Evidence for an Amiloride-Inhibited Mg/2H Antiporter in Lutoid (Vacuolar) Vesicles from Latex of Hevea brasiliensis

Lutoids represent a lysosomal microvacuolar compartment of rubber-tree (Hevea brasiliensis) latex. We observed acidification of isolated vesicles after imposing an outward Mg²⁺ diffusion gradient and dissipation of a preformed pH gradient in the presence of exogenous Mg²⁺. These results suggest the presence of a Mg²⁺/H⁺ antiporter. The maximum Mg²⁺/H⁺ exchange rate was observed at pH 8.5. The K_m values for Mg²⁺ (2.6 mm) were identical for both influx and efflux experiments. When membrane potential was clamped at zero with K⁺ and valinomycin, the response of the membrane potential probe oxonol VI showed that the Mg²⁺/H⁺ exchange was electroneutral. Mg²⁺/H⁺ exchange was inhibited by amiloride and imipramine. Both the inhibiting concentration range and the K_m for Mg²⁺ are similar to those reported for the Mg²⁺/2Na⁺ antiporter in animals cell. These data are consistent with the existence of a Mg²⁺/2H⁺ antiporter in a plant tonoplast.     

The role of calcium ions in phytochrome-controlled swelling of etiolated wheat (Triticum aestivum L.) protoplasts

Protoplasts from dark-grown wheat (Triticum aestivum L.) maintained at a constant osmotic potential at 22°C, were found to swell upon red irradiation (R) and the effect was negated by subsequent far-red light (FR), indicating phytochrome involvement. Swelling only occurred when Ca²⁺ ions were present in the surrounding medium, or were added within 10 min after R. Furthermore, Mg²⁺, Ba²⁺ or K⁺ could not replace this requirement for Ca²⁺. The presence of K⁺ did not enhance the Ca²⁺-dependent swelling response. When the Ca²⁺-ionophore A 23187 was added to the medium, protoplasts swelled in the dark to the same extent as after R. Both the Ca²⁺-channelblocker Verapamil and La³⁺ inhibited R-induced swelling. It is proposed that R causes the opening of Ca²⁺-channels in the plasma membrane. Boyle-van't Hoff analyses of protoplast volume after R and FR are consistent with the conclusion that R irradiation causes changes in membrane properties.Abbreviations EDTA ethylenediaminetetraacetic acid - FR far-red light - nov non-osmotic-volume - Pfr FR-absorbing form of phytochrome - Pr R-absorbing form of phytochrome - R red light     

The effect of sodium on inorganic phosphate- and p-nitrophenyl phosphate-facilitated ouabain binding to (Na+ + K+)-activated ATPase

The effect of the hydrolysis product P_i and the artificial substrate p-nitrophenyl phosphate (p-nitrophenyl-P) on ouabain binding to (Na⁺ + K⁺)-activated ATPase was investigated.The hypothesis that (Mg²⁺ + p-nitrophenyl-P)-supported ouabain binding might be due to P_i release and thus (Mg²⁺ + P_i)-supported could not be confirmed.The enzyme · ouabain complexes obtained with different substrates were characterized according to their dissociation rates after removal of the ligands facilitating binding. The character of the enzyme · ouabain complex is determined primarily by the monovalent ion present during ouabain binding, but, qualitatively at least, it is immaterial whether binding was obtained with p-nitrophenyl phosphate or P_i.The presence or absence of Na⁺ during binding has a special influence upon the character of the enzyme · ouabain complex. Without Na⁺ and in the presence of Tris ions the complex obtained with (Mg²⁺ + P_i) and that obtained with (Mg²⁺ + p-nitrophenyl-P) behaved in a nearly identical manner, both exhibiting a slow decay. High Na⁺ concentration diminished the level of P_i-supported ouabain binding, having almost no effect on p-nitrophenyl phosphate-supported binding. Both enzyme · ouabain complexes, however, now resembled the form obtained with (Na⁺ + ATP), as judged from their dissociation rates and the K⁺ sensitivity of their decay. The complexes obtained at a high Na⁺ concentration underwent a very fast decay which could be slowed considerably after adding a low concentration of K⁺ to the resuspension medium. The most stable enzyme · ouabain complex was obtained in the presence of Tris ions only, irrespective of whether p-nitrophenyl phosphate or P_i facilitated complex formation. The presence of K⁺ gave rise to a complex whose dissociation rate was intermediate between those of the complexes obtained in the presence of Tris and a high Na⁺ concentration.It is proposed that the different ouabain dissociation rates reflect different reactive state of the enzyme. The resemblance between the observations obtained in phosphorylation and ouabain binding experiments is pointed out.     

