Diadenosine 5',5"'-P1,P4-tetraphosphate hydrolase is present in human erythrocytes, leukocytes and platelets.
An asymmetrically-cleaving diadenosine 5',5"'-P1,P4-tetraphosphate hydrolase (Ap4A-->ATP+AMP) is present in all higher eukaryotes and contributes to the regulation of the intracellular level of the alarmone nucleotide diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A). This enzyme has previously been isolated from unfractionated human blood cells. The aim of this report is to determine the contribution made by different blood cell types as part of our study of the roles of Ap4A as an intra- and extracellular signalling molecule. Ap4A hydrolase was partially purified from isolated human erythrocytes, leukocytes and platelets by high performance gel permeation chromatography and characterized by kinetic analysis and by probing immunoblots with an antibody raised against the human placental enzyme. Ap4A hydrolase was clearly present in all three cell types. Each enzyme comprised a single polypeptide of M(r) 19,200. The erythrocyte and platelet enzymes had a Km for Ap4A of 0.70 +/- 0.05 microM (n = 3) while the Km for the leukocyte enzyme was 1.50 +/- 0.20 microM (n = 3). All three enzymes showed substrate inhibition above 10 microM Ap4A. The specific activity of the enzyme in erythrocytes was 0.067 U/10(6) cells, 15-fold lower than that in leukocytes and platelets. However, the erythrocyte hydrolase accounted for 97% of the total activity of unfractionated blood cells (336 U out of 346 U/ml blood). The study shows that leukocytes, platelets and erythrocytes all contain Ap4A hydrolase activity. The last observation is of particular interest given the reported absence of Ap4A from enucleated erythrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
Hankin
S
S
Department of Biochemistry, University of Liverpool, U.K.
Matthew
N
N
Thorne
H
H
McLennan
A G
AG
D017766:Acid Anhydride Hydrolases / Q000032:analysis / Q000097:blood* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001792:Blood Platelets / Q000201:enzymology*; D002645:Chickens / Q000276:immunology; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004912:Erythrocytes / Q000201:enzymology*; D005260:Female; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D007962:Leukocytes / Q000201:enzymology*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000276:immunology; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy
D000906:Antibodies; D009842:Oligopeptides; D017766:Acid Anhydride Hydrolases; C020023:bis(5'-nucleosyl)tetraphosphatase (asymmetrical)