Interaction of heavy chain binding protein (BiP/GRP78) with adenine nucleotides.

Immunoglobulin heavy chain binding protein (BiP/GRP78) is a resident endoplasmic reticulum protein that binds tightly to a number of incompletely assembled or aberrant proteins. BiP also binds ATP and can be purified by ATP affinity chromatography. Here we show that an ATPase activity co-purifies with BiP prepared from canine pancreas. The BiP-associated ATPase has a high affinity for ATP but a low turnover number, suggesting a regulatory, rather than an enzymatic role. We also show that submicromolar levels of ATP or ADP decrease the rate of adsorption of [125I]BiP to nitrocellulose filters coated with protein or non-ionic detergents. In contrast, micromolar levels of AMP increase the rate of adsorption. Furthermore, ATP and ADP decrease the susceptibility of BiP to proteolytic degradation, whereas AMP was found to enhance degradation slightly. Adenine nucleotides may therefore induce or stabilize different conformations of BiP even when ATP hydrolysis does not occur.     

Phosphorylation of ribosomal proteins S3, L1 and L24 during spherulation in Physarum polycephalum

The phosphate content of ribosomal proteins S3, L1 and L24 has been determined in the course of spherulation of Physarum polycephalum. The major phosphoprotein, S3, was completely dephosphorylated after 4 h of differentiation. The phosphate content of L1 and L24 was not altered during the differentiation. The cellular level of ATP remained constant for at least 5 h. A 3-fold reduction of cyclic AMP concentration occurred in the first hour, followed by a slow increase to a final value of twice the level observed in growing cells. The results showed that the phosphorylation of ribosomal proteins is regulated by at least two different mechanisms and that the dephosphorylation of S3 is not induced by a lack of cellular ATP. Although cyclic AMP might trigger the dephosphorylation of S3, the phosphate content of this protein remained at a very low value even when the cellular concentration of cyclic AMP rose significantly. Since the polysome level remains constant during the first 24 h of spherulation, the phosphorylation of S3 is not necessary for active protein synthesis and the phosphorylation of L1 and L24 is not involved in ribosome inactivation, which occurs after 24 h.     

Content of Adenosine Phosphate Compounds in a Long-Day Duckweed, Lemna gibba G3, under Different Light and Nutritional Conditions

The levels of ATP ADP and AMP as well as the energy charge were examined in the long-day duckweed. Lemna gibba G3, under different light and nutritional conditions. ATP and ADP content, but not AMP, decreased slightly when the plant was cultured with a medium depleted of sucrose or was exposed to continuous darkness. The energy charge was not affected either by changes in the light conditions or by the depletion of sucrose from the medium, although increase of fresh weight changed drastically under these conditions. The levels of ADP and AMP and the value of the energy charge remained nearly constant throughout a 1-day period when the plant was exposed to short-day, continuous light and continuous dark. ATP content decreased gradually during the light period of the short day and thereafter remained constant during the dark period. When the plant was exposed to continuous light. ATP content decreased until the 8th hour after beginning of the continuous light, only to recover rapidly to its original level during the phase coinciding with the preceding dark period of the short day. Under continuous dark conditions the ATP content remained constant throughout the day. It was concluded that the diurnal rhythm of physiological activities previously reported cannot be related to the energy charge.     

Variations in the Adenylate Energy Charge During Phased Growth (Cell Cycle) of Candida utilis Under Energy Excess and Energy-Limiting Growth Conditions

