dehydrogenase under the experimental conditions found to be optimal for the enzymes involved in the utilization of ribose-5-phosphate or ribulose-1,5-diphosphate as substrate for CO(2) incorporation. With the exception of ribulose-1,5-diphosphate, all substrates required the addition of adenosine triphosphate (ATP) or adenosine diphosphate (ADP) for CO(2) fixation. Under optimal conditions, with ribose-5-phosphate serving as substrate, each micromole of ATP added resulted in the fixation of 1.5 mumoles of CO(2), whereas each micromole of ADP resulted in 0.5 mumole of CO(2) fixed. These values reflect the activity of adenylate kinase in the extract preparations. The K(m) for ATP in the phosphoribulokinase reaction was 0.91 x 10(-3)m. Kinetic studies conducted with carboxydismutase showed K(m) values of 1.15 x 10(-4)m and 5 x 10(-2)m for ribulose-1,5-diphosphate and bicarbonate, respectively.     

Extracellular Interconversion of Nucleotides Reveals an Ecto-Adenylate Kinase Activity in the Rat Hippocampus

Here, the extracellular interconversion of nucleotides and nucleosides was investigated in rat hippocampal slices and synaptosomes by an HPLC-UV technique. Adenosine 5′-triphosphate (ATP) was converted to adenosine 5′-diphosphate (ADP), adenosine 5′-monophosphate (AMP), adenosine, inosine, and hypoxanthine in the slices, whereas ADP elicited parallel and concentration-dependent formation of ATP and AMP. The specific adenylate kinase inhibitor diadenosine pentaphosphate decreased the rate of decomposition of ADP and inhibited the formation of ATP. No substantial changes in the interconversion of ADP to ATP and AMP were found in the presence of dipyridamole, flufenamic acid, the P2 receptor antagonist pyridoxal-5-phosphate-6-azophenyl-2′,4′-disulphonic acid tetrasodium (PPADS), and the alkaline phosphatase substrate para-nitrophenylphosphate. Negligible levels of nucleotides were generated when uridine 5′-diphosphate (UDP), AMP or adenosine were used as substrates. Ecto-adenylate kinase activity was also observed in purified synaptosomes. In summary, we demonstrate the presence of an ecto-adenylate kinase activity in the hippocampus, which is a previously unrecognized pathway that influences the availability of purines in the central nervous system.     

Inactivation of chicken liver mevalonate 5-diphosphate decar☐ylase by sulfhydryl-directed reagents: evidence of a functional dithiol

Chicken liver mevalonate 5-diphosphate decar☐ylase (ATP:(R)-5-diphosphomevalonate car☐y-lyase (dehydrating), EC 4.1.1.33) is inactivated by methylmethanethiosulfonate and 5,5′-dithiobis(2-nitrobenzoate). The presence of the substrates ATP or mevalonate 5-diphosphate protect very effectively against inactivation. The inactivation is second order with respect to methylmethanethiosulfonate, with an inactivation rate constant of (7.6 ± 0.1) · 10⁻⁵ μM⁻² ·s⁻¹, implying that the modifier may be reacting with more than one thiol in the enzyme. The enzyme is also inactivated by a number of dithiol-specific reagents, suggesting the presence of a functional dithiol. The determined pK_app values for enzyme modification by methyl methanethiosulfonate and phenylarsine oxide are 7.3 ± 0.1 and 7.6 ± 0.3, respectively. From the data presented, it is concluded that the enzyme posseses a functional dithiol that is important for substrate binding.     

Purification, characterization, and regulation of a nicotinamide adenine dinucleotide-dependent lactate dehydrogenase from Actinomyces viscosus.