The relation of the 515 nanometers absorbance change to adenosine triphosphate formation in chloroplasts and digitonin subchloroplast particles

The flash-induced absorbance changes at 515 nanometers has been studied in chloroplasts and in digitonin subchloroplast particles of lettuce. The effect of various conditions and uncouplers was tested on the decay kinetics of this absorbance change and on ATP formation in the presence of phenazine methosulphate, either by continuous or flash illumination. It has been found that in chloroplasts, carbonyl cyanide m-chloromethoxyphenylhydrazone and nigericin in the presence of K⁺ accelerate the decay of the 515 change and inhibit ATP formation. However, under a variety of conditions the rate of decay of the 515 absorbance change was found to be unrelated to ATP formation. Preillumination, addition of valinomycin in the presence of K⁺, addition of Na⁺, or divalent cations accelerate the decay of the 515 absorbance change markedly but have no effect on ATP formation. Addition of phosphorylation reagents has no effect on the decay rate beyond that obtained by Mg²⁺ and inorganic phosphate. NH₄Cl, and to some extent atebrin, while inhibiting ATP formation, do not affect the decay of the 515 absorbance change.     

Different kinetics of membrane potential formation in dark-adapted and preilluminated chloroplasts

The effects of varying dark interval on the kinetics of light-induced formation of the membrane potential were studied on individual chloroplasts of Anthoceros with the use of capillary microelectrodes. Illumination of the chloroplast with 1 s light pulse after 3 min dark period induced the photoelectrical response with two peaks of the potential that were located at 20 and 500 ms after the onset of illumination. The position of the second peak was shifted along the time-scale depending on the preceding dark interval. The repeated illumination of the chloroplast with 1 s light pulse after 30 s dark interval induced the electrical response with only one maximum and a monotonous decay of the potential in the light. Distinctions in the electrical responses induced by the first and the second light pulses were eliminated by the addition of 50 μM dicyclohexylcarbodiimide (DCCD). The results show that the photoinduction kinetics of the membrane potential in chloroplasts is affected by functioning of H⁺-ATPase. The delayed peak of the membrane potential in the photoinduction kinetics is interpreted as a consequence of the photoactivated electron transport supported by Photosystem I.     

Light Response of a Giant Aplysia Neuron

Illumination of an Aplysia giant neuron evokes a membrane hyperpolarization which is associated with a membrane conductance increase of 15%. The light response is best elicited at 490 nM: the neuron also has an absorption peak at this wavelength. At the resting potential (-50 to -60 mV) illumination evokes an outward current in a voltage-clamped cell. This current reverses sign very close to E_K calculated from direct measurements of internal and external K⁺ activity. Increases in external K⁺ concentration shift the reversal potential of the light-evoked response by the same amount as the change in E_K. Decreases in external Na⁺ or Cl^- do not affect the response. Therefore, the response is attributed to an increase in K⁺ conductance. Pressure injection of Ca²⁺ into this neuron also hyperpolarizes the cell membrane. This effect is also due largely to an increase in K⁺ conductance. The light response after Ca²⁺ injection does not appear to be altered. Pressure injection of EGTA abolished or greatly reduced the light response. The effect was reversible. We suggest that light acts upon a single pigment in this neuron, releasing Ca²⁺ which in turn increases K⁺ conductance, thereby hyperpolarizing the neuronal membrane.     