The variations in the levels of adenine nucleotides during the phased growth (cell cycle) of the yeast Candida utilis growing under nitrogen, sulfate, or iron limitation with glycerol as carbon source have been determined. Synchronous cultures were obtained by the continuous phasing technique, and the results were compared with those of chemostat cultures growing at similar growth rates and under the same types of nutrient limitation. Whereas the chemostat experiments indicated only the average energy status of cultures growing at random, results from phased cultures showed that the adenylate energy charge, defined as (ATP + (1/2)ADP)/(ATP + ADP + AMP) (where ATP, ADP, and AMP signify adenosine 5'-triphosphate, -diphosphate, and -monophosphate, respectively), varied during the phased growth of the yeast. These variations were related to the stage of development of the cells and to the type of nutrient limitation. In every case the energy charge dropped to a low value during the first half of the phasing cycle (cell cycle). Whereas the energy charge was maintained at relatively high levels (ranging from 0.78 to 0.94), for sulfate- or nitrogen-limited cultures, it was very low when iron was the growth-limiting nutrient (0.44 to 0.78). In spite of the low energy charge, the yeast continued to grow under iron limitation. The main component of the adenylate pool of the iron-limited culture was ADP and not ATP as observed with other types of nutrient limitation. It is concluded that under iron limitation the growth of the organism is limited by energy and that under energy-limited growth the energy charge of a growing organism is maintained at low levels. The reason for maintaining a low energy charge in an energy-limited culture is discussed.     

Interrelationships between the membrane-bound ATP-dependent energy systems of the gastric mucosa and the gastric cytoprotective effect of beta-carotene on the development of gastric mucosal damage produced by 0.6 M HCl in rats

The effects of different doses (0.01-0.1-1.0-10.0/mg/kg-1) of beta-carotene were studied on gastric secretory responses of 4 hr pylorus-ligated rats: development of gastric mucosal damage (as assessed by number and severity of lesions) produced by intragastric administration of 0.6 M HCl; tissue level of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenylate pool (ATP + ADP + AMP), ratio of ATP X ADP-1, "energy charge" (ATP + 0.5 ADP X X (ATP + ADP + AMP)-1) (during the development of gastric mucosal damage by 0.6 M HCl and of gastric cytoprotection by beta-carotene. It was found that beta-carotene did not decrease the gastric secretory responses of 4 hr pylorus-ligated rats; The development of gastric mucosal damage could be decreased dose-dependently by the administration of beta-carotene; the ATP transformation could be decreased by beta-carotene; the tissue levels of cAMP and AMP could be increased significantly and dose-dependently by beta-carotene; the ratio of ATP X ADP-1 could be increased significantly and dose-dependently by beta-carotene; the values of adenylate pool and "energy charge" remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine triphosphate catabolism in homogenates of rat secretory enamel organs incubated in histochemical lead media.

To investigate how lead, when used as trapping agent, influences the ATP hydrolysis and to study how ATP is catalyzed in histochemical systems, homogenized secretory enamel organs were incubated in histochemical [3H]-ATP media. Aliquots from the media were taken after 3, 10, 20 and 30 min, the ATP and formed metabolites were separated by electrophoresis and radiometrically quantitated. In media lacking both lead and homogenate 2% of the ATP was spontaneously hydrolyzed during 30 min incubation at room temperature. The presence of lead caused an additional 8% hydrolysis at pH 7.2 and an additional 20% hydrolysis at pH 9.4. In the presence of homogenate, however, lead caused a net decrease of the hydrolysis of ATP as well as of ADP and AMP. This enzyme inhibition varied from around zero to some 80%, depending on pH and substrated involved. In homogenate-containing lead media, at both pH 7.2 AND 9.4, ATP was rapidly hydrolyzed primarily to ADP and subsequently to AMP and adenosine and/or inosine. After 5--10 min ADP constituted the predominant substrate at both pH:s. At pH 7.2 ADP remained so for the rest of the incubation, whereas at pH 9.4 AMP was predominant substrate at the end of the incubation. AMP was the finan catabolic product in experiments at pH 7.2, and adenosine and/or inosine at pH 9.4. Inorganic phosphate was liberated almost linearly during the whole incubation period. The results indicate that histochemical studies of substrate specific ATP-ases should be performed with short incubation times and, when high specific activities are present, in large quantities of incubation media to reduce interference by ADP and AMP hydrolyzing enzymes.     

