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Soluble purine-converting enzymes circulate in human blood and regulate extracellular ATP level via counteracting pyrophosphatase and phosphotransfer reactions¹

MediCity Research Laboratory and Department of Medical Microbiology, Turku University and National Public Health Institute, FIN-20520 Turku, Finland

²Correspondence: MediCity, Tykistkatu 6A, FIN-20520 Turku, Finland. E-mail: gennady.yegutkin{at}utu.fi

SPECIFIC AIMS

Extracellular purines mediate diverse proinflammatory (ATP, ADP) and anti-inflammatory (adenosine) events in the vasculature and their turnover are thought to be selectively governed by network of ectoenzymes expressed on endothelial and hematopoietic cells. In contrast to traditional paradigms that focus on cell-associated mechanisms of extracellular purine metabolism, the existence of soluble enzymes in the bloodstream has received little attention, and these studies aimed to evaluate the whole pattern of purine metabolism in human serum.

PRINCIPAL FINDINGS

1. TLC analysis of the metabolism of ³H-labeled purines in human serum
Incubation of human serum with [³H]ATP was accompanied by its progressive decay with concomitant accumulation of [³H]adenosine in the assay medium and there was a little formation of [³H]AMP as an intermediate metabolite. [³H]ATP hydrolysis by serum remained insensitive to inhibitors of ion-exchanging (ouabain) and intracellular (sodium azide, ortho-vanadate) ATPases and was partially inhibited by trypanocidal agent suramin. Serum was also capable of hydrolyzing [³H]AMP, and this nucleotidase reaction can be prevented with specific ecto-5'-nucleotidase inhibitor ,ß-methylene-ADP (APCP) but not with suramin. Serum incubation with [³H]adenosine as initial substrate caused its deamination; this catalytic reaction was inhibited by 95% in the presence of adenosine deaminase inhibitor EHNA.

To test the alternative possibility of backward ATP resynthesis in cell-free serum, we determined the conversion of [³H]AMP into high-energy ³H-phosphoryls. Unlabeled NTP activated [³H]AMP phosphorylation with the following rank order of -phosphate-donating potency: ATP >> UTP > ITP ≥ GTP. Use of [³H]ADP as another phosphate acceptor revealed even more efficient [³H]ADP conversion into [³H]ATP and this phosphotransfer was equally activated by ATP and other NTP. Comparative kinetic analysis revealed that V_max values for nucleotide kinases significantly exceed those of counteracting nucleotidases, whereas the apparent K_m values for most of the serum enzymes were fairly comparable and varied within 40–70 µmol/L range.

2. HPLC and bioluminescent analyses of nucleotide metabolism in human serum
Nucleotide-converting activities were also evaluated by using HPLC. The major peak corresponding to hypoxanthine was detected in the case of control untreated serum (Fig. 1 A). Addition of exogenous ADP to the serum caused its simultaneous conversion into AMP and ATP presumably via reverse adenylate kinase reaction (Fig. 1B ), whereas GTP alone did not induce significant shifts in serum purine metabolism (Fig. 1C ). Combined serum treatment with ADP plus GTP dramatically activated NDP kinase-mediated phosphotransfer reactions through the following scheme: ADP + GTP ATP + GDP (Fig. 1D ). Phosphotransfer reactions were independently confirmed by using a luciferin-luciferase assay where pronounced ATP synthesis was observed under combined serum exposure to ADP and various NTP. The concentration of endogenous ATP in the serum samples was <10^-8 mol/L; HPLC analysis did not reveal any traces of other NTP/NDP (detection limit 5-10 pmol), allowing us to exclude even slight serum hemolysis during blood sampling.

View larger version (22K): [in this window] [in a new window]   Figure 1. HPLC analysis of nucleotide metabolism in human serum. Serum (20 µL) was incubated for 30 min in the absence (A) and presence of ADP (B), GTP (C), and ADP plus GTP (D). Exogenous nucleotide concentrations were 50 µmol/L. Nucleotide substrates and their metabolites were separated by reverse-phase HPLC and indicated in the presented chromatograms. Hyp = hypoxanthine.

3. Metabolism and transphosphorylation of [-³²P]ATP
Serum was incubated with [-³²P]ATP and Fig. 2 illustrates the major interconversion pathways of this nucleotide. Human serum caused progressive hydrolysis of 1-50 µmol/L [-³²P]ATP with concomitant formation of ³²PP_i (lanes 2, 8, 9); this nucleotide pyrophosphatase (NPP) reaction can be prevented by the chelating agent EDTA (lane 10). Likewise, rabbit serum degraded [-³²P]ATP via NPP reactions (lane 13) whereas incubation of [-³²P]ATP with mouse serum caused concurrent appearance of ³²PP_i and ³²P_i (lane 12). The patterns of [-³²P]ATP degradation in human and rabbit serum show clear-cut differences from the E-type NTP-diphosphohydrolase (E-NTPDase) -mediated cleavage of ³²P_i from [-³²P]ATP found in the experiments with Jurkat T lymphocytes (lane 11). In addition to unambiguous identification of soluble NPP as the major ATP-degrading enzyme in human serum, use of [-³²P]ATP allowed us to trace direct transfers of the -terminal ³²P-phosphate during soluble phosphotransfer reactions. Addition of [-³²P]ATP together with UDP, GDP, or ADP to the human serum activated NDP kinase reaction with the formation of corresponding [-³²P]NTP (lanes 3-5). Serum incubation with [-³²P]ATP plus AMP caused their transphosphorylation into [ß-³²P]ADP (lane 6); this reaction can be prevented by specific adenylate kinase inhibitor Ap₅A (lane 7).

