Distribution of the intracellular Ca(2+)-ATPase isoform 2b in pig brain subcellular fractions and cross-reaction with a monoclonal antibody raised against the enzyme isoform.
The presence and distribution of sarco-endoplasmic reticulum Ca(2+)-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. All 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 kDa in the presence of Ca(2+). The observed fragments are typical trypsinized products of the SERCA2b isoform. The monoclonal antibody Y/1F4 raised against the sarcoplasmic reticulum Ca(2+)-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 Ca(2+) suggest that the 115 kDa protein is not a Ca(2+)-ATPase.
Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Ciencias, Universidad de Extremadura, 06071 Badajoz, Spain.
J M
JM
Salvador
M
M
Berengena
M R
MR
Sepúlveda
A M
AM
Mata
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001345:Autoradiography; D015153:Blotting, Western; D001921:Brain / Q000166:cytology / Q000201:enzymology*; D019556:COS Cells; D002118:Calcium / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D003429:Cross Reactions / Q000276:immunology*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology; D008970:Molecular Weight; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D013347:Subcellular Fractions / Q000187:drug effects / Q000201:enzymology*; D013552:Swine; D013570:Synaptic Membranes / Q000201:enzymology; D013574:Synaptosomes / Q000201:enzymology
D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D010750:Phosphoproteins; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium