Adenylate kinase effects

To facilitate conformational transitions in the before-mentioned adenylate kinase, Elamrani and co-workers scaled all atomic masses by a large factor thus allowing the use of a high effective simulation temperature of 2000K ([Elamrani et al. 1996]). To prevent protein unfolding, elements of secondary structure had to be constrained. 

Maker, H.S. Weiss, C. and Brannan. Amine-mediated toxicity The effects of dopamine, norepinephrine, 5-hydroxytryptamine, 6-hydroxy-dopamine, ascorbate, glutathione and peroxide on the in vitro activities of creatine and adenylate kinases in the brain of the rat. 

Treatment of 9-(/ -D-ribofuranosyluronic acid)adenine with diphenylphosphoro-chloridate and orthophosphate or tripolyphosphate yields (62) and (63), which, although unstable, inhibit rabbit AMP aminohydrolase and pyruvate kinase, respectively, with behaviour characteristic of active-site-specific reagents.98 Adenylate kinases from several sources are inactivated by iV6-[2- and 4-fluorobenzoyl]-adenosine-5 -triphosphates, with kinetics characteristic of active-site labelling, although these compounds were without effect on yeast hexokinase and rabbit pyruvate kinase.99... 

Because many cells maintain ATP, ADP, and AMP concentrations at or near the mass action ratio of the adenylate kinase reaction, the cellular content of this enzyme is often quite high. A consequence of such abundance is that, even after extensive purification, many proteins and enzymes contain traces of adenylate kinase activity. The presence of this kinase can confound the quantitative analysis of processes that either require ADP or are carried out in the presence of both ATP and AMP. Furthermore, the equilibrium of any reaction producing ADP may be altered if adenylate kinase activity is present. To minimize the effect of adenylate kinase, one can utilize the bisubstrate geometrical analogues Ap4A and ApsA to occupy simultaneously both substrate binding pockets of this kinase . Typical inhibitory concentrations are 0.4 and 0.2 mM, respectively. Of course, as is the case for the use of any inhibitor, one must always determine whether Ap4A or ApsA has a direct effect on a particular reaction under examination. For example. Powers et al studied the effect of a series of o ,co-di-(adenosine 5 )-polyphosphates (e.g., ApnA, where n =... 

Creatine phosphokinase activity has been reported to be minimally inhibited by hemolysis. Hemoglobin concentrations of 1.25 g/100 ml inhibit 5% and 2.5 g/100 ml, 12% (N5). However, in methods utilizing adenosine diphosphate in the reaction mixture, hemolysates containing 100 mg of hemoglobin per 100 ml may have apparent activities of 5-100 units/liter. The activity is presumably related to adenylate kinase in the erythrocyte (S33). In methods utilizing adenosine diphosphate in a coupled enzyme reaction with hexokinase and glucose-6-phosphatase, the inhibitory effect can be eliminated by adding sufficient adenosine mono-... 

Lin, Y Nageswara Rao, B.D. Structural characterization of adenine nucleotides bound to Escherichia coli adenylate kinase. 1. adenosine conformations by proton two-dimensional transferred nuclear overhauser effect spectroscopy. Biochemistry, 39, 3636-3646 (2000)... 

After these general comments let us further test the idea of thermodynamic buffering on an experimental basis by repeating the above experiment but this time in the presence of an inhibitor of adenylate kinase, namely, diadenosine pentaphosphate. As is depicted in Fig. 6b the buffering effect of the adenylate kinase is abolished by inhibiting this enzyme and it becomes now possible to drive the system beyond the state of optimal efficiency by increasing the hexokinase concentration in the medium. Note that it was not possible to measure points closer to level flow than the ones shown in the figure. This is due to technical reasons. At the lowest phosphate potentials the ATP/ADP ratios where of the order... 

In order to obtain a more intuitive insight into the mechanism of thermodynamic buffering we calculated the effects of thermodynamic buffering on the entropy production of the system. The entropy production of oxidative phosphorylation with an attached load is given in equation (8). A convenient way to introduce the contribution of the adenylate kinase reaction to this system is to consider L/ as an overall load conductance embracing the effects of the adenylate kinase reaction as well as the effects of the true extrinsic load conductance of the irreversible ATP utilizing... 

In this equation the 0.5 in the numerator takes into account the fact that ADP is about half as effective as ATP at carrying chemical energy. Thus, two ADPs can be converted into one ATP and one AMP by a reaction with an equilibrium constant of about l. The highly active enzyme that catalyzes this reaction (adenylate kinase) is universally distributed... 

Relative concentrations of ATP, ADP, and AMP as a function of the adenylate energy charge. The adenylate kinase reaction was assumed to be at equilibrium, and a value of 1.2 was used for its effective equilibrium constant in the direction shown in the equation in the text. 

