A protein kinase is located in the micellar fraction of fresh pasteurized skimmed farm milk.
Recombinant protein kinase CK2 from the yeast Schizosaccharomyces pombe is able to phosphorylate casein in skimmed pasteurized milk. We could incorporate up to 540 pmol of phosphate into 50 microg milk proteins, i.e., 0.26 P/mol caseins. To better understand the action of protein kinase CK2 on milk proteins, we have compared the action of rspCK2alpha on milk, and on different casein micellar subfractions isolated from milk by ultracentrifugation. In contrast to the situation observed with phosphocaseinate, alpha(s) casein was the best substrate for rspCK2alpha, whether milk or micellar fractions were used as substrates. We have characterized the protein content of different micellar fractions obtained by ultracentrifugation of cow milk using capillary zone electrophoresis. We confirm that the kappa casein content of micelles largely decreases when their size increases. In contrast, the alpha(s) casein content slightly increased with micelles size and beta casein content remained constant. All of the micellar fractions were substrates for rspCK2alpha, but a significant amount of intrinsic protein kinase activity was also found. The intrinsic protein kinase used added ATP as phosphate donor, and was only slightly sensitive to high heparin concentration. It could phosphorylate micellar casein in milk ultrafiltrate, in the absence of addition of any metallic cofactor. Its activity was only slightly affected by the addition of either MgCl2 or MnCl2. CaCl2 activated the enzyme significantly. The intrinsic kinase lost its activity with time, and could incorporate from 9 to 26% of the total phosphate incorporated in the presence of rspCK2a. Alpha(s) casein was the best substrate of the intrinsic kinase, followed by beta casein. In the presence of CaCl2, the intrinsic kinase was found to incorporate up to 470 pmol of phosphate into 50 microg of milk proteins.
Lovisi
P
P
Unité Mixte de Recherche de Chimie Biologique INRA INA-PG, Centre de Biotechnologies Agro-Industrielles, 78850 Thiverval-Grignon, France.
Jolivet
P
P
Jagic
F
F
Dalgleish
D
D
Chardot
T
T
D000818:Animals; D002364:Caseins / Q000378:metabolism; D019075:Electrophoresis, Capillary; D008055:Lipids / Q000032:analysis; D008823:Micelles; D008892:Milk / Q000737:chemistry / Q000201:enzymology*; D008894:Milk Proteins / Q000032:analysis / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000032:analysis* / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D013379:Substrate Specificity; D014461:Ultracentrifugation
D002364:Caseins; D008055:Lipids; D008823:Micelles; D008894:Milk Proteins; D011994:Recombinant Proteins; D011494:Protein Kinases