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 (SERCA) isoform 2b in microsomes and other subcellular fractions isolated from pig brain has been demonstrated by the combined use of a specific antibody raised against the SERCA2b isoform and ATP phosphorylation experiments. Ah subcellular fractions show an approximately 110 kDa phosphorylated protein, the band intensity being stronger in microsomes, Preliminary treatment of the samples with trypsin generates two phosphorylated fragments of about 57 and 33 Ma in the presence of Ca2+. The observed fragments are typical trypsinized products of the SERCA2b isoform, The monoclonal antibody Y/ 1F4 raised against the sarcoplasmic reticulum Ca2+-ATPase (isoform 1) binds to the 110 kDa band in membranes isolated from brain. The binding was stronger in microsomes than in other fractions. Furthermore, this antibody also recognizes a clear band at around 115 kDa. This band is always stronger in plasma membrane than in synaptosomes or microsomes and is unaffected by trypsin, Phosphorylation studies in the absence of Ca2+ suggest that the 115 kDa protein is not a Ca2+-ATPase.