Phosphorylation of Proteins with Phosphoric Acid Containing Excess Phosphorus Pentoxide

Ferrel, Robert E.; Olcott, Henry Steel; Fraenkel‐Conrat, H.

doi:10.1021/ja01186a033

Cited by 66 publications

(32 citation statements)

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“…Native human albumin was prepared from plasma by the cold-ether fractionation technique of Kekwick & Mackay (1954). Chemically modified albumins were prepared by standard procedures as follows: esterification, by the method of Chibnall, Mangan & Rees (1958); acetylation, by the method of Marrack & Orlans (1954); bromoacetylation, by the method of Korman & Clarke (1956); phosphorylation, by the method of Ferrel, Olcott & Fraenkel-Conrat (1948).…”

Section: Methodsmentioning

confidence: 99%

Studies on the interaction of magnesium, calcium and strontium ions with native and chemically modified human serum albumin

Irons

Perkins

1962

Biochemical Journal

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The interactions of Ca2+ ions with plasma proteins were first studied by Pribram (1871) Mg2+, Ca2+ > Sr2+> Ba2+ ions, although Mg2+ ion is often irregular in position (Schubert, 1954).In this paper an attempt has been made to extend this work by comparing the binding of the alkaline-earth ions to native and chemically modified human albumin over a range of pH and in the presence of near-physiological concentrations of Na+ ions. Additional information about the nature of the binding sites on albumin for these ions is given. Greenwald (1926) Measurement of radioactivity. The method of isotope dilution was used for the quantitative determination of metal ions. The isotope 28Mg was counted with a wellcrystal system in conjunction with an automatic scaler.The isotopes 45Ca and 89Sr were counted by a p-particle liquid-scintillation technique at -17°. Diphenyloxazole (0-3 %, w/v) in toluene was used as the organic scintillator. Aqueous samples (1 ml.) were blended with a scintillator (5 ml.) with ethanol (10 ml.). Protein solutions were precipitated by the ethanol in the counting solution. When the precipitated protein was allowed to settle in the counting dish it did not appreciably affect the count. In practice, therefore, the protein was precipitated and kept for at least 3-4 hr. in the dish at -17°before counting. Solutions containing buffer ions in the presence and absence of protein were counted with the same efficiency as an aqueous solution containing the same amount of activity. Each sample was counted for a minimum of 150 000 counts.Equilibrium dialysis. All dialyses were carried out with 10 ml. of 0-5-0-7% (w/v) protein-buffer solution inside Visking cellulose membranes. The outer solution (120-140 ml.) was of measured pH and molarity 0-15-0-17.Each dialysis was done in a dialysis rocker overnight at room temperature. Control experiments showed that with this apparatus 0-10m-sodium chloride came into equilibrium with water in 3 hr. Dialysis experiments as a function of pH were carried out at fixeed Ca2+, Mg2+ and Sr2+ ion concentrations of 20, 10 and 40 mg./l. respectively.Outer buffer solutions. The following buffer systems were used: pH 2-4, 0-05M-potassium hydrogen phthalate; pH4-6, 0-05M-sodium acetate-acetic acid; pH6-7-5,0-02M-sodium cacodylate; pH 8-9-5, 0-lm-sodium hydroxideboric acid-potassium chloride. Sodium chloride was added to each buffer solution to bring the molarity to 0-15-0-17.Protein concentration. Protein was estimated by a semimicro-Kjeldahl technique; a protein:N factor of 6-25 was assumed.

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Section: Methodsmentioning

confidence: 99%

Studies on the interaction of magnesium, calcium and strontium ions with native and chemically modified human serum albumin

Irons

Perkins

1962

Biochemical Journal

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“…Peptides corresponding to the 13-amino acid repeat sequence Lys-Ser-Pro-Val-Pro-Lys-SerPro-Val-Glu-Glu-Lys-Gly (designated 1-13nP) in human NF-M (residues 614-626) or a trimer (designated 1-39nP) thereof (residues 614-652) were synthesized on solid phase using a PAM resin and Boc amino acid pentafluorophenyl esters as coupling reagents (11). Phosphopeptides were then generated by chemically phosphorylating (12) the serines in these peptides (termed 1-13P and 1-39P, respectively). We then confirmed that all serine residues in both 1-13P and 1-39P had covalently linked phosphate (13).…”

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confidence: 99%

Identification of the major multiphosphorylation site in mammalian neurofilaments.

Lee

Ötvös

Carden

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

341

238

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The sequence Lys-Ser-Pro-Val-Pro-Lys-SerPro-Val-Glu-Glu-Lys-Gly repeats six times serially in the human midsized neuroframent (NF) protein (NF-M). To establish whether Lys-Ser-Pro-Val(Ala) is the major site for in vivo NF phosphorylation, peptides based on the human NF-M repeat were synthesized and chemically phosphorylated. These synthetic peptides were probed with 515 monoclonal antibodies (mAbs) that were raised to, and distinguished, several differentially phosphorylated forms of NF proteins. Studies with 95 of those mAbs that recognized the peptides before and after chemical phosphorylation demonstrated that a highly immunogenic epitope shared by the peptides is present in NFs from all species tested, including invertebrates. This suggests the phylogenetic conservation of a major NF phosphorylation site. Lastly, a cross-reactive antigenic determinant shared by the peptides and the major NF phosphorylation site was shown to exist in neurofibrillary tangles of patients with Alzheimer disease as well as in two neuron-specific microtubule-associated proteins (MAPs-i.e., MAP2 and tau.

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“…Ferrel et al 14 ) and Mohammad et al 6 ) have accomplished protein phosphorylation under the conditions of minimized water content. The methods of phosphorylation they used may not be·· available in an aqueous system, since P 2 0 S and phosphoric acid rapidly decompose into the free phosphoric ion in water before P 2 0 S and phosphoric acid react with the serine or threonine residues in protein.…”

Section: Discussionmentioning

confidence: 99%

Functionality and Digestibility of a Highly Phosphorylated Soybean Protein

Hirotsuka

Taniguchi

Narita

et al. 1984

Agricultural and Biological Chemistry

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Phosphorylation of Proteins with Phosphoric Acid Containing Excess Phosphorus Pentoxide

Cited by 66 publications

References 0 publications

Studies on the interaction of magnesium, calcium and strontium ions with native and chemically modified human serum albumin

Studies on the interaction of magnesium, calcium and strontium ions with native and chemically modified human serum albumin

Identification of the major multiphosphorylation site in mammalian neurofilaments.

Functionality and Digestibility of a Highly Phosphorylated Soybean Protein

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