Xanthine Phosphoribosyltransferase in Leishmania: Divalent Cation Activation1

ABSTRACT. Xanthine phosphoribosyltransferase (XPRTase; EC 2.4.4.22) was found in the promastigotes of four species of Leishmania (L. mexicana, L. donovani, L. braziliensis and L. tarentolae). In no case was there any transribosylation from 5-phosphoribosyl-1-pyrophosphate (PRibPP), forming XMP, in dialyzed preparations, unless activated by a divalent cation. Magnesium and zinc were very low in activation efficiency in all cases, while manganese was optimally efficient. Cobalt was essentially equal to manganese for activation of the enzyme from L. mexicana and L. braziliensis but much less efficient for the enzyme from L. donovani and L. tarentolae. Gel filtration profiles of cell extracts of L. mexicana on Sephadex G-200 indicated that the enzymes catalyzing the transribosylation from PRibPP to guanine. hypoxanthine, and xanthine were inseparable. All were eluted near the void volume. The enzyme for adenine transribosylation was clearly separate. When cell extracts of L. mexicana were applied to Sephadex G-100 columns, the activity toward XMP formation from xanthine eluted with the void volume, together with a portion of that for the formation of GMP and IMP from guanine and hypoxanthine. A second peak of HGPRTase (EC 2.4.2.8) eluted somewhat later and was devoid of XPRTase activity. XPRTase from promastigotes of L. mexicana is heat labile, has rather a broad pH optima, and is stable to freezing when protected by nonspecific cell protein (40,000 g supernate as opposed to 100.000 g supernates).     

细菌视紫红质的光电响应特性和机制

在ITO导电玻璃上制备定向细菌视紫红质 (BR)电泳沉积膜或LB膜组成光电池系统 ,在短脉冲激光照射下 ,测定其脉冲响应光电压 ;在间断光照射下 ,测定其对光强变化产生的微分响应信号。对脉冲光电响应和微分响应的机理及其关系进行理论分析和解释 ,认为脉冲响应是BR分子内部生色团快速光极化引起的电荷分离和希夫碱及其周围氨基酸去质子化和再质子化过程引起的质子定向运输产生的位移电流 ,是一个快反应过程 ,是微分响应的早期反应和基础。微分响应则是由于菌紫质的光驱动质子泵产生的连续质子流在光开和光关瞬间引起光电池系统充放电以及测量电路的耦合特性引起的 ,是一个慢变化过程     

细菌视紫红质的质子传输机理

细菌视紫红质（bR）是嗜盐菌紫膜中的唯一蛋白质成分, 具有质子泵、电荷分离和光致变色功能. bR分子中的发色团视黄醛通过质子化席夫碱以共价键与Lys216相连. bR分子受可见光照射后, 视黄醛发生从全-反到13-顺式构型的异构化, 导致席夫碱的去质子化,继之以可极化基团位置的改变. 力场的变化引起包括蛋白质三级结构在内的诸多变化, 这些变化促进并保证了质子从细胞质侧向细胞外侧的定向传输.     

The Divalent Cation Pump and its Role in the Regulation of Malic Enzyme Activity in Purified Mitochondria from Potato Tuber

The control of the activity of the matrix-located malic enzyme(EC 1.1.1.39 [EC] ) by Mn²⁺ was investigated in Percoll-purified mitochondriafrom potato (Solarium tuberosum) tuber. Malic enzyme activitywas tightly controlled by the amount of Mn²⁺ available in thematrix space and could be stimulated by the addition of exogenousMn²⁺. A net uptake of Mn²⁺ into the matrix space of energizedmitochondria was measured. The uptake of Mn²⁺ was mediated bythe active cation pump present in the mitochondria. The activityof this cation pump was shown to be dependent on the membranepotential sustained by the activity of the respiratory chain.The uptake of Mn²⁺ was totally abolished in the presence ofan uncoupler and strongly depressed in the presence of rutheniumred, a specific inhibitor of the Ca²⁺-pump which is presentin animal mitochondria. Thus, the effect of Mn²⁺ on matrix-locatedMn²⁺-dependent malic enzyme was strongly influenced by the presenceof an uncoupler or of ruthenium red. In addition, this effectwas reduced in the presence of Ca²⁺. The possible physiologicalsignificance of the presence of this cation pump is discussedin relation to the presence of a matrix-located, NAD⁺-dependentmalic enzyme in plant mitochondria. (Received November 21, 1988; Accepted March 6, 1989)     

