Distributions of Li+, Na+, K+, Rb+, and Cs+ tracer ions in erythrocytes at 38 °C in relation to entry rates of these ions into cells at 0 °C

Forces that are able to transport Na+ and K+ into two compartments were investigated. A modified Nernst-Planck equation for coupled flows of electric current, water, and ions was integrated. The result shows that if alkali ions in the ion channel of the cell membrane are separated by their electric-current-induced inward flows against an electro-osmotic outward flow of water, the logarithms of the stationary cell/medium distributions of these ions should be proportional to the inverse of their diffusion mobilities. The relationship was tested in human erythrocytes. From inward and outward movements of tracer alkali ions, calculations were made to obtain their stationary distributions at infinite time. The cell/medium distributions determined in this way at 38 degrees C are Li+ = 0.59, 22Na+ = 0.044, 42K+ = 10.0, 86Rb+ = 11.9, and 137Cs+ = 3.07. The entry rates of ions into the cell at 0 degrees C are understood to represent their diffusion mobilities in the pump channel. The entry rates are Li+ = 1.44, 2Na+ = 1, 42K+ = 2.22, 86Rb+ = 2.39, and 137Cs+ = 1.72 relative to that of 22Na+. There is an expected negative correlation between the logarithms of the stationary cell/ medium distributions at 38 degrees C and the inverse of the entry rates into the cell at 0 degrees C for the five ions. It is suggested that the proposed physical forces cause the separation of alkali ions in the channel of Na,K-ATPase.     

Loop diuretics may fail to inhibit (Na+, K+, Cl−) ‘cotransport’ in human red cells

The inhibition of passive K+ influx into human red blood cells (RBC) by loop diuretics was found to be dependent on the external Na+ concentration. In the absence of external Na+, there was minimal inhibition but the influx remained dependent on Cl- ions. Thus, raising the external Na+ concentration increased the affinity of the putative (Na+, K+, Cl-) cotransport system in human RBC for loop diuretics.     

Bumetanide-sensitive Ion Fluxes in Vascular Smooth Muscle Cells: Lack of Functional Na+, K+, 2 Cl− Cotransport

To examine the involvement of Na⁺,K⁺,2Cl⁻ cotransport in monovalent ion fluxes in vascular smooth muscle cells (VSMC), we compared the effect of bumetanide on ⁸⁶Rb, ³⁶Cl and ²²Na uptake by quiescent cultures of VSMC from rat aorta. Under basal conditions, the values of bumetanide-sensitive (BS) inward and outward ⁸⁶Rb fluxes were not different. Bumetanide decreased basal ⁸⁶Rb uptake by 70–75% with a K _i of ∼0.2–0.3 μm. At concentrations ranging up to 1 μm, bumetanide did not affect ³⁶Cl influx and reduced it by 20–30% in the range from 3 to 100 μm. In contrast to ⁸⁶Rb and ³⁶Cl influx, bumetanide did not inhibit ²²Na uptake by VSMC. BS ⁸⁶Rb uptake was completely abolished in Na⁺- or Cl⁻-free media. In contrast to ⁸⁶Rb, basal BS ³⁶Cl influx was not affected by Na⁺ _o and K⁺ _o . Hyperosmotic and isosmotic shrinkage of VSMC increased ⁸⁶Rb and ³⁶Cl influx to the same extent. Shrinkage-induced increments of ⁸⁶Rb and ³⁶Cl uptake were completely abolished by bumetanide with a K _i or ∼0.3 μm. Shrinkage did not induce BS ⁸⁶Rb and ³⁶Cl influx in (Na⁺ or Cl⁻)- and (Na⁺ or K⁺)-depleted media, respectively. In the presence of an inhibitor of Na⁺/H⁺ exchange (EIPA), neither hyperosmotic nor isosmotic shrinkage activated ²²Na influx. Bumetanide (1 μm) did not modify basal VSMC volume and intracellular content of sodium, potassium and chloride but abolished the regulatory volume increase in isosmotically-shrunken VSMC. These data demonstrate the absence of the functional Na⁺,K⁺,2Cl⁻ cotransporter in VSMC and suggest that in these cells basal and shrinkage-induced BS K⁺ influx is mediated by (Na⁺ _o + Cl⁻ _o )-dependent K⁺/K⁺ exchange and Na⁺ _o -dependent K⁺,Cl⁻ cotransport, respectively. Received: 30 January 1996/Revised: 20 May 1996     

