Phosphate release

Phosphate release from actin. (a) Monomeric actin with ADP and Pi bound. The protein backbone (tube), ADP (grey spheres), and Ca -Pi (black spheres) are shown. The orientation of the spring indicates the pulling direction during P, unbinding. (b) Force exerted on the deprotonated (solid line) and protonated (dashed line) phosphate during the SMD simulations. 

Wriggers and Schulten, 1998] Wriggers, W., and Schulten, K. Investigating a back door mechanism of actin phosphate release by steered molecular dynamics. Biophys. J. Submitted. 

Orlova, A. Egelman, E.H. (1992). Structural basis for the destabilization of F-actin by phosphate release following ATP hydrolysis. J. Mol. Biol. 227, 1043-1048. 

In summary, therefore, solution and fiber biochemistry have provided some idea about how ATP is used by actomyosin to generate force. Currently, it seems most likely that phosphate release, and also an isomerization between two AM.ADP.Pj states, are closely linked to force generation in muscle. ATP binds rapidly to actomyosin (A.M.) and is subsequently rapidly hydrolyzed by myosin/actomyosin. There is also a rapid equilibrium between M. ADP.Pj and A.M.ADP.Pj (this can also be seen in fibers from mechanical measurements at low ionic strength). The rate limiting step in the ATPase cycle is therefore likely to be release of Pj from A.M.ADP.Pj, in fibers as well as in solution, and this supports the idea that phosphate release is associated with force generation in muscle. 

As mentioned above, pH directly influences substrate uptake rates of PAOs and GAOs. Filipe et al. (2001a) suggested that the ratio of phosphate release to acetate uptake by PAOs is directly influenced by pH with following the relationship [3]. 

Fig. 20.1. Correlation between the air/water partition coefficient, Kaw, determined from measurements of the surface pressure as a function of drug concentration (Gibbs adsorption isotherm) in buffer solution (50 mM Tris/HCI, containing 114 mM NaCI) at pH 8.0 and the inverse of the Michaelis Menten constant, Km obtained from phosphate release...

Negrin MA, Espino-Mesa M, Hernandez-Moreno JM. Effect of water soil ratio on phosphate release P, aluminium and fulvic acid associations in water extracts from Andisols and Andie soils. Eur. J. Soil Sci. 1996 47 385-393. 

Many methods have been developed in which a product of the reaction is chemically modified to produce a substance with a particular spectral property. The inorganic phosphate released by the hydrolysis of phosphate esters may be measured by simple chemical methods (Fiske and Subbarow) after the enzyme reaction has been stopped. Such techniques are often convenient but do not lend themselves to the measurement of initial velocity. 

Treatment with hot organic solvents was the next step in the tissue fractionation, to remove lipid-phosphorous and breakdown lipid-protein interactions. In the Schneider procedure, nucleic acids were then extracted in hot dilute trichloroacetic or perchloric acid, leaving a protein residue with any phosphoprotein links still intact. This method was to become particularly useful when 3H thymidine became the preferred label for DNA in the early 1960s. For investigations where both RNA and DNA were to be examined the Schmidt-Thannhauser process was often chosen. Here the lipid-extracted material was hydrolyzed with dilute sodium hydroxide releasing RNA nucleotides and any hydroxyamino acid bound phosphorus. DNA could be precipitated from the extract but the presence in the alkaline hydrolysate of the highly labeled phosphate released from phosphoprotein complicated... 

Using the standard curve, estimate the amount of phosphate released in the sample assay. 

HP formation may have been promoted by the acidic environment and stabilization of HP precursors through complexation with phosphate released during demineralization. Altogether, therefore, the one-month in situ model appeared unsuitable for simulating the Maillard reaction. Longer exposures would have enhanced the risk of bacterial degradation of the slices. 

Figure 5.8 Active transport is achieved by phosphoiylation and dephosphorylation of the transporter. The dephosphorylated form has a high affinity for the molecules whereas the phosphorylated form has a low affinity. This is achieved by a conformation change resulting from phosphorylation by ATP and dephosphorylation via phosphate release.