Electrically Silent Divalent Cation Entries in Resting and Active Voltage-Controlled Muscle Fibers

Ca²⁺ is known to enter skeletal muscle at rest and during activity. Except for the well-characterized Ca²⁺ entry through L-type channels, pathways involved in these Ca²⁺ entries remain elusive in adult muscle. This study investigates Ca²⁺ influx at rest and during activity using the method of Mn²⁺ quenching of fura-2 fluorescence on voltage-controlled adult skeletal muscle cells. Resting rate of Mn²⁺ influx depended on external [Mn²⁺] and membrane potential. At −80 mV, replacement of Mg²⁺ by Mn²⁺ gave rise to an outward current associated with an increase in cell input resistance. Calibration of fura-2 response indicated that Mn²⁺ influx was too small to be resolved as a macroscopic current. Partial depletion of the sarcoplasmic reticulum induced by a train of action potentials in the presence of cyclopiazonic acid led to a slight increase in resting Mn²⁺ influx but no change in cell input resistance and membrane potential. Trains of action potentials considerably increased Mn²⁺ entry through an electrically silent pathway independent of L-type channels, which provided 24% of the global Mn²⁺ influx at +30 mV under voltage-clamp conditions. Within this context, the nature and the physiological role of the Ca²⁺ pathways involved during muscle excitation still remain open questions.     

Mechanism of Nonlinear Photoinduced Anisotropy in Bacteriorhodopsin and its Derivatives

Polymer films made with photosensitive chromophore protein bacteriorhodopsin (BR) from the extreme halophile Halobacterium salinarium as well as films made with BR derivatives exhibit a nonlinear photoinduced anisotropy. Two different methods can be used to induce anisotropy in polymer BR films. The first method is based on the anisotropic properties of the initial form of the photocycle, BR570 (B-type anisotropy). Another method is based on the anisotropic properties of the longest-lived photocycle intermediate M412 (M-type anisotropy). CW gas lasers were employed to induce a reversible anisotropy in polymer BR films. Nonlinear photoinduced anisotropy is discussed in the context of a model for the anisotropic photoselection of BR molecules under linearly polarized light. A comparison of the experimental dependencies of nonlinear photoinduced anisotropy on laser intensity with similar calculated dependencies enables one to determine the molecular dichroism of BR and its derivatives not only for the initial form of the photocycle, B but also for the longest-lived intermediate M. Here we present the data showing the correlation between the laser induced nonlinear anisotropic properties and chromophore/protein interactions in BR. The effect of polymer binder on the nonlinear photoanisotropic properties of polymer BR films is also described.     

Activation of Divalent Cation Influx into S. cerevisiae Cells by Hypotonic Downshift

Subjecting Saccharomyces cerevisiae cells to a hypotonic downshift by transferring cells from YPD medium containing 0.8 m sorbitol to YPD medium without sorbitol induces a transient rapid influx of Ca²⁺ and other divalent cations into the cell. For cells grown in YPD at 37°C, this hypotonic downshift increases Ca²⁺ accumulation 6.7-fold. Hypotonic downshift-induced Ca²⁺ accumulation and steady-state Ca²⁺ accumulation in isotonic YPD medium are differentially affected by dodecylamine and Mg²⁺. The Ca²⁺-influx pathway responsible for hypotonic-induced Ca²⁺ influx may account for about 10–35% of Ca²⁺ accumulation by cells growing in YPD. Ca²⁺ influx is not required for cells to survive a hypotonic downshift. Hypotonic downshift greatly reduces the ability of S. cerevisiae cells to survive a 5-min exposure to 10 mm Cd²⁺ suggesting that mutants resistant to acute Cd²⁺ exposure may help identify genes required for hypotonic downshift-induced divalent cation influx. Received: 14 January 1997/Revised: 20 June 1997     

Divalent Cation and ATP Dependent Motility of Toxoplasma gondii Tachyzoites After Mild Treatment with Trypsin

