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At least 19 recordsLinked to original sources
PubMed1991

On the interaction of phosvitins with ferric ion: solubility of the Fe(III)-phosphoprotein complex under acidic conditions is a function of the iron/phosphate ratio and the degree of phosvitin phosphorylation.

The interaction of phosvitins, the polyphosphoproteins of the eggs of egg-laying vertebrates, with ferric chloride was investigated under acidic conditions at iron-to-protein phosphorus ratios ranging up to 10. Phosvitins of which all or nearly all serine residues are phosphorylated (P/ser greater than 0.8) precipitate when titrated with the iron salt. As the total Fe/P ratio reaches the value of about 0.5, precipitation becomes maximal. At Fe/P ratios above 0.5, the Fe(III)-phosvitin complex becomes increasingly soluble. At ratios above 2, solubility is essentially fully restored. Phosvitins with an appreciable portion of their serine residues non-phosphorylated (P/ser less than 0.7) show a different dependence of solubility on the Fe/P ratio. The Fe/P ratios of all precipitated complexes themselves vary within a narrow range between about 0.4 and about 1.0; the total Fe/P ratio is varied between 0 and 10. The results imply that phosvitin iron binding sites are non-uniform and that, overall, phosvitin is capable of accommodating iron in different ways, depending on the relative magnitude of the iron load and the availability of phosphoserine clusters in the phosphoprotein.

Animals↗
PubMed1999

Affinity properties of phosvitin: interaction of phosvitin with serine hydroxymethyl transferase.

The affinity of phosvitin with serine hydroxymethyl transferase (SHMT), an acidic multi-subunit protein, was evaluated by measurements of enzyme activity, sedimentation velocity, steady-state fluorescence, circular dichroism and kinetic thermal stability. While the presence of phosvitin had no effect on the SHMT activity, the sedimentation coefficient of SHMT increased from 8.7 S to 12.5 S suggesting the formation of a complex at a SHMT:phosvitin molar ratio of 2:1. Based on steady-state fluorescence quenching measurements an association constant of 2.4 +/- 0.2 x 10(5) M-1 at 25 degrees C was obtained for the interaction of phosvitin with SHMT. The temperature dependency of the association constant in the range 15-35 degrees C suggests the involvement of ionic forces in the interaction. The thermal inactivation of SHMT followed first order kinetics. In the presence of phosvitin the rate constant decreased and half time increased. The circular dichroism measurements suggest that phosvitin interaction does not involve pyridoxal phosphate binding domain of the enzyme. Although minor changes in the secondary structure of the enzyme were observed, the environment around aromatic amino acids did not change significantly.

Fluorescence↗
PubMed1987

Iron binding by phosvitins: variable mechanism of iron release by phosvitins of diverse species characterized by different degrees of phosphorylation.

The rate of reductive iron release from Fe(III) complexes of phosvitins of diverse fish species, at varied initial degrees of saturation with iron, was studied with particular attention to the effect of the degree of phosvitin phosphorylation on the kinetics of iron release. The reaction was followed colorimetrically as phosphorprotein-bound iron was transferred to an excess of o-phenanthroline, in the presence of hydroquinone as a reducing agent. The principal finding was the variability of the kinetic order or iron release by phosvitins, depending on their degree of saturation with iron and the extent to which their serine residues were phosphorylated. Highly phosphorylated proteins, especially at high initial degrees of iron saturation, obey first-order kinetics. Partially phosphorylated proteins, especially at low initial degrees of iron saturation, release their iron in a zero-order fashion. First-order rates imply that the iron binding sites are kinetically independent of each other. Zero-order behavior appears to reflect iron release from hypothetical iron-binding clusters serving as kinetically effective reactive centers of unchanging concentration for most of the time course of the reaction. Variations of the initial degree of iron saturation of given phosvitins produced variations in their kinetic behavior. The results are considered in terms of a dynamic model of phosvitin iron binding sites which may constitute themselves diversely, in response to the amount of iron that is to be accommodated, or may reconstitute themselves as their molecular environment becomes altered.

Animals↗
PubMed1986

Phosvitin isolation from fish eggs: methodological improvements including 'specific' phosvitin precipitation with ferric ion.

