Muscular fatigue: effects of hydrogen ions and inorganic phosphate

During muscular fatigue two metabolites, hydrogen ions (H+) and inorganic phosphate (Pi), increase in concentration. The effect of increase in [H+] has been modeled mathematically for a system containing creatine kinase (EC 2.7.3.2), adenylate kinase (EC 2.7.4.3), and the appropriate concentrations of their substrates. Assuming that no other equilibrium reactions are involved, the result of acidification should be a useful increase in the ratio [ATP]/[ADP]. It is also shown by a reanalysis of earlier 31P NMR studies that the observed combination of increased [H+] and increased [Pi] leads to an increase in the monobasic phosphate concentration [Pi-] that is inversely proportional to the force of contraction. This suggests that Pi- may be a direct inhibitor of the actomyosin ATPase system.     

Regulation of (Na++K+)-ATPase by inorganic phosphate: pH dependence and physiological implications

Inhibition of Na++K+-dependent ATPase activity by Pi was maximal in the pH range of 6.1-7, but decreased with increasing pH in the range of 7-8.5. Ki of Pi was 2.8 mM at pH 7.1, and 12 mM at pH 7.8. K+-dependent phosphorylation of the enzyme by Pi, which is thought to be responsible for inhibition of ATPase activity, also decreased with increasing pH. The data suggest that (a) previously observed requirement of high Pi concentrations for inhibition of ATPase activity and associated pump fluxes may have been due to high pH of the assays; (b) at normal values of intracellular pH the pump may be partially inhibited by intracellular Pi; and (c) this effect of Pi may be amplified or dampened with alterations in intracellular pH and ATP/Pi ratio.     

Improved method for estimation of inorganic phosphate: implications for its application in enzyme assays

The conventional method of Fiske and Subba Row for the estimation of inorganic phosphate (Pi) is although rapid, but suffers from the disadvantage that the color is unstable and hence the optical density (OD) measurements have to be carried out within a short time span of 8-12 min. This poses a restriction on the number of samples, which can be handled in a batch. Although, modified procedures involving use of alternate reducing agents/or increasing the concentration of H2SO4 in conventional method have been subsequently developed, but the problem of color stability could not be solved. In addition, the use of higher concentrations H2SO4 has rendered the methods unsuitable in enzyme assays, especially if the acid labile phosphate containing substrates have been used. In the present study, attempts have been made to suitably modify the method to improve the stability of the color and sensitivity and also for its applicability in enzyme assays, especially when acid labile phosphate containing substrates such as ATP is used. We used the higher concentrations (0.625, 0.8 and 1.0 N) of H2SO4 rather than 0.5 N used in the conventional assay procedures. Under these conditions, the reagent blanks do not develop color for up to 24 h, whereas the intensity of the molybdenum blue color in the standard and/or experimental tubes increased with time reaching optimum value at 24 h. Simultaneously, the absorption maximum shifts from 660 nm to 820 nm. The highest concentration of H2SO4 (1.0 N) is found to be the most effective in the process of color development. The sensitivity of the method is from 1.7 to 2.1 times higher, as compared to the conventional Fiske and Subba Row method for the measurements carried out at the end of 15 min at 820 nm and with the highest concentration of H2SO4 (1.0 N); the sensitivity increased 4.8-fold at the end of 24 h. Presence of glucose and sucrose (1-10 mM), NaCl and KCI (5-100 mM), MgCl2 (1-10 mM) and BSA (10 to 500 microg per assay tube) do not interfere either with color development or with OD measurements. The extent of ATP hydrolysis is 1.6 to 3.4% for up to 1 hi, depending upon the concentration of H2SO4 used. Only negligible hydrolysis of G6P is observed under these conditions. These results suggest that the presently modified method is suitable for Pi analysis in the enzyme assays, in the presence of labile phosphate containing substrates.     

The temperature dependence of shoot hydraulic resistance: implications for stomatal behaviour and hydraulic limitation

Recent soil pressurization experiments have shown that stomatal closure in response to high leaf–air humidity gradients can be explained by direct feedback from leaf water potential. The more complex temperature‐by‐humidity interactive effects on stomatal conductance have not yet been explained fully. Measurements of the change in shoot conductance with temperature were made on Phaseolus vulgaris (common bean) to test whether temperature‐induced changes in the liquid‐phase transport capacity could explain these temperature‐ by‐humidity effects. In addition, shoot hydraulic resistances were partitioned within the stem and leaves to determine whether or not leaves exhibit a greater resistance. Changes in hydraulic conductance were calculated based on an Ohm’s law analogy. Whole‐plant gas exchange was used to determine steady‐ state transpiration rates. A combination of in situ psychrometer measurements, Scholander pressure chamber measurements and psychrometric measurements of leaf punches was used to determine water potential differences within the shoot. Hydraulic conductance for each portion of the pathway was estimated as the total flow divided by the water potential difference. Temperature‐induced changes in stomatal conductance were correlated linearly with temperature‐induced changes in hydraulic conductance. The magnitude of the temperature‐induced changes in whole‐plant hydraulic conductance was sufficient to account for the interactive effects of temperature and humidity on stomatal conductance.     

