Effect Of Phosphate Research Articles

Effect of orthophosphate and bioavailability of dissolved organic phosphorous compounds to typically harmful cyanobacterium Microcystis aeruginosa

Results show that Microcystis aeruginosa can utilize both dissolved organic phosphorus (DOP) and orthophosphate (DIP) even under low phosphorus (P) conditions to sustain its growth. Total P concentrations decreased markedly in all three P source treatments. Alkaline phosphatase activity (APA) in the different P sources tested changed in response to the DOP and DIP. The APA of DOP groups remained low after decreasing significantly, but the APA in the DIP treatments remained high during the period of culture. Changes in APA at different PO43−–P concentrations in a culture medium revealed negative correlations between APA and DIP. However, a positive relationship was observed between APA and DOP under low P concentrations. These findings indicate that M. aeruginosa can regulate its physiological metabolism to acclimate to low ambient DIP environments.

Effects of Inorganic Phosphates on the Thermodynamic, Pasting, and Asian Noodle‐Making Properties of Whole Wheat Flour

ABSTRACTThe effects of four inorganic phosphates on the thermodynamic and pasting properties of whole wheat flour as well as color, cooking quality, textural properties, and structural characteristics of whole wheat noodles were studied. The addition of phosphates increased the gelatinization temperature and enthalpy of melting of starch in whole wheat flour. Rapid visco analysis showed that all phosphates significantly increased whole wheat flour peak viscosity and final viscosity. Moreover, the whole wheat noodles prepared with disodium phosphate, trisodium phosphate, and sodium tripolyphosphate (STPP) exhibited brighter appearance, and the use of STPP and sodium hexametaphosphate reduced the cooking loss of whole wheat noodles. Texture profile analysis of cooked noodles revealed that the addition of phosphates significantly decreased the hardness and slightly increased the springiness, cohesiveness, and resilience. The microstructure of whole wheat noodles showed a larger degree of connectivity of the protein network and coverage of starch granules in the presence of inorganic phosphates. The results suggested that inorganic phosphates exhibited substantial effects on improving the quality of whole wheat noodles. Of the four phosphates studied, STPP appeared to be the most effective one in improving the overall properties of whole wheat noodles when they were normalized to constant phosphate content.

Critical effects of inorganic phosphate at threshold concentrations on cultured aortic valve interstitial cells. Macroautophagocytosis versus procalcific cell degeneration

The conventional threshold values ascribed to inorganic phosphate concentration ([Pi]) in diagnosing normophosphatemia range between 0.8mM and 1.45mM to 2.0mM [Pi]. In cultures mimicking metastatic calcification ([Pi]=3.0mM) a major role was found to be played by [Pi] (Pi-cultures) in priming a procalcific cell degeneration of bovine aortic valve interstitial cells (bAVICs), with mineralization enhancing subsequent to superstimulation with bacterial lipopolysaccharide (LPS) plus conditioned medium from cultured LPS-stimulated macrophages (Pi-LPS-CM-cultures) [1]. Here, bAVIC primary cultures were carried out which contained different [Pi] (0.4, 0.6, and 1.3mM in added solutions, i.e. 0.8, 1.3, and 2.0mM in final cultures), so including border concentrations on respect to hypophosphatemic- and hyperphosphatemic-like conditions. At 0.8mM and 1.3 [Pi] and for each incubation time (3, 9, 15, 21, and 28 days), bAVICs from Pi-cultures and Pi-LPS-CMcultures shared common ultrastructural features showing prominent macroautophagocytosis to occur, consistently with the immunohistochemical detection of the specific marker of mature autophagosomes MAP1-LC3A. Neither cell death signs nor appearance of calcific nodules were observed. At 2.0 [Pi], most bAVICs were affected by degenerative fragmentation as described for severe metastatic-like calcifcation, i.e. the appearence of phthalocianin- positive material outcropping at cell surface, acting as hydroxyapatite nucleator and being source of real calcospherulae. Quantitative spectrophotometric estimation of calcium amounts and alkaline phosphatase activity were consistent with the ultrastructural data, with (i) similar values for Pi-LPS-CM-cultures versus Pi-cultures and control cultures, at 0.8 and 1.3mM [Pi], and (ii) significantly higher values for Pi-LPS-CM-cultures versus Picultures and these latter versus controls, at 2.0mM [Pi]. Restriction of immunopositivity to caspase-8 to very few cells and complete immunonegativity to annexin-V suggested apoptosis to be a negligible epiphenomenon. In conclusion, the propensity of bAVICs to undergo procalcific degeneration resulted to correlate with [Pi] in such a manner that a differential discrimination of this parameter within the conventional normophosphatemic range is suggested for a proper evaluation of the risk for dystrophic valve calcification. Moreover, bacterial-derived inflammation seems to be regarded as an effective trigger for the higher normophosphatemic [Pi].

