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XAS of Zn-peptide binary and ternary complexes prepared using peptides mimicking the potential metal binding sites of rabbit skeletal muscle AMP deaminase (AMPD) strongly suggest that the region 48-61 of the enzyme contains a zinc binding site, whilst the region 360-372 of the enzyme is not able to form 1:1 complexes with zinc, in contrast with what has been suggested for the corresponding region of yeast AMPD. XAS performed on fresh preparations of rabbit skeletal muscle AMPD provides evidence for a dinuclear zinc site in the enzyme compatible with a (mu-aqua)(mu-carboxylato)dizinc(II) core with an average of two histidine residues at each metal site and a Zn-Zn distance of about 3.3 Angstrom. The data indicate that zinc is not required for HPRG/AMPD interaction, both zinc ions being bound to the catalytic subunit of the enzyme, one to the three conserved amino acid residues among those four assumed to be in contact with zinc in yeast AMPD, and the other at the N-terminal region, probably to His-52, Glu-53 and His-57. Tryptic digests of different enzyme preparations demonstrate the existence of two different protein conformations and of a zinc ion connecting the N-terminal and C-terminal regions of AMPD.

The histidine-proline-rich glycoprotein (HPRG) component of rabbit skeletal muscle AMP deaminase under denaturing and reducing conditions specifically binds to a Zn(2+)-charged affinity column and is only eluted with an EDTA-containing buffer that strips Zn(2+) from the gel. The isolated protein is homogeneous showing an apparent molecular weight (MW) of 95000 and the N-terminal sequence L-T-P-T-D-X-K-T-T-K-P-L-A-E-K-A-L-D-L-I, corresponding to that of rabbit plasma HPRG. The incubation with peptide-N-glycosidase F promotes the reduction of the apparent MW of isolated HPRG to 70000, characterizing it as a N-glycosylated protein. The separation from AMP deaminase of an 85-kDa component with a blocked N terminus is observed when the enzyme is applied to the Zn-charged column under nondenaturing conditions. On storage under reducing conditions, this component undergoes an 85- to 95-kDa transition yielding a L-T-P-T-D-X-K-T-T-K-P-L N-terminal sequence, suggesting that the shift in the migration on SDS/PAGE as well as the truncation of the protein at its N terminus are promoted by the reduction of a disulfide bond present in freshly isolated HPRG. The separation of HPRG induces a marked reduction in the solubility of AMP deaminase, strongly suggesting a role of HPRG in assuring the molecular integrity of the enzyme.

AMP deaminase (AMPD) is manifest through a multigene family in higher eukaryotes, including man. The human AMPD1 and AMPD3 genes have been cloned and partially characterized. This study describes the cloning, chromosomal localization, partial-sequence and characterization of the human AMPD2 gene. Composed of nineteen exons and eighteen intervening sequences spanning nearly 14 kb of genomic DNA, the human AMPD2 gene is positioned on the short arm of chromosome 1 near the p13.3 boundary. Two alternative 5′ exons (1A and 1B) are remotely located upstream, whereas the other seventeen are compressed into the 3′ terminal one-half of the gene. Transient transfections of human retinal pigment epithelial (RPE) cells using heterologous constructs containing 5′ flanking and 5′ untranslated sequences cloned upstream of a luciferase reporter gene show that promoter activities are associated with exons 1A and 1B. Inspection of genomic DNA sequence reveals that AMPD2 promoter regions lack readily identifiable TATA boxes and are G + C-rich, particularly in the region of multiple transcription initiation sites in exon 1A. The regulation and evolution of the entire human AMPD multigene family are discussed.

Reactivity of sulfhydryl groups of human uterine smooth muscle AMP-deaminase with DTNB, and the effect of their chemical modification on kinetic and regulatory properties of the enzyme were investigated. (1), Approx. 7 and 5 sulfhydryl groups per mol of the enzyme have been shown to be accessible for DTNB (5,5′-dithiobis(2-nitrobenzoic acid)) titration in denaturated and native AMP-deaminase, respectively. (2), Titrated groups were not homogenous; some of them reacted with DTNB much faster than others. (3), The activity of the modified enzyme was very low, and the modified enzyme manifested unusual hyperbolic saturation kinetics with the substrate. (4), Exhaustive dialysis against a buffer containing 10 mM thioethanol reactivated the modified enzyme, and restored its original regulatory properties. Experimental results obtained indicate that modified sulfhydryl groups play a significant role in the maintenance of the proper, catalytically-efficient conformation of the enzyme.

Deoxycytidylate deaminase was purified about 7000-fold to homogeneity from a human source (HeLa cells). The final step in the purification employed an affinity column, which increased the specific activity of the enzyme from the previous step by 500-fold. Similar to most other dCMP deaminases, this enzyme is allosterically regulated by microM levels of dCTP and dTTP. However, unlike the other enzymes the most dramatic allosteric responses occur at substrate levels of 0.1 mM dCMP or less, where at least a 10-fold increase in activity is effected by dCTP. The enzyme is particularly sensitive to inhibition by dTTP with 50% inhibition being obtained at 1.5 x (10(-6) M in the absence of dCTP. Antibody to the human enzyme did not cross-react with a dCMP deaminase induced in Escherichia coli by T4-bacteriophage, nor did antibody to the phage-induced enzyme cross-react with the human deaminase. A potential transition-state analogue of the substrate, 2'-beta-D-deoxyribose-pyrimidin-2-one 5'-phosphate was prepared, and found to inhibit dCMP deaminase competitively with a Ki of 1.2 x 10(-8) M.

