Characterisation of endoplasmic reticulum and plasma membrane Ca(2+)-ATPases in pancreatic beta-cells and in islets of Langerhans.
We have investigated the plasma membrane (PMCA) and endoplasmic reticulum (SERCA) Ca(2+)-ATPases involved in active transport of Ca2+ in pancreatic beta-cell lines (MIN6, HIT T15, RINm5F) and in islets of Langerhans. Under selective membrane phosphorylation conditions (at low ATP concentration, in the presence of Ca2+ and La3+ and in the absence of Mg2+ at 4 degrees C) the only labelled proteins are the phosphoenzyme intermediates of the Ca(2+)-ATPases. Under these conditions, beta-cell membranes incorporated 32P from [gamma-32P]ATP into two proteins with molecular mass on acidic SDS-polyacrylamide gels of around 115 and 150 kDa. The 150 kDa band was identified as PMCA (i) by reaction with a monoclonal anti-human erythrocyte plasma membrane Ca(2+)-ATPase antibody; (ii) by its typical tryptic cleavage pattern which generated an 80 kDa band; (iii) by lack of inhibition of its autophosphorylation by SERCA-specific inhibitors. The 115 kDa band was identified as SERCA (i) by reaction with a polyclonal anti-rat fast skeletal muscle Ca(2+)-ATPase antibody; (ii) by the concentration-dependent inhibition of its autophosphorylation by thapsigargin and 2,5-di(t-butyl)-1,4-benzohydroquinone (tBHQ), which are specific inhibitors of SERCA. The 115 kDa band was further characterised as the SERCA-2b isoform by reaction with a polyclonal rabbit antibody against the 12 C-terminal amino acids of SERCA-2b.
Váradi
A
A
Nuffield Department of Clinical Biochemistry, John Radcliffe Hospital, Headington, Oxford, UK.
Molnár
E
E
Ashcroft
S J
SJ
D000818:Animals; D000937:Antigen-Antibody Reactions; D000252:Calcium-Transporting ATPases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000276:immunology; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D004721:Endoplasmic Reticulum / Q000201:enzymology; D006873:Hydroquinones / Q000494:pharmacology; D007515:Islets of Langerhans / Q000201:enzymology*; D007527:Isoenzymes / Q000032:analysis; D008297:Male; D010766:Phosphorylation; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D014357:Trypsin
D006873:Hydroquinones; D007527:Isoenzymes; D013729:Terpenes; D019284:Thapsigargin; C018855:2-tert-butylhydroquinone; D014357:Trypsin; D000252:Calcium-Transporting ATPases