Involvement of phosphate carrier as a part of complex with ADP/ATP and aspartate/glutamate antiporters in palmitic acid-induced uncoupling in liver mitochondria

In liver mitochondria, the phosphate carrier is involved in protonophoric uncoupling effect of fatty acids together with ADP/ATP and aspartate/glutamate antiporters (Samartsev et al. 2003. Biochemistry (Moscow). 68, 618–629). Liver mitochondria depleted of endogenous oxidation substrates (exhausted mitochondria) have been used in the present work. In these mitochondria, like in the intact liver mitochondria, the specific inhibitor of ADP/ATP antiporter (carboxyatractylate) and the substrate of aspartate/glutamate antiporter (aspartate) suppress the uncoupling activity of palmitic acid. It is shown that in exhausted mitochondria the substrate of phosphate carrier (inorganic phosphate) and its nonspecific inhibitor mersalyl partially suppress palmitic acid-induced uncoupling due to decrease in the component of uncoupling activity sensitive to carboxyatractylate and aspartate. In the presence of inorganic phosphate or mersalyl, carboxyatractylate and aspartate added separately subsequent to palmitic acid do not suppress its uncoupling activity. They are effective only when added jointly. In the presence of thiourea or pyruvate, such effects of inorganic phosphate and mersalyl are not observed. It is supposed that in the presence of inorganic phosphate or mersalyl and under the condition of oxidation of critical SH-groups in mitochondria, the phosphate carrier, ADP/ATP antiporter, and aspartate/glutamate antiporter are involved in uncoupling function together with the general fatty acid pool as an uncoupling complex. The role of phosphate carrier in this complex may consist in facilitation of lateral transfer of the fatty acid molecules from one antiporter to another.

ATP binding and hydrolysis steps of the uni-site catalysis by the mitochondrial F 1-ATPase are affected by inorganic phosphate

The effect of inorganic phosphate (P i) on uni-site ATP binding and hydrolysis by the nucleotide-depleted F 1-ATPase from beef heart mitochondria (ndMF 1) has been investigated. It is shown for the first time that P i decreases the apparent rate constant of uni-site ATP binding by ndMF 1 3-fold with the K d of 0.38 ± 0.14 mM. During uni-site ATP hydrolysis, P i also shifts equilibrium between bound ATP and ADP + P i in the direction of ATP synthesis with the K d of 0.17 ± 0.03 mM. However, 10 mM P i does not significantly affect ATP binding during multi-site catalysis.

Effect of Inorganic Phosphate on the Rate of ADP Release During Ramp Shortening in Activated Permeabilized Fibers from Rabbit Psoas Muscle

