Kinetics Of Phosphate Release Research Articles

KIF3A accelerates KIF3C within the kinesin-2 heterodimer to generate symmetrical phosphate release rates for each processive step

Heterodimeric KIF3AC is a mammalian kinesin-2 that is highly expressed in the central nervous system and associated with vesicles in neurons. KIF3AC is an intriguing member of the kinesin-2 family because the intrinsic kinetics of KIF3A and KIF3C when expressed as homodimers and analyzed in vitro are distinctively different from each other. For example, the single-molecule velocities of the engineered homodimers KIF3AA and KIF3CC are 293 and 7.5 nm/s, respectively, whereas KIF3AC has a velocity of 186 nm/s. These results led us to hypothesize that heterodimerization alters the intrinsic catalytic properties of the two heads, and an earlier computational analysis predicted that processive steps would alternate between a fast step for KIF3A followed by a slow step for KIF3C resulting in asymmetric stepping. To test this hypothesis directly, we measured the presteady-state kinetics of phosphate release for KIF3AC, KIF3AA, and KIF3CC followed by computational modeling of the KIF3AC phosphate release transients. The results reveal that KIF3A and KIF3C retain their intrinsic ATP-binding and hydrolysis kinetics. Yet within KIF3AC, KIF3A activates the rate of phosphate release for KIF3C such that the coupled steps of phosphate release and dissociation from the microtubule become more similar for KIF3A and KIF3C. These coupled steps are the rate-limiting transition for the ATPase cycle suggesting that within KIF3AC, the stepping kinetics are similar for each head during the processive run. Future work will be directed to define how these properties enable KIF3AC to achieve its physiological functions.

Direct determination of phosphatase activity from physiological substrates in cells.

A direct and continuous approach to determine simultaneously protein and phosphate concentrations in cells and kinetics of phosphate release from physiological substrates by cells without any labeling has been developed. Among the enzymes having a phosphatase activity, tissue non-specific alkaline phosphatase (TNAP) performs indispensable, multiple functions in humans. It is expressed in numerous tissues with high levels detected in bones, liver and neurons. It is absolutely required for bone mineralization and also necessary for neurotransmitter synthesis. We provided the proof of concept that infrared spectroscopy is a reliable assay to determine a phosphatase activity in the osteoblasts. For the first time, an overall specific phosphatase activity in cells was determined in a single step by measuring simultaneously protein and substrate concentrations. We found specific activities in osteoblast like cells amounting to 116 ± 13 nmol min-1 mg-1 for PPi, to 56 ± 11 nmol min-1 mg-1 for AMP, to 79 ± 23 nmol min-1 mg-1 for beta-glycerophosphate and to 73 ± 15 nmol min-1 mg-1 for 1-alpha-D glucose phosphate. The assay was also effective to monitor phosphatase activity in primary osteoblasts and in matrix vesicles. The use of levamisole – a TNAP inhibitor- served to demonstrate that a part of the phosphatase activity originated from this enzyme. An IC50 value of 1.16 ± 0.03 mM was obtained for the inhibition of phosphatase activity of levamisole in osteoblast like cells. The infrared assay could be extended to determine any type of phosphatase activity in other cells. It may serve as a metabolomic tool to monitor an overall phosphatase activity including acid phosphatases or other related enzymes.

An infrared assay of the kinetics of phosphate-release from physiological substrates in living cells

An infrared assay of the kinetics of phosphate-release from physiological substrates in living cells

How myosin VI coordinates its heads during processive movement

A processive molecular motor must coordinate the enzymatic state of its two catalytic domains in order to prevent premature detachment from its track. For myosin V, internal strain produced when both heads of are attached to an actin track prevents completion of the lever arm swing of the lead head and blocks ADP release. However, this mechanism cannot work for myosin VI, since its lever arm positions are reversed. Here, we demonstrate that myosin VI gating is achieved instead by blocking ATP binding to the lead head once it has released its ADP. The structural basis for this unique gating mechanism involves an insert near the nucleotide binding pocket that is found only in class VI myosin. Reverse strain greatly favors binding of ADP to the lead head, which makes it possible for myosin VI to function as a processive transporter as well as an actin-based anchor. While this mechanism is unlike that of any other myosin superfamily member, it bears remarkable similarities to that of another processive motor from a different superfamily--kinesin I.

