Arsenylation of nucleoside diphosphates in Rhodospirillum rubrum chromatophores

Rhodospirillum rubrum chromatophores catalyze the formation of ADP-arsenate during illumination with ADP, Mg²⁺ and arsenate. The reaction was measured with (1) firefly luciferase, (2) a coupled enzyme assay involving hexokinase and glucose-6-phosphate dehydrogenase, and (3) a glass electrode. ADP-arsenate hydrolyzed spontaneously with rate constants ranging from 14 to 43 min^?1. Magnesium, arsenate and phosphate accelerated hydrolysis of ADP-arsenate. From a comparison of the three methods, with ADP as the substrate, it is estimated that φ_R (i.e., the ratio between the quantum yields of ADP-arsenate and ATP for light emission from luciferase) is 0.19–0.23. Furthermore, arsenylation rates were 46–52% of phosphorylation rates in experiments with 30 μ M ADP and 0.8 mM arsenate or phosphate. Similarly, the V_app for arsenylation of GDP or IDP was 47–59% of the V_app for phosphorylation during measurements in the presence of 1 mM arsenate or phosphate. The K_app(GDP) was higher during arsenylation than during phosphorylation; the K_app(IDP) was about the same during arsenylation as during phosphorylation. It is suggested that a shift in the equilibrium of substrates and products on the enzyme, toward hydrolysis, is the main cause of the relatively low arsenylation rates.     

Effects of l, N6-ethenoadenylates on the regulation of photosynthetic activities by adenylates; A study of the nucleotide binding sites on chloroplast coupling factor 1

Effects of l, N⁶-ethenoadenylates (e-adenylates) were testedon phosphorylation, and electron transport under phosphorylation,arsenylation and quasi-arsenylation (stimulation of electrontransport in the presence of ATP, AMP and arsenate) conditionsin isolated spinach chloroplasts. -ATP as well as ATP partially inhibited ferricyanide reductionthrough binding to the chloroplast coupling factor 1 with anapparent dissociation constant (KD^app) of around 5µM,which was remarkably larger than that for ATP (ca. 2µM).e-ATP at below 500 µM had no effect on phosphorylationbut inhibited quasi-arsenylation in competition with ATP withan apparent inhibition constant (K₁^app) of around 60 µM. -ADP as well as ADP partially inhibited ferricyanide reductionwith a KD^app value close to that for -ATP. -ADP was phosphorylated(the apparent Michaelis constant, K_m^app=80µM) accompanyingstimulation of ferricyanide reduction to the magnitude predicted(P/_e=l). -ADP-arsenylation was also detected by stimulationof ferricyanide reduction. -AMP alone caused little inhibition of ferricyanide reductionas AMP, but competitively depressed the electron transport inhibitionby ADP and ATP with a K₁^app value of around 200 µM. -AMPwas not effective for ADP phosphorylation but inhibited stimulationdue to quasi-arsenylation coupling in competition with AMP K₁^app=150µM Among the possible combinations of adenylates and -adenylatesfor quasiarsenylation, only [ATP+AMP] could couple with theenergy transduction mechanism. Based on the specificity of binding sites to adenylates and-adenylates, an attempt was made to distinguish at least four(two pairs) kinds of binding sites (at least six sites in toto)on the chloroplast coupling factor 1 for photosynthetic energytransduction. When one pair of sites is occupied by the designatedadenylates or -adenylates (allosteric effectors), the couplingfactor is thought to be in a conformation for coupling withthe energy transduction mechanism in the presence of phosphateor arsenate. ¹Presented to the 1st Symposium of Japan Bioenergetics Group,December 19, 1975, Osaka. (Received February 17, 1976; )     

Regulation of electron transport in isolated chloroplasts by sequential binding of adenine nucleotides to the coupling factor protein

