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. 700nmoles/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 chromotography resolved two closely eluting peaks of activity, suggesting the existence of two related, but distinct, GCK activities.
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Research Article|
August 23 2000
Purification of Golgi casein kinase from bovine milk
Jennifer S. DUNCAN;
Jennifer S. DUNCAN
1
*The Physiological Laboratory, University of Liverpool, Crown Street, Liverpool L69 3BX, U.K.
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Mark C. WILKINSON;
Mark C. WILKINSON
‡School of Biological Sciences, University of Liverpool, Crown Street, Liverpool L69 3BX, U.K.
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Robert D. BURGOYNE
Robert D. BURGOYNE
2
*The Physiological Laboratory, University of Liverpool, Crown Street, Liverpool L69 3BX, U.K.
2To whom correspondence should be addressed (e-mail burgoyne@liverpool.ac.uk).
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Publisher: Portland Press Ltd
Received:
March 16 2000
Revision Received:
June 08 2000
Accepted:
June 27 2000
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 2000
2000
Biochem J (2000) 350 (2): 463–468.
Article history
Received:
March 16 2000
Revision Received:
June 08 2000
Accepted:
June 27 2000
Citation
Jennifer S. DUNCAN, Mark C. WILKINSON, Robert D. BURGOYNE; Purification of Golgi casein kinase from bovine milk. Biochem J 1 September 2000; 350 (2): 463–468. doi: https://doi.org/10.1042/bj3500463
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