Phosphates modification with

The following protocol describes the modification of DNA or RNA probes at their 5 -phosphate ends with a bis-hydrazide compound, such as adipic acid dihydrazide or carbohydrazide. A similar procedure for coupling the diamine compound cystamine can be found in Section 2.2 (this chapter). 

Among condensed phosphate systems with polyvalent cations, the very complicated calcium salts have been much studied and a great number of compounds have been detected, often with several modifications (5, 27, 28, 34, 54, 79, 186, 137, 191, 198, 204, 211, 276, 805, 830). In this system special interest attaches to the occurrence of crystalline cross-linked phosphates (see Section V) and of calcium pentaphosphate, CayiPsOis (see Section IV,D,/). The end-product obtained by heating Ca(H2P04)2 in the free atmosphere is the high-molecular calcium polyphosphate, which crystallizes in several forms. The condensed strontium phosphates (246, 805) are similarly complicated and cannot readily be made the subject of a brief review. 

The catalytically essential nature of tyrosine 85 and its proximity to the substrate binding site and to tyrosine 115 were demonstrated from studies of modification with tetranitromethane (71) and from studies of intramolecular cross-linking of aminotyrosyl residues (72). The bro-moacetamidophenyl (69) and diazonium (70) reagents obtained from aminophenyl-pdT both react selectively and exclusively with tyrosine 85. This residue is situated, stereochemically, such that its hydroxyl group can interact with the 3 -phosphate of pdTp. 

Dissolve a hydrazide-containing enzyme or other protein at a concentration of 10 mg/ml in 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2. For the preparation of a hydrazide-activated enzyme see Chapter 16, Section 2.4. For modification with a hydrazide-containing probe, such as biotin-hydrazide, use a concentration of 5 mM in the phosphate buffer. For conjugation through the amine groups of a secondary molecule, dissolve the amine-containing protein at 10 mg/ml in 0.2 M sodium carbonate, pH 9.6. 

Figure 2. Low-resolution XPS spectra of a pyrolytic graphite electrode surface. Key A, prior to modification B, following a l-h 02 rf plasma treatment C, after reduction with LiAlHi D, after modification with cyanuric chloride E, after attachment of hydroxymethyl ferrocene and F, following electrolysis in pH 7 phosphate buffer.

The TLR4-MD2 hetero-dimer has complex ligand specificity. It can be activated by structurally diverse LPS molecules, and minor changes in synthetic derivatives of LPS can abolish their endotoxic potency (Raetz and Whitfield, 2002 Rietschel et al., 1994). The diversity in potency of LPS is derived from variance within lipid A, as observed in both the number and the length of fatty acid side chains and the presence of terminal phosphate residues with a variety of modifications. Optimal lipid A potency is achieved with bi-phosphorylated, hexa-acylated, lipid A species (Raetz and Whitfield, 2002). Lipid A moieties that deviate from this pattern often demonstrate a significant decrease in endotoxic activity (Alexander and Rietschel, 2001). 

The role of sulfhydryl groups in phosphorylase activity is not yet known. Modification with p-chloromercuribenzoate certainly causes loss of activity, but it is uncertain whether die sulfhydryl groups form a part of the active center, or are more important for the maintenance of the active conformation of the protein molecule. The second possibility seems the more likely, as p-chloromercuribenzoate causes rabbit-muscle phosphorylase to split up into subunits. Also, the inhibition by this reagent is not parallel to mercaptide formation, and adenosine 5 -phosphate (which causes changes in the conformation of the enzyme) protects against the inhibition. Unmodified sulfhydryl groups are believed to be important for the binding of adenosine 5 -phosphate. ... 

Traditional methods to map posttranslational modification sites, like those of phosphorylation, have been anchored by protein digest and mass spectroscopic (MS) approaches (for a review on the classic evaluation and for MS analyses of O-glycans, see Reference (56)). Unfortunately, like many posttranslational modifications, O-GlcNAcylation occurs routinely on a protein population with substoichiometric frequency, which results in a very small detectable population of a O-GlcNAc-modified product. Also, much like O-phosphate additions, the protein-O-GlcNAc bond is labile and is detached by collision-induced dissociation (CID) during MS analysis. Often, the bond is lost before it can be detected on the peptides analyzed (57, 58). Phosphate modifications, however, can overcome this limitation by emiching the peptide mixtures... 

The whole virus modification with the thiuram disulfides. HIV-1 (MN) (equivalent of 1 mg p24 CA) in 20 ml was treated at 37 C with 50 mM test compoimds for 1 h in sodium phosphate buffer, pH 7. Samples were centrifuged for 1 h at 17,000 g at 4 °C to pellet the virus and remove the drug. Samples for electrophoresis were run under non-reducing conditions. The gels (4-20%) were supplied by NOVEX (San Diego, California). Then proteins were transferred onto PVDF membranes (Towbin et. al., 1979), stained with 0.5% (w/v) Ponceau S and detected by... 

The developments of the Lewis base-modified zirconia and mixed-oxide containing zirconia as stationary phases for high-performance liquid chromatography (HPLC) are reviewed. In this context, the preparation methods of porous spherical zirconia, and zirconia supports for HPLC based on modification with fluoride, phosphate, phos-phonate, carboxylic acid, phenols, and protein, as well as cyclodextrin derivative, are covered. The application of modified-zirconia in capillary electrochromatography (CEC) is also discussed. 

Interaction with the matrix (untreated calcium carbonate in PE decreases tensile strength but after phosphate modification tensile strength is increased glass beads may decrease or increase tensile strength depending on their interfacial adhesion mica and talc give a similar effect in PP polyamide fiber does not reinforce natural rubber because of its lack of interaction)... 

Chen, S.S., and Engel, PC. (1975) The equilibrium position of the reaction of bovine liver glutamate dehydrogenase with pyridoxal5 -phosphate. A demonstration that covalent modification with this reagent completely abolishes catalytic activity. Biochem. J. 147, 351-358. 

Phosphate modifications T7 polymerase does not discriminate significantly between NTP and [aS] NTP (NEN). To obtain the appropriate modification level the NTP is substituted with [aS] NTP to the desired level. A standard transcription protocol, such as the one described in Section 2.3.1, can be used. 

Concurrent base and phosphate modification The current examples of cotranscriptional incorporation of modified bases are rather limited,14-16 and the modified NTPs are generally not commercially available. The standard buffer conditions can be employed, but the modified nucleotides are incorporated with different efficiencies e.g. 5% incorporation of inosine at the G positions requires 40% inosinetriphosphates in the reaction mixture, while 7-methyl-guano-sine is incorporated as readily as GTP. 

I, 4-bisphosphate and inositol 1,4,5-trisphosphate [59,60]. More recent studies, however, have shown that acid conditions are necessary for the extraction of these latter inositol phosphates [238]. With this modification to the experimental protocol it is possible to demonstrate a rapid accumulation of inositol trisphosphate in slices of guinea-pig cerebellum in response to histamine [238]. The rate of accumulation of inositol trisphosphate in this brain region is more rapid than that of the less polar inositol phosphates and the highest rate of accumulation is normally achieved during the first 5 min of... 

---