Substrate kinetics of the tonoplast h-translocating inorganic pyrophosphatase and its activation by free mg

To clarify the kinetic characteristics and ionic requirements of the tonoplast H⁺-translocating inorganic pyrophosphatase (H⁺-PPiase), PPi hydrolysis and PPi-dependent H⁺ transport were studied in tonoplast vesicles isolated from leaf mesophyll tissue of Kalanchoë daigremontiana Hamet et Perrier de la Bâthie. The tonoplast H⁺-PPiase showed an absolute requirement for a monovalent cation and exhibited hyperbolic kinetics with respect to cation concentration. H⁺-PPiase activity was maximal in the presence of K⁺ (K₅₀ approximately 3 millimolar), with PPi-dependent H⁺ transport being more selective for K⁺ than PPi hydrolysis. When assayed in the presence of 50 millimolar KCl at fixed PPi concentrations, H⁺-PPiase activity showed sigmoidal kinetics with respect to total Mg concentration, reflecting a requirement for a Mg/PPi complex as substrate and free Mg²⁺ for activation. At saturating concentrations of free Mg²⁺, H⁺-PPiase activity exhibited Michaelis-Menten kinetics towards MgPPi²⁻ but not Mg₂PPi, demonstrating that MgPPi²⁻ was the true substrate of the enzyme. The apparent K_m (MgPPi²⁻) for PPi hydrolysis (17 micromolar) was significantly higher than that for PPi-dependent H⁺ transport (7 micromolar). Free Mg²⁺ was shown to be an allosteric activator of the H⁺-PPiase, with Hill coefficients of 2.5 for PPi hydrolysis and 2.7 for PPi-dependent H⁺ transport. Half-maximal H⁺-PPiase activity occurred at a free Mg²⁺ concentration of 1.1 millimolar, which lies within the range of accepted values for cytosolic Mg²⁺. In contrast, cytosolic concentrations of K⁺ and MgPPi²⁻ appear to be saturating for H⁺-PPiase activity. We propose that one function of the H⁺-PPiase may be to act as an ancillary enzyme that maintains the proton-motive force across the vacuolar membrane when the activity of the tonoplast H⁺-ATPase is restricted by substrate availability. As ATP levels decline in the cytosol, free Mg²⁺ would be released from the MgATP²⁻ complex, thereby activating the tonoplast H⁺-PPiase.     

Effects of Magnesium on Intact Chloroplasts: I. EVIDENCE FOR ACTIVATION OF (SODIUM) POTASSIUM/PROTON EXCHANGE ACROSS THE CHLOROPLAST ENVELOPE

Exogenous Mg²⁺ (2 millimolar) altered the stromal pH of intact spinach chloroplasts. Without added KCl in the medium, Mg²⁺ decreased the stromal pH in the light by approximately 0.3 pH unit. External KCl (25 millimolar) largely prevented the acidification caused by Mg²⁺. Effects on the stromal pH were not caused by changes in H⁺ pumping across the thylakoid membrane because Mg²⁺ had no effect on the light-induced quenching of atebrin fluorescence by intact chloroplasts. However, Mg²⁺ affected H⁺ fluxes across the envelope. Addition of Mg²⁺ to intact chloroplasts in the dark caused a significant acidification of the medium that was dependent on the presence of K⁺.     

Characterization of a partially purified adenosine triphosphatase from a corn root plasma membrane fraction

The (K⁺,Mg²⁺)-ATPase was partially purified from a plasma membrane fraction from corn roots (WF9 × Mol7) and stored in liquid N₂ without loss of activity. Specific activity was increased 4-fold over that of the plasma membrane fraction. ATPase activity resembled that of the plasma membrane fraction with certain alterations in cation sensitivity. The enzyme required a divalent cation for activity (Co²⁺ > Mg²⁺ > Mn²⁺ > Zn²⁺ > Ca²⁺) when assayed at 3 millimolar ATP and 3 millimolar divalent cation at pH 6.3. When assayed in the presence of 3 millimolar Mg²⁺, the enzyme was further activated by monovalent cations (K⁺, NH₄⁺, Rb⁺ Na⁺, Cs⁺, Li⁺). The pH optima were 6.5 and 6.3 in the absence and presence of 50 millimolar KCl, respectively. The enzyme showed simple Michaelis-Menten kinetics for the substrate ATP-Mg, with a K_m of 1.3 millimolar in the absence and 0.7 millimolar in the presence of 50 millimolar KCl. Stimulation by K⁺ approached simple Michaelis-Menten kinetics, with a K_m of approximately 4 millimolar KCl. ATPase activity was inhibited by sodium orthovanadate. Half-maximal inhibition was at 150 and 35 micromolar in the absence and presence of 50 millimolar KCl. The enzyme required the substrate ATP. The rate of hydrolysis of other substrates, except UDP, IDP, and GDP, was less than 20% of ATP hydrolysis. Nucleoside diphosphatase activity was less than 30% of ATPase activity, was not inhibited by vanadate, was not stimulated by K⁺, and preferred Mn²⁺ to Mg²⁺. The results demonstrate that the (K⁺,Mg²⁺)-ATPase can be clearly distinguished from nonspecific phosphohydrolase and nucleoside diphosphatase activities of plasma membrane fractions prepared from corn roots.     

