Phosphoprotein phosvitin

The phosphoprotein phosvitin may attach a large number of ferric ions/mole, according to Gray (99) [citing work of Saltman and Multani], the number bound may be as large as 46. Ferric ion, although firmly complexed, may be removed from this egg yolk protein by dialysis against solutions of ethylenediamine tetraacetic acid (102). 

The predominating form of iron in animal tissues is haem (particularly myoglobin and haemoglobin). In egg white, iron is bound in conalbumin, and in the yolk to phosphoprotein phosvitin. Milk contains the iron metaUoprotein lactoferrin and part of the iron is bound to casein. Conalbumin and lactoferrin are structurally similar to serum transferrin. In plants, iron is bound in various complexes, especially with phytic acid, aUphatic hydroxycarboxyhc acids, aminocarboxyhc acids, thiols, phenolic substances, nucleotides, peptides and proteins. The iron content in selected foods is shown in Table 6.8. Foods rich in iron are offal dishes, meat, eggs, pulses, tea and cocoa. Moderate amounts of iron are found in fish, poultry, cereals, spinach, parsley and nuts. Low levels of iron are present in milk, dairy products, fats and oils, potatoes and most fruit. 

Phosphoproteins.—A chemical synthesis of partially and fully phosphorylated protamines has been described during the past year,98 and structural requirements for the enzymatic phosphorylation of phosvitin have been delineated.99 Phosphorylated forms of phosphofructokinase100 and fatty acid synthetase101 have been discovered recently both may be concerned with the regulation of their respective enzymes. 

It has been proposed that a low molecular weight phosphoprotein of phosvitin nature (47) which binds iron with high affinity may be involved in the cytosolic transport of iron in liver cells (48, 49), but no energized uptake of iron has been demonstrated such as in isolated rat liver mitochondria using a Fe(III)-phosvitin from avian egg yolk (mol wt 40,000) as the substrate (50). The biochemical significance of the cytosolic iron binding phosphoprotein is therefore still uncertain. 

It is also possible that this phosphoprotein could be involved in the transport of other metals." Isolated phosvitin usually has two to three atoms of iron, with Fe/phosphate ratio about 1 50. Considerably larger amounts of Ca " appears to have no known enzymatic or regulatory role. 

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. 

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. 

Hegenauer, J., Saltman, P., and George, N. (1979). Iron(in)-phosphoprotein chelates stoichiometric equilibrium constant for interaction of iron(lll) and phosphorylserine residues of phosvitin and casein. Biochemistry. 18, 3865-3879. 

The term protein kinase was first used to describe the enzymes of yeast, liver, and brain which phosphorylated casein and phosvitin (85-87). More recently it has been proposed that the term protein kinase be restricted to those enzymes which catalyze the transfer of the y-phos-phate of ATP to serine and threonine hydroxyls of proteins and are regulated by cyclic nucleotides (88). The other class of protein kinases, the phosphoprotein kinases, are not affected by cyclic nucleotides, and they can often utilize GTP as a source of phosphate. Wherever appropriate, we shall refer to these two groups of enzymes as cyclic AMP-dependent or cyclic AMP-independent protein kinases, respectively (89). 

Phosvitin is the principal phosphoprotein of egg yolk. Hen egg yolk phosvitin contains 10% phosphate by weight with 120 of the total 222 residues per mole (MW 35,500) phosphorylated. There are 119 residues of phosphoserine and one residue of phosphothreonine. About 5-7 of the total serine residues of phosvitin are not phosphorylated (97). A distinctive feature of the primary sequence of phosvitin is the repeating units of phosphoserine followed by a basic amino acid residue (72). This is illustrated by the following nine sequences of amino acids found in phosvitin ... 

Dephosphorylation. In vivo, phosphoprotein phosphatases (phos-phoprotein phosphohydrolase EC 3.1.3.16) participate in the regulation of phosphate turnover and in rapid reversal of the phosphorylating reactions, yet there is very little information on this important group of enzymes. Alkaline (104) and acid (105) phosphatases of milk can hydrolyze the phosphate groups of phosphoserine residues in the caseins. E. coli alkaline phosphatase dephosphorylates many phosphoproteins (106,107). A phosphoprotein phosphatase of liver dephosphorylates phosphorylated histones and protamines but has little or no activity on casein or phosvitin (108). The 60-fold purified enzyme had an apparent Km for dephosphorylation of histone I (FI) of 2 X 10"5M and a pH optimum of 7-8. Histone phosphatase activity was detected in all eukaryotic cells examined, but it was not found in the extracts of several prokaryotes. 

The stability of amorphous tricalcium phosphate is reportedly increased by the presence of ATP or certain phosphoproteins such as phosvitin and casein. 

More than a hundred different phosphoproteins have now been recognised. The best known of these include milk casein, the egg proteins phosvitin and ovalbumin, and the iron storage protein ferritin. In addition, nearly all known enzymes are proteins, and enzyme action is usually associated with phosphorylation-dephosphorylation reactions (Chapter 11.4). 

Phosvitin, which is present in egg yolk (Chapter 12.4), has a high P content and a high serine content. It contains at least one phosphate group per two protein amino groups and a chain containing up to eight consecutive serine units is believed to be present in this phosphoprotein [19,20]. 

Phosvitin, the most phosphorus-rich phosphoprotein in egg yolk, contains about 9.6% of P (Chapter 10.2). 

Phosphoproteins, which contain phosphoric acid as the prosthetic group, include the caseins of milk (see p. 406) and phosvitin in egg yolk. 

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]. 

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). 

Phosphoamino acids that are part of proteins known to bind metal ions are posttranslational modifications introduced by specific protein kinases (Meggio et al, 1981 Vogel and Biidger, 1982c). The bovine milk protein casein and the hen egg-white protein ovalbumin, as well as possibly the human saliva acidic proline-iich proteins share sequence homology of their phosphorylated sites. Dephosphorylation of such sites by enzymatic phosphatase treatment usually reduces the affinity of such proteins for metal-ion binding (Bennick et al., 1981). Hence it is likely that dianionic phosphoryl moieties are directly involved in the complexation of metal ions. This seems particularly important for the two polyelectrolyte proteins that contain large amounts of phosphoserine residues, phosvitin purified from egg yolk (Ta-borsky, 1974), and the phosphoprotein purified from dentine (Linde et al, 1980). 

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