Phosphorylation Of Adenosine Research Articles

Cloning and Expression of the Adenosine Kinase Gene from Rat and Human Tissues

Adenosine kinase is ubiquitous in eukaryotes and is a key enzyme in the regulation of the intracellular levels of adenosine, an important physiological effector of many cells and tissues. In this paper we report the cloning of cDNAs encoding adenosine kinase from both rat and human tissues. Two distinct forms of adenosine kinase mRNA were identified in human tissues. Sequence variation between the two forms is restricted to the extreme 5′-end of the adenosine kinase mRNA, including a portion of the coding region, and is consistent with differential splicing of a single transcriptional product. We have expressed both forms inE. coliand produced soluble active enzyme which catalyzes the phosphorylation of adenosine with high specific activityin vitroand is susceptible to known adenosine kinase inhibitors.

Quantitative analysis of similarities and differences in neurotoxicities caused by adenosine and 2'-deoxyadenosine in sympathetic neurons.

These experiments characterize the nucleoside transport and quantify the neurotoxicity of adenosine and 2'-deoxyadenosine (dAdo) in chick sympathetic neurons. We show that [3H]adenosine transport was sensitive to low temperature, specific inhibitors of nucleoside transport, and an excess concentration of adenosine. However, many of these treatments had a marginal effect on [3H]dAdo transport. Total retention of [3H]dAdo over short and long periods was approximately 10 times less than that of [3H]adenosine. These data suggest that adenosine and dAdo enter sympathetic neurons by different routes. Uptake of (3H]norepinephrine ([3H]NE) decreased in neurons damaged by nucleosides and increased to control levels when neurons were protected by various agents against adenosine or dAdo toxicity. These results indicate that [3H]NE uptake serves as a quantitative index of toxicity by the nucleosides. Using this approach we demonstrate that phosphorylation of both nucleosides is essential for their lethal action. For example, iodotubercidin prevented nucleoside-induced neuronal death, but the effect was much more pronounced in the case of dAdo toxicity (IC50 of 0.83 +/- 0.4 vs. 30 +/- 1.6 nM). Another kinase inhibitor, 5'-amino 5'-deoxyadenosine, was effective in protecting neurons against dAdo but had no effect against adenosine toxicity. These results suggest that specific kinases are associated with the phosphorylation of adenosine and dAdo in sympathetic neurons to produce toxic metabolic products. Finally, neurons were susceptible to dAdo toxicity from the time of plating to 4 weeks in culture but were resistant to adenosine toxicity 8 h after plating. In conclusion, our results highlight major differences in the mechanism of neurotoxicity by adenosine and dAdo and provide insights for identification of biochemical pathways leading to neuronal death.

Adenosine-AMP exchange activity is an integral part of the mammalian adenosine kinase.

Purified adenosine kinase (AK) from Syrian hamster and bovine liver was examined for the presence of adenosine (Ad)-AMP exchange activity. The enzyme from both sources, in addition to catalyzing the conventional ATP-dependent phosphorylation of adenosine, supported an Ad-AMP exchange reaction that required ADP. Under optimal conditions both these reactions were found to occur at comparable rates. Several observations strongly indicate that the Ad-AMP exchange activity is an integral part of AK and it is likely associated with its catalytic mechanism. These observations include: (i) Both AK and Ad-AMP exchange activities show a nearly complete dependence upon the presence of pentavalent ions such as phosphate, arsenate or vanadate for catalysis; (ii) Both activities show similar heat-lability and inhibition by 5-iodotubercidin (5-ITu); (iii) In a Chinese hamster cell mutant resistant to adenosine analogs that lacked AK activity, the Ad-AMP activity was also found to be absent. The presence of a phosphoryl-enzyme intermediate, or any exchange between free 32Pi and any of the reactants, however, was not detected under the reaction conditions. Some implications of these observations regarding the catalytic mechanism of AK are discussed.