Fluctuations in Spinach Leaf Nitrate Reductase During Light-Dark Transitions

In vivo nitrate reductase (NR) activity declined gradually either in absence or presence of Mg²⁺ In dark grown plants of spinach. The increased sensitivity of the extracted NR from the dark grown plants to Mg²⁺ and ATP is indicative of the post-translational modification as one of the mechanisms to control NR activity. The response of extracted NR was gradual and not instantaneous suggesting a complex interplay of NR regulation, as the dark acclimatized plants when exposed to light caused significant nitrate reduction within 15 min of light exposures even in the presence of Mg²⁺ and ATP.     

ROLE OF INORGANIC IONS IN CONTROLLING SEDIMENTATION RATE OF A MARINE CENTRIC DIATOM DITYLUM BRIGHTWELLI1,2

The settling rates and intracellular levels of K⁺, Na⁺, Cl^-, Mg²⁺ and Ca²⁺ were measured in Ditylum bright-welli (West) Grunow, grown axenically in an enriched seawater medium at 20 C at 4,000 lx on an 8:16 LD schedule. Cells at the end of the dark period have high Na⁺ (118 mM), low K⁺ (64 mM) and low Cl^- (117 mM) relative to levels at the end of the light period when K⁺ (126 mM) and Cl^- (154 mM) are high and Na⁺ (101 mM) is low. There is no significant change in Mg²⁺ (16–18 mM) or Ca²⁺ (3–4 mM) with time. The net result of the ion changes during the light period is to increase cell density by about 3.4 mg ml^-1. This change can account for the increase in settling rate of ca. 0.3 day^-1 during the same interval. The density of the cell contents, calculated from observed ion concentrations, is 15–18 mg.ml^-1 less than that of the seawater medium. The ion and settling rate changes are light-dependent and do not persist in the dark or under constant light (ca. 850 lx), but cells do exhibit a free-running circadian rhythm in cell division under continuous dim illumination. The cell vacuole expands during the light period and contracts during the dark, apparently in response to the net ion fluxes. D. brightwelli appears to regulate its density by active ion selectivity accompanied by trans-vacuolar water movement.     

Adenosine triphosphatase activity in the neural lobe of the bovine pituitary gland

1. Homogenates of neural lobes of bovine pituitary glands were fractionated by differential and density-gradient ultracentrifugation and the distribution of adenosine triphosphatase (ATPase) activity was studied. It was shown that all the activity was membrane-bound. 2. On the basis of ionic requirements the ATPase activity was grouped into three categories: (a) Mg²⁺-dependent, (b) Ca²⁺-dependent and (c) Mg²⁺+Na⁺+K⁺-dependent (ouabain-sensitive) ATPases. The activity in the absence of bivalent cations was negligible. The ratio between the activities of the three ATPases varied between the different subcellular fractions. 3. Preincubation of the subcellular fractions with deoxycholate increased the activity of the Mg²⁺+Na⁺+K⁺-dependent enzyme, whereas the Mg²⁺- and Ca²⁺-activated ATPases were either unaffected or slightly inhibited. Triton X-100 solubilized the Mg²⁺- and Ca²⁺-ATPases; however, the activity of the Mg²⁺+Na⁺+K⁺-ATPase was abolished by the concentration of Triton X-100 used. 4. All the subfractions displayed unspecific nucleotide triphosphatase activity towards GTP, ITP and UTP. These substrates inhibited the hydrolysis of ATP by all three ATPases. ADP also inhibited the ATPases. 5. Polyacrylamide-gel electrophoresis of extracts containing the Mg²⁺- and Ca²⁺-dependent ATPase activity solubilized by Triton X-100 revealed the presence of two enzymes; one activated by either Mg²⁺ or Ca²⁺ and the other activated only by Ca²⁺. 6. In sucrose density gradients the distribution of vasopressin was different from that of all three types of ATPases. It is therefore suggested that the neurosecretory granules do not possess ATPase activity.     