A coumarin-labeled recombinant phosphorylated nucleoside diphosphate kinase (P∼NDPK-IDCC; West et al., 2009; Biophys.J. 96:3281-3294), was used as a fluorescence probe for time-resolved measurement of changes in [MgADP] during steady shortening of single permeabilized rabbit psoas fibers at 12°C (pCa 4.5, pH 7.1, ATP 5.7mM). A fiber contracted from the relaxed state by immersion into a Ca2+ activation solution at 0°C. Temperature activation was then initiated by immersion of the fiber into silicone oil at 12°C. The activation solutions were prepared with either zero added Pi or with 10 mM added Pi at constant ionic strength (0.15 M). The decline in fluorescence intensity emission (470nm) associated with MgADP-dependent dephosphorylation of P∼NDPK-IDCC (60 μM) was monitored during activation and during a period of isovelocity shortening. Fluorescence emission (580 nm) of a rhodamine dye was measured simultaneously to correct the P∼NDPK-IDCC signal for the effects of fiber movements and volume changes. The rate of MgADP release in the absence of added Pi increased from 0.7 ± 0.07 mM·s−1 at 0.2 fiber-lengths·s−1 to approximately 3.4 ± 0.25 mM·s−1 for shortening velocities between 1 and 2 fiber-lengths·s−1. When 10 mM Pi was added, the rate of ADP release at 0.2 fiber-lengths·s−1 was 0.48 ± 0.05 mM·s−1 and 2.6 ± 0.4 mM·s−1 at 1-2 fiber-lengths·s−1. In the absence of added Pi, the rate of ATP hydrolysis calculated from the appearance of ADP is similar to that calculated previously from the appearance of Pi using MDCC-labeled phosphate binding protein, over the same range of fiber shortening speeds (He et al., 1999, J. Physiol. 517: 839-854). Thus Pi slows the ATPase rate by 25-30%, both in the isometric and isotonic state. The energetic consequences will be discussed.

Effect of Inorganic Phosphate on the Force and Number of Myosin Cross-Bridges During the Isometric Contraction of Permeabilized Muscle Fibers from Rabbit Psoas

The relation between the chemical and mechanical steps of the myosin-actin ATPase reaction that leads to generation of isometric force in fast skeletal muscle was investigated in demembranated fibers of rabbit psoas muscle by determining the effect of the concentration of inorganic phosphate (Pi) on the stiffness of the half-sarcomere (hs) during transient and steady-state conditions of the isometric contraction (temperature 12°C, sarcomere length 2.5μm). Changes in the hs strain were measured by imposing length steps or small 4kHz oscillations on the fibers in control solution (without added Pi) and in solution with 3–20mM added Pi. At the plateau of the isometric contraction in control solution, the hs stiffness is 22.8±1.1 kPa nm−1. Taking the filament compliance into account, the total stiffness of the array of myosin cross-bridges in the hs (e) is 40.7±3.7 kPa nm−1. An increase in [Pi] decreases the stiffness of the cross-bridge array in proportion to the isometric force, indicating that the force of the cross-bridge remains constant independently of [Pi]. The rate constant of isometric force development after a period of unloaded shortening (rF) is 23.5±1.0s−1 in control solution and increases monotonically with [Pi], attaining a maximum value of 48.6±0.9s−1 at 20mM [Pi], in agreement with the idea that Pi release is a relatively fast step after force generation by the myosin cross-bridge. During isometric force development at any [Pi], e and thus the number of attached cross-bridges increase in proportion to the force, indicating that, independently of the speed of the process that leads to myosin attachment to actin, there is no significant (>1ms) delay between generation of stiffness and generation of force by the cross-bridges.

Effect of Inorganic Phosphates on the Adhesion of Mono‐phosphorylated Cornstarch to Fibers

AbstractThe influences of inorganic phosphates, as residuals and/or impurities within starch, on the adhesion of acid‐treated cornstarch phosphates to cotton and polyester fibers were investigated for warp sizing. The phosphate impurities considered included sodium dihydrogenphosphate, sodium hydrogenphosphate, sodium pyrophosphate, and pentasodium triphosphate. The starches with a phosphate degree of substitution (DS) range of 0.004‐0.021 were phosphorylated by heating acid‐treated cornstarch at 125°C for 90 min in a solid‐state reaction with pentasodium triphosphate. The adhesion was evaluated in terms of the breaking strength and work‐to‐break of slightly sized rovings. It was found that the phosphorylation improves the adhesion to both fibers even at a low phosphorylation extent. The phosphate impurities within starch have an adverse effect and reduce the adhesion to both fibers. The adverse effect depends on the type and level of the phosphates, decreasing in the series NaH2PO4 > Na2HPO4 > Na4P2O7 ≈ Na5P3O10. The adhesion enhances as the DS increases and as the free phosphate content decreases. Furthermore, the adverse effect can be offset by the phosphate modification of starch.