View larger version (69K): [in this window] [in a new window]   Figure 2. TLC analysis of the transfer of -phosphate from [-32P]ATP. Human serum (HS; 10 µL) was incubated for 30 min with 1 µmol/L ATP plus tracer [-32P]ATP in the presence of the indicated concentration of certain nucleotide as second substrate. Serum was pretreated with EDTA (2 mmol/L) and Ap5A (30 µmol/L) for 10 min prior to addition of substrates. [-32P]ATP was also incubated with 10 µL of mouse (MS) or rabbit serum (RS) as well as 5 x 104 Jurkat T-cells (Jur). Arrows indicate the positions of inorganic phosphorus (Pi), pyrophosphate (PPi), and nucleotide standards. The first lane is a blank showing the radiochemical purity of [-32P]ATP after a 30 min incubation in the absence of serum.

CONCLUSIONS AND SIGNIFICANCE

Using radio-TLC and HPLC assays, we evaluated the whole pattern of purine metabolism in human serum and further identified the major soluble activities involved in these interconversion pathways. Elements of the purine-inactivating cascade comprise at least three enzymes displaying ATP-pyrophosphohydrolyzing, AMP-hydrolyzing, and adenosine deaminating activities whereas an opposite ATP-regenerating pathway is mediated via sequential adenylate kinase and NDP kinase reactions (Fig. 3 ).

View larger version (11K): [in this window] [in a new window]   Figure 3. A schematic presentation of purine metabolism in human serum. The proposed scheme shows the potential exchange activities of circulating nucleotides in cell-free human serum as well as their conversion into adenosine (Ado) and inosine/hypoxanthine (Ino/Hyp). The following soluble enzymic activities have been identified: nucleotide pyrophosphatase (NPP; EC 3.6.1.9); 5'-nucleotidase (5-NT; EC 3.1.3.5); adenosine deaminase (ADA; EC 3.5.4.4); adenylate kinase (AdK; EC 2.7.4.3); and NDP kinase (NDPK; EC 2.7.4.6).

Identification of a complex mixture of soluble enzymes provides a novel insight into the regulatory mechanisms of purine homeostasis within the vasculature. First, contrary to the endothelial and lymphoid E-NTPDases mediating stepwise ATP hydrolysis via ADP to AMP, serum NPP bypasses generation of a principal platelet-recruiting agent ADP via direct ATP conversion into AMP and PPi. Second, since PPi is one of the major physiological inhibitors of calcification preventing basic calcium phosphate (hydroxyapatite) crystal deposition in bone and cartilage, serum NPPs concurrently serve as powerful physiological inhibitors of mineralization in vivo. Third, soluble adenylate kinase may contribute to the clearance of ADP through its reverse transphosphorylation into ATP and AMP. Last, the existence of highly active soluble NDP kinases capable of indifferently transphosphorylating various nucleoside di- and triphosphates could be particularly pertinent for the maintenance of circulating ATP and other NTP/NDP at certain levels, in this way regulating the pattern of nucleotide receptor stimulation and/or desensitization.

Understanding the role of serum enzymes within the larger framework of purine-converting ectoenzymes is another important issue. Whereas endothelial ecto-nucleotidases are thought to be the major regulators of purinergic signaling in the vasculature, endothelial metabolism prevails only in the microcirculation where the area of endothelial surface exposed to unit volume of blood is very high. Furthermore, soluble nucleotidases can potentially offset the reduced scavenging effectiveness of membrane-bound ecto-nucleotidases under certain inflammatory conditions or traumatic shock where the integrity of a blood vessel wall is compromised. The recombinant soluble form of human NTPDase/CD39 was shown to be a potent inhibitor of thrombosis and tissue injury and is currently considered a promising aspirin-insensitive antithrombotic drug.

In conclusion, the results presented here demonstrate the existence of two opposite, ATP-generating and ATP-consuming, pathways in cell-free human serum. Identification of soluble enzymes contributing, along with membrane-bound ectoenzymes, to the active cycling between circulating ATP and other purines may have significant implications to our understanding of the regulation of the duration and magnitude of purinergic signaling in the blood. Our findings may open up further research to assess the potential diagnostic applications of purine-converting enzymes in clinical biochemistry.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1136fje; doi: 10.1096/fj.02-1136fje

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