The nucleotide sequence of cDNA encoding human erythrocyte AMP deaminase has been determined by screening of human spleen cDNA library and by utilizing polymerase chain reaction (PCR) techniques. The 3.7 kb cDNA contains an open reading frame of 2301 bp which encodes 767 amino acids chain resulting in 89 kDa protein. The polyadenylation consensus signal (5′-AATAAA) located at 1212 bp 3′ downstream from the stop codon. The homologies to human and rat muscle-specific AMP deaminases showed 64.1% and 65.2% identities, respectively, at the nucleotide level in the area of open reading frame, and 60.2% and 59.8% similarities at the deduced amino acid level.

Several adenosine analogs, such as coformycin, 2'-deoxycoformycin and erythro-9-(3-nonyl-p-aminobenzyl)adenine (EHNA), which are strong inhibitors of mammalian adenosine deaminase, are much weaker inhibitors of the Saccharomyces cerevisiae enzyme. The specificity of the yeast enzyme is more restricted than that of mammalian adenosine deaminase, particularly towards the ribose moiety and around position 6 and 1 of the substrate. The sulphydryl group appears to be more masked in the yeast than in the mammalian enzyme. The kinetic effects of pH with adenosine substrate and with the inhibitor purine riboside are reported. The findings on specificity and pH kinetic effects can be interpreted in a model involving proton transfer from the -SH group of the enzyme to the N-1 atom of the substrate.

Adenylate deaminase activity was determined in cultured muscle cells of different maturation grades and muscle biopsies from normal subjects and four patients with a primary myoadenylate deaminase (MAD) deficiency. Adenylate deaminase activity was much lower in cultured human muscle cells than in normal muscle. The activity increased with maturation. The ratio of activities measured at 5 and 2 mM AMP decreased in the order: immature muscle cells greater than more mature muscle cells greater than muscle. Adenylate deaminase activity was detectable in muscle cell cultures of MAD-deficient patients. However, both at 2 and 5 mM AMP this activity was significantly lower than in cultured cells with the same high maturation grade obtained from control subjects, whereas the ratio between the activities at 5 and 2 mM AMP was higher. The observations indicate that transition from a fetal to an adult muscle isoenzyme of adenylate deaminase takes place in human cultured muscle cells during maturation. In cultures obtained from MAD-deficient patients this transition does not occur and only the fetal isoenzyme is present.

Some 2′,3′-dideoxynucleotides, of importance in the enzymology of the anti-HIV compounds, ddA and ddI, have been synthesized and purified by ion-exchange chromatography. 2′,3′-Dideoxyadenylosuccinate, an intermediate in the pathway of ddI to ddATP, is converted to ddAMP by AMPS lyase at 1.85% of the efficiency of the natural substrate, adenylosuccinate. Interestingly, ddAMP and other 2′,3′-dideoxygenated nucleotides are not substrates for AMP deaminase, another relevant enzyme in the conversion of ddA to ddATP via ddl.

Profiles of the catabolism of adenine nucleotides in cultured plant cells were investigated. Adenine nucleotides, prelabelled by incubation of suspension-cultured Catharantus roseus cells with [8- 14 C]adenosine, were catabolized rapidly and most of the radioactivity appeared in 14 CO 2 . Allantoin and allantoic acid, intermediates of the oxidative catabolic pathway of purines, were temporarily labelled. When the cells, prelabelled with [8- 14 C]adenosine, were incubated with high concentrations of adenosine, the rate of catabolism of adenine nucleotides increased. The results suggest that the relative rate of catabolism of adenine nucleotides is strongly dependent on the concentration of adenine nucleotides in the cells. Studies using allopurinol, coformycin and tiazofurin, inhibitors of enzymes involved in purine metabolism, suggest the participation of AMP deaminase and xanthine oxidoreductase in the catabolism of adenine nucleotides in plant cells. AMP deaminase was found in extracts from C. roseus cells and its activity increased significantly in the presence of ATP. In contrast, no adenosine deaminase or adenine deaminase activity was detected. Qualitative differences in the catabolic activity of AMP were observed between suspension-cultured cells from different species of plants.

1. 1.|When brain extracts were chromatographed on a phosphocellulose column, using a NaCl gradient for elution, three peaks and a shoulder with AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) activity were obtained. They were designated as Peaks I, II, III and IV in the order of elution from the column. When Peaks I, II and IV were individually applied to the phosphocellulose column, each was chromatographed as a single peak at the same position as that for the original peak and the mixtures of them yielded only the peaks corresponding to the parental peaks. 2. 2.|Deaminase Peaks I, II and IV were purified by column chromatography using phosphocellulose and DEAE-cellulose. About 130-fold purification was obtained for Peak I, 280-fold for Peak II and 770-fold for Peak IV. 3. 3.|A broad optimum pH was observed in Peak I and a sharp optimum pH, at pH 6, in Peak IV. Peak I was rapidly inactivated when heated at 50 °C. Under the same condition, Peaks II and IV were inactivated more slowly. The rates of deamination of dAMP, relative to AMP, in three deaminase preparations are different from one another. 4. 4.|The molecular sizes of three deaminases are similar. 5. 5.|Deaminase Peak I has apparently a higher affinity for AMP than other types of deaminase both in the absence and presence of activators, while Peak IV has the lowest affinity of all. 6. 6.|The regulatory properties of these deaminases are similar. The velocity vs AMP concentration curves are sigmoidal in the absence of activator such as ATP, KCl or NaCl and the curves become nearly hyperbolic in the presence of activator. The maximum velocities were the same both in the absence and presence of activator, while the enzymes showed a higher affinity for AMP in the presence of activator.

