Inhibitory Effect Of Pi Research Articles

Phosphate has dual roles in cross-bridge kinetics in rabbit psoas single myofibrils.

In this study, we aimed to study the role of inorganic phosphate (Pi) in the production of oscillatory work and cross-bridge (CB) kinetics of striated muscle. We applied small-amplitude sinusoidal length oscillations to rabbit psoas single myofibrils and muscle fibers, and the resulting force responses were analyzed during maximal Ca2+ activation (pCa 4.65) at 15°C. Three exponential processes, A, B, and C, were identified from the tension transients, which were studied as functions of Pi concentration ([Pi]). In myofibrils, we found that process C, corresponding to phase 2 of step analysis during isometric contraction, is almost a perfect single exponential function compared with skinned fibers, which exhibit distributed rate constants, as described previously. The [Pi] dependence of the apparent rate constants 2πb and 2πc, and that of isometric tension, was studied to characterize the force generation and Pi release steps in the CB cycle, as well as the inhibitory effect of Pi. In contrast to skinned fibers, Pi does not accumulate in the core of myofibrils, allowing sinusoidal analysis to be performed nearly at [Pi] = 0. Process B disappeared as [Pi] approached 0 mM in myofibrils, indicating the significance of the role of Pi rebinding to CBs in the production of oscillatory work (process B). Our results also suggest that Pi competitively inhibits ATP binding to CBs, with an inhibitory dissociation constant of ∼2.6 mM. Finally, we found that the sinusoidal waveform of tension is mostly distorted by second harmonics and that this distortion is closely correlated with production of oscillatory work, indicating that the mechanism of generating force is intrinsically nonlinear. A nonlinear force generation mechanism suggests that the length-dependent intrinsic rate constant is asymmetric upon stretch and release and that there may be a ratchet mechanism involved in the CB cycle.

16 Does Inorganic Phosphate inhibit Cardiac Myofibrillar Force Production at 37°C?

In the healthy heart, inorganic phosphate (Pi) is present in millimolar concentrations, rising to 20 millimolar or more in ischaemic heart. Since acute myocardial ischaemia leads to heart failure, there is need to gain better functional understanding of cardiac preparations in the presence of added Pi. Studies with skinned cardiac myocytes over the range 15°C–23°C have shown that Pi suppresses myofibrillar force generation. However, in skinned skeletal fibres a similar inhibitory action of Pi at low temperatures is reduced or abolished at temperatures closer to physiological. We therefore compared the effects of Pi on force development in skinned single cardiomyocytes at 15°C and 37°C. Permeabilised single cardiomyocytes from human ventricular myocardium (either from healthy, unused donor or from failing hearts) were maximally activated (30 μM free Ca 2+ ) in the presence of Pi (0–20 mM added). A rapid 1% stretch was applied during isometric force development to additionally measure crossbridge kinetics. Maximum Ca 2+ activated force decreased exponentially as [Pi] was raised. The sensitivity to Pi was similar for the two temperatures, e.g. 20 mM added Pi decreased force by ~87% at 15°C and ~78% at 37°C (n = 4, P = 0.74). The rates of stretch-induced cross-bridge detachment (krel) and the subsequent force re-development (kdf) increased exponentially as a function of Pi. These findings suggest that the inhibitory effect of Pi on myofibrillar force development is substantial at both 15°C and 37°C. This deleterious effect is therefore likely to contribute to the contractile dysfunction induced by cardiac ischaemia in vivo .

1P160 F_1-ATPaseのP-loop 変異体TF_1(βG158A)に対するリン酸の阻害効果(11. 分子モーター,ポスター,第52回日本生物物理学会年会(2014年度))

1P160 F_1-ATPaseのP-loop 変異体TF_1(βG158A)に対するリン酸の阻害効果(11. 分子モーター,ポスター,第52回日本生物物理学会年会(2014年度))

1PS024 F_1-ATPaseのP-loop変異体に対するリン酸の阻害効果(日本生物物理学会第50回年会(2012年度))

1PS024 F_1-ATPaseのP-loop変異体に対するリン酸の阻害効果(日本生物物理学会第50回年会(2012年度))

Transient receptor potential channels meet phosphoinositides

Transient receptor potential (TRP) cation channels are unique cellular sensors that are involved in multiple cellular functions, ranging from transduction of sensory signals to the regulation of Ca2+ and Mg2+ homoeostasis. Malfunctioning of TRP channels is now recognized as the cause of several hereditary and acquired human diseases. At the time of cloning of the first Drosophila TRP channel, a close connection between gating and phosphatidylinositol phosphates (PIPs) was already recognized. In this review, we summarize current knowledge about the mechanisms of interaction between TRP channels and PIPs, and discuss the possible functional implications of TRP–PIP interactions to human physiology and pathophysiology.

