title: Purification of Golgi casein kinase from bovine milk.
abstract: Caseins and many other secretory proteins are phosphorylated during their transport through the secretory pathway by a protein kinase present within Golgi compartments. Molecular analysis of the Golgi casein kinase (GCK) has not been possible since it has not been purified to homogeneity or been cloned. Previous attempts have been made to purify GCK activity from mammary gland Golgi fractions, but these have not resulted in extensive purification of the enzyme. In the present study, we have demonstrated that substantial amounts of GCK activity, assayed using a specific peptide substrate, can be detected as a soluble form in bovine milk, and we have used milk as a source for purification. A purification protocol was established that allowed>80000-fold purification to a specific activity of GCK (approx. 700 nmoles/min per mg of protein) far higher than previously achieved. These findings cast doubts on previous claims for purification of GCK activity. In addition, ion-exchange chromatography resolved two closely eluting peaks of activity, suggesting the existence of two related, but distinct, GCK activities.
authors.affiliation: The Physiological Laboratory, University of Liverpool, Crown Street, Liverpool L69 3BX, U.K.
authors.forename: J S
authors.initials: JS
authors.lastname: Duncan
authors.forename: M C
authors.initials: MC
authors.lastname: Wilkinson
authors.forename: R D
authors.initials: RD
authors.lastname: Burgoyne
mesh_terms: D000818:Animals; D047388:Casein Kinases; D002417:Cattle; D002847:Chromatography, Agarose; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006056:Golgi Apparatus / Q000201:enzymology*; D008892:Milk / Q000201:enzymology*; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism*; D012685:Sepharose / Q000737:chemistry
chemical_list: D010455:Peptides; D012685:Sepharose; D011494:Protein Kinases; D047388:Casein Kinases