ABSTRACT. Large percentages of Toxoplasma gondii tachyzoites could be induced to display two types of movement associated with active invasive behavior by exposing them for 1 min to 0.002% trypsin in phosphate-buffered saline (PBS). The motile activity, consisting of clockwise rotation around the posterior end (about 20 revolutions per min) and twirling-gliding over a poly-L-lysine substrate (1.2 ± 0.2 μm/s standard deviation), was observed and recorded by video-enhanced contrast microscopy. The number of active tachyzoites reached a maximum 1 min after trypsinization; the motile response of the population lasted for about 5 min. Activation was prevented by soybean trypsin-inhibitor, and could not be induced again in previously treated specimens. Electron-microscopy of trypsinized tachyzoites fixed in the presence of ruthenium-red revealed discrete discontinuities of the plasma membrane, which sealed within 90 min after washing with PBS. Treated tachyzoites were able to invade cultured epithelial cells with a higher relative infectivity than that of untreated parasites. Perfusion of trypsinized tachyzoites with 1 mM of either CaCl₂ or MgCl₂ and 1 mM ATP increased the number of activated parasites to over 60%; on the other hand, all induced motility was inhibited or blocked by agents that chelate divalent cations. The present preparation, which provided the first serial illustrations of T. gondii movements induced by a defined chemical stimulus, may offer a useful experimental model for the study of motility in this parasite.     

Divalent Cation Block of Inward Currents and Low-Affinity K+ Uptake in Saccharomyces cerevisiae

We have used the patch clamp technique to characterize whole-cell currents in spheroplasts isolated from a trk1Δ trk2Δ strain of Saccharomyces cerevisiae which lacks high- and moderate-affinity K⁺ uptake capacity. In solutions in which extracellular divalent cation concentrations were 0.1 mM, cells exhibited a large inward current. This current was not the result of increasing leak between the glass pipette and membrane, as there was no effect on the outward current. The inward current comprised both instantaneous and time-dependent components. The magnitude of the inward current increased with increasing extracellular K⁺ and negative membrane potential but was insensitive to extracellular anions. Replacing extracellular K⁺ with Rb⁺, Cs⁺, or Na⁺ only slightly modulated the magnitude of the inward current, whereas replacement with Li⁺ reduced the inward current by approximately 50%, and tetraethylammonium (TEA⁺) and choline were relatively impermeant. The inward current was blocked by extracellular Ca²⁺ and Mg²⁺ with apparent K_is (at −140 mV) of 363 ± 78 and 96 ± 14 μM, respectively. Furthermore, decreasing cytosolic K⁺ increased the magnitude of the inward current independently of the electrochemical driving force for K⁺ influx, consistent with regulation of the inward current by cytosolic K⁺. Uptake of ⁸⁶Rb⁺ by intact trk1Δ trk2Δ cells was inhibited by extracellular Ca²⁺ with a K_i within the range observed for the inward current. Furthermore, increasing extracellular Ca²⁺ from 0.1 to 20 mM significantly inhibited the growth of these cells. These results are consistent with those of the patch clamp experiments in suggesting that low-affinity uptake of alkali cations in yeast is mediated by a transport system sensitive to divalent cations.     

Energy-linked Adenosine Diphosphate Accumulation by Corn Mitochondria: II. Phosphate and Divalent Cation Requirement

The requirement for phosphate and Mg²⁺ in energy-linked [³H] ADP accumulation by corn mitochondria has been studied. Arsenate will fully substitute for phosphate; sulfate partially substitutes; acetate, bicarbonate, and pyrophosphate are ineffective. Phosphate is also taken up by the mitochondria, but the ADP/Pi ratio varies widely with experimental treatments. ADP does not exchange with endogenous labeled phosphate, although Pi/³²Pi exchange occurs.     

Modulation of Calcitonin Binding by Calcium: Differential Effects of Divalent Cations

Abstract

Binding of salmon calcitonin to bovine hypothalamic membranes is enhanced about 25% by calcium with a half-maximal effect at 15 mM calcium. In contrast, membranes prepared from a cell line expressing a recombinant human calcitonin receptor show no effect of calcium under similar conditions. The hypothalamic calcitonin receptor solubilized with CHAPS detergent retains an apparent Kd of 0.3 nM for salmon calcitonin; however, binding of calcitonin to the detergent-solubilized receptor complex can be inhibited by divalent cations in order of potency Mn>Ca Sr Mg NaCl with Mn and Ca having apparent Ki's of 5 mM and 20 mM respectively. Dixon and Scatchard plots of Mn and Ca inhibition of binding to the soluble receptor complex suggest a noncompetitive mechanism of inhibition. Calcium also inhibits calcitonin binding to a detergent-solubilized recombinant human calcitonin receptor. Inhibition of calcitonin binding is observed using two independent methods for determining soluble receptor-hormone complex and inhibition is reversed by EDTA.     

