Soluble purine-converting enzymes circulate in human blood and regulate extracellular ATP level via counteracting pyrophosphatase and phosphotransfer reactions.
Extracellular ATP and other purines play a crucial role in the vasculature, and their turnover is selectively governed by a network of ectoenzymes expressed both on endothelial and hematopoietic cells. By studying the whole pattern of purine metabolism in human serum, we revealed the existence of soluble enzymes capable of both inactivating and transphosphorylating circulating purines. Evidence for this was obtained by using independent assays, including chromatographic analyses with 3H-labeled and unlabeled nucleotides and adenosine, direct transfer of gamma-terminal phosphate from [gamma-32P]ATP to NDP/AMP, and bioluminescent measurement of ATP metabolism. Based on substrate-specificity and competitive studies, we identified three purine-inactivating enzymes in human serum, nucleotide pyrophosphatase (EC 3.6.1.9), 5'-nucleotidase (EC 3.1.3.5), and adenosine deaminase (EC 3.5.4.4), whereas an opposite ATP-generating pathway is represented by adenylate kinase (EC 2.7.4.3) and NDP kinase (EC 2.7.4.6). Comparative kinetic analysis revealed that the Vmax values for soluble nucleotide kinases significantly exceed those of counteracting nucleotidases, whereas the apparent Km values for serum enzymes were fairly comparable and varied within a range of 40-70 micro mol/l. Identification of soluble enzymes contributing, along with membrane-bound ectoenzymes, to the active cycling between circulating ATP and other purines provides a novel insight into the regulatory mechanisms of purine homeostasis in the blood.
Yegutkin
Gennady G
GG
MediCity Research Laboratory and Department of Medical Microbiology, Turku University and National Public Health Institute, FIN-20520 Turku, Finland. gennady.yegutkin@utu.fi
Samburski
Sergei S
SS
Jalkanen
Sirpa
S
D000243:Adenosine Deaminase / Q000097:blood / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000263:Adenylate Kinase / Q000097:blood / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D006801:Humans; D008163:Luminescent Measurements; D008956:Models, Chemical; D009701:Nucleoside-Diphosphate Kinase / Q000097:blood / Q000378:metabolism; D009711:Nucleotides / Q000097:blood / Q000378:metabolism; D010766:Phosphorylation; D010770:Phosphotransferases / Q000097:blood / Q000378:metabolism; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000097:blood* / Q000378:metabolism; D011687:Purines / Q000097:blood* / Q000378:metabolism*; D011755:Pyrophosphatases / Q000097:blood* / Q000378:metabolism
D009711:Nucleotides; D011687:Purines; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D017856:Phosphotransferases (Phosphate Group Acceptor); D000263:Adenylate Kinase; D009701:Nucleoside-Diphosphate Kinase; D000243:Adenosine Deaminase; D011755:Pyrophosphatases; C021899:nucleotide pyrophosphatase