A nicotinamide adenine dinucleotide-specific L-(+)-lactate dehydrogenase (LDH) (EC 1.11.27) from Actinomyces viscosus T-6-1600 was purified approximately 110-fold by a combination of diethylaminoethyl-cellulose and 0.5 M Agarose A column chromatography. The ldh was stable at 26 C, but was quite labile at temperatures below 5 C. The enzyme had a molecular weight of 100,000 +/- 10,000 as determined by 0.5 M Agarose molecular exclusion chromatography and showed optimum activity between pH 5.5 and 6.2. The A. viscosus LDH exhibited homotropic interactions with its substrate, pyruvate, and its coenzyme, reduced nicotinamide adenine dinucleotide, indicating multiple binding sites on the enzyme for these ligands with some degree of cooperative interaction between them. The enzyme was under negative control by adenosine 5'-triphosphate, and its kinetic response to the negative effector was sigmoidal in nature. Inorganic phosphate reversed the inhibition exerted on the A. viscosus LDH by adenosine. The 5'-triphosphate thermal stability at 65 C of the LDH from A. viscosus was increased in the presence of its negative effector, adenosine 5'-triphosphate, but was markedly decreased in the presence of its coenzyme, reduced nicotinamide adenine dinucleotide. The glycolytic intermediate, fructose-1,6-diphosphate, had no effect on the catalytic activity of the A. viscosus LDH at saturating pyruvate concentrations. However, fructose-1,6-diphosphate was a potent positive effector at low substrate concentrations. Thus the A. viscosus LDH is under positive control by fructose-1,6-diphosphate and inorganic phosphate, but under negative control by adenosine 5'-triphosphate.     

Phosphomevalonate kinase: functional investigation of the recombinant human enzyme

Phosphomevalonate kinase (PMK) catalyzes a key step in isoprenoid/sterol biosynthesis, converting mevalonate 5-phosphate and ATP to mevalonate 5-diphosphate and ADP. To expedite functional and structural study of this enzyme, an expression plasmid encoding His-tagged human PMK has been constructed and recombinant enzyme isolated in an active, stable form. PMK catalyzes a reversible reaction; kinetic constants of human PMK have been determined for both forward (formation of mevalonate 5-diphosphate) and reverse (formation of mevalonate 5-phosphate) reactions. Animal and invertebrate PMKs are not orthologous to plant, fungal, or bacterial PMKs, limiting the information available from sequence alignment analysis. A homology model for the structure of human PMK has been generated. The model conforms to a nucleoside monophosphate kinase family fold. This result, together with sequence comparisons of animal and invertebrate PMKs, suggests an N-terminal basic residue rich sequence as a possible "Walker A" ATP binding motif. The functions of four basic (K17, R18, K19, K22) residues and one acidic (D23) residue in the conserved sequence have been tested by mutagenesis and characterization of isolated mutant proteins. Substrate K(m) values for K17M, R18Q, K19M, and D23N have been measured for forward and reverse reactions; in comparison with wild-type PMK values, only modest (<12-fold) changes are observed. In contrast, R18Q exhibits a V(max) decrease of 100/300-fold (forward/reverse reaction). K22M activity is too low for measurement at nonsaturating substrate concentration; specific activity is decreased by >10000-fold in both forward/reverse reactions, suggesting an active site location and an important role in phosphoryl transfer.     

The interaction of phosphorothioate analogues of ATP with phosphomevalonate kinase. Kinetic and 31P NMR studies

The diastereomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S), adenosine 5'-O-(2-thiotriphosphate) (ATP beta S), and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) could act as substrates for phosphomevalonate kinase in the presence of Mg2+ and Cd2+ as activating divalent metal cations. The Sp diastereomer of ATP alpha S was the preferred substrate regardless of the metal ion used, consistent with the metal ion not binding to the alpha-phosphate. With ATP beta S, the Sp diastereomer was the preferred substrate with Mg2+, and the Rp diastereomer was the preferred substrate with Cd2+. The reversal of specificity establishes that the metal is chelated through the beta-phosphate in the active site of the phosphomevalonate kinase reaction. A comparison of the Vmax values as a function of substitution of oxygen by sulfur showed the order for Mg2+ to be: ATP greater than ATP alpha S(Sp) greater than ATP alpha S(Rp) greater than ATP beta S(Sp) greater than ATP gamma S greater than ATP beta S(Rp). With Cd2+ as the activating metal ion, the order was: ATP greater than ATP alpha S(Sp) greater than ATP alpha S(Rp) greater than ATP beta S(Rp) greater than ATP gamma S greater than ATP beta S(Sp). It is concluded that the chelate structure of metal ATP substrate in the phosphomevalonate kinase reaction is the delta, beta, gamma-bidentate complex. 31P NMR measurements and radioassay with [2-14C] phosphomevalonate were used to measure the equilibrium of the reaction catalyzed by phosphomevalonate kinase with ATP and phosphorothioate analogues of ATP as the phosphoryl group donor. The order as a phosphate donor as determined by both methods in the phosphomevalonate kinase reaction is ATP beta S greater than ATP alpha S greater than ATP greater than ATP gamma S. Except for ATP gamma S, the equilibrium is shifted in the direction of formation of ADP alpha S and ADP beta S relative to ADP formation. Thus, ATP beta S rather than ATP would be effective for the synthesis of diphosphomevalonate. The phosphomevalonate kinase reaction could also be used to synthesize mevalonate 5-(2-thiodiphosphate) using ATP gamma S as the phosphoryl group donor.     

