Cation Binding Sites Research Articles

Architecture of the HCN selectivity filter and control of cation permeation.

Hyperpolarization-activated Cyclic Nucleotide-modulated (HCN) channels are similar in structure and function to voltage-gated potassium channels. Sequence similarity and functional analyses suggest that the HCN pore is potassium channel-like, consisting of a selectivity filter and an activation gate at the outer and inner ends, respectively. In GYG-containing potassium channels, the selectivity filter sequence is ‘T/S-V/I/L/T-GYG’, forming a row of four binding sites through which potassium ions flow. In HCNs, the equivalent residues are ‘C-I-GYG’, but whether they also form four cation binding sites is not known. Here, we focus on the anomalous filter residue of HCNs, the cysteine located at the inner side of the selectivity filter. In potassium channels, this position is occupied by threonine or serine and forms the fourth and most internal ion binding site of the selectivity filter. We find that this cysteine in HCNs does not contribute to permeation or form a fourth binding site.

Molecular epidemiology of hepatitis C virus genotypes in different geographical regions of Punjab Province in Pakistan and a phylogenetic analysis

Hepatitis C virus (HCV) is a causative agent of chronic liver disease, cirrhosis, and hepatocellular carcinoma. In Pakistan, more than 10 million people are living with HCV. Very little is known about the genotype distribution in Punjab Province, the largest province of Pakistan. Pretreatment genotype identification is very important, as different HCV genotypes respond differently to interferon therapy. In this study we performed HCV genotyping for 1537 HCV-infected patients from different districts of Punjab Province, Pakistan. Sequencing of partial HCV NS5B sequences from 14 samples belonging to genotypes 3 and 1 was also done. A sequence comparison was made of our reported sequences with those reported in the National Center for Biotechnology Information (NCBI), and a phylogenetic tree was constructed. Our study showed that the most frequent HCV genotype was 3a (in 88.1% of infected individuals), followed by 1a (3.5%), 3b (3.0%), 1b (0.8%), and 2a (1.0%). A mixed genotype infection was found in 3.6% of infected individuals, with 0.3% living with 1a + 1b co-infection, 3.1% with 3a+3b, and 0.2% suffering from 3a+1b co-infection. The sequence comparison showed that HCV NS5B motif B residues G283, T287, and N291 were highly conserved in both genotype 1 and genotype 3 sequences, while the motif B residue T286 was mutated to proline in all the genotype 3 sequences. The GDD motif, which forms the catalytic pocket and binding site for the divalent cations, was highly conserved in all the reported sequences. The phylogenetic tree suggests clustering of genotype 1 sequences with sequences from the USA, UK, Germany, and France, while genotype 3 sequences are clustered with sequences from Japan and the UK. The major prevalent genotype in Punjab Province of Pakistan was genotype 3a, followed by genotype 1a, and only 3.6% of infected individuals had a mixed genotype infection. Sequencing of the HCV NS5B gene suggested that the active site residues were highly conserved in all the reported sequences. Our sequences, which are clustered with sequences from the USA, UK, France, and Japan, show the diversity in origin of the different genotypes prevalent in Pakistan.

Divalent Cations Define the Structural Requirements for αIIbβ3 Integrin Activation

Abstract 260Platelet αIIbβ3 integrin undergoes structural rearrangements to increase the affinity for ligands. Two major changes in integrin three-dimensional structure are critical for this activation process. One is the straightening of integrin legs (extension), and the other is the swing out movement of the β3 hybrid domain (swing-out). In this study, we have found that this structural requirement for activation differs depending upon the divalent cations present. We previously reported that the swing-out-defective mutant αIIbD319C/β3V359C did not bind fibrinogen in the presence of Ca2+/Mg2+. However, the same mutant bound fibrinogen in the presence of Mn2+, when activated by monoclonal antibody PT25-2 or by forcing the integrin legs to extend by concomitant αIIbQ595NTT mutation. We engineered another mutation that was designed specifically to prevent the swing-out. Likewise, this β3G327C/β3V419C mutation blocked fibrinogen binding in the presence of Ca2+/Mg2+, but not in the presence of Mn2+. On the other hand, when integrin extension was specifically prevented by αIIbD464C/αIIbS728C mutation, fibrinogen binding was observed neither in the presence of Ca2+/Mg2+ nor in Mn2+. Thus, swing-out is not essential for activation in Mn2+, as long as integrin legs are in extended state, while both swing-out and extension are required in Ca2+/Mg2+. To explore the underlying mechanism originating this difference, three distinct cation-binding sites in the βA domain were mutated. Among the three sites, MIDAS is occupied by Mg2+, while ADMIDAS and LIMBS/SyMBS are occupied by Ca2+ in Ca2+/Mg2+ condition. When amino acid residues composing these sites were mutated to Ala, none of them bound fibrinogen, except for D126A and D127A in the presence of Ca2+/Mg2+. Notably, these mutations restored fibrinogen binding in the swing-out-defective mutant. Amino acid residues D126 and D127 compose ADMIDAS together with S123 and M335. Swing-out of the hybrid domain disrupts M335 from ADMIDAS. This allows Ca2+ to move toward MIDAS, bringing the β1-α1 loop closer to ligand to provide contact site. However, similar rearrangement of the cation/ligand binding sites has been shown to take place in Mn2+ without the swing-out. The results suggest partial disruption of ADMIDAS is the key event for activation in Ca2+/Mg2+, while it is not so in Mn2+. Our results may reconcile the dispute over the apparently contradicting findings in the crystal structure that integrin with a closed head with Mn2+ represents an active conformation as one with an open head with Ca2+/Mg2+. Disclosures:Kawai:Daiichi Sankyo: Consultancy; Bayer: Consultancy; Toyama Chemical: Consultancy.