Changes in the ferricyanide-reducing activity in isolated spinach chloroplasts by adenine nucleotides were measured in the presence or absence of phosphate, arsenate or pyrophosphate at 15°C. The activity changes were analyzed and ascribed to the interaction between the nucleotides and the chloroplast coupling factor (CF₁).ADP and ATP (but not AMP) partially inhibited ferricyanide reduction through a 11 binding to the inhibition site on CF₁.When the ferricyanide reduction was coupled to either phosphorylation or arsenylation, the inhibition by ADP was released through a 11 binding of the second ADP molecule to the coupling site on the CF₁ with which the first ADP had been associated.The association constants of ADP for the inhibition and the coupling site were found to be approximately 5×10⁵ and 6×10⁴ M^–1, respectively. TheKm value of ADP for arsenylation (pH 8.3) was around 17 M.The ADP-regulated electron transport was defined based on these results. The ADP-regulated ferricyanide reduction, when coupled with phosphorylation, revealed a stoichiometry of P/e=1 between the amounts of esterified phosphate and reduced ferricyanide.Presented to the annual meeting of the Japanese Society of Plant Physiologists, Sendai, April 1974.     

Rapid Modulation of Spinach Leaf Nitrate Reductase by Photosynthesis : II. In Vitro Modulation by ATP and AMP

Assimilatory nitrate reductase activity (NRA) in crude spinach leaf (Spinacia oleracea) extracts undergoes rapid changes following fluctuations in photosynthesis brought about by changes in external CO₂ or by water stress (WM Kaiser, E Brendle-Behnisch [1991] Plant Physiol 96:363-367). A modulation of NRA sharing several characteristics (stability, response to Mg²⁺ or Ca²⁺, kinetic constants) with the in vivo modulation was obtained in vitro by preincubating desalted leaf extracts with physiological concentrations of Mg²⁺ and ATP (deactivating) or AMP (activating). When nitrate reductase (NR) was inactivated in vivo by illuminating leaves at the CO₂ compensation point, it could be reactivated in vitro by incubating leaf extracts with AMP. For the in vitro inactivation, ATP could be replaced by GTP or UTP. Nonhydrolyzable ATP analogs (β, γ-imido ATP, β, γ-methyl-ATP) had no effect on NR, whereas γ-S-ATP caused an irreversible inactivation. This suggests that NR modulation involves ATP hydrolysis. In contrast to NR in crude leaf extracts, partially purified NR did not respond to ATP or AMP. ATP and AMP levels in whole leaf extracts changed in the way predicted by the modulation of NRA when leaves were transferred from photosynthesizing (low ATP/AMP) to photorespiratory (high ATP/AMP) conditions. Adenine nucleotide levels in leaves could be effectively manipulated by feeding mannose through the leaf petiole. NRA followed these changes as expected from the in vitro results. This suggests that cytosolic ATP/AMP levels are indeed the central link between NRA in the cytosol and photosynthesis in the chloroplast. Phosphorylation/dephosphorylation of NR or of NR-regulating protein factors is discussed as a mechanism for a reversible modulation of NR by ATP and AMP.     

Studies on nucleotidases in plants. Reversible denaturation of the crystalline mung bean nucleotide pyrophosphatase and the effect of adenylates on the native and renatured enzyme

The crystalline mung bean nucleotide pyrophosphatase was inhibited nonlinearly by AMP, one of the products of the reaction. The partially inactive enzyme was specifically reactivated by ADP, and V at maximal activation was the same as that of the native enzyme. ATP was a linear, noncompetitive inhibitor. The kinetic evidence suggested that ADP and ATP might not be reacting at the same site as AMP. The electrophoretic mobility of the enzyme was increased by AMP, whereas ADP and ATP were without effect.The enzyme was denatured on treatment with urea or guanidine hydrochloride. The renatured and the native enzyme had the same pH (9.4) and temperature (49 °C) optimum. The K_m (0.2 mm) and V (3.2) of the native enzyme increased on renaturation to 1.8 mm and 8.0, respectively. In addition, renaturation resulted in desensitization of the enzyme to inhibition by low concentrations of AMP. Renaturation did not affect the reactivation of the apoenzyme by Zn²⁺.     

