Albumin, serum phosphorylation

Ferric ion was immobilized on a Chelating Sepharose Fast Flow column preparatory to the separation of seven enkephalin-related phosphopep-tides.17 Non-phosphorylated peptides flowed through the column, and the bound fraction contained the product. The capacity of the column was found to be 23 pmol/mL by frontal elution analysis. Cupric ion was immobilized on Chelating Superose for the isolation of bovine serum albumin.18 Cupric ion was immobilized on a Pharmacia HiTrap column for the separation of Protein C from prothrombin, a separation that was used to model the subsequent apparently successful separation of Factor IX from prothrombin Factor IX activity of the eluate was, however, not checked.19 Imidazole was used as the displacement agent to recover p-galactosidase from unclarified homogenates injected onto a nickel-loaded IMAC column.20 Pretreatment with nucleases and cleaning in place between injections were required procedures. A sixfold purification factor was observed. 

S ATP -I- [DNA-directed eukaryotic RNA polymerase II subunit Ila] (<4> distinct from other protein phosphokinases, transfers about 20 phosphates to the heptapeptide repeats Pro-Thr-Ser-Pro-Ser-Tyr-Ser in C-terminal domain of MW 220000 subunit of RNA-polymerase II [7] <4> substrates are RNA-polymerase II subunits of wheat germ, soy bean, pea and human [7] phosphorylates predominantly Ser-residues [1-3,5,7] <1> kinase CTDKl almost exclusively phosphorylates Ser-residues [5] <1> kinase CTDK2 phosphorylates to a lesser extent Thr-resi-dues [1] <3-5> phosphorylates to a lesser extent Thr-residues [1,5,7] <1> phosphorylates Ser- and Thr-residues equally [6] <1,3,5> phosphorylates not Tyr-residues [1,6] <1> kinase CTDKl 33 mol phosphate per mol IIA-subunit [5] <1> kinase CTDK2 40-50 mol phosphate per mol IIA-subunit, i.e. 1 phosphate per heptapeptide repeat [5] <4> no substrate is GTP [7] <2,4> no substrates are CTP and UTP [3,7] <2> no substrates are dTTP and AMP-PNP [3] <4> no substrates are bovine serum albumin and calf thymus histone [7] <5> no substrate is phosvitin... 

The peptide substrate was phosphorylated in a reaction mixture (0.25 mL) containing 25 mA/ Tris-HCl (pH 7.5), 0.5 mg/mL bovine serum albumin, 4.0 mA/ MgCl2, 0.2 mA/ CaCl2, 2.6 nM calmodulin, 24 fiM peptide, 1.5 nA/ myosin light chain kinase, and 400 fiM ATP. The reaction was started by adding ATP. After 30 minutes at 28°C, the reaction was terminated by adding 0.1 mL of 10% acetic acid. Aliquots were directly applied to the HPLC column. The reaction was linear for up to 60 minutes. 

The initial event in the utilization of fat as an energy source is the hydrolysis of triacylglycerols by lipases, an event referred to as lipolysis. The lipase of adipose tissue are activated on treatment of these cells with the hormones epinephrine, norepinephrine, glucagon, and adrenocorticotropic hormone. In adipose cells, these hormones trigger 7TM receptors that activate adenylate cyclase (Section 15,1.3 ). The increased level of cyclic AMP then stimulates protein kinase A, -which activates the lipases by phosphorylating them. Thus, epinephrine, norepinephrine, glucagon, and adrenocorticotropic hormone induce lipolysis (Figure 22.6). In contrast, insulin inhibits lipolysis. The released fatty acids are not soluble in blood plasma, and so, on release, serum albumin binds the fatty acids and serves as a carrier. By these means, free fatty acids are made accessible as a fuel in other tissues. 