A basal level of glucosamine-6-phosphate deaminase is detected in yeast cells grown on glucose. However, a burst of enzyme production occurs in the presence of N-acetylglucosamine in pathogenic Candida albicans and non-pathogenic Saccharomyces cervisiae. The enzyme synthesis stops and its concentration in the cells declines rapidly as soon as N-acetylglucosamine is removed from the medium. Experiments with RNA- and protein-synthesis inhibitors indicate that the appearance of new enzyme activity is dependent on concomitant new protein synthesis and the inducer operates at a transcriptional level. However, inhibition of DNA synthesis either by hydroxyurea or by mitomycin-C does not impair the synthesis of glucosamine-6-phosphate deaminase.

We studied the ability of purine compounds to restore the proliferation of concanavalin-A-stimulated rat T-lymphocytes under conditions of purine de novo synthesis inhibition and, on the other hand, the inhibition by purine nucleosides of the response of these cells to a mitogenic stimulation under conditions of normal purine de novo synthesis. The use of 50 microM azaserine, a potent inhibitor of purine de novo synthesis, allowed us to define the physiologically active salvage pathways of purine bases, ribo- and deoxyribonucleosides in concanavalin-A-stimulated rat T-lymphocytes. Except for guanylic compounds, all purines completely restored cell proliferation at a concentration of 50 microM. Guanine, guanosine and 2'-deoxyguanosine at concentrations up to 500 microM did not allow us to restore more than 50% of the cell proliferation. In conditions of normal purine de novo synthesis, the addition of 1000 microM adenine, adenosine, 2'-deoxyadenosine or 100 microM 2'-deoxyguanosine inhibited rat T-lymphocyte proliferation. The differences between the degree of inhibition of cell proliferation could be explained only in part by the differences between the capacities of salvage of these compounds. Furthermore, the fact that 2'-deoxyguanosine toxicity was dependent and 2'-deoxyadenosine toxicity independent on the activation state of the cells provided more evidence that the biochemical mechanisms of inhibition of cell proliferation should be different for these two nucleosides.

A kinetic study has been performed on the inhibition of the chicken erythrocyte AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) reaction by tetraiodofluorescein and Rose Bengal. These dyes inhibited the enzyme by decreasing its affinity for the substrate without affecting the maximum volocity. Kinetic analysis has shown the inhibition constants for tetraiodofluorescein and Rose Bengal to be 350 and 55 μM, respectively, and the presence of 4 binding sites of the enzyme for the inhibitors per enzyme molecule. These results suggest that the fluorescein dyes mimic the AMP binding at the catalytic center for the enzyme, which can be formed by the “dinucleotide fold”.

To evaluate the regulation of adenine nucleotide metabolism in relation to purine enzyme activities in rat liver, human erythrocytes and cultured human skin fibroblasts, rapid and sensitive assays for the purine enzymes, adenosine deaminase (EC 2.5.4.4), adenosine kinase (EC 2.7.1.20), hyposanthine phosphoribosyltransferase (EC 2.4.28), adenine phosphoribosyltransferase (EC 2.4.2.7) and 5'-nucleotidase (EC 3.1.3.5) were standardized for these tissues. Adenosine deaminase was assayed by measuring the formation of product, inosine (plus traces of hypoxanthine), isolated chromatographically with 95% recovery of inosine. The other enzymes were assayed by isolating the labelled product or substrate nucleotides as lanthanum salts. Fibroblast enzymes were assayed using thin-layer chromatographic procedures because the high levels of 5'-nucleotidase present in this tissue interferred with the formation of LaCl3 salts. The lanthanum and the thin-layer chromatographic methods agreed within 10%. Liver cell sap had the highest activities of all purine enzymes except for 5'-nucleotidase and adenosine deaminase which were highest in fibroblasts. Erythrocytes had lowest activities of all except for hypoxanthine phosphoribosyltransferase which was intermediate between the liver and fibroblasts. Erhthrocytes were devoid of 5'-nucleotidase activity. Hepatic adenosine kinase activity was thought to control the rate of loss of adenine nucleotides in the tissue. Erythrocytes had excellent purine salvage capacity, but due to the relatively low activity of adenosine deaminase, deamination might be rate limiting in the formation of guanine nucleotides. Fibroblasts, with high levels of 5'-nucleotidase, have the potential to catabolize adenine nucleotides beyond the control od adenosine kinase. The purine salvage capacity in the three tissues was erythrocyte greater than liver greater than fibroblasts. Based on purine enzyme activities, erythrocytes offer a unique system to study adenine salvage; fibroblasts to study adenine degradation; and liver to study both salvage and degradation.

The problems of whether the kinetic and regulatory properties of AMP deaminase were modified by formation of a deaminase-myosin complex were investigated with an enzyme preparation from rat skeletal muscle. Results showed that AMP deaminase was activated by binding to myosin. Myosin-bound AMP deaminase showed a sigmoidal activity curve with respect to AMP concentration in the absence of ATP and ADP, but a hyperbolic curve in their presence. Addition of ATP and ADP doubled the V value, but did not affect the Km value. Myosin-bound AMP deaminase also gave a sigmoidal curve in the presence of alkali metal ions, whereas free AMP deaminase gave a hyperbolic curve. GTP abolished the activating effects of both myosin and ATP.