Evaluation of a Two-Site, Three-Barrier Model for Permeation in CaV3.1 (α1G) T-Type Calcium Channels: Ca2+, Ba2+, Mg2+, and Na+

We explored the ability of a two-site, three-barrier (2S3B) Eyring model to describe recently reported data on current flow through open Ca_V3.1 T-type calcium channels, varying Ca²⁺ and Ba²⁺ over a wide range (100 nm–110 mm) while recording whole-cell currents over a wide voltage range (−150 mV to +100 mV) from channels stably expressed in HEK 293 cells. Effects on permeation were isolated using instantaneous current–voltage relationships (IIV) after strong, brief depolarizations to activate channels with minimal inactivation. Most experimental results were reproduced by a 2S3B model. The model described the IIV relationships, apparent affinities for permeation and block for Ca²⁺ and Ba²⁺, and shifts in reversal potential between Ca²⁺ and Ba²⁺. The fit to block by 1 mm  \textMg²⁺_\texti {\text{Mg}}^{2+}_{\text{i}} was reasonable, but block by \textMg²⁺_\texto {\text{Mg}}^{2+}_{\text{o}} was described less well. Surprisingly, fits were comparable with strong ion–ion repulsion, with no repulsion, or with intermediate values. With weak repulsion, there was a single high-affinity site, with a low-affinity site near the cytoplasmic side of the pore. With strong repulsion, the net charge of ions in the pore was near +2 over a relatively wide range of concentration and voltage, suggesting a knockoff mechanism. With strong repulsion, Ba²⁺ preferred the inner site, while Ca²⁺ preferred the outer site, potentially explaining faster entry of Ni²⁺ and other pore blockers when Ba²⁺ is the charge carrier.     

Ammonium transport in medullary thick ascending limb of rabbit kidney: Involvement of the Na+, K+, Cl−-cotransporter

Summary In order to investigate the question whether ammonium reabsorption in the thick ascending limb of Henle's loop (TALH) proceeds via the Na⁺,K⁺,Cl^–-cotransporter, plasma membrane vesicles were prepared from TALH cells isolated from rabbit kidney outer medulla and the effect of NH ₄ ⁺ on their transport properties was investigated. It was found that, in the presence of a 78-mmol/liter NaCl gradient, 5 mmol/liter NH ₄ ⁺ inhibited bumetanide-sensitive rubidium flux by 86%; a similar decrease was observed for 5 mmol/liter, K⁺. Inhibition of bumetanide-sensitive rubidium uptake by NH ₄ ⁺ was competitive and an apparentK _i of 1.9 mmol/liter was found Bumetanide-sensitive sodium uptake measured in the presence of a 83 mmol/liter KCl gradient was not inhibited by 5 mmol/liter NH ₄ ⁺ . A 100-mmol/liter NH₄Cl gradient was, however, capable of stimulating bumetanide-sensitive sodium uptake to the same extent as a KCl gradient. These data suggest that NH ₄ ⁺ is accepted by the K⁺ site of the Na⁺,K⁺,Cl-cotransport system and that the transporter can function in a Na⁺, NH ₄ ⁺ ,2Cl mode. Since the affinity of the transporter for NH ₄ ⁺ lies in the concentration range found in the TALH lumen in vivo, it is concluded that Na⁺, NH ₄ ⁺ 2Cl-cotransport can contribute to the NH ₄ ⁺ reabsorption in this tubular segment.     

