Casein dephosphorylation

In accord with experiments on emulsions (Husband et al., 1997), the molecular configurations deduced from SCF calculations have demonstrated the crucial role of the cluster ( blob ) of 5 charged phosphoserine residues in p-casein in maintaining the steric stabilizing layer, whilst also preventing interfacial precipitation (multilayers). The mobility of this blob was demonstrated experimentally by P NMR measurements on P-casein-stabilized emulsions (ter Beek et al., 1996). It was inferred that, when the effective charge on the blob is reduced (by dephosphorylation) or screened (by salt addition), the macromolecular spring relaxes... 

Husband, F.A., Wilde, P.J., Mackie, A.R., Garrood, M.J. (1997). A comparison of the functional and interfacial properties of p-casein and dephosphorylated p-casein. Journal of Colloid and Interface Science, 195, 77-85. 

Although it can dephosphorylate casein under suitable conditions, as far as is known, alkaline phosphatase has no direct technological significance... 

On heating at temperatures above 100°C, lactose is degraded to acids with a concomitant increase in titratable acidity (Figures 9.5, 9.6). Formic acid is the principal acid formed lactic acid represents only about 5% of the acids formed. Acid production is significant in the heat stability of milk, e.g. when assayed at 130°C, the pH falls to about 5.8 at the point of coagulation (after about 20 min) (Figure 9.7). About half of this decrease is due to the formation of organic acids from lactose the remainder is due to the precipitation of calcium phosphate and dephosphorylation of casein, as discussed in section 9.4. 

Decrease in pH. After heating at 140°C for 20 min, the pH of milk has decreased to about 5.8 due to acid production from pyrolysis of lactose, precipitation of soluble calcium phosphate as Ca3(P04)2, with the release of H+, and dephosphorylation of casein with subsequent precipitation of the liberated phosphate as Ca3(P04)2 with the release of H+. The heat-induced precipitation of Ca3(P04)2 is partially reversible on cooling so that the actual pH of milk at 140°C at the point of coagulation is much lower than the measured value and is probably below 5.0. 

Dephosphorylation of casein, which follows first-order kinetics. After heating at 140°C for 60 min, >90% of the casein phosphate groups have been hydrolysed. 

Cobb and Novotny (7) obtained improved separations using C)8 microcolumns as a method for separating quantities on the order of 4 picomoles of tryptic peptides of phosphorylated and dephosphorylated /3-casein. Figure 4 shows two peaks with different retention times, corresponding to the phosphorylated and dephosphorylated forms of the same peptide. The rest of the peptide map is similar. Using this microcolumn, phosphorylation of a single amino acid on a protein can be detected. The method is reproducible with standard deviations smaller than 2%. Characterization of bovine /3-lg tryptic peptides by RP-HPLC on a Nucleosil Cl 8 column was also reported (123). 

Fig. 4 Comparison of tryptic digests from (A) phosphorylated and (B) dephosphorylated forms of /3-casein, separated by microcolumn HPLC. Arrows point to the two peaks that exhibit different retention times between the two forms. Each chromatogram was obtained from a 300-ng protein sample.

Brooks and Landt (1984) and Brooks (1987) isolated a calmodulin-dependent kinase from rat and bovine mammary acini which selectively phosphorylated dephosphorylated K-casein rather than de-phosphorylated asl-casein or P-casein. 

Desdouits F, Siciliano JC, Greengard P, Girault JA (1995a) Dopamine- and cAMP-regulated phosphoprotein DARPP-32 phosphorylation of Ser-137 by casein kinase I inhibits dephosphorylation of Thr-34 by... 

In experiments in which a- and (3-casein are remixed in different proportions, it is noticed that if the relative concentration of the (3-casein in the mixture exceeds 20% the presence of this protein inhibits the enzymatic action, the degree of inhibition being proportional to the concentration of the (3-protein. These results may be taken as an explanation for the failure of previous investigators to dephosphorylate unfractionated casein without preceding transformation to phosphopeptones. 

As in the case of ovalbumin, the dephosphorylation of a-casein is accompanied by a change in the electrophoretic behavior. With the aid of Fig. 5, it is illustrated that the liberation of phosphorus is accompanied by the appearance of several new components with lower mobilities. The mobility decrements of these components at pH 6.8 are 0.5 X 10 or a multiple thereof. If compared with the base binding capacity of a-casein (31) this value corresponds to a change in the net charge of —2. This supports the initial assumption that some of the a-casein phosphorus is present in form of monoesters. 

In a recent communication Sundararajan and Sarma report that a phosphoprotein phosphatase from rat spleen dephosphorylates a-, /3-, and unfractionated casein (90). Since these authors state that their enzyme differs in its action from that of a phosphomonoesterase, their results are in accord with the occurrence of a variety of phosphorus bonds in proteins. In this connection it should be noted that intestinal phosphatase used in our work at pH 9.0 also liberates all of the a-casein phosphorus (72). As discussed earlier, although this enzyme at pH 6.0 hydrolyzes —N—P—... 

If the molecular weights of pepsin and pepsinogen are 35,000 and 38,000, respectively (61), each of these molecules contains one atom of phosphorus (28, 60). Since it had been shown that ovalbumin and a-casein are readily dephosphorylated by certain phosphatases from mammalian tissue, and from potato, the action of these enzymes on pepsin and its precursor w as studied. It was found that only the potato phosphatase at pH 5.6 de-phosphorylates pepsin and pepsinogen, whereas prostate phosphatase does not act on these proteins. The intestinal enzyme, although not active at pH 6.0, liberates phosphorus at pH 8.9 (67). 

From the work carried out by the author it is apparent that a variety of enzymes will dephosphorylate phosphoproteins if the phosphate groups are present as monoesters. As shown by the study of the three ovalbumins and a-casein, together with the results on phosvitin reported in the literature (53), these phosphate groups contribute to the net charge of the proteins. In addition, the protein-phosphorus may be present in form of diester and pyrophosphate bonds. 

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