Journal article

CYTOCHEMICAL IDENTIFICATION OF THE REGULATORY SUBUNIT OF THE CAMP-DEPENDENT PROTEIN-KINASE BY USE OF FLUORESCENTLY LABELED CATALYTIC SUBUNIT - EXAMINATION OF PROTEIN-KINASE DISSOCIATION IN HEPATOMA-CELLS RESPONDING TO 8-BR-CAMP STIMULATION

WH FLETCHER, SM VANPATTEN, HC CHENG, DA WALSH

JOURNAL OF BIOLOGICAL CHEMISTRY | AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC | Published : 1986

Abstract

Homogeneous catalytic subunit from the cAMP-dependent protein kinase, when derivatized with a fluorophore, was used as a cytochemical probe to locate intracellular sites of the protein kinase regulatory subunit. After conjugation, the fluoresceinated catalytic subunit (F:C), derivatized to a stoichiometry of approximately 1 mol/mol, retained near full activity as judged by specific activity and by titration against either regulatory subunit or Inhibitor Protein of the protein kinase. With this molecular probe the dissociated regulatory subunit was localized by direct cytochemistry in Reuber H-35 hepatoma cells that had been exposed, while intact, for 0-120 min to 10(-4) M 8-Br-cAMP. After st..

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