Divalent metal ion, inorganic phosphate, and inorganic phosphate analogue binding to yeast inorganic pyrophosphatase

Four different techniques, equilibrium dialysis, protection of enzymatic activity against chemical inactivation, 31P relaxation rats, and water proton relaxation rates, are used to study divalent metal ion, inorganic phosphate, and inorganic phosphate analogue binding to yeast inorganic pyrophosphatase, EC 3.6.1.1. A major new finding is that the binding of a third divalent metal ion per subunit, which has elsewhere been implicated as being necessary for enzymatic activity [Springs, B., Welsh, K. M., & Cooperman, B. S. (1981) Biochemistry (in press)], only becomes evident in the presence of added inorganic phosphate and that, reciprocally, inorganic phosphate binding to both its high- and low-affinity sites on the enzyme is markedly enhanced in the presence of divalent metal ions, with Mn2+ causing an especially large increase in affinity. The results obtained allow evaluation of all of the relevant equilibrium constants for the binding of Mn2+ and inorganic phosphate or of Co2+ and inorganic phosphate to the enzyme and show that the high-affinity site has greater specificity for inorganic phosphate than the low-affinity site. In addition, they provide. The results obtained allow evaluation of all of the relevant equilibrium constants for the binding of Mn2+ and inorganic phosphate or of Co2+ and inorganic phosphate to the enzyme and show that the high-affinity site has greater specificity for inorganic phosphate than the low-affinity site. In addition, they provide. The results obtained allow evaluation of all of the relevant equilibrium constants for the binding of Mn2+ and inorganic phosphate or of Co2+ and inorganic phosphate to the enzyme and show that the high-affinity site has greater specificity for inorganic phosphate than the low-affinity site. In addition, they provide evidence against divalent metal ion inner sphere binding to phosphate for enzyme subunits having one or two divalent metal ions bound per subunit and evidence for a conformational change restricting active-site accessibility to solvent on the binding of a third divalent metal ion per subunit.     

A simple micro-assay for inorganic phosphate

Modification of two assay procedures (Van Belle, H. (1970) Anal. Biochem.33, 132–142 and Itaya, K., and Ui, M. (1966) Clin. Chim. Acta14, 361) has allowed the development of a manual assay for inorganic phosphate of high simplicity and sensitivity. Total analysis requires only three reagents and is accomplished in less than 5 min, and smaples containing less than 1 μg/ml of inorganic phosphate may be detected. This assay retains a unique principle of the former two, complexation (instead of reduction) of the phosphomolybdate heteropoly complex with an appropriate triphenylmethane dye (malachite green, methyl green). Use of detergents has been eliminated and some further properties of the dyes, the assay, and the latter's applicability to a coupled enzyme system for phosphomonoester and phosphodiester analysis are discussed. Consideration is also given to the associated phenomena of transphosphorylation. (Dayan, J., and Wilson, I. B. (1964) Biochim. Biophys. Acta81, 620–623).     

The temperature dependence of human erythrocyte transport of phosphate, phosphite and hypophosphite

The temperature dependence of the erythrocyte anion transport protein (Band 3 or AE1) mediated influx of three nonspherical substrates, the divalent anions phosphate and phosphite, and the monovalent hypophoshite, were determined. Phase transitions were found in the temperature dependence of the influxes of all three anions. The 95% confidence limits for the transition temperatures were: 34.6-38.1 degrees C, 7.4-9.1 degrees C and 6.7-9.7 degrees C for phosphate, phosphite and hypophosphite, respectively, while the critical influx rates at the transitions were 29-50, 64-102 and 26-58 ions/s per carrier, respectively. That the critical rates rather than the transition temperatures are of similar magnitude indicates that the transitions are related to transport mechanisms rather than to thermal protein conformational changes. These critical rates are two orders of magnitude lower than those reported for the self-exchange of Cl- and Br- (Brahm, J. (1977) J. Gen. Physiol. 70, 283-306). The critical rate of monovalent hypophosphite is similar to that of divalent phosphate and phosphite, but not to that of Cl- indicating that this effect is mediated by the structure of the substrate rather than by its charge. The disparity in the rates rc at which phase transitions occur in AE1-mediated transport of spherical and nonspherical anions indicates a difference in the interaction between the two classes of anions and the protein.     