Adenosine triphosphate catabolism in homogenates of rat secretory enamel organs incubated in histochemical lead media

Summary To investigate how lead, when used as trapping agent, influences the ATP hydrolysis and to study how ATP is catalyzed in histochemical systems, homogenized secretory enamel organs were incubated in histochemical [³H]-ATP media. Aliquots from the media were taken after 3, 10, 20 and 30 min, and ATP and formed metabolites were separated by electrophoresis and radiometrically quantitated.In media lacking both lead and homogenate 2% of the ATP was spontaneously hydrolyzed during 30 min incubation at room temperature. The presence of lead caused an additional 8% hydrolysis at pH 7.2 and an additional 20% hydrolysis at pH 9.4. In the presence of homogenate, however, lead caused a net decrease of the hydrolysis of ATP as well as of ADP and AMP. This enzyme inhibition varied from around zero to some 80%, depending on pH and substrates involved.In homogenate-containing lead media, at both pH 7.2 and 9.4, ATP was rapidly hydrolyzed primarily to ADP and subsequently to AMP and adenosine and/or inosine. After 5–10 min ADP constituted the predominant substrate at both pH:s. At pH 7.2 ADP remained so for the rest of the incubation, whereas at pH 9.4 AMP was the predominant substrate at the end of the incubation. AMP was the final catabolic product in experiments at pH 7.2, and adenosine and/or inosine at pH 9.4. Inorganic phosphate was liberated almost linearly during the whole incubation period.The results indicate that histochemical studies of substrate specific ATP-ases should be performed with short incubation times and, when high specific activities are present, in large quantities of incubation media to reduce interference by ADP and AMP hydrolyzing enzymes.     

The intracellular distribution of free nucleotides in the tobacco leaf. Formation of adenosine 5′-phosphate from adenosine 5′-triphosphate in the chloroplasts

1. A method of studying the free nucleotides of leaf tissue is outlined and the need for a metal ion-binding agent for the complete extraction of certain nucleotides by aqueous ethanol is established. 2. The method was used to study the effect of illumination or darkening of tobacco plants on the free nucleotides present in the chloroplast and non-chloroplast tissue components. 3. When plants that had been in the dark for a prolonged period were given 30sec. of bright light there was a rapid phosphorylation of ADP to ATP in both chloroplast and non-chloroplast components of the tissue. 4. Where plants were moved into the dark from conditions suitable for rapid photosynthesis there was a rapid conversion of ATP into AMP and the AMP was formed only in the chloroplast fraction. In continuing darkness the AMP remained restricted mainly to the chloroplast fraction for at least 2min., but eventually its concentration fell to the low value that is typical of tobacco leaves during conditions of constant illumination. If the plants were returned to the light for 30sec. the AMP was rapidly rephosphorylated.     

Effects of cimetidine administered in cytoprotective and antisecretory doses on the membrane-bound ATP-dependent energy systems in the gastric mucosal lesions induced by HCl in rats

CFY strain rats (both sexes, 180-210 g) were fasted for 24 hr. Different doses of cimetidine (2.5, 10 and 50 mg X kg-1 i.p.) were given 30 min prior to the gastric mucosal lesions induced by the intragastric application of 0.6 M HCl. Animals were sacrificed 1 hr after the administration of the necrotizing agent. The number of gastric lesions was determined and their severity scored. Samples from the gastric fundic mucosa were taken for biochemical analysis. The tissue levels of adenosine-5'-triphosphate (ATP), adenosine-5'-diphosphate (ADP), adenosine-5'-monophosphate (AMP) and L-(+)-lactate were determined enzymatically, while the tissue contents of cyclic 3',5' adenosine monophosphate were measured by radioimmunoassay. The values for adenylate pool (ATP + ADP + AMP)-1 were calculated. All biochemical results were computed for 1.0 mg mucosal protein. We found that (1) the levels of ADP and lactate rose significantly, while ATP, AMP, cAMP, ATP X ADP-1 and energy charge decreased during the development of gastric lesions induced by HCl: (2) cimetidine decreased dose-dependently the number and severity of lesions: (3) the levels of ATP, ADP X ADP-1, and energy charge were increased dose-dependently by cimetidine, while AMP and lactate were decreased: (4) the levels of ADP, adenylate pool and cAMP did not change significantly by cimetidine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes of gastric mucosal biochemistry in ethanol-treated rats with and without acute surgical vagotomy