Table 4.3 Comparison of MurF ASMS screening-based binding constants and MurF activities from the radiolabeled phosphate release assay. Included from [10] with permission from SAGE Publications.

Cells R, Cox L, Hermosin MC, Cornejo J (1996) Retention of metamitron by model and natural particulate matter. Intern J Environ Anal Chem 65 245-260 Chaney RL (1989) Toxic element accumulation in soils and crops protecting soil fertility and agricultural food chains. In Bar Yosef B, Barrow NJ, Goldschmid J (eds) Inorganic contaminants in the vadose zone Springer, Heidelberg, pp 140-159 Charlatchka R, Cambier P (2000) Influence of reducing conditions on solubility of trace metals in contaminated soils. Water, Air Soil PoUut 118 143-167 Chien SH, Clyton WR (1980) Application of Elovich equation to the kinetics of phosphate release and sorption in soils. Soil Sci Am J 44 265-268... 

White et aP measured the rates of phosphate release during a single turnover of actomyosin nucleoside triphosphate (NTP) hydrolysis using a double-mixing stopped-flow spectrofluorometer, at very low ionic strength to increase the affinity of myosin-ATP and myo-sin-ADP-Pi to actin. Myosin subfragment 1 and a series of nucleoside triphosphates were mixed and incubated for approximately 1-10 s to allow NTP to bind to myosin and generate a steady-state mixture of myosin-NTP and myosin-NDP-Pi. The steady-state intermediates were then mixed with actin. 

In all these phosphate-releasing reactions, the several resonance forms available to Pi (Fig. 13-1) stabilize this product relative to the reactant, contributing to an already negative free-energy change. Table 13-6 lists the standard free energies of hydrolysis for a number of phosphorylated compounds. 

Phospholipase C, which initiates the release of phosphatidylinositol derivatives, also requires Ca2+ for activity. It is difficult to determine whether release of Ca2+ is a primary or secondary response. There are three isoenzyme types of phospholipase C-(3, y, and 8- and several sub forms of each with a variety of regulatory mechanisms.298 3"" For example, the y isoenzymes are activated by binding to the tyrosine kinase domain of receptors such as that for epidermal growth factor (see Fig. 11-13). In contrast, the (3 forms are often activated by inhibitory G proteins and also by G, which is specific for inositol phosphate release. 

Initial biochemical studies indicate that agonist binding was regulated by guanyl nucleotides, implying that the receptor belongs to the superfamily of receptors coupled to G proteins. In addition, various intracellular responses were found to be associated with Hi-receptor stimulation inositol phosphate release, increase in Ca2+ fluxes, cyclic AMP or cyclic GMP accumulation in whole cells and arachidonic acid release [1],... 

A much simpler situation is the anion loss from an a-alkoxyalkyl radical with a leaving group in the P-position, such as the phosphate release from the 2-phosphato-methoxyethyl radical (Behrens et al. 1978 for some further reactions of P-(phosphatoxy)alkyl radicals see Whitted et al. 1999 Crich et al. 2000). This reaction only proceeds by a heterolytic cleavage into a radical cation and a phosphate ion. The rate of this reaction strongly depends on the protonation state of the phosphate group [reactions (40)-(44)]. 

Table 6.6. Approximate rate constants of phosphate release from some a-methoxy-p-phosphatoalkyl radicals. (Behrens et al. 1978)...

Phosphate release yields from some 3 - and 5 -mononucleotides are compiled in Table 10.28. The data in N20-saturated solutions indicate that phosphate release from the 3 -position is about twice as efficient than that from the 5 -posi-tion. Correcting for the fact that under 02 the OH yield is about halved, there is a protecting effect for 3 -AMP 3 -dAMP and 3 -GMP but an enhancement in the case of 3 -CMP and 3 -UMP. For the 5 -nucleotides, there is always an increase... 

Table 10.28. Gvalues(unit 10 7molJ ) of inorganic phosphate release in they-radiolysis of aqueous solutions of some 3 - and 5 -monoucleotides (Raleigh et al. 1974). Data from...

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