Purification of and production of an antibody against a 63,000 Mr stimulus-sensitive phosphoprotein in Paramecium.
In vivo labeling of Paramecium cells with 32Pi most heavily labels a minor 63-kDa protein that undergoes a rapid, Ca2+-dependent dephosphorylation when the cell is stimulated to release. This stimulus-sensitive phosphoprotein was isolated and purified to apparent homogeneity. A polyclonal affinity purified antibody made against the purified protein recognizes both the phosphorylated and dephosphorylated forms of the protein. The phosphorylated 63-kDa protein is found in the cytosolic fraction; it is slightly acidic with two isoelectric forms at pI 5.8 and 6.2 and probably exists as a monomeric 60-65-kDa polypeptide in the native state. The labeled phosphoamino acid of the protein is phosphoserine. The affinity purified antibody recognizes a third isoelectric form at pI 6.3 that appears unlabeled. The specificity of the antibody was confirmed by showing that it immunoprecipitates the correct protein, i.e. the stimulus-sensitive 63-kDa phosphoprotein. The availability of purified 63-kDa protein as well as an antibody against it will now allow molecular, biochemical, and immunocytochemical studies into the role of this protein in the mechanism of exocytosis.
Murtaugh
T J
TJ
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461.
Gilligan
D M
DM
Satir
B H
BH
D000818:Animals; D000917:Antibody Formation; D002118:Calcium / Q000378:metabolism; D005089:Exocytosis; D007150:Immunohistochemistry; D007526:Isoelectric Point; D008970:Molecular Weight; D010247:Paramecium / Q000032:analysis*; D010750:Phosphoproteins / Q000276:immunology / Q000302:isolation & purification*
D010750:Phosphoproteins; D002118:Calcium