Modelling of purine nucleoside metabolism during mouse embryonic development: relative routes of adenosine, deoxyadenosine, and deoxyguanosine metabolism.

The individual activities for adenosine kinase, deoxyadenosine kinase, adenosine deaminase, deoxyguanosine kinase, and purine nucleoside phosphorylase were determined during days 7 to 13 of mouse embryonic development. Adenosine deaminase increased 74-fold between days 7 and 9; deoxyadenosine kinase increased 5.4-fold during the same interval. Adenosine kinase, deoxyguanosine kinase, and purine nucleoside phosphorylase exhibited less than 2-fold changes in activity between days 7 and 13. Using Michaelis constants for each enzyme and the maximal velocities determined from enzyme assay, the relative routes of adenosine and deoxyadenosine metabolism via phosphorylation or deamination were modeled as a function of nucleoside concentration for days 7 through 13. For days 7 and 8, phosphorylation of adenosine is the principle route of metabolism at physiological concentrations. A switch occurred at day 9 and following where deamination is at least 5-fold greater than phosphorylation at all substrate concentrations. Deoxyadenosine phosphorylation was at most 10% of deamination at day 7 and then declined to less than 1% for days 9 to 13. Phosphorolysis was the principle route of deoxyguanosine metabolism through the 7 to 13 day period. Thus catabolism rather than phosphorylation was the principle pathway for purine deoxynucleoside metabolism during this period.

Specific effect of magnesium ion on 2',3'-cyclic AMP synthesis from adenosine and trimeta phosphate in aqueous solution.

Phosphorylation of adenosine by trimetaphosphate was investigated using various catalysts in aqueous solution under mild conditions at pH approximately 7.0 and at 41 degrees C. The product was primarily 2',3'-cyclic AMP together with smaller amounts of ATP. Magnesium ion was found to have a remarkable catalytic effect of approximately one hundred times greater than the other chemicals tested. The mechanism for the specific effect of magnesium ion is discussed.

Hyperthyroidism increases adenosine transport and metabolism in the rat heart.

Hyperthyroidism induces a number of metabolic and physiological changes in the heart including hypertrophy, increase in inotropic status, and alterations of myocardial energy metabolism. The effects of hyperthyroidism on adenosine metabolism which is intimately involved in the control of many aspects of myocardial energetics, have not been clarified. The aim of this study was thus to evaluate the potential role of adenosine in the altered physiology of the hyperthyroid heart. Transport of adenosine was studied in cardiomyocytes isolated from hyperthyroid and euthyroid rats. Activities of different enzymes of purine metabolism were studied in heart homogenates and concentrations of nucleotide and creatine metabolites were determined in hearts freeze-clamped in situ. Both transport of adenosine into cardiomyocytes and the rate of intracellular phosphorylation were higher in the hyperthyroid rat. At 10 microM concentration, adenosine transport rates were 275 and 197 pmol/min/mg protein in hyperthyroid and euthyroid cardiomyocytes respectively whilst rates of adenosine phosphorylation were 250 and 180 pmol/min/mg prot. An even more pronounced difference was observed if values were expressed per number of cells due to cardiomyocyte enlargement. Hyperthyroidism was associated with a 20% increase in adenosine kinase, 30% decrease in membrane 5'-nucleotidase and 15% decrease in adenosine deaminase activities measured in heart homogenates. In addition there was a substantial depletion in the total creatine pool from 63.7 to 41.6 mumol/g dry wt, a small decrease in the adenylate pool (from 27.2 to 24.3 mumol/g dry wt) and an elevation of the guanylate pool (from 1.22 to 1.36). These results show that adenosine transport and phosphorylation capacity is enhanced in hyperthyroidism.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine uptake and metabolism in human endothelial cells.

The handling of adenosine by the endothelium is crucial for the effects of this nucleoside in the vascular system. Strategic location of endothelial cells at the vessel wall border indicate its important role in the turnover of adenosine. High endothelial activity of ecto 5’nucleotidase1 implies predominant role in the extracellular formation of adenosine from secreted nucleotides. However, the capacity to release intracellularly produced adenosine, phosphorylation of extracellular adenosine and to some extent adenosine transit between interstitial and intravascular space depend on the membrane transport capacity in this type of cells.

Difference in energy metabolism between fresh and warm ischemic canine pancreases during preservation by the two-layer method.

We have demonstrated that adenosine triphosphate (ATP) is synthesized within a canine pancreas during preservation by the two-layer method and there is a direct correlation between a high ATP tissue level and good posttransplant outcome. The purpose of this study was to examine the difference in energy metabolism between fresh and warm ischemic pancreases during preservation by this method. First, fresh pancreases were preserved with simple cold storage in Euro-Collins solution (EC; group 1A), or by the two-layer method using EC (group 1B), EC with 2,4 dinitrophenol (DNP; group 1C), an uncoupler of oxidative phosphorylation, or modified EC (ECM; group 1d), which contained mannitol in place of glucose for 48 h. ATP tissue concentrations in group 1B were significantly higher than in group 1A (7.91 +/- 1.21 vs. 1.21 +/- 0.31 micromol/g dry weight, P < 0.01) but almost equal to group 1d (7.91 +/- 1.21 vs. 7.59 +/- 0.97 micromol/g dry weight, NS). DNP (group 1C) caused a significant decrease in tissue ATP levels in group 1A (0.61 +/- 0.07 vs. 7.91 +/- 1.21 micromol/g dry weight, P < 0.01). Second, pancreases subjected to 60 min of warm ischemia were preserved by simple cold storage with EC (group 2A) or the two-layer method using EC (group 2B) or EC with adenosine (group 2C) for 24 h. ATP tissue levels in groups 2A and 2B after preservation were 1.40 +/- 0.46 and 1.56 +/- 0.40 micromol/g dry weight and graft survival rates were 0/5 (0%) and 0/3 (0%), respectively. However, tissue ATP levels in group 2C after preservation were significantly higher compared with the value before preservation (7.23 +/- 2.17 vs. 1.90 +/- 0.53/g dry weight, P < 0.01) and graft survival rate was 4/5, 80%. Other nucleosides, hypoxanthine, inosine, and adenine did not substitute for adenosine. In addition, studies with [2-3 H] adenosine demonstrated that almost all of the adenosine was converted to adenine nucleotides. This study clearly demonstrated that fresh grafts synthesize ATP mainly via mitochondrial oxidative phosphorylation using endogenous substrates. However, after significant warm ischemia, pancreases produce ATP mainly via direct phosphorylation of exogenous adenosine during preservation by the two-layer method.

Behavioural sensitization in 6-hydroxydopamine lesioned rats involves the dopamine signal transduction: Changes in DARPP-32 phosphorylation

“Priming” is a phenomenon of behavioural sensitization observed in unilaterally 6-hydroxydopamine lesioned rats following exposure to a dopamine agonist. After priming, a single dose of the D 1 agonist SKF 38393 (3 mg/kg) produces contralateral turning, while the same dose is inactive in drug-naive, lesioned animals. The molecular mechanisms of “priming” were investigated here by studying the phosphorylation of dopamine and adenosine 3′–5′ monophosphate regulated phosphoprotein (DARPP32), a dopamine- and cyclic AMP-regulated phosphoprotein functionally linked to D 1 receptors in striatum. Dephospho-form of DARPP-32 was measured by a back-phosphorylation assay. All assays were performed in striata from both lesioned and unlesioned sides. A significant decrease of dephosphoDARPP-32 (27%) was observed in the denervated striatum of primed rats, indicating an increased Phosphorylation in vivo of DARPP-32 in response to the D 1 agonist. The levels of DARPP-32 protein, as measured by quantitative immunoblotting, remained unchanged in all experimental groups. This study shows that priming is expressed as an increased transduction of the D 1 receptor message.