Purinergic inhibition of Na+,K+,Cl− cotransport in C11-MDCK cells: Role of stress-activated protein kinases

Previously, we observed that sustained activation of P2Y₁ leads to inhibition of Na⁺,K⁺,Cl⁻ cotransport (NKCC) in C11 cells resembling intercalated cells from collecting ducts of the Madin-Darby canine kidney. This study examined the role of stress-activated protein kinases (SAPK) in NKCC inhibition triggered by purinergic receptors. Treatment of C11 cells with ATP led to sustained phosphorylation of SAPK such as JNK and p38. Activation of these kinases also occurred in anisomycin-treated cells. Surprisingly, we observed that compounds SP600125 and SB202190, known as potent inhibitors of JNK and p38 in cell-free systems, activated rather than inhibited phosphorylation of the kinases in C11 cells. Importantly, similarly to ATP, all the above-listed activators of JNK and p38 phosphorylation inhibited NKCC. Thus, our results suggest that activation of JNK and/or p38 contributes to NKCC suppression detected in intercalated-like cells from distal tubules after their exposure to P2Y₁ agonists.     

Na+,K+-ATPase亚单位表达的研究进展

以往研究已发现Na+,K+-ATPase含有α、β和γ亚单位.为了对三种亚单位有一个较为全面的认识,现对亚单位的基本结构、研究简况、生理及病理功能、表达调节等基本情况作一综述.     

Myogenic tone in mouse mesenteric arteries: evidence for P2Y receptor-mediated, Na+, K+, 2Cl− cotransport-dependent signaling

This study examines the action of agonists and antagonists of P2 receptors on mouse mesenteric artery contractions and the possible involvement of these signaling pathways in myogenic tone (MT) evoked by elevated intraluminal pressure. Both ATP and its non-hydrolyzed analog α,β-ATP triggered transient contractions that were sharply decreased in the presence of NF023, a potent antagonist of P2X₁ receptors. In contrast, UTP and UDP elicited sustained contractions which were suppressed by MRS2567, a selective antagonist of P2Y₆ receptors. Inhibition of Na⁺, K⁺, 2Cl⁻ cotransport (NKCC) with bumetanide led to attenuation of contractions in UTP- but not ATP-treated arteries. Both UTP-induced contractions and MT were suppressed by MRS2567 and bumetanide but were insensitive to NF023. These data implicate a P2Y₆-mediated, NKCC-dependent mechanism in MT of mesenteric arteries. The action of heightened intraluminal pressure on UTP release from mesenteric arteries and its role in the triggering of P2Y₆-mediated signaling should be examined further.     

几种抗血吸虫药物对日本血吸虫雄虫体表膜液内K~+,Na~+,H~+转递的影响

本文采用Oak＇s脱膜方法与火焰光度测定经吡喹酮,吐酒石和敌百虫作用后的日本血吸虫体表膜液内的K~+,Na~+的含量实验结果,吡喹酮和吐酒石能刺激K~+从虫体表膜内向外流,分别降低K~+浓度约50%和20%,但对Na~+转运无显著影响.敌百虫的作用是减少膜内K~+外流,导致了膜内K~+浓度升高.这些结果是与体外~(86)Rb渗入虫膜实验一致的.我们也测定了药物作用后虫体表膜液内的H~+,其结果是K~+的外流与H~+的内流有关.     

Rabit distal colon epithelium: II. Characterization of (Na+, K+, Cl−)-cotransport and [3H]-bumetanide binding

Summary Loop diuretic-sensitive (Na⁺,K⁺,Cl^–)-cotransport activity was found to be present in basolateral membrane vesicles of surface and crypt cells of rabbit distal colon epithelium. The presence of grandients of all three ions was essential for optimal transport activity (Na⁺,K⁺) gradien-driven³⁶Cl fluxes weree half-maximally inhibited by 0.14 m bumetanide and 44 m furosimide. While⁸⁶Rb uptake rates showed hyperbolic dependencies on Na⁺ and K⁺ concentrations with Hill coefficients of 0.8 and 0.9, respectively, uptakes were sigmoidally related to the Cl^– concentration, Hill coefficient 1.8, indicating a 1 Na⁺: 1 K⁺:2 Cl^– stoichiometry of ion transport.The interaction of putative (Na⁺, K⁺, Cl^–)-cotransport proteins with loop diuretics was studied from equilibrium-binding experiments using [³H]-bumetanide. The requirement for the simulataneous presence of Na⁺,K⁺, and Cl^–, saturability, reversibility, and specificity for diuretics suggest specific binding to the (Na⁺, K⁺, Cl^–)-cotransporter. [³H]-bumetanide recognizes a minimum of two classes of diuretic receptors sites. high-affinity (K _D1=0.13 m;B _max1 =6.4 pmol/mg of protein) and low-affinity (K _D2=34 m;B _max2=153 pmol/mg of protein) sites. The specific binding to the high-affinity receptor was found to be linearly competitive with Cl^– (K ₁=60mm), whereas low-affinity sites seem to be unaffected by Cl^–. We have shown that only high-affinity [³H]-bumetanide binding correlates with transport inhibition raising questions on the physiological significance of diuretic receptor site heterogeneity observed in rabbit distal colon epithelium.     

