Phosphorylation detection

Group II assays consist of those monitoring cellular second messengers. Thus, activation of receptors to cause Gs-protein activation of adenylate cyclase will lead to elevation of cytosolic or extracellularly secreted cyclic AMP. This second messenger phosphorylates numerous cyclic AMP-dependent protein kinases, which go on to phosphorylate metabolic enzymes and transport and regulatory proteins (see Chapter 2). Cyclic AMP can be detected either radiometrically or with fluorescent probe technology. 

The majority of functional assays involve primary signaling. In the case of GPCRs, this involves activation of G-proteins. However, receptors have other behaviors— some of which can be monitored to detect ligand activity. For example, upon stimulation many receptors are desensitized through phosphorylation and subsequently taken into the cell and either recycled back to the cell surface or digested. This process can be monitored by observing ligand-mediated receptor internalization. For... 

Phosphatidylinositol phosphates (PDPs) are phosphorylated derivatives of PI (phosphatidylinositol). PDPs that have been detected in cells include PI-3-P, PI-4-P (PEP), PI-5-P, PI-3,4-P2, PI-4,5-P2(PEP2), PI-3,5-P2, and PI-3,4,5-P3(PEP3). PEP and PEP2 are the most abundant forms ( 60%). 

DNA sequencing reveals the order in which amino acids are added to the nascent polypeptide chain as it is synthesized on the ribosomes. However, it provides no information about posttranslational modifications such as proteolytic processing, methylation, glycosylation, phosphorylation, hydroxylation of prohne and lysine, and disulfide bond formation that accompany mamra-tion. While Edman sequencing can detect the presence of most posttranslational events, technical hmitations often prevent identification of a specific modification. 

When smooth muscle myosin is bound to F-actin in the absence of other muscle proteins such as tropomyosin, there is no detectable ATPase activity. This absence of activity is quite unlike the situation described for striated muscle myosin and F-actin, which has abundant ATPase activity. Smooth muscle myosin contains fight chains that prevent the binding of the myosin head to F-actin they must be phosphorylated before they allow F-actin to activate myosin ATPase. The ATPase activity then attained hydrolyzes ATP about tenfold more slowly than the corresponding activity in skeletal muscle. The phosphate on the myosin fight chains may form a chelate with the Ca bound to the tropomyosin-TpC-actin complex, leading to an increased rate of formation of cross-bridges between the myosin heads and actin. The phosphorylation of fight chains initiates the attachment-detachment contraction cycle of smooth muscle. 

Creatine phosphate is formed from ATP and creatine (Figure 49-16) at times when the muscle is relaxed and demands for ATP are not so great. The enzyme catalyzing the phosphorylation of creatine is creatine kinase (CK), a muscle-specific enzyme with clinical utility in the detection of acute or chronic diseases of muscle. 

When AMP is heated under reflux in DMF, the 2, 3 -cyclic phosphate is formed, and cyclic phosphates can also be obtained from nucleosides and ortho-, pyro-, or poly-phosphoric acids under the same conditions. Promotion of phosphorylation by DMF is well known and the reaction with AMP is probably intermolecular as no 3, 5 -cyclic AMP can be detected. Minor products in the latter reaction are the 2, 3 -cyclic 5 -diphosphate and the 2 (30,5 -diphosphate. The synthesis of adenosine 2 (3 )-phosphate 5 -pyrophosphate has been achieved by the phosphoromorpholidate method used in a synthesis of Co A. ... 

A glyceryl 2-aminoethylphosphonolipid has been isolated from Tetrahymenapyriformis and (45) has been detected by g.l.c.-mass spectrometry in both the lipid and proteinaceous fractions of human brain. The zwitterionic (45) was converted into volatile (46) by acetylation and methylation. Phosphonolipids derived from A-methyl-(45) have been synthesised by acetylation of A-methyl-(45) and subsequent conversion to the phosphorochloridate for the phosphorylation step. °... 

Acetate kinase is phosphorylated by acetyl phosphate and it has been shown that the phosphoenzyme can synthesise ATP from ADP, and acetyl phosphate from acetate. The mode of decomposition of carbamyl phosphate in aqueous solution is pH dependent and can proceed with either the production of ammonia and carbon dioxide (equation 1), or cyanate (equation 2). No cyanate could be detected during the hydrolysis... 

The reaction starts with the binding of ATP to the H -liganded form of the enzyme, 2H Ei. In the presence of K, this binding is to the K -liganded enzyme form, 2K Ei or 2K E2, or to an occluded form between these two forms. The existence of such an occluded form has not yet been demonstrated, but its detection with filtration or column techniques similar to those used previously to measure occluded transported cations for Na,K-ATPase [113] will be very difficult, because of the rapid dissociation of from the enzyme [96]. Subsequent binding of Mg to 2H E] then leads to phosphorylation at an aspartyl residue [46,114]. The major phosphoenzyme then formed is a K -sensitive intermediate (2H E2-P), whereas a minor part (20%) exists as an ADP-sensitive intermediate (2H Ei-P) [92,93]. With... 