Phosphate deficiency-dependent upregulation of UDP-glucose pyrophosphorylase genes is insensitive to ABA and ethylene status in Arabidopsis leaves

The effects of inorganic phosphate (Pi) deficiency and ABA/ethylene status on expression of UDP-glucose pyrophosphorylase (UGPase) genes (Ugp), involved in sucrose/polysaccharide metabolism, were investigated. Both wild-type (wt), aba and abi mutants (ABA-deficient and -in-sensitive), etr, ein and eto (ethylene resistant and overproducing) grown on Pi-deficient and complete nutrient solution, as well as phol (Pi-deficient) mutants of Arabidopsis thaliana were used for experiments. Generally, Pi-deficiency conditions (including mannose feeding to decrease cytosolic Pi pool) resulted in an increase of Ugp expression in the leaves, under all experimental conditions. Mutant backgrounds reflecting differences in ABA or ethylene status/ sensitivity had no effect on the level of Ugp up-regulation by Pi-stress. Furthermore, feeding ABA to the leaves of wt and pho1 plants had no effect on Ugp expression, regardless of the sucrose status in the leaves. The data suggest that Pi deficiency leading to up-regulation of Ugp acts independently of ABA and ethylene status.

Effect of Phosphate on Cell Growth and Polysaccharide Production by Suspension Cultures of Protocorm-like Bodies of Dendrobium huoshanense

The effect of inorganic phosphate on the cell growth and accumulation of polysaccharides together with nutrient utilization was investigated in suspension cultures of protocorm-like bodies of Dendrobium huoshanense. Thirty-day-old cells were transferred into a liquid medium with the inoculum density of 100 g/L cells. The results indicated that the optimal concentration of phosphate in the medium for the growth of protocorm-like bodies of Dendrobium huoshanense was 2.5 mmol/L and the biomass was 496.5 g/L (fresh weight). The phosphate was a limiting factor for the cell growth and there was a relationship between the levels of intracellular phosphate and the cell growth. A total of 2.5 mmol/L of medium phosphate was beneficial to the absorption of carbon and nitrogen source. A total of 0.312 mmol/L of medium phosphate was better for the accumulation of polysaccharides and the production of polysaccharides was 2.22 g/L on day 36.

Role of inorganic phosphate during electron transfer in the redox system NADH+flavin

We have used 31P NMR and EPR to study the effect of inorganic phosphate (Pi) on the process of electron transfer in oxidation of reduced nicotinamide adenine dinucleotide (NADH) by flavin in aqueous solutions. Observation of a significant (up to 40%) drop in the integrated intensity of the 31P NMR signal for the inorganic phosphate over the course of the reaction is interpreted as a manifestation of the effect of chemically induced polarization of the phosphorus nuclei during singlet-triplet conversion in charge-transfer complexes [NADH…Pi], and is evidence for a possible catalytic role for Pi in processes involving transfer of an electron (hydrogen atom) from NADH to appropriate acceptors. The results presented support the possibility of direct involvement of Pi in a chain of one-electron conversions in chemical systems.

Expression of several genes involved in sucrose/starch metabolism as affected by different strategies to induce phosphate deficiency in Arabidopsis

The effects of inorganic phosphate (Pi) deficiency on expression of genes encoding ADP-glucose pyrophosphorylase small and large subunits (ApS and ApL1, ApL2, ApL3 genes), UDP-glucose pyrophosphorylase (Ugp gene), sucrose synthase (Sus1), soluble and insoluble acid invertases (Inv and Invcw) and hexokinase (Hxk1 gene), all involved in carbohydrate metabolism, were investigated in Arabidopsis thaliana (L.) Heynh. We used soil-grown pho mutants affected in Pi status, as well as wild-type (wt) plants grown under Pi deficiency conditions in liquid medium, and leaves of wt plants fed with D-mannose. Generally, ApS, ApL1, Ugp and Inv genes were upregulated, although to a varied degree, under conditions of Pi-stress. The applied conditions had differential effects on expression of other genes studied. For instance, Sus1 was downregulated in pho1 (Pi-deficient) mutant, but was unaffected in wt plants grown in liquid medium under P-deficiency. Mannose had distinct concentration-dependent effects on expression of genes under study, possibly reflecting a dual role of mannose as a sink for Pi and as glucose analog. Feeding Pi (at up to 200 mM) to the detached leaves of wt plants strongly affected the expression of ApL1, ApL2, Sus1 and Inv genes, possibly due to an osmotic effect exerted by Pi. The data suggest that ADP-glucose and UDP-glucose pyrophosphorylases (enzymes indirectly involved in Pi recycling) as well as invertases (sucrose hydrolysis) are transcriptionally regulated by Pi-deficiency, which may play a role in homeostatic mechanisms that acclimate the plant to the Pi-stress conditions.