Mevalonate-5-diphosphate decarboxylase: stereochemical course of ATP-dependent phosphorylation of mevalonate 5-diphosphate

Chicken liver mevalonate-5-diphosphate decarboxylase catalyzes the reaction of mevalonate 5-diphosphate (MVADP) with ATP to produce isopentenyl diphosphate, ADP, CO2, and inorganic phosphate. The overall reaction involves an anti elimination of the tertiary hydroxyl and carboxyl groups. To investigate the mechanism for transfer of the terminal phosphoryl group of ATP to the C-3 oxygen of MVADP, we have carried out the reaction using stereospecifically labeled (Sp)-adenosine 5'-O-(3-thio[3-17O2,18O]triphosphate) [( gamma-17O2,18O]ATP gamma S) in place of ATP. The configuration of the [17O,18O]thiophosphate produced was found to be Rp, corresponding to overall inversion of configuration at phosphorus in the thiophosphoryl group transfer step. This result is consistent with the direct transfer of the thiophosphoryl group from (Sp)-[gamma-17O2,18O]ATP gamma S to MVADP at the active site. Our result does not indicate the involvement of a covalent thiophosphoryl-enzyme on the reaction pathway.     

Effect of clofibrate on brain mevalonate-5-pyrophosphate decarboxylase

The effect of clofibrate on the activity of the three mevalonate-activating enzymes has been studied for the first time in brain by reactions carried out using [2-¹⁴C] mevalonic acid as substrate and 105,000g supernatants from 14-day-old chick brain. Mevalonate-5-pyrophosphate decarboxylase was clearly inhibited, while mevalonate kinase and mevalonate-5-phosphate kinase were not significantly affected. The effect of clofibrate on decarboxylase activity was progressive with increasing concentrations (1.25–5.00 mM) of the inhibitor. A transient inhibition and a subsequent activation as a function of clofibrate concentration seemed to occur for mevalonate kinase. Direct measurements of decarboxylase activity utilizing [2-¹⁴C] pyrophosphomevalonate as the specific substrate of this enzyme corroborated these results. Kinetic studies showed that clofibrate competes with the substrate ATP.     

Microplate enzyme-coupled assays of mevalonate and phosphomevalonate kinase from Catharanthus roseus suspension cultured cells

A microplate assay for mevalonate and 5-phosphomevalonate kinase activities has been developed using an enzyme-coupled system of pyruvate kinase and lactate dehydrogenase. Mevalonate and 5-phosphomevalonate kinase activities were measured in crude and partially purified enzyme preparations from Catharanthus roseus suspension-cultured plant cells. The assay was validated with respect to protein and substrate concentration. Mevalonate and 5-phosphomevalonate kinase showed Michaelis-Menten kinetics with respect to ATP and their specific substrates; the apparent Km values of mevalonate kinase for ATP and mevalonate were 210 and 65 microM, respectively, and of 5-phosphomevalonate kinase for ATP and 5-phosphomevalonate were 0.41 and 0.4 mM, respectively. Considering mevalonate kinase, the relative standard deviation of enzyme activity within a determination (n = 3) is always less than 2.5% and in between determinations (n = 9) is less than 2%. The method can be used in a continuous assay as well as in a discontinuous assay.     