Evidence suggesting that AMP deaminase (EC 3.5.4.6) is responsible for the stimulation of threonine dehydratase (EC 4.2.1.16) activity in situ is presented using yeast cells which have been rendered permeable. The addition of polyamine, an activator of AMP deaminase, resulted in the increase in ammonia concentration, which can stimulate the activity of yeast threonine dehydratase. Polyamine may regulate the synthesis of isoleucine and valine, and of the intermediates of citric acid cycle through the activation of AMP deaminase-threonine dehydratase system as a ‘cascade system’ in yeast.

The kinetic and regulatory properties of purified rat heart AMP deaminase were investigated. In the presence of 100 mM KCl, the enzyme exhibited a slightly sigmoid-shaped plot of reaction rate, vs. substrate concentration, which shifted to a more hyperbolic form when ATP, ADP or GTP were added. ATP was the most potent activator of the enzyme, whereas GTP at low (less than 0.25 mM) concentrations increased the enzyme activity. The activation effect was negligible at higher concentrations of GTP. The calculated value of K 0.5 of approx. 3 mM for unactivated enzyme decreased to approx. 0.6 mM and 1.1 mM when 0.5 mM ATP or 1.5 mM ADP were present in the incubation mixture, respectively. The theoretical model (Monod J., Wyman, J. and Changeux, J.P. (1965) J. Mol. Biol. 12, 88–118) gave a partial explanation of these results.

In human, there are four AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) isozymes: E1, E2, M and L. Chromatographic, electrophoretic and immunological studies showed the existence of isozymes E1 and E2 in erythrocytes, isozyme M in muscle and isozyme L in liver and brain. The tissues such as heart, kidney and spleen contained isozymes E1, E2 and L. Isozymes E1, M and L were isolated as apparently homogeneous preparations. The three isozymes were all tetramers composed of identical subunits, but differing slightly in molecular weight; isozyme E1 showed a subunit molecular weight of 80,000, isozyme M 72,000 and isozyme L 68,000. They were immunologically different from one another. The antisera precipitated only the corresponding enzyme and did not precipitate any other isozyme. The three isozymes were also different in kinetic and regulatory properties. Isozyme E2 was very similar to isozyme E1 in immunological an kinetic properties, although isozyme E2 could be separated from isozyme E1 by phosphocellulose chromatography, and zonal electrophoresis.

The amino acid pool composition and its concentration ratios with respect to blood and plasma, as well as the activities of alanine, aspartate and branched chain amino acid transaminases, glutamine synthetase, adenylate deaminase and glutamate dehydrogenase have been studied in the interscapular brown adipose tissue of control, 12-h cold-exposed and 15-day cold-acclimated rats. Cold temperature affected the amino acid metabolism and pool composition more intensely after 15 days than after 12-h cold-exposure, even though the patterns of change were very similar in both groups. Cold temperatures induced a decrease in glutamine and an increase in glutamate concentration in the tissue. This probably increased the metabolism of branched chain amino acids and caused a decrease in adenylate deaminase activity. It also seemed to increase alanine utilization. We concluded that amino acid metabolism in brown adipose tissue is enhanced by cold temperature acclimation.

The effects of adenosine, N6-phenylisopropyl adenosine and 2',5'-dideoxyadenosine on lipolysis and cyclic AMP accumulation, in hamster adipocytes treated with cholera toxin, were studied. Cholera toxin caused an increase in lipolysis and cyclic AMP accumulation that was dependent upon the concentration of toxin and the length of time cells were exposed to the toxin. When N6-phenylisopropyl adenosine or 2',5'-dideoxyadenosine were present, the lipolytic and cyclic AMP responses to cholera toxin were inhibited. The adenosine analogues were equally effective inhibitors of lipolysis and cyclic AMP accumulation, when they were added 1 or 2 h after exposure to the toxin. Enzymatic removal of endogenously produced adenosine with adenosine deaminase potentiated both the lipolytic and cyclic AMP responses to cholera toxin. In addition, the inhibitory effects of N6-phenylisopropyl adenosine, 2'5'-dideoxyadenosine and clonidine on lipolysis and cyclic AMP were enhanced consequent to enzymatic removal of adenosine. These data show responses of intact fat cells to N6-phenylisopropyl adenosine, 2',5'-dideoxyadenosine or removal of endogenous adenosine and provide evidence for an adenosine sensitivity of fat cells exposed to cholera toxin.

The deamination of AMP by AMP aminohydrolase (EC 3.5.4.6) serves as the major source of ammonia production in skeletal muscle. It has been suggested that the ammonia may serve either in a buffering capacity to combat acidosis due to the accumulation of lactic acid produced during prolonged muscular activity, or as a substrate for glutamine formation which can ultimately be utilized by the kidney in adapting to metabolic acidosis. In view of this proposal, the properties of the enzyme obtained from skeletal muscle of acidotic rats have been compared with the enzyme from normal muscle. The specific activity of AMP deaminase in crude homogenates of acidotic muscle was not significantly different from normal levels. The enzyme from acidotic muscle was purified to homogeneity and was found to be identical to the enzyme obtained from normal muscle by the criteria of electrophoretic mobility, pH optimum, molecular weight, sedimentation coefficient, subunit composition, amino acid composition, monovalent cation requirement, substrate saturation, and inhibition by ATP, P i and creatine- P . Thus, if the enzyme functions to prevent acidosis, the ability to respond to changes in the intracellular environment which accompany acidosis must be built into the structure of the enzyme normally found in skeletal muscle. Three lines of evidence strongly support this viewpoint: (a) the rate of deamination is approximately 2-fold higher at pH 6.5 than at pH 7.0, (b) the activity increases linearly with a decrease in the adenylate energy charge, and (c) within the normal physiological range of the adenylate energy charge, the enzyme is operating at only 10–20% of its maximum capacity.