Kinetic Mechanism of Human Myosin IIIA

Myosin IIIA is specifically expressed in photoreceptors and cochlea and is important for the phototransduction and hearing processes. In addition, myosin IIIA contains a unique N-terminal kinase domain and C-terminal tail actin-binding motif. We examined the kinetic properties of baculovirus expressed human myosin IIIA containing the kinase, motor, and two IQ domains. The maximum actin-activated ATPase rate is relatively slow (k(cat) = 0.77 +/- 0.08 s(-1)), and high actin concentrations are required to fully activate the ATPase rate (K(ATPase) = 34 +/- 11 microm). However, actin co-sedimentation assays suggest that myosin III has a relatively high steady-state affinity for actin in the presence of ATP (K(actin) approximately 7 microm). The rate of ATP binding to the motor domain is quite slow both in the presence and absence of actin (K(1)k(+2) = 0.020 and 0.001 microm(-1).s(-1), respectively). The rate of actin-activated phosphate release is more than 100-fold faster (85 s(-1)) than the k(cat), whereas ADP release in the presence of actin follows a two-step mechanism (7.0 and 0.6 s(-1)). Thus, our data suggest a transition between two actomyosin-ADP states is the rate-limiting step in the actomyosin III ATPase cycle. Our data also suggest the myosin III motor spends a large fraction of its cycle in an actomyosin ADP state that has an intermediate affinity for actin (K(d) approximately 5 microm). The long lived actomyosin-ADP state may be important for the ability of myosin III to function as a cellular transporter and actin cross-linker in the actin bundles of sensory cells.

Effect of soil properties on the kinetics of phosphate release

The kinetics of the phosphate release by 35 non‐calcareous soil samples were investigated by consecutive extraction of the soil. The soil samples covered a wide range of texture, pH and degree of phosphate saturation. For all soils, phosphate release initially followed first order kinetics. With time, the process became linear to the logarithm of time. These relationships were used to distinguish between the initial fast process of phosphate desorption and the much slower diffusion‐dissolution process. The influence of physico‐chemical soil properties on the parameters describing the two processes was investigated using multiple regression analysis. The total amount of reversibly adsorbed phosphate was highly significant correlated to the oxalate extractable phosphate of the soil: the former approximated 33 % of the latter. The rate constant of the initial desorption process was mainly influenced by the pH of the soil: increasing the pH positively affects the desorption rate. The kinetics of the final diffusion‐dissolution process was most related to the oxalate extractable phosphorus content of the soil. Under the prevailing experimental conditions, the reversibly adsorbed phosphate is completely released after eight days only whereas it will take almost two years to solubilize all the precipitated P. Using the degree of phosphate saturation (DPS) instead of Pox for estimating the reversibly adsorbed phosphate results in regression models with about the same determination coefficients.

Long-Term Kinetics of Phosphate Release from Soil

Long-term phosphate (P) desorption from soil is described using two discrete P pools in the soil : one available and one strongly fixed pool. The P release kinetics for each pool are described with a first-order rate equation. A new desorption method is used with hydrous iron oxide inside dialysis tubing acting as a P sink. The widely used iron-impregnated filter paper desorption method overestimates initial P desorption by a factor of up to 4 and underestimates the quantitative progression of desorption as a function of time. P desorption continued with substantial rates for periods longer than 1600 h. A wide range in P desorbability was observed : 15-70% of oxalate-extractable P (P ox ) desorbed after 1600 h. P desorbability decreased with increasing Fe ox +Al ox content of the sample. The relative size of the quickly desorbing pool increased with increasing initial degree of P saturation α 0 =P 0X [Fe 0X + Al 0X ] of the soils. This fact is of direct importance for the estimation of P losses from phosphate-rich soils. This study furthermore provides evidence that all oxalate-extractable P potentially is desorbable : no irreversibly fixed P OX exists.

Effects of Ca2+ on the kinetics of phosphate release in skeletal muscle.

The process of phosphate dissociation during the muscle cross-bridge cycle has been investigated by photoliberation of inorganic phosphate (Pi) within skinned fibers of rabbit psoas muscle. This permitted a test of the idea that Ca2+ controls muscle contraction by regulating the Pi release step of the cycle. Photoliberation of Pi from structurally distinct "caged" Pi precursors initiated a rapid tension decline of up to 12% of active tension, and this was followed by a slower tension decline. The apparent rate constant of the fast phase, kPi, depended on both [Pi] and [Ca2+], whereas the slow phase generally occurred at 2-4 s-1. At maximal Ca2+, kPi increased in a nonlinear manner from 43 +/- 2 s-1 to 118 +/- 7 s-1, as Pi was raised from 0.9 to 12 mM. This was analyzed in terms of a three-state kinetic model in which a force-generating transition is coupled to Pi dissociation from the cross-bridge. As Ca(2+)-activated tension was reduced from maximal (Pmax) to 0.1 Pmax, (i) kPi decreased by up to 2.5-fold, (ii) the relative amplitude of the rapid phase increased 2-fold, and (iii) the relative amplitude of the slow phase increased about 6-fold. Changes in the rapid phase are compatible with Ca2+ influencing an apparent equilibrium constant for the force-generating transition. By comparison, kPi was faster than the rate constant of tension redevelopment, ktr, and was influenced less by Ca2+. Ca2+ effects on the caged Pi transient cannot account for the large effects of Ca2+ on actomyosin ATPase rates or cross-bridge cycling kinetics but may be a manifestation of reciprocal interactions between the thin filament and force-generating cross-bridges, and may represent Ca2+ regulation of the distribution of cross-bridges between non-force-and force-generating states.

