title: Calcium-activated, phospholipid-dependent protein kinases from rat liver: subcellular distribution, purification, and characterization of multiple forms.
abstract: Three forms of Ca2+- and phospholipid-dependent protein kinase (protein kinase C) were extensively purified from rat liver homogenate. Subcellular fractionation analysis indicated that the majority (approximately 85%) of the activity was associated with particulate fractions of the liver. Among these, the microsomal and nuclear fractions accounted for approximately 63% and approximately 10% of total activity. The remaining 15% of protein kinase C was recovered in the soluble fraction following differential centrifugation. It was also found that most of the membrane-associated protein kinase C was latent, with 4-6-fold stimulation with detergents such as 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate, octyl beta-glucoside, or Triton X-100. The activity of both the bound form and the soluble enzyme was enhanced by the addition of Ca2+ and phosphatidylserine, when histone H1 was used as substrate. The bound protein kinase C activity was dissociated by homogenization of liver in buffer containing ethylene glycol bis(beta-aminoethyl ether)-N,-N,N',N'-tetraacetic acid, ethylenediaminetetraacetic acid, and various proteolytic inhibitors, and the solubilized extract was used to purify multiple forms of the enzyme. The purification procedure sequentially utilized (NH4)2SO4 fractionation, ion-exchange chromatography on DEAE-cellulose, gel permeation chromatography on Fractogel TSK HW-55 (F), ion-exchange chromatography on hydroxylapatite, gel permeation chromatography on Ultrogel AcA34, and affinity chromatography on polyacrylamide-immobilized phosphatidylserine. On hydroxylapatite columns, protein kinase C activity was resolved into three isoenzymic forms designated C-I, C-II, and C-III. The molecular weights of the three isoenzymic forms were in the range of 208,000-225,000 as shown by chromatography on calibrated Ultrogel AcA34 columns and sucrose density gradient centrifugation. Furthermore, all three isoenzymes demonstrated a single peak with a sedimentation coefficient (s20.w) in the range of 9.0-9.2. However, with polyacrylamide gel electrophoresis, all the forms showed a single protein component with average molecular weight of 64K, suggesting that the native isoenzymes may be composed by subunits. Finally, all three isoenzymes exhibited nearly identical enzymatic properties.
authors.affiliation: Department of Medicine, Stanford University School of Medicine, California.
authors.forename: S
authors.initials: S
authors.lastname: Azhar
authors.forename: J
authors.initials: J
authors.lastname: Butte
authors.forename: E
authors.initials: E
authors.lastname: Reaven
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology; D002845:Chromatography; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003600:Cytosol / Q000201:enzymology; D017886:Durapatite; D006657:Histones / Q000378:metabolism; D006882:Hydroxyapatites; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008862:Microsomes, Liver / Q000201:enzymology; D008970:Molecular Weight; D010766:Phosphorylation; D011487:Protein Conformation; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism*; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D013347:Subcellular Fractions / Q000201:enzymology
chemical_list: D000596:Amino Acids; D006657:Histones; D006882:Hydroxyapatites; D007527:Isoenzymes; D017886:Durapatite; D011494:Protein Kinases; D011493:Protein Kinase C