Modifications of the method of Wallace et al. [Can. J. Biochem. 44, 1647-1655 (1966)] for phosvitin isolation from vertebrate eggs were devised to enhance the method's general effectiveness. Phosvitins which do not precipitate on dilution from solutions of their lipovitellin complexes may be selectively adsorbed onto, and desorbed from, DEAE-cellulose. Phosvitins which are too small for dialysis or ultrafiltration may be concentrated or desalted by precipitation with stoichiometric amounts of ferric ions, followed by iron removal with EDTA on gel filtration. Since phosvitin distribution among ammonium sulfate fractions depends on initial protein and salt concentrations in a species-specific manner, pilot experiments are needed to establish conditions for optimal fractionation.

Amino Acids↗
PubMed1990

Coadsorption onto hydroxyapatite of human salivary mucins and phosvitin, a phosphoprotein from egg yolk.

The adsorption of human salivary mucins (HWSM, 0.4 mg/ml) onto hydroxyapatite (HAP) was studied in the presence of varying amounts of the phosphoprotein phosvitin by three different procedures. a. Preadsorption of HWSM onto HAP for 20 h, followed by 4 h coadsorption with phosvitin, resulted in a decrease of 50% in HWSM binding to HAP with 0.3 mg/ml phosvitin and a complete desorption with 1.0 mg/ml phosvitin. b. Preincubation of HAP with phosvitin for 20 h, followed by 4 h coadsorption with HWSM, resulted in decrease of 50% in HWSM binding to HAP with 0.15 mg/ml phosvitin and the adsorption of HWSM was prevented completely with 1.0 mg/ml phosvitin. c. Simultaneous incubation of HWSM and phosvitin gave the least adsorption of HWSM to HAP: a decrease of 50% with as little as 0.025 mg/ml phosvitin and a nearly complete desorption with 0.3 mg/ml phosvitin. Similarly, the adsorption of phosvitin was strongly inhibited by HWSM after either simultaneous adsorption or preadsorption with HWSM. However, after preincubation of HAP with phosvitin, desorption of phosvitin by HWSM was not achieved. Release of phosphate increased by preadsorption with HWSM followed by incubation with phosvitin, but was lowered by about 50% after preadsorption with phosvitin. After simultaneous incubation of HAP with both species, the adsorption did not result in release of phosphate ions. Calcium release was only substantial when phosvitin was in excess in solution. The smallest release of calcium ions was observed when HAP was preincubated with phosvitin, followed by coadsorption with HWSM.

Adsorption↗
PubMed1975

Structural requirements for the enzymatic phosphorylation of phosvitin.

Some structural features required for the enzymatic phosphorylation of phosvitin by purified rat liver cytosol phosvitin kinase have been investigated by testing the activity of such an enzyme toward phosphopeptides differing in size and chemical composition, obtained by pronase or acid hydrolysis of phosvitin. The results obtained can be summarized as follows: (a) Phosvitin kinase phosphorylates even fairly simple phosphopeptides (mol.wt 1000-2000) at rates comparable with intact phosvitin. (b) Acetylation of both phosvitin and pronase phosphopeptides completely prevents their phosphorylation indicating that some lysine residues are strictly required for the phosvitin kinase reaction. (c) Accordingly polyphosphorylserine blocks Ser(P)n which are very actively phosphorylated in phosvitin and pronase phosphopeptides, do not undergo any more enzymatic phosphorylation once isolated as such in a form free of other amino acids. (d) The activity of phosvitin kinase toward substrates probably devoid of Ser(P)n blocks suggests that there are not required for the protein kinase reaction. However, they apparently enhance the phosphorylation rate of the peptide substrates, likely by making easier their binding to the enzyme. It is proposed therefore that the peptidic unit able to undergo phosphorylation by rat liver cytosol phosvitin kinase consists of one or more phosphorylserine residues having in their close proximity a lysine residue playing a critical role in the mechanism of transphosphorylation.

Amino Acids↗
PubMed1982

Dephosphorylation of chicken riboflavin-binding protein and phosvitin decreases their uptake by oocytes.

Riboflavin-binding protein (RBP) and phosvitin are phosphoglycoproteins transferred from the plasma of laying hens into the yolk of developing oocytes. We have examined the effect of phosphate removal on this yolk deposition process. Unmodified yolk RBP and phosvitin contain, respectively, 8.3 and 109 residues of phosphate/molecule. Complete dephosphorylation of yolk RBP caused a 20-min decrease in the plasma clearance half-life an 87% decrease in the uptake of the protein into oocytes in vivo. Although partially desialylated, dephospho-yolk RBP was identical with the native protein by several criteria, including riboflavin-binding capacity, mobility on SDS-polyacrylamide gels, and circular dichroism. A series of partially dephosphorylated yolk RBP samples, prepared by limited enzymatic hydrolysis, was indistinguishable from native yolk RBP by all criteria except phosphate content. Removal of the 1st phosphate residue decreased uptake of yolk RBP into oocytes by about 60%. Uptake into oocytes could not be restored to dephospho-yolk RBP by addition of anionic groups by succinylation. However, succinylation of native yolk RBP decreased its deposition into oocytes to the same extent as dephosphorylation. Partial dephosphorylation of phosvitin also had marked effects. The plasma clearance of dephosphophosvitin (70% of phosphate removed) was much faster than native phosvitin. After 4 h, 15% of injected 125I-phosvitin remained in circulation compared with only 3.8% of 125I-dephosphophosvitin. The uptake of dephosphovosvitin into oocytes was 79% less than that of native phosvitin. In vitro, 125I-phosvitin bound specifically to a preparation of oocyte plasma membranes as indicated by competition with unlabeled phosvitin but not with RBP. The specific binding of dephosphophosvitin was 96% less than that of native phosvitin and it could be displaced equally well by phosvitin or yolk RBP.

Acid Phosphatase↗
PubMed1999

Molecular mechanism of the excellent emulsifying properties of phosvitin-galactomannan conjugate.

The emulsifying properties of native and N- and C-terminal-deleted phosvitin (protease digests) were compared after conjugation with galactomannan. The emulsifying properties of Maillard-type phosvitin-galactomannan conjugates were greatly improved, whereas those of the protease-digested phosvitin-galactomannan conjugates were not so dramatically improved. Phosvitin was highly glycosylated with galactomannan, whereas the protease-digested phosvitin conjugate consisting of a highly phosphorylated core peptide fragment was not. The results suggest that both N and C termini of the peptide moiety, digested by protease, were essential for the improvement of emulsifying properties of phosvitin-galactomannan conjugates. In addition, the role of N and C termini as anchors in oil droplets was supported from the comparative studies of native phosvitin, phosvitin-galactomannan conjugates, and protease-digested phosvitin-galactomannan conjugates.

Amino Acid Sequence↗
PubMed1986

Selective repression of RNA polymerase II by microinjected phosvitin.

We have used a microinjection technique to examine whether injected phosvitin, in its capacity as substrate for casein kinase NII, could compete out the endogenous phosphorylation of some nuclear phosphoproteins with regulatory potential and thereby interfere with the activity of RNA polymerase II. Phosphorylation, which utilizes ATP as phosphate donor, was separated from phosphorylation which uses GTP. Phosvitin introduced into nuclei of salivary gland cells becomes phosphorylated by the endogenous nuclear protein kinase(s) and incorporates phosphates from ATP as well as from GTP. The phosphorylation of nuclear proteins and phosvitin is heparin-sensitive, indicating that they are phosphorylated by casein kinase NII. Microinjected phosvitin does not seem to affect the incorporation of phosphate groups from ATP into nuclear proteins, but protein phosphorylation by GTP is influenced. Apart from a minor overall reduction of 32P-incorporation, the phosphorylation of a 42 kDa nuclear protein, a putative transcription stimulatory factor, and of a 115 kDa nuclear protein was competed out by 70%-80% compared with the control value obtained in the absence of phosvitin. Parallel analyses of DNA transcription in phosvitin-injected nuclei showed that the RNA polymerase II-mediated synthesis of hnRNA and Balbiani ring RNA was diminished by 80% and 90%, respectively. In contrast, the transcription of nucleolar pre-ribosomal 38 S RNA by RNA polymerase I remained unaffected. The inhibitory effect of injected phosvitin could be reversed by in vitro phosphorylation of phosvitin prior to injection, using isolated nuclei as source of protein kinase(s). Taken together, the results suggest a causal relationship between the modification of the GTP-dependent phosphorylation of specific non-histone proteins and the activity of RNA polymerase II.