Chorismate synthase. Pre-steady-state kinetics of phosphate release from 5-enolpyruvylshikimate 3-phosphate

Chorismate synthase. Pre-steady-state kinetics of phosphate release from 5-enolpyruvylshikimate 3-phosphate

Chorismate synthase. Pre-steady-state kinetics of phosphate release from 5-enolpyruvylshikimate 3-phosphate

The pre-steady-state kinetics of phosphate formation from 5-enolpyruvylshikimate 3-phosphate catalysed by Escherichia coli chorismate synthase (EC 4.6.1.4) were studied by a rapid-acid-quench technique at 25 degrees C at pH 7.5. No pre-steady-state 'burst' or 'lag' phase was observed, showing that phosphate is released concomitant with the rate-limiting step of the enzyme. The implications of this result for the mechanism of action of chorismate synthase are discussed.

Kinetics of phosphate release from soil and its uptake by wheat

SummaryLaboratory and glasshouse experiments were conducted to study P release kinetics and uptake of P by wheat (Trilicum, aestivum L.) in ten benchmark soils of Punjab (India). Phosphorus desorption, determined by successive extraction with 0·01 m-CaCl2 solution, was lower in calcareous soils (group I) than in non-calcareous soils (group II). Desorption of surface P in soils followed pseudo-first-order kinetics. The values of the kinetic constant of desorption and the radial diffusion coefficient of P increased with the amount of added P in soils.A significant linear relationship between cumulative desorbed P and both dry-matter yield and P uptake indicated that the rate of release of P from the solid matrix is important in determining P uptake by wheat crop. Phosphate uptake was less dependent on the initial solution P concentration than on the rate of phosphate release.

Manifold effects of sodium butyrate on nuclear function. Selective and reversible inhibition of phosphorylation of histones H1 and H2A and impaired methylation of lysine and arginine residues in nuclear protein fractions.

In addition to its known effect in suppressing the deacetylation of the nucleosomal core histones, sodium butyrate in the concentration range 0.5 to 15 mM causes a selective inhibition of [32P]phosphate incorporation into histones H1 and H2A of cultured HeLa S3 cells. No commensurate general inhibition of phosphorylation is seen in the non-histone nuclear proteins of butyrate-treated cells, but phosphorylation patterns are altered and 32P-uptake may be stimulated, as well as inhibited, depending upon the protein fraction analyzed. The degree of inhibition of histone phosphorylation in intact cells increases progressively as the butyrate concentration is raised from 0.5 to 15 mM. The effect is time-dependent and fully reversible. Butyrate has no effect on the kinetics of phosphate release from previously phosphorylated histones of cultured cells, nor does it significantly alter the rate of dephosphorylation of 32P-labeled histone H1 by endogenous phosphatases in vitro. Despite the suppression of [32P]phosphate incorporation into histones H1 and H2A of butyrate-treated cells, Na-butyrate does not inhibit the in vitro activities of either type I or type II cyclic AMP-dependent protein kinases, or the cAMP-independent H1 kinase associated with cell cycle progression. This suggests that the butyrate effect on histone phosphorylation in vivo is indirect and may involve an alteration in substrate accessibility or a modulation of systems affecting kinase activities. The poly(ADP)-ribosylation of HeLa histones is not inhibited by 5 mM Na-butyrate. Cells exposed to butyrate show an impaired methylation of lysine and arginine residues in their histones and nuclear hnRNP particles, respectively.

Application of Elovich Equation to the Kinetics of Phosphate Release and Sorption in Soils

AbstractExperimental data on the phosphate release and sorption in soils, when inadequately described by a first‐order kinetic reaction, are often interpreted as a combination of two or three simultaneous first‐order reactions. A simple modified Elovich equation in the form: q = (1/β) ln (αβ) + (1/β) ln t was derived to fit the reported experimental data in literature that failed to conform to a single first‐order kinetic equation. In this equation, q is the amount of phosphate released or sorbed, and α and β are constants. The equation successfully described the data as a single straight line that covers the entire course of reaction time. It also appears that constants of α and β may be used for comparison of reaction rates of phosphate release or sorption in different soils.