Phosphorylation of β-Casein and α-Lactalbumin by Casein Kinase from Lactating Bovine Mammary Gland

Abstract

Two milk proteins, beta-casein and alpha-lactalbumin, were compared as substrates for casein kinase from bovine mammary gland. beta-Casein could be rephosphorylated after removal of its phosphate groups, whereas alpha-lactalbumin was an effective substrate after the protein had been reduced and carboxymethylated. The native proteins could not be phosphorylated. Magnesium2+, Ca2+, and Mn2+ stimulated phosphorylation of the modified proteins. Calcium2+ was the most effective cation for alpha-lactalbumin and Mn2+ for beta-casein. Michaelis constants were 144 microM for alpha-lactalbumin in the presence of Ca2+ and 142 microM for beta-casein in the presence of Mn2+; however, the maximum velocity for alpha-lactalbumin was three times that of beta-casein. After phosphorylation with [gamma-32P] ATP, partial hydrolysis showed that only serine residues were phosphorylated in both proteins. Chymotryptic peptides of phosphorylated alpha-lactalbumin and tryptic peptides of phosphorylated beta-casein were examined by HPLC and selected peptides were analyzed for amino acid content. Comparison of the analyses with sequence data showed that serine at position 47 in alpha-lactalbumin is the major site of phosphate incorporation. Dephosphorylated beta-casein was only partially rephosphorylated. However, the sites identified correspond to the phosphorylated residues in native beta-casein, namely, serine at position 35 and the cluster of four serines between residues 15 and 20.

Purification of Golgi casein kinase from bovine milk

“…Previous work has shown that GCK activity present within Golgi fractions is exposed to the lumen and can be only assayed with exogenous substrate following detergent solubilization of the membranes [6,[9][10][11]. This resulted in the assumption that GCK is an integral Golgi membrane protein [25,26].…”

“…This resulted in the assumption that GCK is an integral Golgi membrane protein [25,26]. In our initial experiments, GCK activity was solubilized from lactating mammary tissue using 1 % Triton X-100 as previously described [6,[9][10][11]. Extensive dialysis to remove detergent led to massive precipitation of solubilized protein.…”

“…The phosphorylation of caseins is important to allow them to bind Ca# + and for the subsequent formation of casein micelles, which remain stable in milk [3]. The protein kinase responsible for this physiological casein phosphorylation is present within Golgi compartments [4][5][6][7][8], and is only accessible in in itro assays after disruption of the Golgi membranes [6,[9][10][11]. The use of caseins as substrates for protein kinases has resulted in the identification and detailed characterization of two protein kinases : casein kinase (CK) 1 and CK2 [12].…”

“…These are cytosolic enzymes and are, therefore, misnamed (since they would not encounter casein as a physiological substrate). The basic properties of the Golgi casein kinase (GCK) have been characterized in Golgi fractions and membrane extracts [6,[9][10][11], but this enzyme has not been convincingly purified or cloned. In addition, it is neither clear whether GCK activity encompasses single or multiple enzymes, nor is it known if a mammary-gland-specific GCK exists, or if instead GCK is a single, ubiquitously expressed enzyme.…”

Two physiological substrate‐specific casein kinases are present in the bovine mammary gland

“…Recent work of Bingham et al [13] demonstrated that all sites of phosphorylation previously described by sequence analysis can be phosphorylated by a Ca +-and CM-independent casein kinase preparation. The preparation of the membranes was performed in 1 mM EDTA, suggesting that the Ca 2+-and CM-dependent enzyme may not participate in phosphorylation of the familiar sites.…”