ROLE OF ADENOSINE IN PRESERVATION BY THE TWO-LAYER METHOD OF ISCHEMICALLY DAMAGED CANINE PANCREAS

The purpose of this study was to clarify the role of adenosine in preservation of ischemically damaged pancreas by the two-layer (Euro-Collins solution [EC]/perfluorochemical [PFC]) method using a canine model. Twenty-four-hour preservation of the pancreas graft subjected to 60-min warm ischemia was successful by the two-layer (EC with adenosine/PFC) method (4/5, 80%), but neither simple cold storage in EC (0/5, 0%), nor EC with adenosine (1/5, 20%), nor the two-layer (EC/PFC) method (0/3, 0%) was successful. Tissue ATP concentrations at the end of preservation by the two-layer (EC with adenosine/PFC) method were significantly higher compared with the two-layer (EC/PFC) method (7.23 +/- 2.17 vs. 1.56 +/- 0.40 mumol/g dry weight, P < 0.01). Studies with [2-3H]adenosine demonstrated that only part of adenosine was converted to inosine, hypoxanthine, and adenine, whereas the remainder was incorporated into adenine nucleotides in the pancreas graft. In addition, hypoxanthine, inosine, and adenine did not substitute for adenosine. We conclude that provision of adenosine to ischemically damaged pancreas during preservation by the two-layer (EC/PFC) method allows ATP synthesis within the graft via direct phosphorylation of adenosine. Metabolic processes vital to repair damaged cells and maintain cellular integrity can be maintained, which makes it possible to preserve ischemically damaged pancreas.

Phosphorylation of adenosine in renal brush-border membrane vesicles by an exchange reaction catalysed by adenosine kinase.

Uptake of [3H]adenosine in brush-border membrane (BBM) vesicles from either rat or pig kidney leads to an accumulation of intravesicular [3H]AMP. The lack of significant levels of ATP and the presence of AMP in BBM indicated that a phosphotransfer between [3H]adenosine and AMP occurs. The phosphotransfer activity is inhibited by iodotubercidin, which suggests that it is performed by adenosine kinase acting in an ATP-independent manner. The existence of a similar phosphotransferase activity was demonstrated in membrane-free extracts from pig kidney. From the compounds tested it was shown that a variety of mononucleotides could act as phosphate donors. The results suggest that phosphotransfer reactions may be physiologically relevant in kidney.

Phosphorylation of adenosine in anoxic hepatocytes by an exchange reaction catalysed by adenosine kinase.

The elevation of adenosine levels induced by anoxia in isolated rat hepatocytes has been shown to result mainly from an arrest of the recycling of the nucleoside by adenosine kinase [Bontemps, Vincent and Van den Berghe (1993) Biochem. J. 290, 671-677]. To assess the activity of the latter enzyme in intact hepatocytes, incorporation of radioactive adenosine into the cells' adenine nucleotides was measured. Unexpectedly, despite the near-absence of ATP in anoxic cells, 40% of 50 microM [8-14C]adenosine was still incorporated into adenylates over 5 min. Moreover, whereas unlabelled and labelled adenosine were utilized in parallel in normoxic cells, uptake of [8-14C]adenosine did not correspond to a net disappearance of adenosine in anoxic cells. Addition of 1 mM unlabelled adenosine to anoxic hepatocytes in which the adenine nucleotides had been prelabelled with [U-14C]adenine induced an immediate loss of their radioactivity. The latter was recovered in the form of adenosine, but the size of the adenylate pool was not modified. Taken together, these results suggest the occurrence of an exchange reaction between AMP and adenosine. Incubation of Sephadex G-25-filtered high-speed supernatants of rat liver with 20 microM [8-14C]adenosine, 10 mM MgCl2 and 1 mM AMP resulted in the labelling of AMP in the total absence of ATP. This labelling was influenced by effectors of both adenosine kinase and cytosolic IMP-GMP 5'-nucleotidase; the latter is known to catalyse an exchange reaction [Worku and Newby (1982) Biochem. J. 205, 503-510]. Chromatography of cytosolic fractions of rat liver on DEAE-Sepharose, followed by Sephacryl S-200 and AMP-Sepharose, demonstrated that the exchange reaction between adenosine and AMP co-purified with adenosine kinase. It is concluded that incorporation of labelled adenosine into adenine nucleotides should not be considered to be proof of adenosine kinase activity in anoxia.

