Biochemical and immunochemical characterisation of human diadenosine triphosphatase provides evidence for its identification with the tumour suppressor Fhit protein.
We describe here the purification and characterisation of the human enzyme diadenosine triphosphatase isolated from human platelets and leukocytes, offering biochemical and immunochemical evidence to identify this enzyme with the novel tumour suppressor Fhit protein, a homodimer composed of approximately 17 kDa monomers. It catalyses the Mg(2+)-dependent hydrolysis of diadenosine triphosphate, Ap(3)A, to AMP+ADP. The fluorogenic substrate di-ethenoadenosine triphosphate, epsilon-(Ap(3)A), and Fhit antibodies were used for enzymatic and immunochemical characterisations, respectively. Human Ap(3)Aase presents a native molecular mass of approximately 32 kDa and no significant differences were found in K(m) values (2 microM), activating effects by Mg(2+), Ca(2+), and Mn(2+), optimum pH (7.0-7.2) or inhibition by Zn(2+) and diethyl pyrocarbonate between the human enzyme and the recombinant Fhit protein. Suramin is a very potent competitive inhibitor of both human Ap(3)Aase and Fhit protein with K(i) values in the range 20-30 nM. Both human and rat Ap(3)Aase activity co-purifies with Fhit immunoreactivity under gel filtration, ion-exchange and affinity chromatography. Homogeneous human Ap(3)Aase preparations analysed by SDS-PAGE and Western blot analysis with Fhit antibodies elicit immunochemical responses corresponding to a approximately 17 kDa polypeptide, indicating a dimeric structure for the enzyme Ap(3)Aase. The strong inhibition of Fhit enzyme by the drug suramin, supports the need to investigate the therapeutic potential of Fhit-Ap(3)Aase mediated by its interaction with suramin or related drugs.
Asensio
Aaron C
AC
Departamento de Bioquímica y Biología Molecular, Universidad de La Laguna, 38206 La Laguna, Canary Islands, Spain.
Rodríguez-Ferrer
Carmen R
CR
Oaknin
Sol
S
Rotllán
Pedro
P
D017766:Acid Anhydride Hydrolases / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001792:Blood Platelets / Q000201:enzymology; D001921:Brain / Q000201:enzymology; D002384:Catalysis / Q000187:drug effects; D002850:Chromatography, Gel / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D015226:Dinucleoside Phosphates / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005470:Fluorometry; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D007962:Leukocytes / Q000201:enzymology; D008297:Male; D009363:Neoplasm Proteins / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013379:Substrate Specificity; D013498:Suramin / Q000494:pharmacology; D025521:Tumor Suppressor Proteins / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism*; D000099170:Bis(5'-adenosyl)triphosphatase
D017766:Acid Anhydride Hydrolases; D015226:Dinucleoside Phosphates; D009363:Neoplasm Proteins; D013498:Suramin; D025521:Tumor Suppressor Proteins; D000099170:Bis(5'-adenosyl)triphosphatase; C011440:diadenosine triphosphate