Controversy exists concerning the cause and effect relationships between deficiencies of the enzymes adenylate kinase (AK EC 2.7.4.3) and adenosine triphosphatase (ATPase EC 3.6.1.3) and hemolytic disease. Since the consensus seems to be that there is no association, these enzymes will not be discussed here. 

Still, there seems to be something to this surface-to-volume ratio idea, because a number of normally monomeric enzymes oligomerize when they are found in hyperthermophilic organisms. Adenylate kinase from S. acidocaldarius is a trimer, for example, whereas nearly all other adenylate kinases are monomeric. Perhaps the best evidence that this effect may be important comes from an engineered protein, the repressor of primer (ROP). ROP is normally an all helical homod-imeric protein that denatures at 71°. Removal of five amino acids from a surface... 

These data demonstrate a marked abnormality in the ability of intact uremic RBC to metabolize adenosine (deoxyadenosine) to inosine (deoxyinosine) and hypoxanthine. The equivalent labelling by radioactive adenosine and deoxyadenosine of ATP pools suggests that the adenylate kinase pathway in uremic RBC is normal despite markedly elevated ATP levels. The latter may reflect decreased utilization of ATP in uremic RBC or an effect of high inorganic phosphate on ATP turnover (8). 

Figure 5. Effect of Protease Treatment of Intact Mitochondria on Adenylate Kinase content.

Murant, R.S., Gibson, S.L., and Hilf, R., Photosensitizing effects of Photofrin II on the site-selected mitochondrial enzymes adenylate kinase and monoamine oxidase. Cancer Res., 47, 4323, 1987. 

Sephadex G-75 and SDS polyacrylamide gel electrophoresis. There was no evidence for a subunit structure of the three similar enzymes due to SDS polyacrylamide gel electrophoresis after treatment with 1% 2-mercapto-ethanol and 1% SDS in 250 mM phosphate buffer, pH 7.1, at 90°C for 15 min. The apparent Km-values for ADP corresponded to 0.26 mM for R. palustris, 0.27 mM for R. sphaeroides and 0.24 mM for R. rubrum. ADP in excess had a strong inhibitory effect (Table 2). Competitive product inhibition was found for AMP, with Ki-values of 0.017 mM for R. palustris, 0.018 mM for R. sphaeroides and 0.014 mM for R. rubrum. A competitive inhibitor likewise was pi,P -di(adenosine-5 )pentaphosphate (=DAPP) with K -values of 0.020 jjM for R. palustris and R. sphaeroides, and 0.15 jjM for R. rubrum. Sulfhydryl reacting reagents like p-chloromercuribenzoate and iodoacetic acid were found to be non-inhibitory. Several divalent cations were effective in potentiating the enzymatic activity. The order of decreasing effectiveness was Mg2 > Ca > Co > Mn " " > Zn " ". The adenylate kinases from all three bacteria studied were activated maximally by a molar ratio of Mg2+ ADP of 1.0. 

The numbers calculated for the apparent Km(ADP) values, determined in the presence of 1 mM MgCl2 from Lineweaver-Burk plots revealed through their linear appearance that the velocity of the reaction was a function of ADP concentrations. This implies only one active absorption site for ADP on the enzyme by the substrate Mg-ADP as it has been concluded for spinach chloroplasts by Murakami and Strotmann (1978). The adenylate kinases were sensitive to AMP which functioned as an product inhibitor, as expressed by the obtained K (AMP) values, and ADP exhibited a substrate inhibition effect indicated by the Ki(ADP) values obtained (Table 2). DAPP likewise functioned as a competitive inhibitor in rather low concentrations (Table 2) in contrast to its action in beef heart mitochondria (Tomasselli and Noda, 1980). 

Since the adenylate kinases studied were not sensitive towards sulfhydryl reacting reagents, one may conclude that the action center of the enzyme does not hold free sulfhydryl groups. Since Mg " " was found to be most effective among the metal ions in stimulating the enzymatic activity one again may conclude that the Mg-ADP complex represents the real substrate. The molecular masses of the adenylate kinases of the three bacteria were calculated to be 33,500, 34,400 and 32,100 whereas reported numbers for the enzyme from other sources range from 52,000 to 21,000 (Albrecht, 1970). 

P-glycerate kinase reaction. Fig. 11 also shows the effect of sulfate ion on the spectrum of the stoichiometric equilibrium mixture. That is nearly equal to 1 for arginine kinase, creatine kinase and adenylate kinase for which indicates significant catalysis in either direction, or for 3-P-glycer-... 

It is perhaps not surprising that DA produces such mixed effects. The Di receptor is primarily linked to the activation of adenylate cyclase and then protein kinase A. The response to its activation will therefore depend on the ion channels and other proteins modulated by the kinase which can vary from one neuron to another. Since the D2 receptor is not so closely associated with just one G-protein, this gives it the potential for even more effects (see Greenhoff and Johnson 1997). 

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