ATP-Induced Inhibition of Na+, K+, Cl− Cotransport in Madin-Darby Canine Kidney Cells: Lack of Involvement of Known Purinoceptor-Coupled Signaling Pathways

P_2U/2Y-receptors elicit multiple signaling in Madin-Darby canine kidney (MDCK) cells, including a transient increase of [Ca²⁺] _i , activation of phospholipases C (PLC) and A₂ (PLA₂), protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). This study examines the involvement of these signaling pathways in the inhibition of Na⁺,K⁺,Cl⁻ cotransport in MDCK cells by ATP. The level of ATP-induced inhibition of this carrier (∼50% of control values) was insensitive to cholera and pertussis toxins, to the PKC inhibitor calphostin C, to the cyclic nucleotide-dependent protein kinase inhibitors, H-89 and H-8 as well as to the inhibitor of serine-threonine type 1 and 2A phosphoprotein phosphatases okadaic acid. ATP led to a transient increase of [Ca²⁺]_i that was abolished by a chelator of Ca²⁺ _i , BAPTA. However, neither BAPTA nor the Ca²⁺ ionophore A231287, or an inhibitor of endoplasmic reticulum Ca²⁺-pump, thapsigargin, modified ATP-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport. An inhibitor of PLC, U73122, and an inhibitor of MAPK kinase (MEK), PD98059, blocked ATP-induced inositol-1,4,5-triphosphate production and MAPK phosphorylation, respectively. However, these compounds did not modify the effect of ATP on Na⁺,K⁺,Cl⁻ cotransport activity. Inhibitors of PLA₂ (AACOCF₃), cycloxygenase (indomethacin) and lypoxygenase (NDGA) as well as exogenous arachidonic acid also did not affect ATP-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport. Inhibition of the carrier by ATP persisted in the presence of inhibitors of epithelial Na⁺ channels (amiloride), Cl⁻ channels (NPPB) and Na⁺/H⁺ exchanger (EIPA) and was insensitive to cell volume modulation in anisosmotic media and to depletion of cells with monovalent ions, thus ruling out the role of other ion transporters in purinoceptor-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport. Our data demonstrate that none of the known purinoceptor-stimulated signaling pathways mediate ATP-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport and suggest the presence of a novel P₂-receptor-coupled signaling mechanism. Received: 29 July 1998/Revised: 19 October     

拟南芥内膜Na+,K+/H+反向转运体研究进展

拟南芥内膜Na,K~+/H~+反向转运体(Endosomal NHX)的亚细胞定位、离子转运特性及生物学功能阐释取得了重要进展。拟南芥内膜Na~+,K~+/H~+反向转运体包含AtNHX5和AtNHX6两个成员,它们的氨基酸序列相似性为78.7%。研究表明,AtNHX5和AtNHX6具有功能冗余,它们都定位在高尔基体(Golgi)、反面高尔基体管网状结构(TGN)、内质网(ER)和液胞前体(PVC),参与调控耐盐胁迫、pH平衡和K~+平衡等。有报道显示内膜NHXs跨膜结构域存在能够调控自身离子活性的酸性保守氨基酸残基,对其自身功能具有决定性作用。最新研究结果表明,拟南芥内膜NHXs影响囊泡运输和蛋白存贮,并参与生长素介导的植物生长和发育。文中主要对拟南芥内膜NHXs的亚细胞定位、离子转运、功能及应用进展进行了概述。     

Kinetic mechanism of Na+, K+, Cl−-cotransport as studied by Rb+ influx into HeLa cells: Effects of extracellular monovalent ions