Channel Activated by Histidine Phosphorylation

Previous work had indicated that the calcium-activated K + channel KCa3.1, which is important for T and B cell activation, was indirectly activated by phosphatidylinositol 3-phosphate [PI(3)P]. Srivastava et al . now show that KCa3.1 interacts with and is stimulated by nucleoside diphosphate kinase B (NDPK-B), which is a histidine kinase. A 14-amino-acid sequence in the C-terminal region of KCa3.1 was known to mediate the regulation by PI(3)P, and using this region in a yeast two-hybrid assay, the authors identified NDPK-B as an interacting partner. KCa3.1 and NDPK-B were also coimmunoprecipitated from transfected cells, and the endogenous proteins were coimmunoprecipitated from T cells. Overexpression of active NDPK-B, but not a kinase inactive mutant or the related protein NDPK-A, stimulated KCa3.1 channel activity in whole cells and inside-out patches. Furthermore, depletion of PI(3)P from the cells with wortmannin inhibited the KCa3.1 channel activity, and full activation by NDPK-B required the addition of PI(3)P in these patches from the PI(3)P-depleted cells. KCa3.1 has a histidine at position 358 within the C-terminal 14-amino-acid sequence that is required for PI(3)P regulation. The related channels KCa2.1, KCa2.2, and KCa2.3 have an aspartic acid, and these channels are not regulated by PI(3)P (previous work), nor were they stimulated by NDPK-B. The C-terminal domain of KCa3.1 was phosphorylated in vitro by NDPK-B. Mutation of the His 358 eliminated the stimulation of channel activity by NDPK-B, and these mutant channels were resistant to the inhibitory effects of PI(3)P depletion. Finally, mRNA analysis of activated CD4 + T cells showed that the transcription of gene encoding NDPK-B, as was already known for gene encoding KCa3.1, was increased. Down-regulation of NDPK-B activity with siRNA reduced KCa3.1 channel activity in stimulated T cells without altering the activity of an unrelated K + channel. Because KCa3.1 hyperpolarizes the membrane potential to maintain a driving force for Ca 2+ entry, siRNA-mediated down-regulation of NDPK-B also decreased Ca 2+ influx in stimulated T cells, thus confirming the biological importance of NDPK-B activity in regulation of T cell function. S. Srivastava, Z. Li, K. Ko, P. Choudhury, M. Albaqumi, A. K. Johnson, Y. Yan, J. M. Backer, D. Unutmaz, W. A. Coetzee, E. Y. Skolnik, Histidine phosphorylation of the potassium channel KCa3.1 by nucleoside diphosphate kinase B is required for activation of KCa3.1 and CD4 T cells. Mol. Cell 24 , 665-675 (2006). [Online Journal]

Immunosuppressive components of Ascaris suum down‐regulate expression of costimulatory molecules and function of antigen‐presenting cells via an IL‐10‐mediated mechanism

High-molecular-weight components (PI) of Ascaris suum suppress both cell-mediated and humoral responses against ovalbumin (OVA) via an IL-4/IL-10-dependent mechanism. The aim of this work was to investigate the effect of PI on the ability of APC to activate T cells and the role of IL-10 in this process. Flow cytometry analyses of MHC class II, CD80, CD86 and CD40 molecules on LN cells from mice immunized with OVA or OVA+PI showed that PI inhibits expression of these molecules on unfractionated cells and on purified CD11c(+) cells. A low proliferative response was obtained when OVA-specific TCR-Tg T cells were incubated with CD11c(+) cells from OVA+PI-immunized mice pulsed with OVA, when compared to those incubated with cells from OVA-immunized mice. Similar results were obtained using as APC CD11c(+) cells from OVA-immunized mice pulsed with OVA+PI, which also expressed less of the four markers. The inhibitory effect of PI on both the expression of costimulatory molecules and the induction of T cell proliferation was abolished in IL-10-deficient mice. Our data indicate that the potent immunosuppressive effect of A. suum extract components on the host immune system is primarily related to their property of down-regulating the Ag-presenting ability of DC via an IL-10-mediated mechanism.