Effect of Inorganic Phosphate on FMN Binding and Loop Flexibility in Desulfovibrio desulfuricans Apo-flavodoxin

The complex between flavin mononucleotide (FMN) and apo-flavodoxin is dominated by isoalloxazine-stacking interactions and 5'-phosphate hydrogen bonds. We show here that FMN binding to Desulfovibrio desulfuricans apo-flavodoxin is faster and the affinity is higher in the presence of inorganic phosphate as compared to in its absence (I=110 mM, pH 7, 20 degrees C). The transition-state of complex formation was investigated by phi-value analysis using Trp60Ala and Tyr98Ala apo-flavodoxin variants. We find that Tyr98 is highly involved in the FMN/protein transition state independent of inorganic phosphate, whereas the participation of Trp60 is modulated by inorganic phosphate. The phi-value for Trp60 is higher without phosphate, implying that at this condition stronger binding of Trp60 is required in the transition state to assure successful complex formation. Consistent with the experimental data, all-atom molecular dynamic simulations reveal that the presence of an anion in the phosphate subsite restricts the mobility of the Trp60-containing loop in terms of both backbone and side-chain movements, but has no effect on the Tyr98-containing loop. The overall thermodynamic stability of apo-flavodoxin is higher in the presence of inorganic phosphate as compared to in its absence (I=110 mM, pH 7, 20 degrees C). Kinetic experiments reveal that the additional stability originates in slower unfolding. The combined experimental and computational observations demonstrate that phosphate has an ordering effect on the Trp60-containing loop, which positions Trp60 favorably for FMN binding and increases the barrier for protein unfolding.

Comparison of stannous chloride and phosphate for lead corrosion control

Because of health hazards associated with lead toxicity, the U.S. Environmental Protection Agency in 1991 promulgated the Lead and Copper Rule (LCR), which established an action level for lead at 15 μg/L as Pb. Even though lead pipes are no longer installed in U.S. distribution systems, many cities still have lead service connections that can release lead into the water. To help control lead concentrations, many water utilities add corrosion inhibitors to the treated water to create a protective scale layer on pipe walls. In this research, a 13‐month study investigated the effects of corrosion control chemicals on lead release and bacterial growth in a pipe‐loop system of new ductile‐iron and lead pipes. The five parallel lines in the study consisted of a control loop plus four loops testing orthophosphate, polyphosphate, an orthophosphate‐polyphosphate blend, and stannous chloride. Total lead concentrations in the treated loops were significantly lower than in the untreated control, with orthophosphate yielding the lowest lead concentrations of all of the chemicals tested. However, none of the chemicals consistently maintained lead concentrations below the 15‐μg/L action level for the 8‐hour stagnation time sample. The microbiological results suggest that utilities should exercise caution when adding corrosion control chemicals in order to ensure that reduced corrosion is not achieved at the expense of other water quality indicators. For utilities concerned about phosphate's effects on microbiological water quality, stannous chloride may represent a viable option to help them achieve LCR compliance.

Interactive effects of phosphate deficiency, sucrose and light/dark conditions on gene expression of UDP-glucose pyrophosphorylase in Arabidopsis