Three AMP deaminase isozymes (EC 3.5.4.6 AMP aminohydrolase) were purified from rat heart, kidney and muscle. These enzyme preparations contained only the required isozyme. Antisera to individual isozymes were prepared and immunological relationships were tested. There was no cross-reactivity as tested by precipitation experiments. The antisera precipitated only the corresponding isozyme and there was no effect on other isozymes. These isozymes were also different in Km values for AMP and in substrate specificity. From the present studies, combined with previous results, it seems clear that the heart, kidney and muscle enzymes are different basic types. It is proposed that the muscle enzyme be designated as AMP deaminase A; the enzyme in kidney and liver, AMP deaminase B; the enzyme in heart, AMP deaminase C. Brain extracts contained five isozymes; two parent isozymes (B and C) and presumably their three hybrids.

Primary rat cardiomyocyte cultures were utilized as a model for the study of purine nucleotide metabolism in the heart muscle, especially in connection with the mechanisms operating for the conservation of adenine nucleotides. The cultures exhibited capacity to produce purine nucleotides from nonpurine molecules (de novo synthesis), as well as from preformed purines (salvage synthesis). The conversion of adenosine to AMP, catalyzed by adenosine kinase, appears to be the most important physiological salvage pathway of adenine nucleotide synthesis in the cardiomyocytes. The study of the metabolic fate of IMP formed from [14C]formate or [14C]hypoxanthine and that of AMP formed from [14C]adenine or [14C]adenosine revealed that in the cardiomyocyte the main flow in the nucleotide interconversion pathways is from IMP to AMP, whereas the flux from AMP to IMP appeared to be markedly slower. Following synthesis from labeled precursors by either de novo or salvage pathways, most of the radioactivity in purine nucleotides accumulated in adenine nucleotides, and only a small proportion of it resided in IMP. The results suggest that the main pathway of AMP degradation in the cardiomyocyte proceeds through adenosine rather than through IMP. About 90% of the total radioactivity in purines effluxed from the cells during de novo synthesis from [14C]formate or following prelabeling of adenine nucleotides with [14C]adenine were found to reside in hypoxanthine. The activities in cell extracts of AMP 5′-nucleotidase and IMP 5′-nucleotidase, which catalyze nucleotide degradation, and of AMP deaminase, a key enzyme in the purine nucleotide cycle, were low. The nucleotidase activity resembles, and that of the AMP deaminase contrasts the respective enzyme activities in extracts of cultured skeletal-muscle myotubes. The results indicate that in the cardiomyocyte, in contrast to the myotube, the main mechanism operating for conservation of nucleotides is prompt phosphorylation of AMP, rather than operation of the purine nucleotide cycle. The primary cardiomyocyte cultures are a plausible model for the study of purine nucleotide metabolism in the heart muscle.

The release and metabolism of adenosine was examined using rat fat cells in which the nucleotide pool had been labeled by incubation with radioactive adenine. The accumulation of adenosine in the medium was near maximal at the start of the incubation and increased only slightly thereafter. Adenosine was rapidly deaminated to inosine and subsequently oxidized to uric acid. In the presence of allopurinol, an inhibitor of xanthine dehydrogenase, hypoxanthine accumulated in the medium as the end-product of adenosine catabolism. Adenosine accumulated in the medium only if fat cells were incubated in the presence of erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of adenosine deaminase. Even in the presence of this inhibitor there was no acceleration of adenosine release by norepinephrine in the presence of theophylline. However, there was an increase in labeled intracellular AMP accumulation by norepinephrine plus theophylline. The increase in labeled AMP correlated with the final free fatty acid to albumin ratio suggesting that the rise in AMP was related to an accumulation of intracellular free fatty acids. The addition of sodium oleate to the medium mimicked the effect of norepinephrine plus theophylline on the accumulation of labeled AMP. These results indicate that AMP rather than adenosine accumulates in isolated fat cells during incubation with lipolytic agents.

The effect of polyamines and divalent cations including alkaline earth metals and transition metals on the AMP deaminase (AMP aminohydrolase EC 3.5.4.6) purified from baker's yeast was investigated. (1) Polyamines and alkaline earth metals activated the enzyme in the absence of ATP: these cations largely enhanced the maximal velocity without alteration of S0.5 and nH (Hill coefficient) values. However, transition metals acted as potent inhibitors, which decreased the maximal velocity of the enzyme in the absence of ATP. (2) All of the divalent cations showed an activation of the enzyme in the presence of ATP, followed by a progressive decrease in activity as the concentrations of transition metals increased. (3) The increase in the concentrations of polyamines or alkaline earth metals showed no more activating effect when the enzyme was fully activated by the addition of excess alkali metals in the absence of ATP, but divalent cation-activation was observed in the presence of ATP even if alkali metals were saturating. These results suggest the presence of two types of binding sites for cations: 1, the sites for free cations and 2, those for ATP-metal complexes. The former sites include the activating sites for alkali metals, polyamines and free alkaline earth metals, and the inhibitory sites for free transition metals. The latter sites are the activating sites for ATP-metal complexes, which are suggested to be commonly occupied by alkaline earth metals and transition metals and to form an ATP bridge (E-ATP-M) complex.

