Phosphorylation rate

Hafher, R.P., Brown, G.C.. Brand, M.D. (1990). Analysis of the control of respiration rate, phosphorylation rate, proton leak rate and proton motive force in isolated mitochondria using the top-down approach of metabolic control theory. Eur. J. Biochem. 188,313-319. 

It has been established by substitution of for Mg that, prior to phosphorylation, the divalent cation binds at a cytosolic site with a stoichiometry of about 1 mol per phosphorylation site [124,125]. These experiments also demonstrated that the phosphorylation rate is sensitive to the nature of the divalent cation bound. With Mg bound, the phosphorylation rate is about 20 times faster than with Ca bound. The divalent cation dissociates after dephosphorylation, suggesting that it is tightly bound to the phosphoenzyme during the reaction cycle. It was also demonstrated that the type of divalent cation that occupies the divalent cation site required for phosphorylation is important for the step 2K E2-P to 2K E2 P to 2K E2 [124,125]. With Mg bound, the 2K E2-P conformer is -sensitive, whereas with Ca bound, the intermediate is -insensitive. 

As with Km, the effect of pH on Fmax cannot be described by a simple ionization curve. With calf intestinal phosphatase, the log ym8X curve for a monoester substrate is sigmoid (143, 162) or, in the case of synovial phosphatase, extremely shallow (76). Both curves approach a maximum value at alkaline pH. Barman and Gutfreund, however, found that milk phosphatase had an optimum at pH 10 with only 60% activity at pH 11 (83). This is by no means typical since placental phosphatase has been shown to be fully active with the same substrate, p-nitrophenyl phosphate at pH 11.5 (85). With PP as substrate there is evidence that an optimum in Vmax is reached at considerably lower pH values (8.5-9.2) (116, 117, 164). A pH-activity curve for calf intestinal phosphatase is given in Fig. 3. Features to note are the plateau in activity around pH 7, corresponding to a minimum in the phosphorylation rate constant, and a change in rate determining step at about pH 6 (165). 

The phosphorylation rate (v) of the isolated glucokinase enzyme is given by the Michaelis-Menten expression [27] ... 

Fig. 2. (Continued) method (regression model) third row SUV image (SUV), global metabolic rate (influx), and distribution volume (DV). The parametric images are obtained by applying the Patlak model to the data fourth row SUV image (SUV), phosphorylation rate (k3), and transport rate (kl). The images are obtained by a voxel-based application of the two-compartment model.

The mitochondrial Mg2+ ATPase activity from liver and brain was inhibited by about 50-60% after an administration of 100 mg Al kg 1 body weight in the diet for a period of 90-120 days. By contrast, in the heart mitochondria, the ATPase activity increased from 73 to 212% after this treatment [38]. In this work, it was also found that ADP phosphorylation rates were decreased by 46% and that the changes in the ATPase activity, in general, were paralleled to those of the respiratory rates. The author suggested that these results imply that the effects of Al3+ treatment on respiratory activity and the ATPase activity go hand in hand. Curiously, liver and brain mitochondria presented doubled aluminum concentration and impaired respiration rate, whereas the heart mitochondria, that accumulated 11 times higher amount of aluminum, presented stimulation of respiration. Thus, an indirect action of aluminum in this tissue could be suggested. 

Fractionation of proteins by strong cation exchange (SCX) chromatography, followed by IMAC enrichment of phosphopeptides from SCX fractions, led to a comprehensive identification of phosphoproteins of PSD isolated from mouse brain using LC-MS/MS (Trinidad et al. 2006). In this study, phosphorylation site(s) were mapped to 287 proteins from a total of 1,264 unique proteins identified. This translates into a 23% phosphorylation rate, comparable to an expected 33% rate in the general proteome (Johnson et al. 2005). The 287 phosphoproteins were derived from a total of 998 unique phosphorylated peptides, and the phosphorylations were mapped to 723 unique sites. Most of these occurred on serines, to a lesser extent on threonines, and only minimally on tyrosines (Figure 5A). 

In order to enhance affinity and selectivity for Brc-Abl, we modified the inhibitor methylating at positions I and II (Fig. 7.5d). The synthesis of the wrapping prototype recapitulates imatinib synthesis [38], as described in [39], To test whether the specificity and affinity for Brc-Abl improved, we conducted a spectrophotometric kinetic assay to measure the phosphorylation rate of peptide substrates in the presence of the kinase inhibitor at different concentrations. This assay couples production of adenosine diphosphate (ADP), the byproduct of downstream phosphorylation, with the concurrent detectable oxidation of reduced nicotinamide adenosine dinucleotide (NADH). The oxidation results upon transfer of phosphate from PEP (phospho-enolpyruvate) to ADP followed by the NADH-mediated reduction of PEP to lactate. Thus, phosphorylation activity is monitored by the decrease in 340 nm absorbance due to the oxidative conversion NADH->-NAD+ [34, 39]. 