Summary Ouabain-insensitive, furosemide-sensitive Rb⁺ influx (J _Rb) into HeLa cells was examined as functions of the extracellular Rb⁺, Na⁺ and Cl^– concentrations. Rate equations and kinetic parameters, including the apparent maximumJ _Rb, the apparent values ofK _m for the three ions and the apparentK _i for K⁺, were derived. Results suggested that one unit molecule of this transport system has one Na⁺, one K⁺ and two Cl^– sites with different affinities, one of the Cl^– sites related with binding of Na⁺, and the other with binding of K⁺(Rb⁺). A 11 stoichiometry was demonstrated between ouabain-insensitive, furosemidesensitive influxes of²²Na⁺ and Rb⁺, and a 12 stoichiometry between those of Rb⁺ and³⁶Cl^–. The influx of either one of these ions was inhibited in the absence of any one of the other two ions. Monovalent anions such as nitrate, acetate, thiocyanate and lactate as substitutes for Cl^– inhibited ouabain-insensitive Rb⁺ influx, whereas sulfamate and probably also gluconate did not inhibitJ _Rb. From the present results, a general model and a specialized cotransport model were proposed: 1) In HeLa cells, one Na⁺ and one Cl^– bind concurrently to their sites and then one K⁺ (Rb⁺) and another Cl^– bind concurrently. 2) After completion of ion bindings Na⁺, K⁺(Rb^–) and Cl^– in a ratio of 112 show synchronous transmembrane movements.     

Purinergic Modulation of Na+,K+,Cl− Cotransport and MAP Kinases is Limited to C11-MDCK Cells Resembling Intercalated Cells from Collecting Ducts

We demonstrated recently that in renal epithelial cells from collecting ducts of Madin-Darby canine kidneys (MDCK), Na⁺,K⁺,Cl⁻ cotransport is inhibited up to 50% by ATP via its interaction with P_2Y purinoceptors (Biochim. Biophys. Acta 1998. 1369:233–239). In the present study we examined which type of renal epithelial cells possesses the highest sensitivity of Na⁺,K⁺,Cl⁻ cotransport to purinergic regulation. We did not observe any effect of ATP on Na⁺,K⁺,Cl⁻ cotransport in renal epithelial cells from proximal and distal tubules, whereas in renal epithelial cells from rabbit and rat collecting ducts ATP decreased the carrier's activity by ∼30%. ATP did not affect Na⁺,K⁺,Cl⁻ cotransport in C7 subtype MDCK cells possessing the properties of principal cells but led to ∼85% inhibition of this carrier in C11-MDCK cells in which intercalated cells are highly abundant. Both C7- and C11-MDCK exhibited ATP-induced IP₃ and cAMP production and transient elevation of [Ca²⁺] _i . In contrast to the above-listed signaling systems, ATP-induced phosphorylation of ERK and JNK MAP kinases was observed in C11-MDCK only. Thus, our results reveal that regulation of renal Na⁺,K⁺,Cl⁻ cotransport by P_2Y receptors is limited to intercalated cells from collecting ducts and indicate the involvement of the MAP kinase cascade in purinergic control of this ion carrier's activity. Received: 10 June 1999/Revised: 23 August 1999     

Na+, K+, Cl− cotransport and its regulation in Ehrlich ascites Tumor cells. Ca2+/Calmodulin and protein kinase C dependent pathways