Different relationships between cellular adenosine or 3'-deoxyadenosine phosphorylation and cellular adenine ribonucleotide catabolism may be obtained.

Treatment of BALB/c-3T3 mouse fibroblasts with 3'-deoxyadenosine led to a rapid accumulation of 3'-deoxyadenosine phosphates and the kinetics of this process has been determined. Concomitant with accumulation of these compounds, the adenine ribonucleotide pool was reduced. The kinetics of the two processes suggested that they were tightly coupled. The inhibitory effect of relatively high concentrations of coformycin indicated that IMP was an intermediate in the catabolic pathway. Similar experiments with Ehrlich ascites tumor cells were performed in Ringer-Hepes solution at pH 6.5 or 7.5 and with varying concentrations of orthophosphate. The experiments were performed with cells where ATP was [3H]-labeled. This allowed the determination of the catabolism of adenine ribonucleotides to labeled nucleosides under conditions where added adenosine was phosphorylated. The results showed that at low phosphate concentration (5.8 mM) at pH 6.5 adenosine may be phosphorylated at a rate that was completely balanced to the concomitant catabolism of adenine ribonucleotides; that is, there was apparently a tight kinetic coupling between anabolism of adenosine and catabolism of adenine ribonucleotides. With 3'-deoxyadenosine a corresponding effect was obtained although the apparent coupling between phosphorylation of 3'-deoxyadenosine and catabolism of adenine ribonucleotides was not complete. When experiments were performed at the same pH but at high concentration of phosphate (45 mM) there was in contrast no coupling between the two processes; that is, ATP was present in constant amounts while 3'-deoxyadenosine phosphates accumulated at a high rate. In experiments with adenosine under these conditions there was still some although a relatively limited degree of apparent coupling between phosphorylation of adenosine and catabolism of adenine ribonucleotides. In both lines of cells used and with both adenosine and 3'-deoxyadenosine, the main products of the catabolism of adenine ribonucleotides were inosine and hypoxanthine. With 3'-deoxyadenosine there was in addition (about 20%) formation of xanthosine, suggesting that IMP dehydrogenase had also been activated. These results lead to the suggestion that adenosine (or 3'-deoxyadenosine) may be phosphorylated in two ways. 1) Phosphorylation may depend on an adenosine kinase unrelated to catabolism of adenine ribonucleotides. 2) Phosphorylation may be tightly coupled to catabolism of adenine ribonucleotides. A nucleoside phosphotransferase may catalyze the transfer of a phosphoryl group from IMP to adenosine (or 3'-deoxyadenosine) to form AMP (or 3'-dAMP) and inosine, a process that may be tightly coupled to an AMP deaminase reaction. The IMP formed in the latter reaction may not be released but transferred to the phosphotransferase.(ABSTRACT TRUNCATED AT 400 WORDS)

Phosphorylation of cyclic adenosine 3',5'-monophosphate (cAMP) response element-binding protein isoforms by the cAMP-dependent protein kinase

Phosphorylation of cyclic adenosine 3',5'-monophosphate (cAMP) response element-binding protein isoforms by the cAMP-dependent protein kinase