The effect of various fatty acids on the purified and in situ AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) was investigated: both the purified AMP deaminase and the permeabilized system of yeast cells were used as the enzyme sources. (1) All the saturated fatty acids, longer than 10 in the hydrocarbon chain, were inhibitors of the purified enzyme in the absence of ATP, whereas no or little inhibition of the enzyme was observed in the presence of ATP. Unsaturated fatty acids acted as more potent inhibitors of the purified enzyme, although the addition of ATP increased the I0.5 values for these fatty acids. Fatty acids acted as non-competitive inhibitors without alteration of the affinity for the substrate in the absence and presence of ATP. (2) Unsaturated fatty acids showed a powerful inhibition of the in situ AMP deaminase, and the presence of ATP could scarcely affect the inhibition of the in situ enzyme by these fatty acids. On the other hand, no or little inhibition of the in situ enzyme by saturated fatty acids was observed in the absence and presence of ATP. The difference in the kinetics properties between the in situ and the purified enzyme suggests that there is difference in protein interactions for AMP deaminase in situ and in vitro.

In view of its vasodilatory effect on the coronary circulation (probably mediated by adenosine) and its metabolic compartmentalization (intramitochondrial activation to form acetyl-CoA), the metabolic effects of acetate were studied in isolated rat heart mitochondria. Acetate caused conversion of adenylates to AMP and the formation of adenosine. Adenylate efflux was inhibited by carboxyatractyloside but not by N-ethylmaleimide. The intramitochondrial accumulation of AMP was enhanced by carboxyatractyloside during acetate metabolism and the formation of extramitochondrial adenosine inhibited. A carboxyatractyloside-sensitive unidirectional AMP influx with a Km of 50 microM and Vmax of 11 nmol/min per mg mitochondrial protein was also observed. The mitochondrial adenosine content was high and constant during the experiments. The steep apparent concentration gradient of adenosine indicates that most of the mitochondrial adenosine is tightly bound to protein. Adenosine formation was proportional to the extramitochondrial AMP concentration, showing that the 5'-nucleotidase activity of cardiac mitochondrial preparations is extramitochondrial in origin. The data suggest that the mitochondrial ATP/ADP carrier is capable of transporting AMP and that intramitochondrial AMP is recycled during acetate metabolism in the myocardium partially by means of the ATP/ADP translocator, leading to an increase in extramitochondrial AMP and adenosine formation.

Enzyme activities of purine catabolism and salvage, the concentrations of high-energy phosphates adn the reutilisation of purine bases and purine nucleosides were studied in rat heart myoblasts and myocytes. Rat heart myoblasts H9c2(2-1) were grown in Dulbecco's modified Eagle's minimum essential medium supplemented with 10% fetal calf serum. Reduction of fetal calf serum to 2% for 1 week resulted in a differentiation into myocytes with respect to their morphological features and their enzyme pattern. In differentiated myocytes, activity of 5′-nucleotidase was increased more than 2-fold, and AMP deaminase and creatine kinase activities were more than 10-fold elevated. The concentration of creatine phosphate in differentiated myocytes was doubled compared to that in myoblasts. The uptake into myoblasts and myocytes and the incorporation into adenine nucleotides was highest using adenosine, inosine and adenine uptake rates were intermediate, and hypoxanthine was utilised least. Differentiation of myoblasts into myocytes resulted in a slightly lower overall uptake of adenosine and adenine, whereas about 40% more inosine and hypoxanthine were utilised by myocytes. Increasing the phosphate concentration in the incubation medium up to 50 mmol/1 resulted in a stimulation of uptake of all purine compounds tested. This stimulation was more pronounced in myoblasts.

The variations of kinetic parameters with pH of the activity of 14-day-old chicken embryo and adult hen skeletal muscle AMP-deaminase in the presence and in the absence of adenine nucleotide effectors have been examined. The results obtained indicate that the kinetic and regulatory properties of the two developmental forms of AMP-deaminase are different.

Limited proteolysis of rabbit skeletal muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) with trypsin results in conversion of the enzyme to a form which is no longer inhibited by ATP and exhibits hyperbolic kinetics even at low K+ concentration and in the absence of ADP. The interaction with troponin T from white skeletal muscle or with the phosphorylated 42-residue N-terminal peptide of troponin T restores in the trypsin-treated AMP deaminase the sensitivity to adenine nucleotides and increases the KA for K+ activation of the enzyme from 1 mM to 12 mM, this effect being diametrically opposite to that exerted by limited proteolysis on the native enzyme. Treatment of the N-terminal peptide of troponin T with alkaline phosphatase abolishes the modulating properties of the peptide, suggesting that phosphorylation-dephosphorylation processes may be involved in the regulation of the enzyme.

AMP deaminase was completely solubilized from rat skeletal muscle with 50 mM Tris-HCl buffer (pH 7.0) containing KCl at a concentration of 0.3 M or more. The purified enzyme was found to be bound to rat muscle myosin or actomyosin, but not to F-actin at KCl concentrations of less than 0.3 M. Kinetic analysis indicated that 1 mol of AMP deaminase was bound to 3 mol of myosin and that the dissociation constant (Kd) of this binding was 0.06 micrometer. It was also shown that AMP deaminase from muscle interacted mainly with the light meromyosin portion of the myosin molecule. This finding differs from that of Ashby and coworkers on rabbit muscle AMP deaminase, probably due to a difference in the properties of rat and rabbit muscle AMP deaminase. AMP deaminase isozymes from rat liver, kidney and cardiac muscle did not interact with rat muscle myosin. The physiological significance of this binding of AMP deaminase to myosin is discussed.

The differential effects of polyamines on the activity of AMP deaminase isozyme A (from rat muscle) and isozyme B (from rat liver) are reported. Polyamines activate isozyme B but inhibit isozyme A.