HCO(3) Influx Across the Plasmalemma of Chara corallina: Divalent Cation Requirement

An absolute requirement for divalent cations is reported for H(14)CO(3) (-) influx in Chara corallina. Effective substitution of eluted Ca(2+) by Mg(2+) and Sr(2+) was observed, but Mn(2+) was completely ineffective in restoring H(14)CO(3) (-) transport activity. Similarly, La(3+) could not substitute for Ca(2+) in this system. Low concentrations of ethylenediaminetetraacetate (0.01 to 0.06 mm) significantly enhanced the rate at which H(14)CO(3) (-) transport capacity was lost.Examination of the response of OH(-) efflux, during Ca(2+)-free treatment, indicated that the cellular control over OH(-) efflux remained unaffected until membrane integrity became severely affected. This conclusion was supported by the response of OH(-) efflux to 10 mm K(+). Therefore, assimilation of H(14)CO(3) (-) is not rate-limited by an effect of Ca(2+) elution on the OH(-) transport system. Kinetic experiments indicated that Ca(2+) removal from the membrane resulted in noncompetitive inhibition of H(14)CO(3) (-) assimilation; the apparent Michaelis constant remained unaltered over a wide range of conditions. An hypothesis is presented which suggests that membrane integrity is necessary for HCO(3) (-) transport to occur, but Ca(2+) (Mg(2+), Sr(2+)), per se, must be bound to the transport complex before activity is established.     

Divalent Cation Interactions with Na,K-ATPase Cytoplasmic Cation Sites: Implications for the <Emphasis Type="Italic">para</Emphasis>-Nitrophenyl Phosphatase Reaction Mechanism

The interactions of divalent cations with the adenosine triphosphatase (ATPase) and para-nitrophenyl phosphatase (pNPPase) activity of the purified dog kidney Na pump and the fluorescence of fluorescein isothiocyanate (FITC)-labeled pump were determined. Sr²⁺ and Ba²⁺ did not compete with K⁺ for ATPase (an extracellular K⁺ effect). Sr²⁺ and Ba²⁺ did compete with Na⁺ for ATPase (an intracellular Na⁺ effect) and with K⁺ for pNPPase (an intracellular K⁺ effect). These results suggest that Ba²⁺ or Sr²⁺ can bind to the intracellular transport site, yet neither Ba²⁺ nor Sr²⁺ was able to activate pNPPase activity; we confirmed that Ca²⁺ and Mn²⁺ did activate. As another measure of cation binding, we observed that Ca²⁺ and Mn²⁺, but not Ba²⁺, decreased the fluorescence of the FITC-labeled pump; we confirmed that K⁺ substantially decreased the fluorescence. Interestingly, Ba²⁺ did shift the K⁺ dose-response curve. Ethane diamine inhibited Mn²⁺ stimulation of pNPPase (as well as K⁺ and Mg²⁺ stimulation) but did not shift the 50% inhibitory concentration (IC₅₀) for the Mn²⁺-induced fluorescence change of FITC, though it did shift the IC₅₀ for the K⁺-induced change. These results suggest that the Mn²⁺-induced fluorescence change is not due to Mn²⁺ binding at the transport site. The drawbacks of models in which Mn²⁺ stimulates pNPPase by binding solely to the catalytic site vs. those in which Mn²⁺ stimulates by binding to both the catalytic and transport sites are presented. Our results provide new insights into the pNPPase kinetic mechanism as well as how divalent cations interact with the Na pump.     

Divalent Cation Activation of Deoxycholate-Solubilized and -Inactivated Membrane Reduced Nicotinamide Adenine Dinucleotide Oxidase of Bacillus megaterium KM

After treating Bacillus megaterium KM membranes with 0.2% sodium deoxycholate, most of the membrane reduced nicotinamide adenine dinucleotide (NADH) oxidase was inactivated, and all of the membrane NADH-2,6 dichlorophenol indophenol oxidoreductase was solubilized. Dilution of the deoxycholate-treated membranes in the presence of divalent cations restored almost all of the original membrane NADH oxidase. The effectiveness of the divalent cation activation decreased in the order Ba(2+) > Ca(2+) > Mg(2+) > Mn(2+). After centrifugation, the deoxycholate-treated membranes at 100,000 x g for 1 hr, all of the NADH oxidase that was activated by a divalent cation was soluble. Cation-activated oxidase, however, was insoluble. The results show that 0.2% deoxycholate at least partially solubilizes the total electron chain from NADH to O(2) in an inactive from which can be reactivated by divalent cations with the formation of active, insoluble NADH oxidase.     

