Identification of two distinct E-NTPDases in liver of goldfish

K.E. ALLEVA, M.V. ESPELT, G. KRUMSCHNABEL, P.J. SCHWARZBAUM

COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY. PART B, BIOCHEMISTRY & MOLECULAR BIOLOGY.

ELSEVIER SCIENCE INC

Año: 2002 p. 725 - 731

1096-4959

We have recently reported the existence of ATPase activity capable of hydrolyzing extracellular ATP and localized at the external cell membrane of goldfish hepatocytes wAm. J. Physiol. (1998) 274 R1031x. In the present study, we investigated whether one or more enzymes of the ATP diphosphohydrolase family (called E-NTPDases) are responsable for the hydrolysis of extracellular ATP and other nucleotides. Using soluble extracts from goldfish liver, enzyme activity was detected in the presence of ATP (32.1"4.0 nmol Pi liberated mg proteiny1 miny1), ADP (20.7"3.3 nmol Pi liberated mg proteiny1 miny1) and UTP (20.7"1.2 nmol Pi liberated mg proteiny1 miny1). In line with the presence of this hydrolytic activity, liver samples separated by non-denaturing gel electrophoresis and subsequently exposed to either ATP, ADP or UTP yielded a single band with enzyme activity and similar electrophoretic mobility. Subsequent SDS-PAGE electrophoresis of the active bands resulted in the appearance of two protein bands with molecular masses of 70 and 64 kDa. Inmunoblotting of soluble extracts and microsomes obtained from goldfish liver, using a monoclonal antibody against CD39 (a well-known E-NTPDase), detected a single 97-kDa protein. The enzyme activity measured in solution and in native gels, together with structural information from denaturing gels plus immunoblots, points to the existence, in goldfish liver, of at least two different E-NTPDases.