Purification of and production of an antibody against a 63,000 Mr stimulus-sensitive phosphoprotein in Paramecium.

T. J. Murtaugh, D. M. Gilligan, B. H. Satir

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)15734-15739
Number of pages6
JournalThe Journal of biological chemistry
Volume262
Issue number32
StatePublished - Nov 15 1987

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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