A microtiter plate assay for inorganic phosphate

A microtiter assay for the detection of picomolar quantities of inorganic phosphate has been described. The assay, linear between 50 and 1000 pmol of inorganic phosphate, is simple and rapid, with results obtainable in several minutes. Results from 5'-nucleotidase and (Ca2+ + Mg2+)ATPase assays using this method were compared with conventional phosphate assays and showed a high degree of correlation. The high sensitivity of this assay and the small sample size needed allows its widespread use in biochemical studies involving the generation of inorganic phosphate.     

Polynucleotide phosphorylase-based photometric assay for inorganic phosphate

Polynucleotide phosphorylase is a prokaryotic enzyme that catalyzes phosphorolysis of polynucleotides with release of nucleotide diphosphates. By taking advantage of this property, we developed a photometric assay for inorganic phosphate. In the presence of polyadenylic acid, phosphate is converted into adenosine 5'-diphosphate (ADP) by this enzyme. ADP then reacts with phosphoenolpyruvate in a pyruvate kinase-catalyzed reaction, thus giving rise to adenosine 5'-triphosphate and pyruvate. Finally, pyruvate oxidizes reduced nicotinamide adenine dinucleotide (NADH) through the action of L-lactate dehydrogenase, with concomitant decrease in absorbance at 340 nm. As expected, in this detection system 1 mol of NADH was oxidized per mole of phosphate. The assay showed an excellent reproducibility, as the standard deviations never exceeded 5%. It also was shown to be unaffected by several compounds that are regarded as major interferents of the traditional colorimetric assays. Absence of interference was also demonstrated when determining phosphate content in different biological samples, such as human serum and perchloric acid extracts from Escherichia coli, yeast, and bovine liver. An E. coli strain overexpressing His-tagged polynucleotide phosphorylase developed in our laboratories allowed quick and straightforward purification of enzyme, making the assay feasible and convenient. Since all other reagents required are inexpensive, the assay represents a cheaper alternative to commercially available phosphate assay kits.     

A biosensor for inorganic phosphate using a rhodamine-labeled phosphate binding protein

A novel biosensor for inorganic phosphate (Pi) has been developed based on the phosphate binding protein of Escherichia coli. Two cysteine mutations were introduced and labeled with 6-iodoacetamidotetramethylrhodamine. When physically close to each other and correctly oriented, two rhodamine dyes interact to form a noncovalent dimer. In this state, they have little or no fluorescence, unlike the high fluorescence intensity of monomeric rhodamine. The labeling sites were so placed that the distance and orientation between the rhodamines change as a consequence of the conformational change associated with Pi binding. This movement alters the extent of interaction between the dyes. The best mutant of those tested (A17C, A197C) gives rise on average to approximately 18-fold increase in fluorescence intensity as Pi binds. The kinetics of interaction with Pi were measured at 10 degrees C. Under these conditions, the fluorescence increase associated with Pi binding has a maximum rate of 267 s-1. The Pi dissociation rate is 6.6 s-1, and the dissociation constant is 70 nM. An application of the sensor is demonstrated for measuring ATP hydrolysis in real time as a helicase moves along DNA. Advantages of the new sensor are discussed, both in terms of the use of a rhodamine fluorophore and the potential of this double labeling strategy.     

Solubilization of inorganic phosphate byRhizobium

A large number of strains ofRhizobium were able to solubilize the insoluble phosphate compound, hydroxy-apatite, in liquid culture. Solubilization of hydroxyapatite byRhizobium was not mediated by an enzyme but acidity developed in the cultures was involved in the process. An inverse relationship between the level of soluble phosphate and medium pH was evident. The ability to solubilize hydroxyapatite varied among the strains. In a medium without NH ₄ ⁺ , some of the strains showed better activity than when NH ₄ ⁺ was present, suggesting involvement of different mechanisms for phosphate solubilization.R. meliloti SU 47 produced 2-ketogluconic acid along with an unidentified acid in the medium containing NH ₄ ⁺ . 2-Ketogluconic acid was identified as the major factor in inorganic phosphate solubilization. Initial presence of soluble phosphate in the medium had no discernible influence on the extent of hydroxyapatite solubilization. Initial presence of calcium reduced solubilization of phosphate and addition of EDTA to stationary phase cultures caused an increase in the level of soluble phosphate.     