In Vitro ATP Regeneration from Polyphosphate and AMP by Polyphosphate:AMP Phosphotransferase and Adenylate Kinase from Acinetobacter johnsonii 210A

In vitro enzyme-based ATP regeneration systems are important for improving yields of ATP-dependent enzymatic reactions for preparative organic synthesis and biocatalysis. Several enzymatic ATP regeneration systems have been described but have some disadvantages. We report here on the use of polyphosphate:AMP phosphotransferase (PPT) from Acinetobacter johnsonii strain 210A in an ATP regeneration system based on the use of polyphosphate (polyP) and AMP as substrates. We have examined the substrate specificity of PPT and demonstrated ATP regeneration from AMP and polyP using firefly luciferase and hexokinase as model ATP-requiring enzymes. PPT catalyzes the reaction polyP_n + AMP → ADP + polyP_n−1. The ADP can be converted to ATP by adenylate kinase (AdK). Substrate specificity with nucleoside and 2′-deoxynucleoside monophosphates was examined using partially purified PPT by measuring the formation of nucleoside diphosphates with high-pressure liquid chromatography. AMP and 2′-dAMP were efficiently phosphorylated to ADP and 2′-dADP, respectively. GMP, UMP, CMP, and IMP were not converted to the corresponding diphosphates at significant rates. Sufficient AdK and PPT activity in A. johnsonii 210A cell extract allowed demonstration of polyP-dependent ATP regeneration using a firefly luciferase-based ATP assay. Bioluminescence from the luciferase reaction, which normally decays very rapidly, was sustained in the presence of A. johnsonii 210A cell extract, MgCl₂, polyP_n=35, and AMP. Similar reaction mixtures containing strain 210A cell extract or partially purified PPT, polyP, AMP, glucose, and hexokinase formed glucose 6-phosphate. The results indicate that PPT from A. johnsonii is specific for AMP and 2′-dAMP and catalyzes a key reaction in the cell-free regeneration of ATP from AMP and polyP. The PPT/AdK system provides an alternative to existing enzymatic ATP regeneration systems in which phosphoenolpyruvate and acetylphosphate serve as phosphoryl donors and has the advantage that AMP and polyP are stabile, inexpensive substrates.     

Further studies on the properties of the rabbit reticulocyte adenosine 3',5'-cyclic monophosphate-dependent protein kinase I

The properties of cyclic AMP-dependent protein kinase I isolated from rabbit reticulocytes were further investigated. The enzyme catalyzes the phosphorylation of histone in the presence of ATP and Mg²⁺ and this reaction is stimulated by cyclic AMP. The pH optimum of the reaction was between 8.5 and 9.0, when assayed in the presence of cyclic AMP. No distinct pH optimum was observed in the absence of the cyclic nucleotide. The K_m values for ATP appeared to be very similar whether it was determined in the presence (K_m = 1.7 × 10⁻⁴m) or absence (K_m = 2.5 × 10⁻⁴m) of cyclic AMP. The rate of heat inactivation of the catalytic activity and the cyclic AMP binding activity of kinase I were found to be dependent on the presence of Mg²⁺, ATP, and/or cyclic AMP. In the presence of cyclic AMP, the rate of inactivation of the catalytic activity of kinase I at 53 ° was accelerated. On the other hand, the cyclic AMP binding activity appeared to be protected from heat inactivation by the cyclic nucleotide. When both ATP and Mg²⁺ were present in the heating mixture, no loss of catalytic and binding activities of kinase I were observed even up to 8 min of heating at 53 °. The cyclic AMP binding activity of kinase I was almost completely inhibited by mercuric acetate at a concentration of 1 mm, while the loss in catalytic activity was only 50%. These results substantiate our previous observation that kinase I contains two nonidentical subunits, a catalytic subunit and a cyclic AMP binding subunit.     

Biochemical and Kinetic Characterization of Xylulose 5-Phosphate/Fructose 6-Phosphate Phosphoketolase 2 (Xfp2) from Cryptococcus neoformans

Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp), previously thought to be present only in bacteria but recently found in fungi, catalyzes the formation of acetyl phosphate from xylulose 5-phosphate or fructose 6-phosphate. Here, we describe the first biochemical and kinetic characterization of a eukaryotic Xfp, from the opportunistic fungal pathogen Cryptococcus neoformans, which has two XFP genes (designated XFP1 and XFP2). Our kinetic characterization of C. neoformans Xfp2 indicated the existence of both substrate cooperativity for all three substrates and allosteric regulation through the binding of effector molecules at sites separate from the active site. Prior to this study, Xfp enzymes from two bacterial genera had been characterized and were determined to follow Michaelis-Menten kinetics. C. neoformans Xfp2 is inhibited by ATP, phosphoenolpyruvate (PEP), and oxaloacetic acid (OAA) and activated by AMP. ATP is the strongest inhibitor, with a half-maximal inhibitory concentration (IC₅₀) of 0.6 mM. PEP and OAA were found to share the same or have overlapping allosteric binding sites, while ATP binds at a separate site. AMP acts as a very potent activator; as little as 20 μM AMP is capable of increasing Xfp2 activity by 24.8% ± 1.0% (mean ± standard error of the mean), while 50 μM prevented inhibition caused by 0.6 mM ATP. AMP and PEP/OAA operated independently, with AMP activating Xfp2 and PEP/OAA inhibiting the activated enzyme. This study provides valuable insight into the metabolic role of Xfp within fungi, specifically the fungal pathogen Cryptococcus neoformans, and suggests that at least some Xfps display substrate cooperative binding and allosteric regulation.     