Fig. 8.6 Kinetic inhibitory impact of compounds WBZ 4 and imatinib determined by measuring phosphorylation rates through spectrophotometric assays of C-Kit and Bcr-Abl kinase activity. The kinases are inhibited by WBZ 4 (squares) and by the parental compound (triangles). Phosphorylation rate plots are given for Bcr-Abl (red) and C-Kit (blue). The open red symbols correspond to inhibition of unphosphorylated Bcr-Abl, while the full red symbols correspond to the Tyr412-phosphorylated form. Error bars represent dispersion over five runs for each kinetic assay. Reprinted from the [14], copyright 2007 with permission from the American Society for Clinical Investigation...

Fig. 9 NMR spectral changes revealed by a 5 mm solution of hyperpolarized choline upon undergoing phosphorylation by 0.5 units of choline kinase, (a) Emergence of the new phosphocholine resonance shown by directly detected single-pulse N NMR spectroscopy experiments, (b) Emergence of the H NMR resonance associated with the methylenes in the C2-position of phosphocholine, (c) Comparison between the expected enzyme kinetics of kinase with results afforded by the N- ( ) and H-detected ( ) hyperpolarized experiments, as derived from the relative peak ratios of the NMR peaks in (a) and (b). The straight line illustrates the best fit of the combined set of data points, and corresponds to an initial phosphorylation rate of 0.3 mM min under these conditions. Reproduced with permission from [55]...

The ability of CheY P to generate clockwise rotation was demonstrated in vivo by expressing CheY from a low-copy-number plasmid (under the control of the lac promoter) in an E. coli strain deleted for the genes cheB, cheY, and cheZ [18, 169]. The absence of CheB resulted in fully methylated MCPs and, consequently, in highly active CheA and in rapid phosphorylation rate of CheY. The absence of CheZ greatly reduced the rate of CheY dephosphorylation. As a result of the enhanced... 

FIGURE 2. Normalized phosphorylation rate vs. ADP concentration. P 8 mM, hexylamine 300 yM. The lines are computed hyperbolas (see text). 

FIGURE 3. ApH-dependent ADP inhibition of phosphorylation rate Vp. The theoretical V is extrapolated from computed Michaelis curve. ApH varied by light, P 8 mM, hexylamine 300-500 yM. Inset direct illustration of inhibition (only closed symbols are used to draw Michaelis curves). 

Among purple bacteria, energy transformation rates are rather low in Rhodopseudomonas palustris (ATCC 11168) due to a weakly membrane-bound RpFi-ATPase. Cell breakage by high pressure or sonication treatment resulted in a crude chromatophore preparation with a lag phase of about 6 h where no energy induced uptake or phosphorylation rate could be observed (Fig.1), caused by RpFi-solubilization and passive H+ flow through the RpFi-leaks (1). The light-induced H+ uptake could be restored by N,N dicyclohexylcarbodiimide (DCCD) which seals the RpFi-leaks. 

A 100% depletion of RpFi from washed chromatophores could be achieved by sonication in the presence of EDTA and washing the membranes again (Tab.1). The totally depleted chromatophores did not perform any ATPase activity or phosphorylation rate. - Depleted chromatophores recoupled the isolated and purified RpFi-ATPase protein, and thereby restored light-induced H+ uptake (in the presence of cytochrome c) by 90% and photophosphorylation by 75%. NADH-dependent oxidative phosphorylation was reconstituted by 65%. 

The three sites of phosphorylation suspected to exist in intact mitochondria have also been recognized in the phosphorylating particle where these sites do not function at full capacity. Although the first of these sites (between NAD and the flavoprotein) operates at 90% of its full capacity, the second (between cytochrome c and cytochrome c ) is active at only 20% of its normal phosphorylating ability, and the third (between cytochrome c and oxygen) reaches 70% of the normal phosphorylating rates. 

Figure 27.10 A double-reciprocal plot for the initial phosphorylation rate v of glycerol kinase against the concentration of the substrate aminopropanediol, at various concentrations of MgATP. (o) 0.126mM ( ) 0.504 mM ( ) 2.52 mM. Source J Kyte, Mechanism in Protein Chemistry, Garland, New York, 1995. Adapted from WB Knight and WW Cleland, Biochemistry 28, 5728 (1989).

Similar results were obtained by Dallam and Anderson (1957) on rat liver mitochondria. After inactivation by irradiation, the oxidative phosphorylation was reduced to a third of the initial value, while the oxidation was not impaired. Addition of vitamin Ki restores the phosphorylation. From the observation that two-thirds of the phosphorylation rate is dependent on vitamin Ki, they conclude that two of the three phosphorylation steps are concerned with vitamin Ki. On this point they do not agree with Martius and Nitz-Litzow (1953,1954a,b), who considered vitamin K to he involved in only one step. As phosphorylation coupled with oxidation of reduced cytochrome c is not impmred by irradiation (Dallam and Anderson, 1958), vitamin K action seems to be related to the phosphorylating S3rstem of the chain between DPN and cytochrome c. 

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