Thermodynamics, kinetics, and mechanism in yeast inorganic pyrophosphatase catalysis of inorganic pyrophosphate: inorganic phosphate equilibration

We have developed two methods for quantitatively measuring inorganic pyrophosphate (PPi) in the presence of 10(3)--10(4) molar excesses of inorganic phosphate (Pi) and used them to measure the extent of enzyme-bound pyrophosphate (EPPi) formation in solutions of yeast inorganic pyrophosphatase and Pi. We have also measured the rate of enzyme-catalyzed H2O--phosphate oxygen exchange. We find both processes to have essentially identical dependence on Mg2+ and Pi concentrations, thus providing important confirmation for the recent proposal by Janson et al. (1979) that oxygen exchange proceeds via EPPi formation. Our results are consistent with a model in which three Mg2+ per active site are required for EPPi formation but inconsistent with a model requiring only two Mg2+ per active site and permit the formulation of an overall scheme for inorganic pyrophosphatase catalysis of PPi--Pi equilibration as well as the evaluation of equilibrium and rate constants in this scheme. The major results and conclusions of our work are the following: (a) the equilibrium constant for PPi (enzyme-bound) in equilibrium with 2Pi (enzyme-bound) is 4.8; (b) following PPi hydrolysis, the first released Pi contains an oxygen from solvent water; (c) the steps for PPi hydrolysis on the enzyme and for release of both product Pi's are all partially rate determining in overall enzyme-catalyzed PPi hydrolysis; (d) PPi formation on the enzyme is rate determining for H2O--Pi oxygen exchange; (e) PPi dissociation from the enzyme is very slow and is the rate-determining step in Pi--PPi exchange (Cohn, 1958; Janson et al., 1979). This also accounts for the observation that the calculated dissociation constant for MgPPi complex binding to enzyme is considerably lower than the measured Km for enzyme-catalyzed MgPPi hydrolysis.     

Exercise-induced respiratory muscle fatigue: implications for performance.

It is commonly held that the respiratory system has ample capacity relative to the demand for maximal O(2) and CO(2) transport in healthy humans exercising near sea level. However, this situation may not apply during heavy-intensity, sustained exercise where exercise may encroach on the capacity of the respiratory system. Nerve stimulation techniques have provided objective evidence that the diaphragm and abdominal muscles are susceptible to fatigue with heavy, sustained exercise. The fatigue appears to be due to elevated levels of respiratory muscle work combined with an increased competition for blood flow with limb locomotor muscles. When respiratory muscles are prefatigued using voluntary respiratory maneuvers, time to exhaustion during subsequent exercise is decreased. Partially unloading the respiratory muscles during heavy exercise using low-density gas mixtures or mechanical ventilation can prevent exercise-induced diaphragm fatigue and increase exercise time to exhaustion. Collectively, these findings suggest that respiratory muscle fatigue may be involved in limiting exercise tolerance or that other factors, including alterations in the sensation of dyspnea or mechanical load, may be important. The major consequence of respiratory muscle fatigue is an increased sympathetic vasoconstrictor outflow to working skeletal muscle through a respiratory muscle metaboreflex, thereby reducing limb blood flow and increasing the severity of exercise-induced locomotor muscle fatigue. An increase in limb locomotor muscle fatigue may play a pivotal role in determining exercise tolerance through a direct effect on muscle force output and a feedback effect on effort perception, causing reduced motor output to the working limb muscles.     

Inhibition of inorganic pyrophosphatase from Escherichia coli with inorganic phosphate

The interaction of inorganic pyrophosphatase from E. coli with inorganic phosphate (Pi) was studied in a wide concentration range of phosphate. The apoenzyme gives two inactive compounds with Pi, a product of phosphorylation of the carboxylic group of the active site and a stable complex, which can be detected in the presence of the substrate. The phosphorylation occurs when Pi is added on a millimole concentration scale, and micromole concentrations are sufficient for the formation of the complex. The formation of the phosphorylated enzyme was confirmed by its sensitivity to hydroxylamine and a change in the properties of the inactive enzyme upon its incubation in alkaline medium. The phosphorylation of pyrophosphatase and the formation of the inactive complex occur upon interaction of inorganic phosphate with different subsites of the enzyme active sites, which are connected by cooperative interactions.