Summary Net Cl^– uptake as well as unidirectional³⁶Cl influx during regulatory volume increase (RVI) require external K⁺. Half-maximal rate of bumetanide-sensitive³⁶Cl uptake is attained at about 3.3mm external K⁺. The bumetanide-sensitive K⁺ influx found during RVI is strongly dependent on both Na⁺ and Cl^–. The bumetanide-sensitive unidirectional Na⁺ influx during RVI is dependent on K⁺ as well as on Cl^–. The cotransporter activated during RVI in Ehrlich cells, therefore, seems to transport Na⁺, K⁺ and Cl^–. In the presence of ouabain and Ba⁺ the stoichiometry of the bumetanide-sensitive net fluxes can be measured at 1.0 Na⁺, 0.8 K⁺, 2.0 Cl^– or approximately 1 : Na, 1 : K, 2 : Cl. Under these circumstances the K⁺ and Cl^– flux ratios (influx/efflux) for the bumetanide-sensitive component were estimated at 1.34 ±0.08 and 1.82 ± 0.15 which should be compared to the gradient for the Na⁺, K⁺, 2Cl^– cotransport system at 1.75 ± 0.24.Addition of sucrose to hypertonicity causes the Ehrlich cells to shrink with no signs of RVI, whereas shrinkage with hypertonic standard medium (all extracellular ion concentrations increased) results in a RVI response towards the original cell volume. Under both conditions a bumetanide-sensitive unidirectional K⁺ influx is activated. During hypotonic conditions a small bumetanide-sensitive K⁺ influx is observed, indicating that the cotransport system is already activated.The cotransport is activated 10–15 fold by bradykinin, an agonist which stimulates phospholipase C resulting in release of internal Ca²⁺ and activation of protein kinase C.The anti-calmodulin drug pimozide inhibits most of the bumetanide-sensitive K⁺ influx during RVI. The cotransporter can be activated by the phorbol ester TPA. These results indicate that the stimulation of the Na⁺, K⁺, Cl^– cotransport involves both Ca²⁺/calmodulin and protein kinase C.     

Thiol-Dependent passive K/Cl transport in sheep red cells: IV. Furosemide inhibition as a function of external Rb+, Na+, and Cl−

Summary The effect of the loop diuretic furosemide (4-chloro-N-furfuryl-5-sulfamoyl-anthranilic acid) on the thiol-dependent, ouabain-insensitive K(Rb)/Cl transport in low K⁺ sheep red cells was studied at various concentrations of extracellular Rb⁺, Na⁺ and Cl^–. In Rb⁺-free NaCl media, 2×10^–3 m furosemide inhibited only one-half of thiol-dependent K⁺ efflux. In the presence of 23mm RbCl, however, the concentration of furosemide to produce 50% K⁺ efflux inhibition (IC₅₀) was 5×10^–5 m. In Rb⁺ containing NaCl media, the inhibitory effect of 10^–3 m furosemide was equal to that caused by NO ₃ ^– replacement of Cl^– in the medium. The apparent synergistic action of furosemide and external Rb⁺ on K⁺ efflux was also seen in the ouabain-insensitive Rb⁺ influx. A preliminary kinetic analysis suggests that furosemide binding alters both maximal K⁺(Rb⁺) transport and apparent external Rb⁺ affinity. In the presence of external Rb⁺, Na⁺ (as compared to choline) exerted a small but significant augmentation of the furosemide inhibition of K⁺(Rb⁺) fluxes. There was no effect of Cl^– on the IC₅₀ value of furosemide. As there is no evidence for coupled Na⁺K⁺ cotransport in low K⁺ sheep red cells, furosemide may modify thiol-dependent K⁺(Rb⁺/Cl flux or Rb⁺ (and to a slight degree Na⁺) modulate the effect of furosemide.     

Comparison of bone and osteosarcoma adenylate cyclase. Effects of Mg2+, Ca2+, ATP4? and HATP3? in the assay mixture

The effects of Mg(2+) and Ca(2+) on bone and osteosarcoma adenylate cyclase were investigated. The concentrations of the cations and other ionic species in the assay mixture were calculated by solving the simultaneous equations describing the relevant ionic interactions (multiple equilibria). We re-examined the effects of HATP(3-) and ATP(4-) on enzyme activity and found that (i) the concentration of the minor ATP species is less than 1% of that of MgATP(2-), and their ratio to MgATP(2-) is constant if Mg(2+) and H(+) concentrations are unchanged; (ii) Mg(2+) addition decreased the ratio of the minor species to MgATP(2-) and increased the enzyme activity, but no meaningful kinetic model could attribute this effect of HATP(3-) or ATP(4-). On the other hand, kinetic analysis of Mg(2+) effects showed: (i) stimulation via two metal sites, separate from the catalytic (MgATP(2-)) site, with apparent K(m) values of approximately 1 and 8mm; (ii) that the low affinity increased towards the higher one when the enzyme activity rose as a result of increased substrate or guanine nucleotide concentrations, this effect being less pronounced in tumour; (iii) conversely, that two apparent affinities for MgATP(2-) merged into one at high Mg(2+) concentration; (iv) kinetically, that this relationship is of the mixed con-competitive type, which is consistent with a role for Mg(2+) as a requisite activator, and binding occurring in non-ordered sequence. Analysis of the Ca(2+) effects showed: (i) competition with Mg(2+) at the metal site (K(i) 20mum for bone and 40mum for tumour); (ii) that relative to the substrate the inhibition was uncompetitive, i.e. velocity decreased and affinity increased proportionally, which is consistent with Ca(2+) binding after substrate binding. These findings support the existence of interacting enzyme complexes, losing co-operativity at increased enzyme activity. They also indicate a potential physiological role for Ca(2+) in enzyme regulation and point to quantitative differences between bone and tumour with regard to these properties.     