The biochemical background of ethanol-(ETOH) induced gastric mucosal damage was studied in rats with intact vagus and after acute surgical vagotomy. Observations were carried out on Sprague-Dawley (CFY) strain rats of both sexes. Gastric mucosal lesions were produced by intragastric administration of 1 ml 96% ethanol. Bilateral truncal surgical vagotomy was carried out 30 min before ETOH administration. The number and severity of gastric mucosal lesions was noted 1 h after ETOH administration. Biochemical measurements (gastric mucosal level of ATP, ADP, AMP, cAMP and lactate) were carried out from the total homogenized gastric mucosa. The adenylate pool (ATP + ADP + AMP), energy charge ((ATP + 0.5 ADP)/(ATP + ADP + AMP)) and ratio of ATP/ADP were calculated. It was found that: 1) ATP transformation into ADP increased, while ATP transformation in cAMP decreased in ethanol-treated animals with intact vagus nerve, while these transformations were quite the opposite in vagotomized animals; 2) no significant changes were found in the tissue level of lactate: and 3) the extent of biochemical changes was significantly less after surgical vagotomy. It is concluded that an intact vagus is basically necessary for the metabolic adaptation of gastric mucosa.     

Implication of guanosine 3′,5′-cyclic monophosphate,adenosine 3′,5′-cyclic monophosphate,adenosine 5′-mono-, di- and triphosphate and fructose-2,6-bisphosphate in the regulation of the glycolytic pathway in hypoxic/anoxic mussel,Mytilus galloprovincialis

The change in the content of cyclic GMP, cyclic AMP, ATP, ADP, AMP and fructose-2,6-bisphosphate that occurred in the mantle of the mussel Mytilus galloprovincialis Lmk when specimens of this mollusk were subjected to a hypoxia/anoxia situation were assessed. After the early 24 h in anaerobiosis, a clear decrease was observed in the ATP content, which remained close to that value for the rest of the time. AMP content doubled during the early 24 h in anaerobiosis and, from that time on, it remained close to that value. Fructose-2,6-bisphoshate and cyclic GMP showed a similar behavior. The levels of these compounds rose significantly during the early hours in anaerobiosis, and then fell to values similar to those of aerobiosis, remaining constant for the rest of the time. Neither ADP nor cAMP showed significant variations.     

5''-ADENINE MONONUCLEOTIDES TN SYNAPTOSOMAL PREPARATIONS FROM GUINEA PIG NEOCORTEX: THEIR CHANGE ON INCUBATION, SUPERFUSION AND STIMULATION

–(l) The contents of potassium and of ATP, ADP and AMP of homogenates of guinea pig neocortex in 0.32 M-SUCROSE, and of synaptosomal preparations derived therefrom, were determined and effects of incubation, superfusion and stimulation of the preparation were examined. The synaptosome preparations in M-SUCTOSe carried a smaller content of 5′-nucleotide per unit protein than did the homogenate from which they were derived. However, the proportion of synaptosomal nucleotides present as ATP was markedly greater than in the homogenate as a whole, the adenylate energy-charge being 83% greater in the synaptosomes than in the homogenate. Dilution of the synaptosomal preparation from the 1 M-sucrose to isotonic sucrose, decreased the contcnt of ATP per unit protein, but did not change the sum ATP + ADP + AMP. (2) Examined as deposited beds during 5 to 20 min incubation in oxygenated glucose-bicarbonate salines, synaptosomal K content and adenylate energy charge increased. These changes were sustained during a subsequent 40 min of incubation and also during superfusion. During such continued superfusion, electrical stimulation caused diminution of the ATP and the adenylate energy charge of the beds, as also did superfusion with fluids of increased K-content. (3) Lactate formation by the superfused beds was relatively stable during 30 min incubation. By electrical stimulation, the rate of lactate formation was increased by up to 30%. Increase in the potassium content of superfusion fluids could however increase lactate production 2.4-fold. The basis for these actions is discussed.     

