Proteins phosphate reactions

Phosphates, which react with calcium to reduce the calcium ion activity, assist in stabilizing calcium-sensitive proteins, eg caseinate and soy proteinate, during processing. Phosphates also react with milk proteins. The extent of the reaction depends upon chain length. Casein precipitates upon addition of pyrophosphates, whereas whey proteins do not. Longer-chain polyphosphates cause the precipitation of both casein and whey proteins. These reactions are complex and not fully understood. Functions of phosphates in different types of dairy substitutes are summarized in Table 9 (see also Food additives). 

Purify the modified protein from reaction by-products by dialysis or gel filtration using 50mM sodium phosphate, 0.15M NaCl, pH 7.2. 

Purify the modified protein from reaction by-products by dialysis or gel filtration using 50 mM sodium phosphate, 0.15 M NaCl, 10 mM EDTA, pH 7.2. Alternatively, centrifugal spin columns containing a desalting resin may be used for rapid purification (Thermo Fisher). 

The epoxy-activated dendrimer may be conjugated to thiol-containing proteins by reaction in 50 mM sodium phosphate, pH 7.2. The reaction can be done at 4°C or at room temperature for 8-16 hours to form thioether linkages. 

The transporting protein in the sarcoplasmic membrane can be phosphorylated by ATP as well as by inorganic phosphate (cf.2,174 ). In the forward running mode of the pump, i. e. when the calcium pump accumulates calcium and concomitantly hydrolyzes ATP, the terminal phosphate residue of ATP is transferred to the transport protein. The reaction depends on the presence of calcium ions in the external medium. In the reverse mode of the pump inorganic phosphate is incorporated into the transport protein. This reaction is inhibited when calcium ions are present in the external medium,... 

One category of intramolecular RNA catalysis produces a 2, 3 -cyclic phosphate and 5 -OH terminus on the reaction products. A number of small plant pathogenic RNAs (viroids, satellite RNAs, and virusoids), a transcript from a Neurospora mitochondrial DNA plasmid, and the animal vims hepatitis delta vims (HDV) undergo a self-cleavage reaction in vitro in the absence of protein. The reactions require neutral pH and Mg2... 

The reaction mixture contained in a final volume of 0.1 mL, 0.1 M sodium phosphate buffer (pH 6.8), 1.0 mM tryptamine, 5 mM secologanin, and 3 mM dithiothreitol. To inhibit glucosidase activity, 100 mM D(+)-gluconic acid-5-D-gluconolactone was included. The incubation was started by addition of 10 /xL of enzyme. After 30 minutes of incubation at 30°C, the reaction was stopped by addition of 0.1 mL of 5% trichloroacetic acid. Before centrifugation, 25 /xL of 8 mM codeine hydrochloride was added as the internal standard. HPLC analysis was performed on 4 /xL aliquots. With enzyme purified to a specific activity of 710 pkat per milligram of protein, the reaction was linear with time for 1 hour and with protein up to at least 50 /xg of protein during a 20-minute incubation period. 

Staphylococcus aureus V8 protease deaves proteins specifically after glutamate residues, provided that the following residue is not proline, or a further glutamate or aspartate. In ammonium bicarbonate buffer at pH 7.8, the enzyme is very specific for glutamate in Na-phosphate buffer at the same pH it also cleaves adjacent to aspartate. V8 protease is added at a ratio of 1-2% (w/w) to the protein substrate. Reaction can be stopped by freezing, or by the addition of PMSF or a2-rtiacroglobu-lin. 

Fumarase. Fumarase is determined by measuring the conversion to malate to fumarate. This assay was used in the opposite direction by Racker. The mixture contains, in a volume of 1.3 ml, 8.0 x 10 phosphate buffer (adjusted to pH 7.5), 4.0mM dithiothreitol, 8.0mM sodium malate, and 2 to 25 fxg of protein. The reaction is initiated with malate and is followed at 240 nm. The molar extinction coefficient of fumarate is 2.6 X lO cm /mole. 

The method described by Morrison and Bayse (1970) for the enzymic iodination of tyrosine can be readily adapted to the modification of proteins. The reaction mixture contains, in order of addition, L-tyrosine (8.1x10 M), KI (1.0 xlO M), lactoperoxidase (7.4 X 10 M), in 0.05 M K-phosphate buffer, containing 1 x 10 M EDTA, at pH 7.4. The iodination is initiated by the addition of H2O2 to a concentration of 1.0 x 10 M. The specific activity observed for lactoperoxidase under these conditions was 1.05 x 10 moles of L-3-iodotyrosine per min per mole of enzyme at 25°C. At pH 7.4, the rate of enzymatic conversion of L-3-iodotyrosine to L-3,5-diiodotyrosine was 0.34 that of monosubstitution (Morrison and Bayse 1970). The desired level of iodination can be attained by successive equimolar additions of KI and HjOj to the reaction mixture. In this manner, only a low concentration of H2O2 is maintained, minimizing oxidation reactions. The concentration of lactoperoxidase may be calculated from the millimolar extinction coefficient of 114 at 412 run, while the concentrations of stock H2O2 solutions may be determined from the absorbance at 230 nm and a molar extinction coefficient of 72.4 (Phillips and Morrison 1970). 

The adsorption of salivary proteins to enamel may include exchange reactions in which the protein phosphate groups replace surface phosphate in the enamel hydroxyapatite. (20.21)... 

Proteins that have tightly bound cofactors, such as heme proteins, photosynthetic reaction centers and antenna proteins, flavoproteins, and pyridoxal phosphate- and NAD-dependent enzymes, provide a variety of chromophores which have absorption bands in the visible and UV region. The CD bands associated with the chromophoric groups are frequently quite intense, despite the fact that the isolated chromophores are achiral in many cases, and therefore have no CD, or are separated from the nearest chiral center by several bonds about which relatively free rotation can occur, and therefore have only weak CD. The extrinsic or induced CD observed in the visible and near-UV spectra of the proteins can provide useful information about the conformation and/or environment of the bound chromophore, which usually plays a critical role in the function of the protein. 

Phosphorylation, or the attachment of a phosphate group to amino acid side chains, is one of the most abundant posttranslational modifications (PTMs) of proteins. Phosphorylation reactions are mediated by phosphotransferase enzymes, termed kinases, with ATP as the typical source of the transferred phosphoryl group. Ser, Thr, and Tyr are the most commonly phosphorylated residues in eukaryotes, while His and Asp phosphorylation has also been observed, predominantly in prokaryotes. Protein activity, localization, and structure as well as protein-protein interactions are all affected by protein phosphorylation [1, 2]. As kinases play integral roles in cellular signaling, dysregulated kinase function has emerged as a driver for many different disease states, including... 

The volume of protein extract solution was adjusted to 5.0 ml with a phosphate reaction buffer consisting of 20 mM phosphates at pH 7.4 and then incubated with 5.0 mg ultra clean carbon/cellobiose core (0.5 ml of stock solution) at 4o C for 24 hours. The final colloid consisted of carbon ceramic particulates coated with a layer of cellobiose and a more superficial layer of adsorbed HIV proteins. These colloidal ceramic viral decoys were prepared for use by clearing unadsorbed material by ultrafiltration dialysis with a stir cell [Filtron, Northborough, MA] mounted with a 100 kd filter and flushed with 100 ml of sterile phosphate buffered saline (injection grade) at 4°C under a N2 pressure head of 10 psi. 

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