Peptide phosphorylation

This amide, readily formed from an amine and the anhydride or enzymatically using penicillin amidase, is readily cleaved by penicillin acylase (pH 8.1, A -methylpyrrolidone, 65-95% yield). This deprotection procedure works on peptides, phosphorylated peptides, and oligonucleotides, as well as on nonpeptide substrates. The deprotection of racemic phenylacetamides with penicillin acylase can result in enantiomer enrichment of the cleaved amine and the remaining amide. An immobilized form of penicillin G acylase has been developed. ... 

Scheme 27 Synthesis of a Tyr(P)-Peptide via On-Line Peptide Phosphorylation 136 ...

S ATP -I- 338-SKTETSQVAPA-348 <1, 12> (<1, 12> peptide containing the last 11 amino acids of the C-terminal of bovine rhodopsin [20, 24] <1> phosphorylated at Ser-343, about 11% of the rate with rhodopsin, photo-activated rhodopsin-dependent, soluble active kinase catalyzes photoacti-vated rhodopsin-independent peptide phosphorylation [20] <12> only in the presence of photoactivated rhodopsin, which activates RK for peptide phosphorylation, also activated by metarhodopsin III, but not by opsin, up to 60% of the rate with photoactivated rhodopsin, light-dependent phosphorylation [24]) (Reversibility <1,12> [20,24]) [20, 24]... 

S ATP -I- peptide <3> (<3> synthetic [9,13,14] <3> e.g. Leu-Glu-Glu-Ser-Ser-Ser-Ser-Asp-His-Ala-Glu-Arg-Pro-Pro-Gly or Arg-Arg-Arg-Glu-Glu-Glu-Glu-Glu-Ser-Ala-Ala-Ala, role of acidic amino acids in peptide substrates, preference for negatively charged amino acids localized to the N-terminal side of a Ser- or Thr-residue, Ser-containing peptides are 4fold better than Thr-containing [9] <3> /1-ARK 1 and 2 prefer peptide substrates with acidic amino acids N-terminal to a Ser-residue [13] <3> /)-ARK 1 prefers peptides containing acidic residues on the N-terminal side of a serine or threonine, presence of activated receptor enhances peptide phosphorylation [14]) (Reversibility <3> [9,13,14]) [9, 13, 14]... 

Additional information <3> (<3> kinetics of peptide phosphorylation, influence of activated rhodopsin and /I2-AR on the value for different peptide substrates [14]) [14]... 

CPL spectroscopy may also be used to signal the nature of the ternary anion adduct. In the series of phospho-anion complexes of [Eu.la]3+, Figure 5, complexes of HP042 and glucose-6-phosphate give identical CPL spectra - consistent with their very similar NMR spectral profile. Adducts with phospho-tyrosine, N-acetyl phospho-Tyr and a short peptide (phosphorylated at the Pyr residue) are also near-identical, consistent with chemoselective binding of the Tyr-OP phospho-anion. 

Houseman et al. prepared a peptide chip by Diels-Alder-mediated reaction of kinase-peptide substrates with a self-assembled monolayer of alkanethiolates on gold surface. Peptide phosphorylation was determined by incubating the peptide microarrays with c-src protein-tyrosine kinase followed by quantitation with a phos-phoryl imager. In the presence of soluble inhibitors at a range of concentrations, dose-dependent inhibition of phosphorylation against a number of peptide substrates could be determined on a single chip. ... 

Shen TY, Wong CF, McCammon JA. 2001. Atomistic Brownian dynamics simulation of peptide phosphorylation. J. Am. Chem. Soc. 123 9107-11... 

Signal peptide, phosphorylation and glycosylation site analysis)... 

The importance of Ser-19 phosphorylation for actin-activated smooth muscle myosin MgATPase activity and for contraction of smooth muscle stimulated research to find out which amino acids, surrounding the serine, are required for the phosphorylation. Using synthetic peptide analogs of the native phosphorylation site comprising residues from Lys-11 to Ala-23, it was shown that Arg-16 had a strong influence on the kinetics of peptide phosphorylation (Kemp and Pearson, 1985). The location of Arg-16 in relation to Ser-19, as well as the distance between Arg-13 and Arg-16, was found to be important. Placement of Arg-16 at position 15 caused a complete switch in specificity from the natural Ser-19 phosphorylation site to Thr-18. Increasing the number of alanine residues between Arg-13 and Arg-16 in the model peptide also influenced the kinetics and site specificity of peptide phosphorylation. On the carboxyl side of Ser-19, Val-21 and Phe-22 influenced the of peptide phosphorylation, whereas Ala-23 was found not to be essential (Pearson et ah, 1986). 

Add 200 pL of cell lysate to mouse anti-human phosphorylated MBP antibody-coated 96-well microtiter plates at 4°C overnight. For the standards, add 0, 1, 2,4, and 8 pg of the custom-made peptide (phosphorylated MBP). Add IX PBS to wells for background signal detection. 

A CZE procedure for detection and assay of protein kinase and phosphatase activities in complex biological mixtures has been developed by Dawson et al. Thephosphorylated and dephosphorylated forms of several peptides were resolved using an uncoated capillary and a 150 mM phosphoric acid buffer at pH 2.0 or pH 5.0. Furthermore, the CZE-based assay was capable of resolving a peptide phosphorylated on different sites and permitted the quantification of each peptide. Since this application, much research has been done on protein kinase assays based on CE. 

Figure 2. Peptide phosphorylation. Quantitation of peptide phosphorylation was carried out as 2.4.2. Each point represents the mean and standard error of three determinations (s3nnbol designation is the same as for Fig. Ic).

Spengler, B. and Hester, A. (2008) Mass-based classification (MBC) of peptides highly accurate precnusor ion mass values can be used to directly recognize peptide phosphorylation. J. Am. Soc. Mass Spectrom., 19, 1808-1812. 

Phospholization of peptide may also affect the flrrx due to its additiorral negative charge. However, the transport rates of ANS were 1.6, 1.4 and 1.5 mmol/cm per h for the peptide, phosphorylated peptide and dephosphorylated peptide, respectively, indicating that phosphorylation and dephosphorylation has practically no effect on the transport rate. 

Gillece-Castro, B. L. Arnott, D. P. Bier, M. E. Land, A. P. Stults, J. T. Peptide phosphorylation site identification A new method based on ion trap mass spectrometry. In Proceedings of the 43rd Conference on Mass Spectrometry and Allied Topics, Atlanta, GA, May 21-26,19 >S, p. 302. 

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