Acid-soluble purine and pyrimidine derivatives of growing, encysting and encystment-inhibited amoebae

The intracellular acid-soluble purine and pyrimidine derivatives of myxamoebae-swarm cells of Physarum flavicomum were investigated during growth, microcyst formation, and during adenine-inhibition of encystment, using high performance liquid chromatography (HPLC). We also studied the incorporation of exogenous radioactive adenine into the acid soluble purine derivatives and S-adenosyl-sulphur compounds separated by HPLC. The most abundant ribonucleoside monophosphate was AMP in the growing and 15 h encysting cells (NC), while it was UMP in the 15 h adenine-inhibited cells (AIC). ADP was the nucleoside diphosphate present in the greatest quantity in the growing and NC cells but it was CDP in the AIC. The nucleoside triphosphate in highest concentration was ATP, UTP, and GTP in growing, NC, and AIC, respectively. Guanosine was the most abundant nucleoside in all cells. The nucleobase occurring in greatest concentration was cytosine, cytosine and guanine, and adenine in the growing, NC, and AIC, respectively. The AMP content in the 15 h AIC was 2.1-fold higher than that of adenosine. The 15 h NC had the lowest adenylate energy charge, a value of 0.54 +/- 0.02, while the values for growing cells and the AIC were 0.62 +/- 0.02 and 0.76 +/- 0.01, respectively. [14C]-Adenine labelling studies (15 h) revealed the occurrence of purine nucleotide interconversion, as the label was detected not only in adenosine, AMP, ADP, ATP, but also in guanine, guanosine, GMP, GDP, GTP, as well as, in inosine monophosphate and xanthosine monophosphate. The percentage incorporation of the radiolabelled adenine into AMP was higher than into adenosine. An increased intracellular level of guanine nucleotides is associated with the inhibition of encystment. The extracellular adenine, rather than internal adenine sources, appears to be the primary precursor of nucleotide for S-adenosylmethionine synthesis during adenine-inhibition of encystment.     

Adenylic nucleotides and energy charge during the embryonic development of Bufo arenarum

The contents of ATP, ADP and AMP were determined by HPLC and adenylic energy charge (AEC) was estimated during different stages of the embryonic development of Bufo arenarum up to the tailbud stage. All the developmental stages studied showed a high ATP content (about 1.04-1.48 nmol/emb.). The concentration of ADP was low (0.025-0.041 nmol/emb.) but rose slightly at the neural tube stage. AMP was undetectable before the tailbud stage. AEC values were almost constant (about 0.987-0.992) throughout the period studied. Only a fall at the tailbud stage could be detected which can be related to this more advanced cellular differentiation stage.     

Levels of adenylic system components (ATP, ADP, AMP) and cAMP during development of Blastocladiella emersonii

Concentrations of ATP, ADP, AMP, cAMP as well as pyruvate and glucose-6-phosphate were measured in B. lastocladiella emersonii cells developing via RS morphogenetic pathway. They varied significantly in the course of development (1.3-14.8 mumole/g dry weight for the sum of ATP+ADP+AMP; 0.012-5.3 nmole for cAMP; 0.47-1.9 mumole for pyruvate; 0.36-4.78 mumole for glucose-6-phosphate). At the same time the adenylate energy charge remained essentially unchanged (about 0.8) from the middle of exponential growth till the end of the stationary phase. At the late stages of RS-sporangia formation the concentration of all the above compounds decreased by about 10 times, and the adenylate energy charge only by 30%. Positive correlation between the levels of ATP and cAMP in RS cells was demonstrated. The concentration of adenylic nucleotides and cAMP showed the most noticable changes at the end of exponential growth; transition of the point of no return was not accompanied by significant changes in the pools of adenylic system, cAMP or energy charge.     

