Phosphoprotein casein

Serine was first isolated from sericin by Cramer in 1865. Most proteins contain about 4-8% serine. In phosphoproteins (casein, phosvitin) serine, like threonine, is a carrier of phosphoric acid in the form of 0-phosphoserine. The carbohydrate component of glycoproteins may be bound 0-glycosidically through the hydroxyl group of serine and/or threonine [cf. 10.1.2.1.1 (K-casein) and 13.1.4.2.4]. 

The main components of the casein fraction in bovine milk are 0(5-caseins. Bovine milk contains phosphoprotein -casein and a52 Casein, both occurring in four genetic variants, identified by the letters A, B, C and D, which differ somewhat in their primary structure. The most common variant is B, which is a polypeptide chain composed of 199 amino acids (molecular weight 23.6 kDa, p/= 4.92-5.35). aj-Caseins comprise from 44 to 55% of the total caseins and ajj-casein comprises approximately 80% of the total aj-caseins. The polypeptide chain contains eight phosphoserine residues located mainly at positions 43-80, which makes this part of the molecule polar. Non-polar side chains of amino adds are located in positions 100-199. ajj-Casein forms insoluble calcium salts in the presence of calcium ions. Fragments of aji-casein are considered to be. X-casein. a52-Casein has a similar structure (molecular weight 25.2 kDa), but is less sensitive to the presence of calcium ions. 

P-Eliminations from serine phosphates have been observed on dephosphorylation of phosphoproteins (casein, phosvitin) (10, 261, 299, 361). P-Elimination from serine phosphate esters by means of dilute aqueous alkali or diethylamine in aprotic solvents has occasionally been used for the synthesis of dehydroamino acid units (302, 331, 332). 

PHOSPHOPROTEINS. These proteins have phosphate groups esterified to the hydroxyls of serine, threonine, or tyrosine residues. Casein, the major protein of milk, contains many phosphates and serves to bring essential phosphorus to the growing infant. Many key steps in metabolism are regulated between states of activity or inactivity, depending on the presence or absence of phosphate groups on proteins, as we shall see in Chapter 15. Glycogen phospho-rylase a is one well-studied example. 

Phosphoproteins contain phosphate Casein Phosphate groups ... 

Animal glue is a complex colloidal mixture of proteins. The related gelatins are also complex heterogeneous mixtures of proteins. They are strongly hydrophilic and rich in the amino acids glycine, proline, lysine, hydroxyproline and hydroxylysine. Casein is a phosphoprotein obtained from the milk of mammals. 

Skim milk can be considered as a two-phase system consisting of casein-colloidal calcium phosphate micelles in quasi-equilibrium with an aqueous solution of salts and proteins the phase boundary is ill-defined because of the intimate association between the calcium phosphate and the caseins (phosphoproteins). 

Milk acid phosphatase has been purified to homogeneity by various forms of chromaotgraphy, including affinity chromatography purification up to 40 000-fold has been claimed. The enzyme shows broad specificity on phosphate esters, including the phosphoseryl residues of casein. It has a molecular mass of about 42 kDa and an isoelectric point of 7.9. Many forms of inorganic phosphate are competitive inhibitors, while fluoride is a powerful non-competitive inhibitor. The enzyme is a glycoprotein and its amino acid composition is known. Milk acid phosphatase shows some similarity to the phosphoprotein phosphatase of spleen but differs from it in a number of characteristics. 

Originally, the caseins were defined as those phosphoproteins which precipitate from raw skim milk upon acidification to pH 4.6 at 20°C, and the individual families were identified by alkaline urea gel electrophoresis (Whitney et al 1976). With the resolution of their primary structure, it became possible to classify them according to their chemical structure, rather than on the basis of an operational definition. When one does this, it is apparent that not all of the caseins contain phosphorus (Table 3.1) some are also found in the acid whey after removal of the precipitated caseins. 

Casein is not coagulated by heat. It is precipitated by acids and by rennin. a proteolytic enzyme obtained from the stomach or calves. Casein is a conjugated protein belonging lo the group uf phosphoproteins. The enzyme trypsin can hydrolyze off a phosphorus-containing peptone. 