K stimulation of ATPase activity associated with the chloroplast inner envelope

Studies were conducted to characterize ATPase activity associated with purified chloroplast inner envelope preparations from spinach (Spinacea oleracea L.) plants. Comparison of free Mg²⁺ and Mg·ATP complex effects on ATPase activity revealed that any Mg²⁺ stimulation of activity was likely a function of the use of the Mg·ATP complex as a substrate by the enzyme; free Mg²⁺ may be inhibitory. In contrast, a marked (one- to twofold) stimulation of ATPase activity was noted in the presence of K⁺. This stimulation had a pH optimum of approximately pH 8.0, the same pH optimum found for enzyme activity in the absence of K⁺. K⁺ stimulation of enzyme activity did not follow simple Michaelis-Menton kinetics. Rather, K⁺ effects were consistent with a negative cooperativity-type binding of the cation to the enzyme, with the K_m increasing at increasing substrate. Of the total ATPase activity associated with the chloroplast inner envelope, the K⁺-stimulated component was most sensitive to the inhibitors oligomycin and vanadate. It was concluded that K⁺ effects on this chloroplast envelope ATPase were similar to this cation's effects on other transport ATPases (such as the plasmalemma H⁺-ATPase). Such ATPases are thought to be indirectly involved in active K⁺ uptake, which can be facilitated by ATPase-dependent generation of an electrical driving force. Thus, K⁺ effects on the chloroplast enzyme in vitro were found to be consistent with the hypothesized role of this envelope ATPase in facilitating active cation transport in vivo.     

Surface Charge-Mediated Effects of Mg on K Flux across the Chloroplast Envelope Are Associated with Regulation of Stromal pH and Photosynthesis

Studies of Spinacia oleracea L. were undertaken to characterize further how Mg²⁺ external to the isolated intact chloroplast interacts with stromal K⁺, pH, and photosynthetic capacity. Data presented in this report were consistent with the previously developed hypothesis that millimolar levels of external, unchelated Mg²⁺ result in lower stromal K⁺, which somehow is linked to stromal acidification. Stromal acidification directly results in photosynthetic inhibition. These effects were attributed to Mg²⁺ interaction (binding) to negative surface charges on the chloroplast envelope. Chloroplast envelope-bound Mg²⁺ was found to decrease the envelope membrane potential (inside negative) of the illuminated chloroplast by 10 millivolts. It was concluded that Mg²⁺ effects on photosynthesis were likely not mediated by this effect on membrane potential. Further experiments indicated that envelope-bound Mg²⁺ caused lower stromal K⁺ by restricting the rate of K⁺ influx; Mg²⁺ did not affect K⁺ efflux from the stroma. Mg²⁺ restriction of K⁺ influx appeared consistent with the typical effects imposed on monovalent cation channels by polyvalent cations that bind to negatively charged sites on a membrane surface near the outer pore of the channel. It was hypothesized that this interaction of Mg²⁺ with the chloroplast envelope likely mediated external Mg²⁺ effects on chloroplast metabolism.     

Effect of inhibitors on the sigmoidicity of the calcium ion transport kinetics in rat liver mitochondria

The kinetic plot (initial rate of Ca²⁺ transport versus concentration) of mitochondrial Ca²⁺ transport is hyperbolic in a sucrose medium. The plot becomes sigmoidal in the presence of competitive inhibitors of Ca²⁺ binding to low affinity sites of the membrane surface such as Mg²⁺ and K⁺. The plot also becomes sigmoidal in the presence of Ba²⁺. Ba²⁺ is a competitive inhibitor of both Ca²⁺ transport and Ca²⁺ binding to the low affinity sites. The K_i for the inhibition of Ca²⁺ transport by Ba²⁺ increases in the presence of K⁺ and Mg²⁺, which suggests a competition for the low affinity sites between the cations. The plot is still hyperbolic in the presence of La³⁺, which inhibits Ca²⁺ transport competitively. Ruthenium red which is a pure non-competitive inhibitor of mitochondrial Ca²⁺ transport, does not affect the shape of the kinetic plot. These results indicate that the surface potential, which depends on the ions bound to the low affinity sites, determines whether the kinetics of Ca²⁺ uptake in mitochondria is sigmoidal or hyperbolic.     