The effects of inorganic phosphate (Pi) status, light/dark and sucrose on expression of UDP-glucose pyrophosphorylase (UGPase) gene (Ugp), which is involved in sucrose/ polysaccharides metabolism, were investigated using Arabidopsis wild-type (wt) plants and mutants impaired in Pi and carbohydrate status. Generally, P-deficiency resulted in increased Ugp expression and enhanced UGPase activity and protein content, as found for wt plants grown on P-deficient and complete nutrient solution, as well as for pho1 (P-deficient) mutants. Ugp was highly expressed in darkened leaves of pho1, but not wt plants; daily light exposure enhanced Ugp expression both in wt and pho mutants. The pho1 and pho2 (Pi-accumulating) mutations had little or no effect on leaf contents of glucose and fructose, regardless of light/dark conditions, whereas pho1 plants had much higher levels of sucrose and starch in the dark than pho2 and wt plants. The Ugp was up-regulated when leaves were fed with sucrose in wt plants, but the expression in pho2 background was much less sensitive to sucrose supply than in wt and pho1 plants. Expression of Ugp in pgm1 and sex1 mutants (impaired in starch/sugar content) was not dependent on starch content, and not tightly correlated with soluble sugar status. Okadaic acid (OKA) effectively blocked the P-starvation and sucrose-dependent expression of Ugp in excised leaves, whereas staurosporine (STA) had only a small effect on both processes (especially in -P leaves), suggesting that P-starvation and sucrose effects on Ugp are transmitted by pathways that may share similar components with respect to their (in) sensitivity to OKA and STA. The results of this study suggest that Ugp expression is modulated by an interaction of signals derived from P-deficiency status, sucrose content and dark/light conditions, and that light/sucrose and P-deficiency may have additive effects on Ugp expression.

Phosphate Increases Mitochondrial Reactive Oxygen Species Release

The effects of inorganic phosphate (Pi), the main intracellular membrane permeable anion capable of altering mitochondrial pH gradients (ΔpH), were measured on mitochondrial H2O2 release. As expected, Pi decreased ΔpH and increased the electric membrane potential (ΔΨ). Mitochondrial H2O2 release was stimulated by Pi and also by its structural analogue arsenate. However, acetate, another membrane-permeable anion, did not stimulate mitochondrial H2O2 release. The stimulatory effect promoted by Pi was prevented by CCCP, which decreases transport of Pi across the inner mitochondrial membrane, indicating that Pi must be in the mitochondrial matrix to stimulate H2O2 release. In conclusion, we found that Pi and arsenate stimulate mitochondrial reactive oxygen release, an effect that may contribute towards oxidative stress under conditions such as ischemia/reperfusion, in which high-energy phosphate bonds are hydrolyzed.

Effect of Orthophosphate, Nucleotide Analogues, ADP, and Phosphorylation on the Cytoplasmic Domains of Ca2+-ATPase from Scallop Sarcoplasmic Reticulum

The effects of orthophosphate, nucleotide analogues, ADP, and covalent phosphorylation on the tryptic fragmentation patterns of the E1 and E2 forms of scallop Ca-ATPase were examined. Sites preferentially cleaved by trypsin in the E1 form of the Ca-ATPase were detected in the nucleotide (N) and phosphorylation (P) domains, as well as the actuator (A) domain. These sites were occluded in the E2 (Ca(2+)-free) form of the enzyme, consistent with mutual protection of the A, N, and P domains through their association into a clustered structure. Similar protection of cytoplasmic Ca(2+)-dependent tryptic cleavage sites was observed when the catalytic binding site for substrate on the E1 form of scallop Ca-ATPase was occupied by Pi, AMP-PNP, AMP-PCP, or ADP despite the presence of saturating levels of Ca2+. These results suggest that occupation of the catalytic site on E1 can induce condensation of the cytoplasmic domains to yield a unique structural intermediate that may be related to the form of the enzyme in which the active site is prepared for phosphoryl transfer. The effect of Pi on the E2 form of the scallop Ca-ATPase was also investigated, when it was found that formation of E2-P led to extreme resistance toward secondary cleavage by trypsin and stabilization of enzymatic activity for long periods of time.

Inorganic phosphate affects the pCa-force relationship more than the pCa-ATPase by increasing the rate of dissociation of force generating cross-bridges in skinned fibers from both EDL and soleus muscles of the rat.