Casein or Caseinogen. A white phosphoprotein occurring as a colloidal suspension in milk. It can be pptd by heating milk with an acid or by other methods. Casein is the principal ingredient of cottage or other cheeses. It is also used in manuf of plastics, paints, paper, synthetic fibers, etc... 

Caseins were once defined as the phosphoproteins that precipitate from milk at pH 4.6 but are now defined by their amino acid sequences. In the elucidation of the primary structures of the caseins a French group has taken a prominent part (Ribadeau Dumas et al., 1975). The four polypeptide chains of bovine casein are the aSi-, as2-, P-, and K-caseins. Of these, the two as-caseins and K-casein are each phosphorylated to different extents and K-casein also exists in a number of glycosylated forms. 

According to Bloomfield and Mead (1974), The ultimate goal of all workers on casein is to reconstitute micelles with native properties from the separated constituents of skim-milk. This assertion reflects the large number of studies in the literature on the precipitation and association properties of the caseins.There are, however, legitimate scientific goals in this kind of work other than the creation of artificial casein micelles, such as the elucidation of the mechanisms by which phosphoproteins profoundly influence the nucleation and growth of calcium phosphate phases. 

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... 

Desdouits F, Cohen D, Nairn AC, Greengard P, Girault JA (1995b) Phosphorylation of DARPP-32, a dopamine-and cAMP-regulated phosphoprotein, by casein kinase I in vitro and in vivo. J Biol Chem 270 8772-8778. 

Phosphoproteins. An important group that includes many major food proteins. Phosphate groups are linked to the hydroxyl groups of serine and threonine. This group includes casein of milk and the phosphoproteins of egg yolk. 

Casein is a phosphoprotein. It is the main protein of milk, and the name "casein often refers to milk casein. Pure casein is white, but it is sold on the market as creamy or slightly greyish brown grains. It is insoluble in water, alcohol or ether, but it slowly dissolves in alkali, carbonate and strong acid. The material which passes 60 mesh is the best for paste. 

This amino acid CsHsOeNP (P = 16.7%) and with an [a]n of -f7.2 (1) was first obtained by Lipmann and Levene from vitellinic acid (44). Lip-mann subsequently succeeded in isolating this substance from an acid hydrolyzate of casein (45) and thus established that phosphoserine may occur as a constituent in phosphoproteins. More recently Agren, de Verdier, and Glomset crystallized 0-phosphorylserine (1). 

Following this discussion of some of the properties of three naturally occurring phosphoamino acids and of the dipeptide, 0-phosphorylseryl-glutamic acid, a few examples of phosphoproteins, i.e., casein, vitellin, vitellenin, and phosvitin, and of the phosphopeptones derived from these materials, will be considered. 

Whereas casein has been investigated rather extensively, little is known about the phosphoproteins from eggs, i.e., vitellin, vitellenin, and phos-vitin, respectively. Two of these proteins are present in combination with phospholipids representing 25% of the egg yolk solids (2, 14, 62). After removal of the lipids with 80% ethanol, vitellin and vitellenin are obtained with a phosphorus content of 1% and 0.29%, respectively. Nothing is known about the homogeneity of these preparations. 

One point of interest emerges from these experiments, namely, that ovalbumin is the second protein from which phosphoserine has been isolated. As in the case of the dipeptide, SerP.Glu, isolated from casein (38,77) ovalbumin also contains an amino acid sequence with phosphoserine adjacent to a dicarboxylic acid. Moreover, the close association of this amino acid with aspartic and glutamic acids, isoleucine and alanine in ovalbumin (22, 68) and casein (78-80) suggests the existence of a systematically recurring sequence in phosphoproteins. 

Although it has been reported in the literature (3, 79, 85) that casein is resistant to the action of purified phosphomonoesterases from mammalian tissues, this protein was nevertheless chosen as an example of a typical phosphoprotein for the reasons outlined below ... 

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—... 

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. 

---