The mode of activation by potassium of potassium-dependent and ouabain-sensitive phosphatase activity.

A new simple procedure has been developed for the purification of plasma membranes from rabbit kidney microsomes which yields a three- to fourfold increase in the specific activity of Na⁺-K⁺-adenosine triphosphatase (ATPase). The procedure differs from previous methods with deoxycholate or other detergents and does not change the molecular activity of the ATPase. The K⁺-dependent p-nitrophenylphosphatase activity of the native Na⁺-K⁺-ATPase is controlled more effectively by Mg²⁺ in the presence of K⁺ at concentrations higher than that of Mg²⁺, and by K⁺ in the presence of Mg²⁺ at concentrations higher than that of K⁺. The enzyme in its Mg²⁺-regulating state, which shows K⁺-saturation curves with a Hill coefficient of 1, is less sensitive to ouabain (I_0.5 = 90 μM) and corresponds to the enzyme conformation reported previously which is inhibited by the concurrent presence of Na⁺ and ATP or of Na⁺ and oligomycin (I_0.5 is the midpoint of the saturation curve). The enzyme in its K⁺-regulating state, which shows K⁺-saturation curves with a Hill coefficient of 2, is more sensitive to ouabain inhibition (I₀₅ = 8 μM) and corresponds to the enzyme conformation which is stimulated by the concurrent presence of Na⁺ and ATP or of Na⁺ and oligomycin. There appear to be two conformations of the enzyme that are regulated by Mg²⁺ binding on the inhibitory sites of the enzyme.     

Properties and nature of (Na+ + Mg2+)-dependent adenosine triphosphatase in the gills of the eel, angvilla anguilla (L.)

The specific activity of (Na⁺ + Mg²⁺)-dependent ATPase is three times greater in the microsomes of sea-water eels than in freshwater eels; the specific activity is one quarter of that of (Na⁺ + K⁺ + Mg²⁺)-dependent ATPase in both cases.(Na⁺ + Mg²⁺)-dependent ATPase is optimally active in a medium containing 8 mM NaCl, 4 mM MgCI₂, 4 mM ATP, pH 8.8 and at 30 °C; the enzyme is inhibited by ouabain, by NaCl concentrations > 100 mM and by treatment with urea.It is concluded that the (Na⁺ + Mg²⁺)-dependent ATPase activity of gills arises from the presence of a (Na⁺ + K⁺ + Mg²⁺)-dependent ATPase.     

Hydrolysis of adenylyl imidodiphosphate in the presence of Na+ + Mg+ by (Na+ + K+)-activated ATPase

(1) Contrary to what has usually been assumed, (Na⁺ + K⁺)-ATPase slowly hydrolyses AdoPP[NH]P in the presence of Na⁺ + Mg²⁺ to ADP-NH₂ and P_i. The activity is ouabain-sensitive and is not detected in the absence of either Mg²⁺ or Na²⁺. The specific activity of the Na⁺ + Mg²⁺ dependent AdoPP[NH]P hydrolysis at 37°C and pH 7.0 is 4% of that for ATP under identical conditions and only 0.07% of that for ATP in the presence of K⁺. The activity is not stimulated by K⁺, nor can K⁺ replace Na⁺ in its stimulatory action. This suggests that phosphorylation is rate-limiting. Stimulation by Na⁺ is positively cooperative with a Hill coefficient of 2.4; half-maximal stimulation occurs at 5–9 mM. The K_m value for AdoPP[NH]P is 17 μM. At 0°C and 21°C the specific activity is 2 and 14%, respectively, of that at 37°C. AMP, ADP and AdoPP[CH₂]P are not detectably hydrolysed by (Na⁺ + K⁺)-ATPase in the presence of Na⁺ + Mg²⁺. (2) In addition, AdoPP[NH]P undergoes spontaneous, non-enzymatic hydrolysis at pH 7.0 with rate constants at 0, 21 and 37°C of 0.0006, 0.006 and 0.07 h^?1, respectively. This effect is small compared to the effect of enzymatic hydrolysis under comparable conditions. Mg²⁺ present in excess of AdoPP[NH]P reduces the rate constant of the spontaneous hydrolysis to 0.005 h^?1 at 37°C, indicating that the MgAdoPP[NH]P complex is virtually stable to spontaneous hydrolysis, as is also the case for its enzymatic hydrolysis. (3) A practical consequence of these findings is that AdoPP[NH]P binding studies in the presence of Na⁺ + Mg²⁺ with enzyme concentrations in the mg/ml range are not possible at temperatures above 0°C. On the other hand, determination of affinity in the (Na⁺ + K⁺)-ATPase reaction by competition with ATP at low protein concentrations (μg/ml range) remains possible without significant hydrolysis of AdoPP[NH]P even at 37°C.     