Modulation of Binding at Opioid Receptors by Monoand Divalent Cations and by Cholinergic Compounds

Abstract

In membrane suspensions from guinea-pig brain, NaCl, LiCl, NH₄Cl and KCl, inhibit the equilibrium binding (25°C) of the selective μ-agonist [³H]-[D-Ala²,MePhe⁴,Gly-ol⁵]enkephalin, the selective δ-agonist [³H]-[D-Pen²,D-Pen⁵]enkephalin and the selective δ-agonist [³H]-dynorphin A (1-9). Choline chloride inhibits the binding of the μ- and δ-agonists but not of the δ-agonist; the choline derivative, methacholine, inhibits also the binding of the δ-agonist. Binding of the δ-agonist is potentiated by CaCl₂, MgCl₂ and MnCl₂; these salts inhibit binding of the δ-agonist. As far as binding of the μ-agonist is concerned, MgCl₂ and MnCl₂ may potentiate or inhibit whereas CaCl₂ is only inhibitory. The binding of the μ-antagonist [³H]-naloxone is potentiated by NaCl; while the threshold of inhibition by LiCl is increased there is no potentiation. In membrane suspensions of the rabbit cerebellum about 80% of the opioid binding sites are of the μ-type; the binding of the μ-agonist [³H]-[D-Ala², MePhe⁴, Gly-ol⁵]enkephalin is inhibited by NaCl, LiCl, KCl and choline chloride whereas that of the μ-antagonists [³H]-naloxone and [³H]-(-)-bremazocine is potentiated at low concentrations but inhibited at higher concentrations of NaCl. In membranes of the guinea-pig cerebellum about 80% of the opioid binding sites are of the δ-type; they are particularly effective for assays of K-receptors when the selective K-agonist [³H]-dynorphin A (1-9) is used as ligand.     

Epithelial Barrier Resistance is Increased by the Divalent Cation Zinc in Cultured MDCKII Epithelial Monolayers

Topical zinc applications promote wound healing and epithelialization. "Leaky" MDCKII epithelia exposed to apical ZnCl? (10 mM) showed a time-dependent increase (t (0.5) 22.2 ± 2.7 min) of transepithelial resistance (R (t)) from 82.3 ± 2.4 Ω cm2 to 1,551 ± 225.6 Ω cm2; the increase was dose-dependent, being observed at 3 mM but not at 1 mM. Basal Zn2+ applications also increased epithelial resistance (at 10 mM to 323 ± 225.6 Ω cm2). The linear current-voltage relationship in control epithelia changed after apical 10 mM ZnCl? to show rectification. Voltage deflections resulting from inward currents showed time-dependent relaxation (basal potential difference (p.d.)-positive), with outward currents being time-independent. Cation selectivity was tested after apical ZnCl? elevated resistance; both the NaCl:mannitol (basal replacement) dilution p.d. and the choline:Na bi-ionic p.d. decreased (P(Na)/P(Cl) from 4.9 to 2.3 and P(Na)/P(choline) from 3.8 to 2.1, respectively). Transepithelial paracellular basal to apical ??Ca fluxes increased approximately twofold when driven by a basal positive Na:NMDG bi-ionic p.d., but with basal 10 mM ZnCl?, ??Ca fluxes decreased approximately twofold. Neither ZO-1 nor occludin distribution was altered after ~2-h exposure to apical 10 mM ZnCl?. However, claudin-2, though present at the tight junction, increased within the cell. Increased epithelial barrier resistance by Zn2+ is due to modification of the paracellular pathway, most probably by multiple mechanisms.     

Proton Transport Mechanism of Bacteriorhodopsin as Revealed by Site-Specific Mutagenesis and Protein Sequence Variability

A large share of the current ideas about the mechanism of proton transport by bacteriorhodopsin has emerged from studies of site-specific mutants. This review is an attempt to check some of these ideas against the natural variability in the primary structure of the protein.