Inhibition of Na+, K+-ATPase activity by δ-aminolevulinic acid

-Aminolaevulinic acid (ALA) has been shown to be toxic to cultured neurons and glia at concentrations as low as 10 M. In an attempt to elucidate the mechanism of toxicity, the effects of ALA on membrane ATPase activity were investigated. Exposure of neuron cultures to 1 mM ALA for 7 days caused a substantial decrease in both Na⁺, K⁺-ATPase and Mg²⁺-ATPase activities. At lower concentrations, ALA affected only the Na⁺, K⁺-component. ALA appeared to act directly, inhibiting Na⁺, K⁺-ATPase activity in rat brain cortex membrane preparations at 10 M Although this effect was slight, it may well represent the mechanism of action of ALA, since ouabain, a potent inhibitor of Na⁺, K⁺-ATPase activity, proved to be more toxic to cultured neurons than ALA. Furthermore, cardiac glycoside overdosage causes neurological disturbances which are very similar to those observed in the acute attack of porphyria.     

A UB3LYP and UMP2 theoretical investigation on unusual cation–π interaction between the triplet state HB=BH ( {}^3\Sigma_g^{-} ) and H+, Li+, Na+, Be2+ or Mg2+

The nature of the unusual cation–π interactions between cations (H⁺, Li⁺, Na⁺, Be²⁺ and Mg²⁺) and the electron-deficient B=B bond of the triplet state HB=BH ( $ {}^3\Sigma_g^{-} $ ) was investigated using UMP2(full) and UB3LYP methods at 6–311++G(2df,2p) and aug-cc-pVTZ levels, accompanied by a comparison with 1:1 and 2:1 σ-binding complexes between BH and the cations. The binding energies follow the order HB=BH...H⁺ > HB=BH...Be²⁺ > HB=BH...Mg²⁺ ? HB=BH...Li⁺ > HB=BH...Na⁺ and HB=BH (¹Δ_g)...M⁺/M²⁺ > H₂C=CH₂...M⁺/M²⁺ > HC≡CH...M⁺/M²⁺ > HB=BH ( $ {}^3\Sigma_g^{-} $ )...M⁺/M²⁺. Furthermore, except for HB...H⁺, the σ-binding interaction energy of the 1:1 complex HB...M⁺/M²⁺ is stronger than the cation–π interaction energy of the C₂H₂...M⁺/M²⁺, C₂H₄...M⁺/M²⁺, B₂H₂ (¹Δ_g)...M⁺/M²⁺ or B₂H₂ ( $ {}^3\Sigma_g^{-} $ )...M⁺/M²⁺ complex, and, for the 2:1 σ-binding complexes, except for HBBe²⁺...BH, they are less stable than the cation–π complexes of B₂H₂ (¹Δ_g) or B₂H₂ ( $ {}^3\Sigma_g^{-} $ ). The atoms in molecules (AIM) theory was also applied to verify covalent interactions in the H⁺ complexes and confirm that HB=BH ( $ {}^3\Sigma_g^{-} $ ) can be a weaker π-electron donor than HB=BH (¹Δ_g), H₂C=CH₂ or HC≡CH in the cation–π interaction. Analyses of natural bond orbital (NBO) and electron density shifts revealed that the origin of the cation–π interaction is mainly that many of the lost densities from the π-orbital of B=B and CC multiple bonds are shifted toward the cations.