Metabolic and functional cardiac impairment after reperfusion with persantine

The effect of dipyridamole (DYP) on postischemic myocardial function and metabolism was studied using the isolated rabbit heart model. Twenty-one isolated rabbit heart preparations were divided into two groups: KH (control N = 10) were reperfused after 24 min normothermic hyperkalemic arrest with modified Krebs-Henseleit buffer (KH) while DYP ( N = 11) were reperfused with KH and 5 × 10 −6 M DYP. Hearts were analyzed for myocardial function (DP, developed pressure, +dp/dt, -dp/dt) and metabolic function (ATP, CrP, ADP, AMP, purines, and lactate levels). Data analysis revealed significant reperfusion depression in DYP myocardial function compared with KH ( P < 0.05): DP (42 ± 6 vs 89 ± 7 mm Hg), +dp/dt (390 ± 21.6 vs 1227 ± 48.4), and -dp/dt (280 ± 20.1 vs 677 ± 19.8). Comparison of DYP to KH metabolic parameters was also significantly different ( P < 0.05): ATP (5.8 ± 0.7 vs 9.5 ± 1.4), ADP (2.1 ± 0.2 vs 3.2 ± 0.6), CrP (9.6 ± 0.3 vs 17.2 ± 1.3). Tissue purines (adenosine and inosine) were significantly elevated ( P < 0.01) in the DYP group, while coronary sinus purines and lactate loss were similar. Thus, the data suggest that DYP, present during postischemic reperfusion, depresses myocardial function by inhibiting adenosine phosphorylation, thereby decreasing the generation of high-energy phosphates without increased substrate loss or ischemia.

Mycoplasma contamination alters 2′‐deoxyadenosine metabolism in deoxycoformycin‐treated mouse leukemia cells

Deoxycoformycin-treated P388 and L1210 mouse leukemia cells salvage 2'-deoxyadenosine from the medium only inefficiently, because deoxyadenosine deamination is blocked and its phosphorylation is limited by feedback controls. Mycoplasma contamination at a level that had no significant effect on the growth of the cells increased the salvage of deoxyadenosine greater than 10 fold over a 90 min period of incubation at 37 degrees C, but in this case deoxyadenosine was mainly incorporated into ribonucleotides and RNA via adenine formed from deoxyadenosine by mycoplasma adenosine phosphorylase. Deoxyadenosine was an efficient substrate for this enzyme, in contrast to 2',3'-dideoxyadenosine which was not phosphorolyzed. Mycoplasma infection was confirmed by the presence of uracil phosphoribosyltransferase activity and by culture isolation. The contaminant has been identified as Mycoplasma orale. Mycoplasma infection had no effect on the deamination and phosphorylation of deoxyadenosine and adenosine, on the salvage of hypoxanthine and adenine, or on the degradation of dAMP and dATP by the cells or on their acid and alkaline phosphatase activities.

Effects of glucose and adenosine on the ATP content of hamster spermatids

Effects of glucose and adenosine on ATP metabolism were studied using isolated round spermatids from hamsters. The ATP content of the spermatids was strongly decreased after 1 h of incubation of the cells in the presence of 0.1 mM D-glucose. Glucose (1 mM) had no effect during 18 h of incubation in the presence of 12 mM sodium DL-lactate. However, 10 mM glucose caused an almost complete loss of cellular ATP in the presence of lactate. The effect of adenosine was estimated in the absence of glucose with lactate as the energy-yielding substrate. The cellular ATP content was approximately 4 and 8 nmol/10(6) cells, after 18 h of incubation in the absence and presence of 0.1 mM adenosine, respectively. This two-fold increase was prevented by inhibitors of adenosine uptake and phosphorylation and was slowly reversed after removal of the exogenous adenosine. Treatment of the cells with adenosine had no effect on the energy charge, which was higher than 0.90, and did not alter the cellular cyclic AMP content. The suggestion that the physiological ATP content of the round spermatids is probably stabilized in the region of 4 nmol/10(6) cells is discussed.