Nucleotide interconversions in microtubule protein preparations, a significant complication for accurate measurement of GTP hydrolysis in the presence of adenosine 5'-(beta, gamma-imidotriphosphate)

Pursuing the observation of Carlier and Pantaloni [Carlier, M.-F., & Pantaloni, D. (1982) Biochemistry 21, 1215-1224] that adenosine 5'-(beta, gamma-imidotriphosphate) (pNHppA) strongly inhibited tubulin-independent phosphatases in microtubule protein preparations, we observed with a number of commercial preparations of pNHppA that a major proportion of the terminal phosphate of [gamma-32P]GTP added to microtubule protein preparations was rapidly converted into ATP. Initially postulating degradation of pNHppA to AMP followed by stepwise conversion of AMP to ATP, we isolated two nucleoside monophosphate kinase activities from microtubule protein capable of generating ATP from AMP + GTP. The amounts of these enzymes in microtubule protein preparations, however, are probably too low to account for rapid ATP formation. Instead, ATP formation most likely is caused by nucleoside diphosphate kinase acting on ADP contaminating commercial pNHppA preparations. Such ADP contamination was demonstrated by high-performance liquid chromatography, with the amount of ATP formed with different pNHppA preparations proportional to the amount of ADP contamination. Repurification of commercial pNHppA until it was free of contaminating ADP also resulted in the elimination of ATP formation. The repurified pNHppA potently inhibited GTP hydrolysis in microtubule protein preparations. In addition, especially when supplemented with equimolar Mg2+, the repurified pNHppA strongly inhibited GTP hydrolysis and microtubule assembly in reaction mixtures containing purified tubulin and heat-treated microtubule-associated proteins (which contain negligible amounts of tubulin-independent phosphatase activity). We conclude that studies of microtubule-dependent GTP hydrolysis which make use of pNHppA must be interpreted with extreme caution.     

The Effect of the Stimulation Regime on Palaemon serratus Tail Muscle Energy Metabolism

The evolution of phosphometabolites was determined in the abdominal muscle of a crustacean Palaemon serratus during intermittent electrical stimulation at 1, 2 and 4 Hz and during natural escape behavior.The changes in AMP, IMP, phosphomonoesters, adenylate energy charge and ATP/ADP ratio were not affected by the frequency of electrical stimulation. On the contrary, changes in ATP, ADP and sum of adenylates depended on the stimulation protocol: degradation of ATP and accumulation of ADP were not significantly different after electrical stimulation at 2 and 4 Hz as compared to manual stimulation, but differed from the 1 Hz stimulation protocol values. The sum of adenylates decreased similarly after 2 and 4 Hz stimulation and manual protocols. The different exercise protocols did not produce any changes in AMP and IMP accumulation, ATP/ADP ratio and A.E.C. After manual stimulation, the phosphomonoester and phosphoarginine concentrations were similar to the variations observed in the all electrical stimulation protocols, while the Pi levels were similar to the variations observed in the 4 Hz stimulation protocol only. The NMR index decrease was significantly higher after the manual and 4 Hz stimulation protocols.     

