Photoaffinity labelling of the ATP-binding sites of two Ca 2+,Mg-ATPase isoforms in pancreatic endoplasmic reticulum

Abstract

Pancreatic rough ER ATP-binding proteins, including two isoforms of SERCA-2b Ca 2+,Mg-ATPase, were identified using specific photoaffinity labelling with 8-azido-ATP. 8-Azido-ATP irreversibly inhibited Ca 2+, Mg-ATPase activity only after UV irradiation and the inhibition was prevented by inclusion of 5 mM ATP in the labelling reaction. Rough ER proteins of apparent molecular masses 141, 111, 100, 84, 69, 55 and 47 kDa were detected following photoaffinity-labelling with 8-azido-[α- 32P]ATP. The two bands at 111 kDa and 100 kDa corresponded in molecular mass to the two SERCA-2b Ca 2+,Mg-ATPase isoforms previously demonstrated immunologically [1]. Immunoprecipitation of rough ER proteins by a SERCA-2b-specific antibody showed that the two ATPase bands were photoaffinity-labelled. Photoaffinity labelling of the 111 and 100 kDa proteins was: (a) abolished when Ca 2+,Mg-ATPase activity was inactivated by EDTA-treatment of rough ER membranes; (b) inhibited by the Ca 2+,Mg-ATPase inhibitor vanadate; (c) not affected by thapsigargin. The data demonstrate that pancreatic rough ER contains two isoforms of the SERCA-2b Ca 2+,Mg-ATPase whose ATP-binding properties are susceptible to inhibition by vanadate but not thapsigargin.