1. Rat skeletal muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) at optimal KCl concentrations shows a biphasic response to increasing levels of the allosteric inhibitor ATP. 2. Up to 10 micrometer, ATP appears to convert the enzyme to a form exhibiting sigmoidal kinetics while at higher concentrations its inhibitory effect is manifested by an alteration of AMP binding to AMP deaminase indicative of negative homotropic cooperativity at about 50% saturation. 3. AMP deaminase is inactivated by incubation with the periodate oxidation product of ATP. The (oxidized ATP)--AMP deaminase complex stabilized by NaBH4 reduction shows kinetic properties similar to those of the native enzyme in the presence of high ATP concentrations. 4. A plausible explanation of the observed cooperativity is that ATP induces different conformational state of AMP deaminase subunits, causing the substrate to follow a sequential mechanism of binding to enzyme. 5. Binding of the radioactive oxidized ATP shows that 3.2 mol of this reagent bind per mol AMP deaminase.

Purified rat muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) binds tightly to rat myosin. The binding is abolished in the presence of low concentrations of various ligands. Pyrophosphate and GTP at concentrations as low as 0.1 micrometer were effective in abolishing the interaction between two proteins. Other nucleoside triphosphates were less effective than GTP and the concentrations required for 50% inhibition were approximately 0.3 to 0.7 micrometer. ADP and AMP are effective in inhibiting the interaction between two proteins, but they are less effective than the nucleoside triphosphates; 50% inhibition occurred at 34 micrometer with ADP and at 1 mM with AMP. Creatine phosphate and inorganic phosphate showed 50% inhibition at 5 to 6 mM. All of the compounds, which affected AMP deaminase activity, were effective in abolishing the interaction of the enzyme with myosin; however, the interaction-abolishing effects of the compounds are not parallel with their inhibitory effects on the deaminase activity. Although there exist three parental isozymes of AMP deaminase in the rat, all three enzymes interacted with myosin.

The effect of adenosine on the mouse thymocyte adenylate cyclase-adenosine 3′:5′-monophosphate (cyclic AMP) system was examined. Adenosine, like prostaglandin E1, can cause 5-fold or greater increases in thymocyte cyclic AMP content in the presence but not in the absence of certain cyclic phosphodiesterase inhibitors. Two non-methylxanthine inhibitors potentiated the prostaglandin E1 and adenosine responses, while methylxanthines selectively inhibited the adenosine response. Adenosine increased cyclic AMP content significantly wihtin 1 min and was maximal by 10 to 20 min with approx. 2 and 10 μM adenosine being minimal and half-maximal effective doses, respectively. Combinations of prostaglandin E1, isoproterenol and adenosine were near additive and not synergistic. Of the adenosine analogues tested, only 2-chloro- and 2-fluoroadenosine significantly increased cyclic AMP. Thymocytes prelabeled with [14C] adenine exhibited dramatic increases in cyclic [14C]AMP 10 min after addition of adenosine or prostaglandin E1 which corresponded to simultaneously determined increases in total cyclic AMP. Using [14C]adenosine, the percent of total cyclic AMP increase due to adenosine was only 16%. Adenosine was also shown to elicit a 40% increase in particulate thymocyte adenylate cyclase activity. Therefore, the increased content of cyclic AMP seen in mouse thymocytes after incubation with adenosine was due primarily to stimulation of adenylate cyclase and only partially to conversion of adenosine to cyclic AMP. The increased cellular content of cyclic AMP may be, in part, responsible for various immunosuppressive effects of adenosine.

The effects of fructose on purine nucleotide synthesis and degradation were studied in isolated rat hepatocytes. Incubation of the hepatocytes with fructose resulted in dece;eration of the rate of de novo purine synthesis, gauged by the rate of incorporation of precursor [14C]formate into total purines produced, and in acceleration of purine nucleotide degradation, as measured by the rate of conversion of prelabelled purine nucleotides into end-product allantoin. These effects were found to be associated with decreases in cellular content of ATP and Pi and in the metabolic availability of 5-phosphoribosyl 1-pyrophosphate. The results support the suggestion that the fructose-induced acceleration of purine degradation is mediated through activation of AMP deaminase. However, the results also suggest that decreased reutilization of hypoxanthine for IMP synthesis, due to the decreased PP-Rib-P availability, is an additional mechanism for the acceleration of purine degradation. The decreased PP-Rib-P availability is also suggested to be the main mechanism for the fructose-induced deceleration of purine synthesis.

The specific activities of nucleoside deaminase (cytidine aminohydrolase, EC 3.5.4.5) and adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) were determined in extracts of mouse liver following 70 % hepatectomy. There was an increase in the levels of these enzymes which preceded the onset of DNA synthesis by many hours. Enzyme levels remained elevated during the period of rapid DNA synthesis and for more than a week after the cessation of DNA synthesis. It is suggested that the increases in nucleoside deaminase and adenosine deaminase may be related to altered RNA metabolism in regenerating liver.

1. 1.AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) activities of dialyzed and undialyzed hemolyzates of human erythrocytes in the presence of optimal concentrations of K+ were measured. Whereas the activity of the dialyzed hemolyzate was a linear function of the hemolyzate concentration, that of the undialyzed hemolyzate was not. These results suggested the presence of a dialyzable inhibitor of the enzyme in the hemolyzate. 2. 2.Effects of several organic phosphates of erythrocytes on AMP deaminase were studied, 2,3-Diphosphoglyceric acid, at concentrations usually found in erythrocytes, was found to be an inhibitor of the enzyme. 3. 3.The substrate-velocity curve of AMP deaminase is a sigmoid. In the presence of 2,3-diphosphoglyceric acid the curve became more sigmoidal in shape. The effects of varying concentrations of 2,3 diphosphoglyceric acid on AMP deaminase in the presence and absence of ATP were also determined. In the presence of ATP, when the per cent inhibition of velocity was plotted against the concentration of 2,3-diphosphoglyceric acid a sigmoidal curve was obtained. 4. 4.The data suggest that 2,3-diphosphoglyceric acid may control the adenine nucleotide content of the intact erythrocyte through an allosteric regulation of the AMP deaminase activity.