ATP inhibits insulin-degrading enzyme activity

We studied the ability of ATP to inhibit in vitro the degrading activity of insulin-degrading enzyme. The enzyme was purified from rat skeletal muscle by successive chromatographic steps. The last purification step showed two bands at 110 and 60 kDa in polyacrylamide gel. The enzyme was characterized by its insulin degradation activity, the substrate competition of unlabeled to labeled insulin, the profile of enzyme inhibitors, and the recognition by a specific antibody. One to 5 mM ATP induced a dose-dependent inhibition of insulin degradation (determined by trichloroacetic acid precipitation and insulin antibody binding). Inhibition by 3 mM adenosine 5'-diphosphate, adenosine 5'-monophosphate, guanosine 5'-triphosphate, pyrophosphate, beta-gamma-methyleneadenosine 5'-triphosphate, adenosine 5'-O-(3 thiotriphosphate), and dibutiryl cyclic adenosine 5'-monophosphate was 74%, 4%, 38%, 46%, 65%, 36%, and 0%, respectively, of that produced by 3 mM ATP. Kinetic analysis of ATP inhibition suggested an allosteric effect as the plot of 1/v (insulin degradation) versus ATP concentration was not linear and the Hill coefficient was more than 1 (1.51 and 2.44). The binding constant for allosteric inhibition was KiT = 1.5 x 10(-7) M showing a decrease of enzyme affinity induced by ATP. We conclude that ATP has an inhibitory effect on the insulin degradation activity of the enzyme.     

Mutation and inhibition studies of mevalonate 5-diphosphate decarboxylase

Mevalonate 5-diphosphate decarboxylase plays an important role in regulating cholesterol biosynthesis, which was studied through incubation with various synthetic substrate analogs and characterization of mutated enzymes. The results are potentially useful for further developing inhibitors that block the mevalonate pathway which is a drug target for treating cardiovascular disease and cancer.     

Kinetic effects of ATP, divalent metal ions and pH on chicken liver mevalonate 5-diphosphate decarboxylase

The activity of chicken liver mevalonate 5-diphosphate decarboxylase was measured over a wide range of Mg2+ and ATP concentrations. It was found that free ATP activated the enzyme, whereas free Mg2+ had no effect on the enzyme activity. Computed analyses of free species concentrations and pH studies indicated that MgATP2- is the true substrate. The relative efficiencies of Mg2+, Mn2+, Cd2+, and Zn2+ as activating metal ions were evaluated in terms of V/Km for the corresponding (metal-ATP)2- complexes, and the relative ratios were: Mn2+ 100, Cd2+ 37, Mg2+ 14, Zn2+ 1.7. Inhibitory effects were demonstrated for all free divalent cations tested, except for Mg2+, and were in the order Zn2+ greater than Cd2+ greater than Mn2+.     

Level of photosynthetic intermediates in isolated spinach chloroplasts

The level of intermediates of the photosynthetic carbon cycle was measured in intact spinach chloroplasts in an attempt to determine the cause of the induction lag in CO₂ assimilation. In addition, transient changes in the level of the intermediates were determined as affected by a light-dark period and by the addition of an excess amount of bicarbonate during a period of steady photosynthesis. Assayed enzymically were: ribulose 1,5-diphosphate, pentose monophosphates (mixture of ribose 5-phosphate, ribulose 5-phosphate and xylulose 5-phosphate, hexose monophosphates (mixture of glucose 6-phosphate, glucose 1-phosphate, and fructose 6-phosphate), glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, glycerate acid 3-phosphate, a mixture of fructose 1,6-diphosphate and sedoheptulose 1,7-diphosphate, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP).     

N,N′-Dicyclohexylcarbodiimide Inhibition of Dunaliella Growth,and Restoration by Some Adenine Nucleotides and Plant Growth Regulators

N,N′-Dicyclohexylcarbodiimide (DCCD), an inhibitor of membrane-bound ATPase, strongly inhibited the growth, as measured by an increase in cell number, of Dunaliella tertiolecta. However, this inhibition was reversed by simultaneous application of adenosine 5′-triphosphate (ATP) or adenosine 2′-monophosphate (2′-AMP). Adenosine and adenosine 5′-diphosphate (ADP) were ineffective in restroration of the DCCD-inhibited growth. Gibberellin A₃ (GA₃) and 2,4- dichlorophenoxyacetic acid (2,4-D) also reversed the inhibition of DCCD on D. tertiolecta growth, although these plant growth regulators did not promote an increase in cell number.