Animals↗
PubMed1993

Comparative studies of phosvitin from chicken and salmon egg yolk.

1. A method developed for the isolation of phosvitin from chicken egg yolk was successfully applied to the isolation of phosvitin from salmon eggs. 2. Salmon roe phosvitin is smaller in molecular size than chicken egg phosvitin. 3. Circular dichroism spectra of all phosvitins investigated displayed good similarities with spectra showing characteristics of unordered and beta-sheet secondary structure. 4. The main component in the Fourier transform infrared spectra of chicken egg phosvitin is indicative of unordered conformation, whereas the Fourier infrared data of the salmon egg phosvitin are consistent with more of beta-sheet structure compared to the chicken egg phosvitin.

Amino Acids↗
PubMed1986

A Raman spectroscopic study of hen egg yolk phosvitin: structures in solution and in the solid state.

Laser Raman spectroscopy has been employed to study the structure of the hen egg yolk protein phosvitin in H2O and D2O solutions at neutral and acidic pH (pD) and in the solid state. The Raman data indicate an unusual conformation for phosvitin in neutral aqueous solution, which is deficient in both alpha-helix and conventional beta-sheet conformations. This unusual pH 7 structure is, however, largely converted to a beta-sheet conformation in strongly acidic media (pH less than 2). beta-Sheet is also the predominant secondary structure for phosvitin in the solid state, obtained by lyophilization of the protein from aqueous solution at neutral pH. The imidazolium rings of histidyl residues remain significantly protonated near neutrality, which suggests substantial elevation of the pK for imidazolium ring ionizations of phosvitin in aqueous solution. This may result from extensive ion-pair interactions involving positively charged histidines and negatively charged phosphoserines, which are prevalent in the phosvitin sequence. The present results suggest that antiparallel beta-sheets may not be the secondary structure most characteristic of native phosvitin (physiological pH), even though beta-sheet is the predominant conformation for phosvitin in acidic solutions (pH 1.5) and in the lyophilized solid. Phosvitin appears to be the first protein for which the major component to the Raman amide I band is centered near 1685 cm-1, which is 10-40 cm-1 higher than proteins heretofore examined in aqueous solution by Raman spectroscopy.

Animals↗
PubMed1986

Characteristics of egg yolk phosvitin as an antioxidant for inhibiting metal-catalyzed phospholipid oxidations.

A study was conducted to determine the antioxidant activity of phosvitin in an egg yolk phospholipid emulsion system. Various inorganic and organic metals (Fe2+, Cu2+, and hemin) in several different concentrations were added individually to the emulsions to induce lipid oxidation. Characteristics of phosvitin for inhibiting these metal-catalyzed lipid oxidations were investigated. The effect of heat treatments, both pasteurization (61.1 C, 4 min) and autoclaving (121.1 C, 10 min), on phosvitin was examined to find out if there was any effect on its antioxidant characteristics. Lipid oxidations were measured by thiobarbituric acid assays. Phosvitin effectively inhibited Fe2+ and Cu2+-catalyzed phospholipid oxidations as shown by low malondialdehyde values over the entire reaction period; however, it did not exert similar effects on the hemin-catalyzed oxidation reaction. Phosvitin demonstrated higher capacity to inhibit iron catalysis of phospholipid oxidations (up to 30:1 Fe2+-to-phosvitin molar ratio) than copper catalysis (1:1 molar ratio). Pasteurization did not change the antioxidant activities of phosvitin; however, autoclaving decreased phosvitin's inhibitory capacity on iron catalysis.

Antioxidants↗
PubMed1985

Phosvitins in Fundulus oocytes and eggs. Preliminary chromatographic and electrophoretic analyses together with biological considerations.