1. RNA content of control breast muscle increased between 19 days en ovo and 1 week after hatching and then declined. In dystrophic muscle RNA content did not decrease until 2 weeks after hatching and at all ages from 2 weeks to 3–5 months was greater than that of corresponding controls. The dystrophic condition did not affect the RNA or DNA content of other tissues investigated. 2. AMP deaminase (adenylate aminohydrolase, EC 3.5.4.6) activity of control breast muscle increased 4-fold between 19 days en ovo and 1–2 months after hatching. In the dystrophic group there was a delayed, temporary increase in enzyme activity at one month and values were always less than those of corresponding controls from 1 week old and older chickens. 3. Inosine phosphorylase (purine nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) activity of control breast muscle decreased after the first week ex ovo, while in dystrophic muscle activity remained at essentially embryonic levels. Enzyme activity in pancreas and liver increased during development and was unaffected by the dystrophic condition. 4. Xanthine dehydrogenase activity in liver and pancreas from control chickens increased during development. In pancreas from the dystrophic group enzyme activity did not increase appreciably and was significantly less than that of adult (3–5 months) controls. The dystrophic condition did not affect the development of enzyme activity in liver or activity in adult kidney. 5. Hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) activity decreased in control muscle after hatching while remaining at essentially embryonic levels in breast muscle from the dystrophic group. 6. Incorporation in vivo of [8-14C]hypoxanthine into breast muscle RNA was greater in 1–6-week-old dystrophic chickens than in comparable controls.

A specific pterin deaminase has been detected in rat liver. The enzyme was purified 23-fold. During its purification, guanine deaminase (EC 3.5.4.3) activity of the protein was increased 39-fold. Evidence is given which establishes the nonidentity of pterin deaminase and guanine deaminase: changes in the ratio of their activities during preparation; differences in stability during storage and inhibition by CN− and by azaguanine and guanine; differences in the pH maxima and in the distribution of enzyme activities in various organisms. The reaction maximum is at pH 6.5 and Km is 30 μM. The enzyme specifically deaminates pterin, isoxanthopterin and tetrahydropterin. Xanthopterin, biopterin, neopterin, pterin-6-carboxylic acid, and 6-hydroxymethylpterin are not deaminated. In the honeybee (Apis mellifica), pterin deaminase activity has also been detected. The highest activity has been found in 4-day-old larvae.

1. 1. The metabolism of [8-14C]adenosine was examined in human erythrocytes and erythrocyte lysates. 2. 2. The metabolism of [8-14C]adenosine in erythrocytes is largely dependent upon its concentration. 3. 3. Adenosine deaminase and adenosine kinase activities were assayed in crude erythrocyte lysates. No adenosine phosphorylase activity was detected. 4. 4. Adenosine deaminase exhibited a Km for adenosine of 4 · 10−5 M. Substrate inhibition of the enzyme was exhibited at concentrations of adenosine greater than 2 · 10−4 M. No inhibition of deaminase activity by equimolar or greater concentration of AMP, ADP, ATP, GDP, GTP, Pi, guanylic acid or 2,3-diphosphoglycerate was noted, but 20 % inhibition by high concentrations of adenine and inosine was observed. 5. 5. Adenosine kinase required ATP (Km = 4 · 10−4 M), adenosine (Km = 1.9 · 10−6 M), and Mg2+ for activity. No substrate inhibition by adenosine occurred at concentrations 20 times its Km. Adenosine 5′-triphosphate and Mg2+ were inhibitory at concentrations greater than 1.25 mM and 0.50 mM, respectively. The reaction of ATP with adenosine kinase was competitively inhibited by AMP, ADP, guanylic acid, GDP, IMP, and adenine. Adenosine 5′-triphosphate was variably replaceable as the phosphate donor by a wide range of triphosphates.

1. 1. The ATP analog 5′-adenylyl methylenediphosphonate (AMP-PCP) added to the serosal bathing solution of the chambered bullfrog gastric mucosa inhibits acid secretion, raises the transepithelial potential difference, and causes an increase in lag time before resumption of secretion after anoxia. These effects are similar in direction and magnitude to those caused by ATP. 2. 2. While AMP-PCP stimulates the production of inosine in this tissue, it is apparently not itself a precursor for inosine, and seems to act by stimulating catabolism of endogenous purines. 3. 3. AMP-PCP is not a substrate for adenosine deaminase or AMP deaminase, has no marked effects on mitochondrial respiration or phosphorylation, and is not catabolised by gastric mucosa. 4. 4. Washout kinetics of AMP-PCP loaded tissues show this material to be exiting with a single exponential time constant of about 10 min. By contrast, inosine shows 2 washout exponentials of 5 and 30 min. The appropriate analysis suggests that the cells may be more permeable to AMP-PCP than to inosine. 5. 5. It is suggested that the inhibitory action of AMP-PCP (and by extension, ATP and ADP) is due to the possession by these compounds of a nitrogen with an unshared pair of electrons, as has been suggested for other secretory inhibitors.