Stabilization of a Native Protein Mediated by Ligand Binding Inhibits Amyloid Formation Independently of the Aggregation Pathway

The acylphosphatases from Sulfolobus solfataricus and Drosophila melanogaster (Sso AcP and AcPDro2) were previously shown to form amyloid-like aggregates without the need to unfold initially. Inorganic phosphate (Pi), a competitive inhibitor binding specifically to the active site of these proteins, was found to stabilize, upon binding, the native state of AcPDro2 and to inhibit its conversion into amyloid-like fibrils. The inhibitory effect of Pi is suppressed only in a variant in which the Arg residue responsible for Pi binding is mutated. The study on Sso AcP shows that Pi retards both the formation of the initial nativelike oligomers and their subsequent conversion into protofibrils. Thus, stabilization of the native structure mediated by specific binding with small molecules can be an effective therapeutic strategy against protein deposition diseases that originate from initially folded proteins, independently of the structure of the protein, its aggregation pathway, and the particular aggregated species responsible for pathogenesis.

Photosynthetic control of the plasma membrane H+-ATPase in Vallisneria leaves. II. Presence of putative isogenes and a protein equipped with a C-terminal autoinhibitory domain.

In vitro treatment with trypsin of plasma membrane (PM) vesicles isolated from the leaves of Vallisneria gigantea Graebner, an aquatic monocot, produced a marked decrease in the Km for ATP and an increase in the Vmax of H+-transporting activity. Concomitantly, the removal of 8 kDa of the C-terminal domain from the 94-kDa PM H+-ATPase was confirmed by immunoblotting using different kinds of polyclonal antibody. Three partial clones of putative PM H+-ATPase genes (Vga1, 2, and 3) were isolated from leaves by reverse transcription polymerase chain reaction. Northern blotting analysis revealed that the expression level of Vga3 was high and that of the other two genes was much lower. The H+-transporting activity of PM vesicles was substantially suppressed in the presence of inorganic phosphate (Pi), which has been supposed to be a noncompetitive inhibitor of the PM H+-ATPase, coincident with an increase in the Km for ATP and a decrease in the Vmax. After treatment of the isolated PM vesicles with trypsin, the inhibitory effect of Pi was no longer evident. This result indicates that Pi inhibited the activity through the C-terminal autoinhibitory domain of the PM H+-ATPase. Furthermore, Pi increased the Km for ATP of the H+-transporting activity in the PM vesicles isolated from both dark-adapted and red-light-irradiated leaves. The results suggest that regulation of the Km for ATP through the operation of photosynthesis is independent of regulation through the cytoplasmic level of Pi.

Effects of pH on spontaneous tension oscillation in skinned bovine cardiac muscle.

Skinned cardiac muscle preparations exhibit spontaneous tension oscillations (spontaneous oscillatory contractions; SPOCs) in the absence of Ca2+, and in the presence of MgATP, MgADP and inorganic phosphate (Pi; ADP-SPOC). Similar oscillations occur in the presence of sub-micromolar concentrations of Ca2+ under normal activating conditions without MgADP and Pi (Ca-SPOC). In the study presented here, we investigated the effects of pH on both types of SPOC in skinned bovine cardiac ventricular muscle. First, a decrease in pH increased the MgADP concentration required to induce the half-maximal isometric tension that is obtained in the absence of Ca2+ and in the presence of MgATP (ADP-contraction). The inhibitory effect of Pi on ADP-contractions was not affected by pH. Second, ADP-SPOCs occurred upon the addition of Pi to the solution that resulted in ADP-contraction, and the relative amplitude and the period of the tension oscillation in the presence of 2 mM MgATP, 10 mM MgADP and 10 mM Pi were unchanged under all pH conditions examined (6.6, 7.0, 7.4). On the contrary, the relative amplitude and the period of the Ca-SPOCs were markedly diminished at pH 6.6. Finally, we constructed state diagrams showing the effects of pH on SPOC conditions. The state diagram shows that SPOCs occur less frequently under acidic conditions than at neutral pH. We suggest that the intermediate state of crossbridges that is required for SPOCs is more difficult to attain at a low pH.