The effect of inorganic phosphate (Pi) on Ca2+ -activation of actomyosin ATPase activity and force in permeabilized (skinned) single extensor digitorum longus (EDL) and soleus muscle fibers of the rat were investigated. Increasing concentrations of Pi decreased force more than ATPase rate at all Ca2+ concentrations and this effect was more pronounced at submaximal Ca2+ -activation. Increasing Pi caused both the normalized pCa-ATPase and pCa-force relationship to be shifted to a higher Ca2+ concentration. At all Ca2+ concentrations ATPase was activated at a lower concentration of Ca2+ than force and this difference in Ca2+ concentration required for the activation of ATPase and force was greater in fast-twitch (EDL) than in slow twitch (soleus) muscle. Soleus muscle pCa-ATPase and pCa-force curves were more sensitive to Ca2+ (pCa50 = 5.97 and 5.89, respectively) than EDL (pCa50 = 5.68 and 5.54, respectively). Finally the shape of the pCa-ATPase and pCa-force curves was similar and not affected by Pi. Analysis shows that Pi increases the rate of dissociation of force generating myosin cross-bridges (ratio of ATPase/force (g(app at all Ca2+ concentration, especially at submaximal Ca2+ -activation levels. Pi effects on g(app) are discussed in terms Pi interacting with the isomerization high force AM*ADP states to form high force transitional AM*ADP*Pi* states which facilitate the dissociation of ADP from AM*ADP. Increasing Ca2+ during Ca2+ -activation of the fibers is associated with a progressive decrease in rate of dissociation of force generating myosin cross-bridges g(app).

The effects of inorganic phosphate and arsenate on both passive muscle visco-elasticity and maximum Ca2+ activated tension in chemically skinned rat fast and slow twitch muscle fibres.

The effects of adding either 25 mM inorganic phosphate (Pi) or its structural analogue arsenate (ASi) on both the maximum Ca2+ activated tension (Po) and passive muscle visco-elasticity (P2 tension) were investigated at 10 degrees C, using segments of single, chemically skinned rat muscle fibres. Whilst the results confirmed some previous findings on the effects of Pi on Po, they also showed that the addition of 25 mM ASi led to a large (approximately 50%) but completely reversible depression of Po in both the fast and slow twitch rat muscle fibres. Moreover, the depression of Po by ASi was greater at low than at high pH values. Examined in the presence of Dextran T-500, the passive tension and sarcomere length responses to a ramp stretch were found to be qualitatively and quantitatively similar to those previously reported in intact rat muscle fibres. Thus, the tension response to a ramp stretch, in the presence and absence of either 25 mM Pi or ASi, consisted of a viscous (P1), a visco-elastic (P2) and an elastic (P3) tension. However, the addition of either 25 mM Pi or ASi led to approximately 15-18% increase in the amplitude of the visco-elastic (P2) tension but had little or no effect on the amplitudes of the other two tension components (viscous, P1 and elastic, P3 tensions). Furthermore, neither compound significantly altered the relaxation rate of the passive muscle visco-elasticity (P2 tension). These results show that Po (arising from cycling cross-bridges) and passive muscle visco-elasticity (P2 tension) are affected differently by both Pi and ASi and suggest that they may not share a common structural basis. The possibility that passive muscle visco-elasticity (P2 tension) arises from the gap-(titin) filament (as suggested previously by Mutungi and Ranatunga, 1996b J Physiol 496: 827-837) and that Pi and ASi increase its amplitude by interacting with the PEVK region of the filament are discussed.

Effects of inorganic phosphate on cross-bridge behavior after photorelease of ATP in permeabilized cells of rat skeletal muscle.

Effects of 20 mM inorganic phosphate on the cross-bridge behavior after photorelease of ATP from caged ATP was studied by X-ray diffraction in rat skinned psoas muscle fibers at 16 degrees C. In the first 30 ms after the photorelease, tension was similar in the presence and absence of phosphate. The tension development was then suppressed in the presence of phosphate. At 500 ms after the photolysis, it was lower by about 30% in the presence of phosphate. In the presence of phosphate, the intensity of the third meridional reflection from the thick filament at 1/14.4 nm(-1) increased more slowly and was 60% of the level without phosphate at 500 ms after the photolysis. The intensities of the equatorial (1,1) reflection and the actin layer-line at 1/36 nm(-1) decreased to a lower level in the presence of phosphate. These results suggest that phosphate does not affect dissociation of myosin heads from actin, but decreases the number of myosin heads in force-generating conformation and reduces tension. Phosphate may also shift the equilibrium between the detached and attached states towards the former.