Mechanism of action of ATP on intestinal epithelial cells: Cyclic AMP-mediated stimulation of active ion transport

ATP, ADP and AMP but not adenosine increased cyclic AMP in dispersed enterocytes prepared from guinea pig small intestine. This action of ATP was augmented by IBMX and was reproduced by App(NH)p or App(CH₂)p. ATP also increased the formation of cyclic [¹⁴C]AMP in enterocytes that had been preincubated with [¹⁴C]adenine. Gpp(NH)p and NaF each caused persistent activation of adenylate cyclase in plasma membranes from enterocytes and ATP caused significant augmentation of this persistent activation. In addition to increasing cellular cyclic AMP and agumenting Gpp(NH)p and NaF-stimulated persistent activation of adenylate cyclase, ATP increased the I_sc across mounted strips of small intestine and inhibited net absorption of fluid and electrolytes in segments of everted small intestine. These results indicate that intestinal epithelial cells possess a receptor that interacts with ATP and other adenine nucleotides and that receptor occupation by ATP causes activation of adenylate cyclase, increased cyclic AMP and changes in active ion transport across intestinal mucosa.     

Effect of exercise on the properties of AMP-deaminase from trout white muscle

AMP-deaminase was purified to homogeneity from white skeletal muscle of control (resting) and exercised (1 min burst swimming) rainbow trout, Oncorhynchus mykiss. The enzyme showed a subunit molecular weight of 71,600 ± 550 kD, a K_m AMP of 1.6–1.8 mM at pH 7, and was affected by allosteric inhibitors (GTP, IMP) amd activators (ADP, ATP). AMP-deaminase was inhibited by MgSO₄ but activated by low concentrations of NaCl and KCl (100–150 mM); higher KCl was inhibitory. Exercise resulted in a stable modification of some properties (possibly via reversible phosphorylation); I₅₀ values for IMP decreased by 65% and activation energies (from Arrhenius plots) changed significantly. Other properties were affected by assay pH: K_m AMP decreased by 50% and K_a, ADP decreased by 70% when pH was lowered from pH 7.3 (typical of resting muscle) to pH 6.6 (muscle pH after exhaustive exercise). The data suggest that a stable modification of AMP-deaminase during exercise, coupled with effects of reduced cytosolic pH, could enhance enzyme function in the rapid conversion of AMP to IMP in working fish muscle.     

Regulatory properties of 6-phosphofructo-1-kinase of the mid-gut of the grasshopper,Poekilocerus bufonius

The fine structure of the mid-gut of Poekilocerus bufonius has been examined and three types of epithelial cells were identified; normal epithelial cells with their apical part possessing well developed microvilli, goblet-like cells containing myelin-like figures and the small basal cells with small and round nuclei, nidi. The regulation of 6-phosphofructo-1-kinase (PFK-1) prepared from the mid-gut of the grasshopper, Poekilocerus bufonius, was studied. Mid-gut PFK-1 displayed cooperativity with respect to fructose-6-phosphate at pH 7.0, and the enzyme was inhibited by high concentrations of ATP. The affinity of the enzyme for fructose-6-phosphate was increased by fru-2,6-P₂ whereas the inhibition of the enzyme by high concentrations of ATP was relieved by fru-2,6-P₂. The activity of mid-gut PFK-1 was highly stimulated in a simultaneous presence of low concentrations of fru-2,6-P₂ and AMP. ADP, AMP and c-AMP were all shown to be activators of the mid-gut PFK-1 with AMP being the greatest effector. The enzyme was not inhibited by citrate either in the presence of low or high concentrations of ATP. These results suggest that the PFK-1 of the mid-gut of the grasshopper is highly regulated with positive stimulators, specially fru-2,6-P₂, whereas the enzyme is not regulated by citrate or glucose-1,6-bisphosphate.     