Vitellogenin serves as the plasma precursor for the yolk proteins, lipovitellin and phosvitin, in nonmammalian vertebrates. 32P-Vitellogenin was isolated from the plasma of the teleost, Fundulus heteroclitus, and was used both to label phosvitin in the ovary and to indicate the phosvitin region in preparative chromatographs of ovarian extracts on DEAE-cellulose. Crude [32P]phosvitin could be resolved further into two labeled components with shallow gradients on DEAE-cellulose and into eight labeled components by electrophoresis on 12% polyacrylamide gels. Only the two largest electrophoretically resolved components could be correlated with Coomassie Blue staining bands, but several of the smaller components could be indicated with the cationic carbocyanine dye, Stains-all. Stains-all-dyed components were also generally indicated as multiple bands. The ovary of a reproductively active female contains vitellogenic oocytes, postvitellogenic oocytes undergoing maturation prior to ovulation, and ovulated eggs. Examination of various types of follicles and eggs on polyacrylamide gels revealed that during maturation, the largest phosvitin components formed during vitellogenesis either disappear or diminish, while smaller phosvitin components appear. The transformation of phosvitin components can also be achieved in vitro by incubating prematurational follicles in a saline medium containing deoxycorticosterone. These preliminary results demonstrate that a complex array of phosvitin-like components are present within a single ovary of F. heteroclitus. We also postulate that one reason for the anomalous yolk proteins generally found thus far in teleost eggs is that some of the proteins derived from vitellogenin during vitellogenesis undergo further proteolysis during oocyte maturation.

Animals↗
PubMed1996

Oligophosphopeptides of varied structural complexity derived from the egg phosphoprotein, phosvitin.

Phosvitins are the principal phosphoproteins in the eggs of oviparous vertebrates. They have an exceptionally high serine content and most, or even all, of the serine residues are esterified to phosphate. The phosphorylated residues tend to occur in uninterrupted runs of as many as 28 phosphoserines (as in Xenopus phosvitin). This unique structural feature gives phosvitins extraordinary properties and can be expected to play a key role in phosvitin function. For example, the concentration of phosphate groups provides for numerous highly efficient metal-binding sites in clusters. The mode of binding had been shown to be affected by the size of the protein and the degree to which serine residues are phosphorylated. For structure-function studies of phosvitins (and other polyphosphoproteins), phosphopeptides of differentiated structural complexity are desirable. Such model peptides were produced in this work by limited proteolysis of chicken phosvitin, and oligophosphopeptides of widely varying sizes, phosphoserine content, and sequence were purified and characterized. These include phosvitin segments containing one, two, or several oligophosphoserine runs, corresponding to segments of the N-terminal, C-terminal, and core sequence of the protein.

Amino Acid Sequence↗
PubMed1985

Oxidation and reduction of cytochrome c bound to the phosphoprotein phosvitin.

The oxidation-reduction reactions and structural characteristics of phosvitin-bound cytochrome c were examined at various ratios of cytochrome c to phosvitin. At binding ratios below half the maximum, the rate constants for the oxidation reactions with cytochrome c oxidase and ferricyanide and the rate constants for the reduction reactions with cytochrome b2 and ascorbate were low, but at higher ratios these rate constants gradually increased to that of free cytochrome c and, in particular, the rate constant for oxidation by cytochrome c oxidase was raised to two to three times that of the free form. This binding-ratio dependence of the rate constants for the oxidation and reduction reactions was different from that of the net charge of the cytochrome c-phosvitin complex, implying that the negative charges of phosvitin are unlikely to modulate the rates. In contrast, the broadening of the NMR signals for the heme and methionine-80 methyl groups and the conformational transition in the vicinity of the heme moiety on change from the native to the cyanide-bound or urea-denatured form of cytochrome c showed a similar binding-ratio dependence to the rate constants for the oxidation and reduction reactions. Since the conformation and electronic structure in the heme environment of ferric and ferrous cytochromes c were not changed significantly by binding to phosvitin, and since the binding strength of cytochrome c to phosvitin at binding ratios below half the maximum is different from that at higher ratios, these findings suggest that a difference in the movement of cytochrome c in its complex with phosvitin may modulate its oxidation-reduction reactions.

Circular Dichroism↗