Immunochemical and catalytic characteristics of adenosine kinase from Leishmania donovani

Polyclonal antibodies to homogeneous preparation of adenosine kinase from Leishmania donovani were raised in rabbit. The antiserum was inhibitory and precipitated enzyme activity from both homogeneous and partially purified adenosine kinase from the parasite. However, the antiserum did not immunoprecipitate adenosine kinase of other higher eukaryotic sources tested so far. Immunoblot analysis of extracts from L. donovani and other sources revealed specific reaction of the antiserum with only the parasite enzyme. Under similar conditions, the enzyme monophosphorylated adenosine and 7-amino-3[beta-D-ribofuranosyl]-1H-pyrazolo[4,3-d]pyrimidine (formycin A) with almost equal efficiency, exhibiting Km values of 16 and 24 microM, respectively. The turnover number (Kcat) of the enzyme with both adenosine and formycin A was 24 s-1, whereas Kcat/Km yielded values of 1.5 and 1.0 microM-1 s-1, respectively. Substrate competition experiments indicated strong inhibition of [3H]formycin A phosphorylation by adenosine. In contrast, [3H]adenosine phosphorylation was insensitive to formycin A except at very high concentrations. The inhibitions of [3H]formycin A and [3H]adenosine phosphorylation by adenosine and formycin A were noncompetitive with respect to each other. Of the two nucleosides, adenosine was found to be effective in eluting the enzyme from the 5'-AMP Sepharose 4B column. Phosphorylation of [3H]formycin A was strongly inhibited by N-ethylmaleimide at concentrations which exerted minimal effect on [3H]adenosine phosphorylation. Adenosine exclusively, but not formycin A, protected the enzyme from N-ethylmaleimide-mediated inactivation. Taken together the results suggest that (a) adenosine kinase from L. donovani is immunologically distinct and (b) the enzyme possibly has two discrete catalytically active nucleoside interacting sites.

Fermentative production of inosine 5′-phosphate from adenosine by Baker's yeast

IMP was formed by the addition of glucose to a mixture for phosphorylation of adenosine to ATP with dried baker's yeast cells. IMP was isolated by column chromatography on Dowex 1 × 2 (formate type), and identified by various methods. The effects of several factors on the IMP production were examined to establish optimum conditions: 155 mM IMP was accumulated 4 h after the feeding of 1200 mM glucose at 6 h to the initial reaction mixture consisting of 300 mM adenosine, 700 mM glucose, 1200 mM potassium phosphate buffer (pH 7.0), 50 mM MgSO 4, and 400 mg/ml dried baker's yeast cells. The mechanism of the IMP formation was discussed.

Production of ATP from adenine by a combination of bacterial and baker's yeast cells

A new process for ATP production from adenine was designed involving the use of intact bacterial cells containing nucleoside phosphorylases and dried baker's yeast cells containing an energy generating system: it consists of (1) the formation of adenosine through trans- N-ribosylation between adenine and uridine, cytidine, or inosine catalyzed by the nucleoside phosphorylases and (2) the phosphorylation of adenosine to ATP through alcoholic fermentation of the yeast cells. Erwinia carotovora was better than Escherichia coli or Aerobacter aerogenes as the source of nucleoside phosphorylases. Inosine and uridine were effective as ribosyl donors, and cytidine was also usable, if the reactions were done with cell-free extracts. ATP production was greatly influenced by the concentrations of inorganic phosphate and glucose in the mixture. The immobilized phosphorylase preparation was better than the cell-free extract in the ATP production. The nucleoside phosphorylases were stable at 60°C but adenosine deaminating enzymes were not, and large amounts of adenine could be used due to the increase in solubility at that high temperature. Through a two-step reaction, i.e., transribosylation with intact cells of Er. carotovora at 60°C followed by fermentation with baker's yeast cells at 28°C, 98 mM ATP (about 50 g/ l) was obtained from 150 mM adenine.