Distribution of the intracellular Ca2+-ATPase isoform 2b in pig drain subcellular fractions and cross-reaction with a monoclonal antibody raised against the enzyme isoform 1

The presence and distribution of sarco-endoplasmic reticulum Ca2+-ATPase (S ERCA) isoform 2b in microsomes and other subcellular fractions isolated fro m pig brain has been demonstrated by the combined use of a specific antibod y raised against the SERCA2b isoform and ATP phosphorylation experiments. A h subcellular fractions show an approximately 110 kDa phosphorylated protei n, the band intensity being stronger in microsomes, Preliminary treatment o f the samples with trypsin generates two phosphorylated fragments of about 57 and 33 Ma in the presence of Ca2+. The observed fragments are typical tr ypsinized products of the SERCA2b isoform, The monoclonal antibody Y/ 1F4 r aised against the sarcoplasmic reticulum Ca2+-ATPase (isoform 1) binds to t he 110 kDa band in membranes isolated from brain. The binding was stronger in microsomes than in other fractions. Furthermore, this antibody also reco gnizes a clear band at around 115 kDa. This band is always stronger in plas ma membrane than in synaptosomes or microsomes and is unaffected by trypsin , Phosphorylation studies in the absence of Ca2+ suggest that the 115 kDa p rotein is not a Ca2+-ATPase.