It is generally accepted that a relationship exists between the structural complexity of a compound, i.e., the variety of its components, and its ability to induce an immune response. For example, homopolymers of amino acids by themselves are very poor antigens, however, when used in a complex (e.g., with phosphorylated serum albumin), they induce normal levels of antibodies. The same effect can be accomplished by the introduction of a second or third different amino acid. Analogous situations exist in some naturally occurring macromolecules. Thus, the low level of antibodies induced by gelatin could be greatly elevated by the introduction of tyrosyl residues. - At the present time, we cannot explain... 

The [Cr(OH)(OH2)x(Solv)]2+ ions (.v 0 3, Solv = MeOH or MeCN) have been detected by ESMS in the gas phase, on fast evaporation of solutions of [Cr(OH2)6](C104)3 in MeOH or MeCN.47 The [Cr(NH3)5(OH)]2+ complex promotes the phosphorylation (by ATP) of serine and threonine OH-residues in bovine serum albumin in vitro.511... 

The ability of some Cr(III) complexes to activate ATP dependent kinases (496, 497) may seem surprising, as stable Cr -ATP complexes (such as XIV in Chart 2 one of the many possible stereoisomers is shown) (629) are well known to act as kinase inhibitors. Chromium(III) in these complexes is a kinetically inert replacement of a natural ATP binding ion, Mg(II) (629-631). However, a Cr(III) complex, [Cr(NH3)5(OH)] +, has recently been shown to promote the in vitro phosphorylation by ATP of the hydroxo groups of Ser and Thr residues in bovine serum albumin at pH 7.4, while several other Cr(III) complexes with N- and O-donor ligands did not possess such activity (632). The proposed phosphorylation mechanism (Scheme 12) (632) involves the formation of a ternary complex of Cr(III), ATP, and the amino acid residue. This mechanism may be related to the kinase-catalyzed phosphorylation of protein Tyr residues. 

Scheme 12. Proposed mechanism of phosphorylation of Ser and Thr residues in bovine serum albumin by ATP, promoted by [Cr(NH3)5(OH)] +. [Adapted from (632).]...

Fig. 7 Selective phosphoprotein detection in SDS-PAGE using 36. Each lane includes two phosphoproteins (ovalbumin (45.0 kDa) and a-casein (24.0 kDa)), and four non-phosphorylated proteins (/S-galactosidase) (MW = 116.0 kDa), bovine serum albumin (MW = 66.2 kDa), avidin (MW = 18.0 kDa), and lysozyme (MW = 14.4 kDa). Lane 1 CBB staining of the six proteins. Lane 2, 3 Detection of the phosphoproteins with UV transilluminator after staining with 36. The amount of each protein in lane 2 and lane 3 is 5.0 and 2.5 xg, respectively...

Human serum albumin consists of a single polypeptide chain of 584 amino acids, which is stabilized by 17 disulfide bridges. Ovalbumin contains one oligosaccharide chain, coupled via an aspartate residue, as well as a phosphorylated serine residue. 

Mayer and Heidelberger (353) report the phosphorylation of crystalline serum albumin. They found that the use of potassium borate held the pH relatively constant during phosphorylation, but fewer phosphoryl... 

The titration curves of a phosphorylated serum albumin preparation showed an additional 60 X 10 equivalent acid groups per gram protein in the region of pH 2.0-2.5 and a similar increase between pH 5.0 and pH 8.0. Since there had been an increase of 56 X 10 equivalents of phosphorus per gram protein in this preparation, the authors point out that this increase of two titratable equivalents per phosphorus means that most of the phosphorus is linked to the protein as a mono ester. 

Insulin, lyso2yme, human serum albumin (Sigma-Aldrich) and tyrosine phosphorylated and unphosphorylated peptides (Bachem) were used with out... 

Sogorb, M.A., Monroy, A., Vilanova, E., 1998. Chicken serum albumin hydrolyzes dichlorophenylphosphoramidates by a mechanism based on transient phosphorylation. Chem. Res. Toxicol. 11 (12), 1441-1446. 

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