title: Purification of three forms of chromatographically distinct protein kinase C from the swine ovary.
abstract: A calcium-sensitive, lipid-dependent protein kinase (protein kinase C) modulates physiological function in a variety of cells. In certain tissues, multiple forms of this key regulatory enzyme exist. To examine the nature of ovarian protein kinase C, swine luteal cytosol (1000 mg protein) was applied and eluted from DE-52 cellulose. The pooled active fractions were sequentially purified further by threonine- (TS) and phenyl-Sepharose (PS) affinity chromatography in a buffer of 20 mM Tris, 0.5 mM EDTA, 0.5 mM EGTA, 5 mM dithiothreitol and 250 micrograms/l leupeptin. Protein kinase C activity was eluted with NaCl gradients of 0.075-125 M (DE-52), 0.1-1 M (TS) and 0.6-0.0 M (PS). This purification scheme yielded three distinct peaks of highly purified protein kinase C activity and an overall enrichment in protein kinase C activity of approximately 1000-fold. Inasmuch as control of steroidogenesis or peptide hormone production in the ovary via the Ca2+-protein kinase C pathway could occur at several different levels, we postulate that the demonstrated presence of isoforms of this enzyme in ovarian tissue offers a means for selective spatial and temporal compartmentalization of the endocrine stimulus with correspondingly distinct changes in cellular function.
authors.affiliation: Department of Internal Medicine, University of Virginia School of Medicine, Charlottesville 22908.
authors.forename: M B
authors.initials: MB
authors.lastname: Wheeler
authors.forename: J D
authors.initials: JD
authors.lastname: Veldhuis
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007527:Isoenzymes / Q000302:isolation & purification; D010053:Ovary / Q000201:enzymology*; D011493:Protein Kinase C / Q000302:isolation & purification*; D012685:Sepharose; D013552:Swine; D013912:Threonine
chemical_list: D007527:Isoenzymes; D013912:Threonine; D012685:Sepharose; D011493:Protein Kinase C