Variations of ATP and its metabolites in the hippocampus of rats subjected to pilocarpine-induced temporal lobe epilepsy

Although purinergic receptor activity has lately been associated with epilepsy, little is known about the exact role of purines in epileptogenesis. We have used a rat model of temporal lobe epilepsy induced by pilocarpine to study the dynamics of purine metabolism in the hippocampus during different times of status epilepticus (SE) and the chronic phase. Concentrations of adenosine 5′-triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine in normal and epileptic rat hippocampus were determined by microdialysis in combination with high-performance liquid chromatography (HPLC). Extracellular ATP concentrations did not vary along 4 h of SE onset. However, AMP concentration was elevated during the second hour, whereas ADP and adenosine concentrations augmented during the third and fourth hour following SE. During chronic phase, extracellular ATP, ADP, AMP, and adenosine concentrations decreased, although these levels again increased significantly during spontaneous seizures. These results suggest that the increased turnover of ATP during the acute period is a compensatory mechanism able to reduce the excitatory role of ATP. Increased adenosine levels following 4 h of SE may contribute to block seizures. On the other hand, the reduction of purine levels in the hippocampus of chronic epileptic rats may result from metabolic changes and be part of the mechanisms involved in the onset of spontaneous seizures. This work provides further insights into purinergic signaling during establishment and chronic phase of epilepsy.     

Crowding within synaptic junctions influences the degradation of nucleotides by CD39 and CD73 ectonucleotidases

Synapsed cells can communicate using exocytosed nucleotides like adenosine triphosphate (ATP). Ectonucleotidases localized to synaptic junctions degrade nucleotides into metabolites like adenosine monophosphate (AMP) or adenosine. Oftentimes nucleotide degradation occurs in a sequential manner, of which ATP degradation by CD39 and CD73 is a representative example. Here, CD39 first converts ATP and adenosine diphosphate (ADP) into AMP, after which AMP is dephosphorylated into adenosine by CD73. Hence, the concerted activity of CD39 and CD73 can help shape cellular responses to extracellular ATP. In a previous study, we demonstrated that coupled CD39 and CD73 activity within synapse-like junctions is strongly controlled by the enzymes' co-localization, their surface charge densities, and the electrostatic potential of the surrounding cell membranes. In this study, we demonstrate that crowders within synaptic junctions, which can include globular proteins like cytokines and membrane-bound proteins, impact coupled CD39 and CD73 ectonucleotidase activity and, in turn, the availability of intrasynapse ATP. Specifically, we developed a spatially explicit, reaction-diffusion model for the coupled conversion of ATP → AMP and AMP → adenosine in a model synaptic junction with crowders that is solved via the finite element method. Our modeling results suggest that the association rate for ATP to CD39 is strongly influenced by the density of intrasynaptic protein crowders, as increasing crowder density generally suppressed ATP association kinetics. Much of this suppression can be rationalized based on a loss of configurational entropy. The surface charges of crowders can further influence the association rate, with the surprising result that favorable crowder-nucleotide electrostatic interactions can yield CD39 association rates that are faster than crowder-free configurations. However, attractive crowder-nucleotide interactions decrease the rate and efficiency of adenosine production, which in turn increases the availability of ATP and AMP within the synapse relative to crowder-free configurations. These findings highlight how CD39 and CD73 ectonucleotidase activity, electrostatics, and crowding within synapses influence the availability of nucleotides for intercellular communication.     

Rapid Increase in Adenosine 5'-Triphosphate during Early Wheat Embryo Germination

The ATP content of isolated wheat (Triticum aestivum L. var. Polk) embryos increases 5-fold during the first 30 minutes and 10-fold during the first hour of germination to 80% of maximum. The ATP level remains at approximately 800 nanomoles per gram of tissue during the next 15 hours. ADP, AMP, and total adenosine phosphates decrease between 1 and 6.5 hours, while adenylate energy charge increases from 0.6 to 0.8 and remains constant. The rapid increase in ATP during imbibition is consistent with the energy requirement for polyribosome formation and protein synthesis during the first hours of germination. A method for determining nanomole quantities of ATP in tissue extracts by isotopic dilution of γ-³²P-ATP in the hexokinase reaction is outlined.