In conclusion, the steady-state kinetics of mannitol phosphorylation catalyzed by II can be explained within the model shown in Fig. 8 which was based upon different types of experiments. Does this mean that the mechanisms of the R. sphaeroides II " and the E. coli II are different Probably not. First of all, kinetically the two models are only different in that the 11 " model is an extreme case of the II model. The reorientation of the binding site upon phosphorylation of the enzyme is infinitely fast and complete in the former model, whereas competition between the rate of reorientation of the site and the rate of substrate binding to the site gives rise to the two pathways in the latter model. The experimental set-up may not have been adequate to detect the second pathway in case of II " . The important differences between the two models are at the level of the molecular mechanisms. In the II " model, the orientation of the binding site is directly linked to the state of phosphorylation of the enzyme, whereas in the II" model, the state of phosphorylation of the enzyme modulates the activation energy of the isomerization of the binding site between the two sides of the membrane. Steady-state kinetics by itself can never exclusively discriminate between these different models at the molecular level since a condition may be proposed where these different models show similar kinetics. The II model is based upon many different types of data discussed in this chapter and the steady-state kinetics is shown to be merely consistent with the model. Therefore, the II model is more likely to be representative for the mechanisms of E-IIs. 

A more recently introduced format is the AlphaScreen assay. The assay principal behind this technology has previously been described above. In the kinase format a biotinylated peptide is bound to a streptavidin donor bead, and a phopshospecific antibody is bound to the acceptor bead. When the substrate is phosphorylated, the beads come in close proximity and a signal is generated. An example using the assay for the detection of inhibitors of serine kinases is presented by Von Leo-prechting [26]. 

The second method also relies on site-specific chemical modification ofphosphoproteins (Oda et al., 2001). It involves the chemical replacement of phosphates on serine and threonine residues with a biotin affinity tag (Fig. 2.7B). The replacement reaction takes advantage of the fact that the phosphate moiety on phosphoserine and phosphothreonine undergoes -elimination under alkaline conditions to form a group that reacts with nucleophiles such as ethanedithiol. The resulting free sulfydryls can then be coupled to biotin to create the affinity tag (Oda et al., 2001). The biotin tag is used to purify the proteins subsequent to proteolytic digestion. The biotinylated peptides are isolated by an additional affinity purification step and are then analyzed by mass spectrometry (Oda et al., 2001). This method was also tested with phosphorylated (Teasein and shown to efficiently enrich phosphopeptides. In addition, the method was used on a crude protein lysate from yeast and phosphorylated ovalbumin was detected. Thus, as with the method of Zhou et al. (2001), additional fractionation steps will be required to detect low abundance phosphoproteins. 

Petruzzelli L, Herrera R, Arenas-Garcia R, Fernandez R, Bimbaum MJ, Rosen OM 1986 Isolation of a Drosophila genomic sequence homologous to the kinase domain of the human insulin receptor and detection of the phosphorylated Drosophila receptor with an anti-peptide antibody. Proc Natl Acad Sci USA 83 4710-4714 Podskalny J, McElduff A, Gorden P 1984 Insulin receptors on Chinese hamster ovary (CHO) cells altered insulin binding to glycosylation mutants. Biochem Biophys Res Commun 125 70-75... 

The monomeric metaphosphate ion itself commands a fair amount of attention in discussions of metaphosphates. It is postulated as an intermediate of numerous hydrolysis reactions of phosphoric esters 52 S4,S5) and also of phosphorylation reactions S6> kinetic and mechanistic studies demonstrate the plausibility of such an assumption. In addition, the transient formation of ester derivatives of meta-phosphoric acid — in which the double-bonded oxygen can also be replaced by thio and imino — has also been observed they were detected mainly on the basis of the electrophilic nature of the phosphorus. 

The extent to which 151 phosphorylates the aromatic amine in the phenyl ring is highly dependent upon the solvent. For instance, aromatic substitution of N-methylaniline is largely suppressed in the presence of dioxane or acetonitrile while pho.sphoramidate formation shows a pronounced concomitant increase. The presence of a fourfold excess (v/v) or pyridine, acetonitrile, dioxane, or 1,2-di-methoxyethane likewise suppresses aromatic substitution of N,N-diethylaniline below the detection limit. It appears reasonable to assume that 151 forms complexes of type 173 and 174 with these solvents — resembling the stable dioxane-S03 adduct 175 — which in turn represent phosphorylating reagents. They are, however, weaker than monomeric metaphosphate 151 and can only react with strong nucleophiles. 

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