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"'-P1, P4-tetraphosphate (Ap4A) 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 Ap4A and with the non-specific PDase-I substrate thymidine 5'-monophosphate p-nitrophenyl ester had Km and Vmax values of 2.0 microM 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-Ap4A and 4-methylumbelliferyl phenylphosphonate co-electrophoresed on native polyacrylamide gels. No activity was detected which exclusively hydrolysed Ap4A. 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-ApnAase) is a PDase-I, probably bovine PC-1.
Gasmi
L
L
Cellular Regulation and Signalling Group, School of Biological Sciences, Life Sciences Building, University of Liverpool, Crown St, Liverpool L69 7ZB, UK.
Cartwright
J L
JL
McLennan
A G
AG
D017766:Acid Anhydride Hydrolases / Q000302:isolation & purification / Q000378:metabolism; D000313:Adrenal Medulla / Q000201:enzymology / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D019439:Chromaffin Cells / Q000201:enzymology / Q000378:metabolism; D015226:Dinucleoside Phosphates / Q000378:metabolism*; D006868:Hydrolysis; D007120:Immunochemistry; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D013379:Substrate Specificity
D015226:Dinucleoside Phosphates; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I; D017766:Acid Anhydride Hydrolases; C020023:bis(5'-nucleosyl)tetraphosphatase (asymmetrical)