Photoaffinity labelling of the ATP-binding sites of two Ca2+,Mg-ATPase isoforms in pancreatic endoplasmic reticulum.
Pancreatic rough ER ATP-binding proteins, including two isoforms of SERCA-2b Ca2+,Mg-ATPase, were identified using specific photoaffinity labelling with 8-azido-ATP. 8-Azido-ATP irreversibly inhibited Ca2+,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-[alpha-32P]ATP. The two bands at 111 kDa and 100 kDa corresponded in molecular mass to the two SERCA-2b Ca2+,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 Ca2+,Mg-ATPase activity was inactivated by EDTA-treatment of rough ER membranes; (b) inhibited by the Ca2+,Mg-ATPase inhibitor vanadate; (c) not affected by thapsigargin. The data demonstrate that pancreatic rough ER contains two isoforms of the SERCA-2b Ca2+,Mg-ATPase whose ATP-binding properties are susceptible to inhibition by vanadate but not thapsigargin.
Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff, UK.
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D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000345:Affinity Labels; D000818:Animals; D001386:Azides; D001665:Binding Sites; D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000378:metabolism*; D010179:Pancreas / Q000201:enzymology*; D010777:Photochemistry; D011233:Precipitin Tests; D051381:Rats; D017208:Rats, Wistar
D000345:Affinity Labels; D001386:Azides; D007527:Isoenzymes; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase