The hydrolytic activity of bovine adrenal medullary plasma membranes towards diadenosine polyphosphates is due to alkaline phosphodiesterase-I

A hydrolase activity directed against diadenosine 5',5"'-P-1,P-4-tetraphosphate (Ap(4)A) has been solubilised and partially purified from the plasma membrane fraction of bovine adrenal medullary chromaffin tissue in order to determine its relationship to alkaline phosphodiesterase-I/nucleotide pyrophosphatase (PDase-I, EC 3.1.4.1). Activity with the specific dinucleoside tetraphosphatase (EC 3.6.1.17) substrate Ap(4)A and with the non-specific PDase-I substrate thymidine 5'-monophosphate p-nitrophenyl ester had K-m and V-max values of 2.0 mu M and 600 pmol/min/mg protein and 0.2 mM and 26 nmol/min/mg protein respectively and co-chromatographed upon gel filtration and ion-exchange chromatography. Activity with the fluorescent substrates etheno-Ap(4)A and 4-methylumbelliferyl phenylphosphonate co-electrophoresed on native polyacrylamide gels. No activity was detected which exclusively hydrolysed Ap(4)A. Immunoblotting of the most purified fraction with an antibody against mouse PC-1, one of the major PDase-I family members, detected bands of 240, 120 and 62 kDa corresponding to PC-1 dimer, monomer and proteolytic fragment. Therefore, the activity previously described as bovine adrenal chromaffin cell ecto(diadenosine polyphosphate hydrolase) (ecto-Ap(n)Aase) is a PDase-I, probably bovine PC-1. (C) 1998 Elsevier Science B.V. All rights reserved.