Influence of adenosine phosphates and magnesium on photosynthesis in chloroplasts from peas, sedum, and spinach

¹⁴CO₂ photoassimilation in the presence of MgATP, MgADP, and MgAMP was investigated using intact chloroplasts from Sedum praealtum, a Crassulacean acid metabolism plant, and two C₃ plants: spinach and peas. Inasmuch as free ATP, ADP, AMP, and uncomplexed Mg²⁺ were present in the assays, their influence upon CO₂ assimilation was also examined. Free Mg²⁺ was inhibitory with all chloroplasts, as were ADP and AMP in chloroplasts from Sedum and peas. With Sedum chloroplasts in the presence of ADP, the time course of assimilation was linear. However, with pea chloroplasts, ADP inhibition became progressively more severe, resulting in a curved time course. ATP stimulated assimilation only in pea chloroplasts. MgATP and MgADP stimulated assimilation in all chloroplasts. ADP inhibition of CO₂ assimilation was maximal at optimum orthophosphate concentrations in Sedum chloroplasts, while MgATP stimulation was maximal at optimum or below optimum concentrations of orthophosphate. MgATP stimulation in peas and Sedum and ADP inhibition in Sedum were not sensitive to the addition of glycerate 3-phosphate (PGA).

PGA-supported O₂ evolution by pea chloroplasts was not inhibited immediately by ADP; the rate of O₂ evolution slowed as time passed, corresponding to the effect of ADP on CO₂ assimilation, and indicating that glycerate 3-phosphate kinase was a site of inhibition. Likewise, upon the addition of AMP, inhibition of PGA-dependent O₂ evolution became more severe with time. This did not mirror CO₂ assimilation, which was inhibited immediately by AMP. In Sedum chloroplasts, PGA-dependent O₂ evolution was not inhibited by ADP and AMP. In chloroplasts from peas and Sedum, the magnitude of MgADP and MgATP stimulation of PGA-dependent O₂ evolution was not much larger than that given by ATP, and it was much smaller than MgATP stimulation of CO₂ assimilation. Analysis of stromal metabolite levels by anion exchange chromatography indicated that ribulose 1,5-bisphosphate carboxylase was inhibited by ADP and stimulated by MgADP in Sedum chloroplasts.

The appearance of label in the medium was measured when [U-¹⁴C] ADP-loaded Sedum chloroplasts were challenged with ATP, ADP, or AMP and their Mg²⁺ complexes. The rate of back exchange was stimulated by the presence of Mg²⁺. This suggests that ATP, ADP, and AMP penetrate the chloroplast slower than their Mg²⁺ complexes. A portion of the CO₂ assimilation and O₂ evolution data could be explained by differential penetration rates, and other proposals were made to explain the remainder of the observations.

     

Steady-state kinetics of ADP-arsenate and ATP-synthesis in Rhodospirillum rubrum chromatophores

(1) Rates of ATP synthesis and ADP-arsenate synthesis catalyzed by Rhodospirillum rubrum chromatophores were determined with the firefly luciferase method and by a coupled enzyme assay involving hexokinase and glucose-6-phosphate dehydrogenase. (2) V_m for ADP-arsenate synthesis was about 2-times lower than V_m for ATP-synthesis. With saturating [ADP], K(As_i) was about 20% higher than K(P_i). With saturating [anion], K(ADP) was during arsenylation about 20% lower than during phosphorylation. (3) Plots of $\text{1}\text{v}$ vs. $\text{1}\text{[}\text{substrate}\text{]}$ were non-linear at low concentrations of the fixed substrate. The non-linearity was such as to suggest a positive cooperativity between sites binding the variable substrate, resulting in an increased $\text{V}{}_{\mathrm{<mtext>m</mtext>}}\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ ratio. High concentrations of the fixed substrate cause a similar increase in $\text{V}{}_{\mathrm{<mtext>m</mtext>}}\text{K}{}_{\mathrm{<mtext>m</mtext>}}$, but abolish the cooperativity of the sites binding the variable substrate. (4) Low concentrations of inorganic arsenate (As_i) stimulate ATP synthesis supported by low concentrations of P_i and ADP about 2-fold. (5) At high ADP concentrations, the apparent K_i of As_i for inhibition of ATP-synthesis was 2–3-times higher than the apparent K_m of As_i for arsenylation; the apparent K_i of P_i for inhibition of ADP-arsenate synthesis was about 40% lower than the apparent K_m of P_i for ATP synthesis. (6) The results are discussed in terms of a model in which P_i and As_i compete for binding to a catalytic as well as an allosteric site. The interaction between these sites is modulated by the ADP concentration. At high ADP concentrations, interaction between these sites occurs only when they are occupied with different species of anion.     