Balance between Metabolite Accumulation and Transport in Relation to Photosynthesis by Isolated Spinach Chloroplasts

The relationship between the rate of orthophosphate (Pi) transport into the stroma and the rate of CO(2) fixation by intact chloroplasts was investigated. High Pi concentrations in the medium lead to a depletion of stromal metabolites, due to excessive Pi transport into the stroma, resulting in the inhibition of CO(2) fixation. This inhibitory effect of Pi is released by inhibitors of Pi transport, such as pyrophosphate, citrate or pyridoxal-5-phosphate. The latter compound appeared to be specially valuable in inhibiting Pi transport without affecting stromal reactions.The Pi optima of CO(2) fixation were studied when the rate of CO(2) fixation or the rate of Pi transport was varied by the application of specific inhibitors. For optimal performance the rates of CO(2) fixation and Pi transport have to be matched in such a way, that transport neither limits nor exceeds CO(2) fixation. This accounts for the large variation in the Pi optima for CO(2) fixation by different chloroplast preparations.

63: Mechanism of fructose-induced hyperuricemia

To investigate the mechanism whereby fructose provokes a loss of total, adenine nucleotides, the kinetics of purified rat liver AMP-aminohydrolase was studied with a new method based on the separation by column chromatography of radioactive IMP formed from [14C] AMP. The sensitivity of this method greatly facilitated the study of the enzyme in the presence of physiological concentrations of its substrate (0.2mM) and effectors (3mM for ATP. 0.5mM for GTP, 5mM for Pi). ATP 3mM increased the enzyme activity 200-fold due to a change of the kinetics from sigmoidal to hyperbolic. The inhibitory effect of Pi on the ATP-activated enzyme became only apparent at ATP concentrations below 1mM or if 0.3 to 0.5mM of. the inhibitor GTP was also present. At physiological concentrations of substrate and effectors, the combined inhibitory effects reached 95%. Within 10 sec. after the administration of fructose (2.5 mg/g I.V.) to mice, the concentration of Pi decreased abruptly, reaching 50% of its control value at 30 sec. A similar decrease of the level of GTP was found. The concentration of ATP declined progressively, down to 1/2-1/3 of its normal value at 2 min. These findings may explain the transient purine catabolism observed in these conditions since the decrease of the inhibitors becomes offset by the depletion of the activator.

The interaction of adenosinetriphosphate and inorganic phosphate with the sodium pump in red cells.

1. An increase in the intracellular concentration of inorganic phosphate (Pi) reduces the rate of the Na:K exchange catalysed by the Na pump in red cells. The inhibitory effect of Pi is exerted on the maximum rate of flux, Pi having no appreciable effect on the apparent affinity of the Na pump for either internal Na or external K. The effect of Pi is exerted along a rectangular hyperbola which tends to zero as Pi tends to infinity and is half-maximal at about 17 mM internal Pi. 2. Pi does not modify the rate of Na:Na exchange catalysed by the Na pump. 3. A reduction in the intracellular concentration of ATP reduces the maximum rate of Na:K exchange having no effect on the apparent affinity for either internal Na or external K. 4. The effects of ATP and Pi are mutually independent. 5 The lack of effect of ATP and Pi on the apparent affinity for internal Na is compatible with the idea that the affinity of the inner sites of the Na pump remains constant during a pump cycle. 6. The lack of effect of ATP on the apparent affinity for external K and the independence between the effects of ATP and Pi are difficult to explain if the only effect of ATP were its combination at a phosphorylating site. 7. The apparent affinities for K and phosphate become independent of the concentration of ATP, if it is assumed that in our experimental range the phosphorylating site is fully saturated with ATP, the rate of pumping being controlled by the state of occupation of a second non-phosphorylating site whose affinity for ATP is much lower. 8. The lack of effect of Pi on the apparent affinity for external K seems to indicate that during Na:K exchange the conformations of the pump that predominate are endowed with a reactivity towards inorganic phosphate and have the same high affinity for K in both their phospho and their dephospho states. 9. The kinetic behaviour of the Na pump in regard to its interactions with inner and outer cations, ATP and Pi seems to indicate that, in contrast with what happens with soluble allosteric proteins, in the active transport system ligand-induced changes in the reactivity are more important than ligand-induced changes in affinity. In this respect therefore the Na pump behaves as an allosteric 'V system'.