Kinetic and Structural Properties of NADP-Malic Enzyme from Sugarcane Leaves

Oligomeric structure and kinetic properties of NADP-malic enzyme, purified from sugarcane (Saccharam officinarum L.) leaves, were determined at either pH 7.0 and 8.0. Size exclusion chromatography showed the existence of an equilibrium between the dimeric and the tetrameric forms. At pH 7.0 the enzyme was found preferentially as a 125 kilodalton homodimer, whereas the tetramer was the major form found at pH 8.0. Although free forms of l-malate, NADP⁺, and Mg²⁺ were determined as the true substrates and cofactors for the enzyme at the two conditions, the kinetic properties of the malic enzyme were quite different depending on pH. Higher affinity for l-malate (K_m = 58 micromolar), but also inhibition by high substrate (K_i = 4.95 millimolar) were observed at pH 7.0. l-Malate saturation isotherms at pH 8.0 followed hyperbolic kinetics (K_m = 120 micromolar). At both pH conditions, activity response to NADP⁺ exhibited Michaelis-Menten behavior with K_m values of 7.1 and 4.6 micromolar at pH 7.0 and 8.0, respectively. Negative cooperativity detected in the binding of Mg²⁺ suggested the presence of at least two Mg²⁺ - binding sites with different affinity. The K_a values for Mg²⁺ obtained at pH 7.0 (9 and 750 micromolar) were significantly higher than those calculated at pH 8.0 (1 and 84 micromolar). The results suggest that changes in pH and Mg²⁺ levels could be important for the physiological regulation of NADP-malic enzyme.     

Ionic Selectivity and Permeation Properties of Human PIEZO1 Channels

Members of the eukaryotic PIEZO family (the human orthologs are noted hPIEZO1 and hPIEZO2) form cation-selective mechanically-gated channels. We characterized the selectivity of human PIEZO1 (hPIEZO1) for alkali ions: K⁺, Na⁺, Cs⁺ and Li⁺; organic cations: TMA and TEA, and divalents: Ba²⁺, Ca²⁺, Mg²⁺ and Mn²⁺. All monovalent ions permeated the channel. At a membrane potential of -100 mV, Cs⁺, Na⁺ and K⁺ had chord conductances in the range of 35–55 pS with the exception of Li⁺, which had a significantly lower conductance of ~ 23 pS. The divalents decreased the single-channel permeability of K⁺, presumably because the divalents permeated slowly and occupied the open channel for a significant fraction of the time. In cell-attached mode, 90 mM extracellular divalents had a conductance for inward currents carried by the divalents of: 25 pS for Ba²⁺ and 15 pS for Ca²⁺ at -80 mV and 10 pS for Mg²⁺ at -50 mV. The organic cations, TMA and TEA, permeated slowly and attenuated K⁺ currents much like the divalents. As expected, the channel K⁺ conductance increased with K⁺ concentration saturating at ~ 45 pS and the K_D of K⁺ for the channel was 32 mM. Pure divalent ion currents were of lower amplitude than those with alkali ions and the channel opening rate was lower in the presence of divalents than in the presence of monovalents. Exposing cells to the actin disrupting reagent cytochalasin D increased the frequency of openings in cell-attached patches probably by reducing mechanoprotection.