Affinity labeling of AMP-ADP sites in heart phosphofructokinase by 5-p-fluorosulfonylbenzoyl adenosine.

The purine nucleotide derivative, 5′-p-fluorosulfonylbenzoyl adenosine (5′-FSO₂B_ZAdo) functions as an affinity label for the allosteric sites of phosphofructokinase. The modified enzyme at pH 6.9 is insensitive to allosteric inhibition by ATP, activation by AMP, c-AMP, ADP and shows no sigmoidal kinetics for fructose-6-P. The reaction does not appear to occur at the catalytic site since modification of the enzyme does not significantly affect its specific activity nor its Michaelis constant at pH 8.2. ADP, and to a much lesser degree AMP and ATP, protects the enzyme from modification by the adenosine reagent. The modified enzyme essentially does not bind significant amounts of AMP, c-AMP, ADP, but still binds an analog of ATP, AppNHp. The adenosine affinity label will be of value in studies on the nature of the AMP-ADP allosteric sites.     

NMR studies of the interaction of cis-diamminedichloro-platinum(II) and corresponding hydrolysis products with adenosine phosphates

Complexes formed in aqueous solution between cisplatin or hydrolysis species and 5′ adenosine monophosphate (AMP) or 5′ adenosine triphosphate (ATP), the latter with and without chloride ions, have been determined using ¹⁹⁵Pt, ³¹P, ¹³C and ¹H NMR. The present results lead to the conclusion that the only monodentate complexes with AMP are cis-Pt(NH₃)₂(AMP-N7)Cl at acid pH and cis-Pt(NH₃)₂(AMP-N7)OH at neutral and basic pH. Other bidentate complexes were identified as cis-Pt(NH₃)₂(AMP-N7)₂ and cis-Pt(NH₃)₂(AMP-N7)(AMP-PO). Also discussed herein are the binding of platinum to the phosphate group Pγ with ATP and at acid pH, and the formation of the [cis-Pt(NH₃)₂(ATP-N7)H₂O]⁺ complex. In neutral and basic pH ranges, the phosphate moiety of ATP is the most reactive site. In the presence of an excess of chloride ions, the complexation rates between the ATP and the cisplatin are decreased. Furthermore, in the experimental conditions used neither the ATP nor the AMP have shown binding to N1.     

Continuous pH stat assay technique for glutamine and asparagine synthetase enzyme systems, involving ATP conversion to ADP plus Pi and AMP plus PPi, respectively

Glutamine synthetase and asparagine synthetase systems with reactions involving lysis of ATP to ADP and P_i or AMP and PP_i are usually assayed by discontinuous sampling and analysis or by coupled enzymic systems. Experimental results confirm theoretical predictions that such reactions may be continuously and directly monitored by pH stat devices. Sample volumes of 0.5–1.0 ml and buret volumes of 0.05–0.25 ml, with ATP levels near 1 mm can be used routinely. The number of enzyme reactions involving ATP to which this technique can be applied is quite large.     

Pathways of adenine nucleotide catabolism in primary rat muscle cultures

The pathways of AMP degradation and the metabolic fate of adenosine were studied in cultured myotubes under physiological conditions and during artificially induced enhanced degradation of ATP. The metabolic pathways were gauged by tracing the flow of radioactivity from ATP, prelabelled by incubation of the cultures with [¹⁴C]adenine, into the various purine derivatives. The fractional flow from AMP to inosine through adenosine was estimated by the use of the adenosine deaminase (EC 3.5.4.4) inhibitors, coformycin and 2′-deoxycoformycin. The activities of the enzymes involved with AMP and adenosine metabolism were determined flow of label from ATP to diffusible bases and nucleosides, most of which are effluxed to the incubation medium. This catabolic flow is mediated almost exclusively by the activity of AMP deaminase (EC 3.5.4.6), rather than by AMP 5′-nucleotidase (EC 3.1.3.5), reflecting the markedly higher V_max/K_m ratio for the deaminase. Enhancement of ATP degradation by inhibition of glycolysis or by combined inhibition of glycolysis and of electron transport resulted in a markedly greater flux of label from adenine nucleotides to nucleosides and bases, but did not alter significantly the ratio between AMP deamination and AMP dephosphorylation, which remained around 19:1. Combined inhibition of glycolysis and of electron transport resulted, in addition, in accumulation of label in IMP, reaching about 20% of total AMP degraded. In the intact myotubes at low adenosine concentration, the anabolic activity of adenosine kinase was at least 4.9-fold the catabolic activity of adenosine deaminase, in accord with the markedly higher V_max/K_m ratio of the kinase for adenosine. The results indicate the operation in the myotube cultures, under various rates of ATP degradation, of the AMP to IMP limb of the purine nucleotide cycle. On the other hand, the formation of purine bases and nucleosides, representing the majority of degraded ATP, indicates inefficient activity of the IMP to AMP limb of the cycle, as well as inefficient salvage of hypoxanthine under these conditions.     

Role of CFTR’s intrinsic adenylate kinase activity in gating of the Cl− channel

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl^?channel in the ATP-binding cassette (ABC) transporter protein family. CFTR features the modular design characteristic of ABC transporters, which includes two membrane-spanning domains forming the channel pore, and two ABC nucleotide-binding domains that interact with ATP and contain the enzymatic activity coupled to normal gating. Like other ABC transporters CFTR is an ATPase (ATP + H₂O → ADP + P_i). Recent work has shown that CFTR also possesses intrinsic adenylate kinase activity (ATP + AMP ? ADP + ADP). This finding raises important questions: How does AMP influence CFTR gating? Why does ADP inhibit CFTR current? Which enzymatic activity gates CFTR in vivo? Are there implications for other ABC transporters? This minireview attempts to shed light on these questions by summarizing recent advances in our understanding of the role of the CFTR adenylate kinase activity for channel gating.     

Content of adenosine phosphate compounds in pea roots grown in saline media

The levels of ATP, ADP and AMP, the activity of phosphatases, and the ability for oxidative phosphorylation were studied in roots of pea (Pisum sativum) plants grown in media salinized either with NaCl or Na₂SO₄. In response to salinity, the ATP level in the roots decreased, whereas the ADP level increased slightly. As a result, the ADP:ATP ratio in the tissue increased with increasing salinity in the growth medium. The AMP level in the tissue was not affected by salinity.     

Effect of administration of the fructose on the glycogenolytic action of glucagon. An investigation of the pathogeny of hereditary fructose intolerance

1. The mechanism by which the administration of fructose to patients with hereditary fructose intolerance makes them unresponsive to the hyperglycaemic action of glucagon was studied. In four patients, a 10-fold increase in the urinary excretion of cyclic AMP was induced by glucagon, but this effect was drastically decreased by the previous administration of fructose (250mg/kg). Further, the intravenous injection of 6-N,2′-O-dibutyryl cyclic AMP did not cause an increase in the blood glucose during fructose-induced hypoglycaemia. 2. The administration of a large dose of fructose (5g/kg) to mice decreased markedly both the concentration of ATP and the increase in the concentration of cyclic AMP caused by glucagon in the liver. Other ATP-depleting agents had a similar effect and a linear correlation could be drawn between the concentration of ATP and the change in cyclic AMP concentration; a half-maximal effect was obtained for a concentration of ATP close to the K_m value of adenylate cyclase. 3. The administration of fructose to mice caused the inactivation of phosphorylase in the liver, but this effect was easily reversed by glucagon. 4. At a concentration of 10mm-fructose 1-phosphate and 1.5mm-P_i, purified liver phosphorylase a was inhibited by 70%. This inhibition appears to be a likely explanation for the unresponsiveness to glucagon of patients with hereditary fructose intolerance.