Nonselective Conduction in a Mutated NaK Channel with Three Cation-Binding Sites

The NaK channel is a cation-selective protein with similar permeability for K+ and Na+ ions. Crystallographic structures are available for the wild-type and mutated NaK channels with different numbers of cation-binding sites. We have performed a comparison between the potentials of mean force governing the translocation of K+ ions and mixtures of one Na+ and three K+ ions in a mutated NaK channel with only three cation-binding sites (NaK-CNG). Since NaK-CNG is not selective for K+ over Na+, analysis of its multi-ion potential energy surfaces can provide clues about how selectivity originates. Comparison of the potentials of mean force of NaK-CNG and K+-selective channels yields observations that strongly suggest that the number of contiguous ion binding sites in a single-file mechanism is the key determinant of the channel’s selectivity properties, as already proposed by experimental studies. We conclude that the presence of four binding sites in K+-selective channels is essential for highly selective and efficient permeation of K+ ions, and that a key difference between K+-selective and nonselective channels is the absence/presence of a binding site for Na+ ions at the boundary between S2 and S3 in the context of multi-ion permeation events.

Cryo-EM structure of gastric H + ,K + -ATPase with a single occupied cation-binding site

Gastric H(+),K(+)-ATPase is responsible for gastric acid secretion. ATP-driven H(+) uptake into the stomach is efficiently accomplished by the exchange of an equal amount of K(+), resulting in a luminal pH close to 1. Because of the limited free energy available for ATP hydrolysis, the stoichiometry of transported cations is thought to vary from 2H(+)/2K(+) to 1H(+)/1K(+) per hydrolysis of one ATP molecule as the luminal pH decreases, although direct evidence for this hypothesis has remained elusive. Here, we show, using the phosphate analog aluminum fluoride (AlF) and a K(+) congener (Rb(+)), the 8-Å resolution structure of H(+),K(+)-ATPase in the transition state of dephosphorylation, (Rb(+))E2~AlF, which is distinct from the preceding Rb(+)-free E2P state. A strong density located in the transmembrane cation-binding site of (Rb(+))E2~AlF highly likely represents a single bound Rb(+) ion, which is clearly different from the Rb(+)-free E2AlF or K(+)-bound (K(+))E2~AlF structures. Measurement of radioactive (86)Rb(+) binding suggests that the binding stoichiometry varies depending on the pH, and approximately half of the amount of Rb(+) is bound under acidic crystallization conditions compared with at a neutral pH. These data represent structural and biochemical evidence for the 1H(+)/1K(+)/1ATP transport mode of H(+),K(+)-ATPase, which is a prerequisite for generation of the 10(6)-fold proton gradient in terms of thermodynamics. Together with the released E2P-stabilizing interaction between the β subunit's N terminus and the P domain observed in the (Rb(+))E2~AlF structure, we propose a refined vectorial transport model of H(+),K(+)-ATPase, which must prevail against the highly acidic state of the gastric lumen.

Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP.

Sodium-coupled substrate transport plays a central role in many biological processes. However, despite knowledge of the structures of several sodium-coupled transporters, the location of the sodium-binding site(s) often remains unclear. Several of these structures have the five transmembrane-helix inverted-topology repeat, LeuT-like (FIRL) fold, whose pseudosymmetry has been proposed to facilitate the alternating-access mechanism required for transport. Here, we provide biophysical, biochemical, and computational evidence for the location of the two cation-binding sites in the sodium-coupled betaine symporter BetP. A recent X-ray structure of BetP in a sodium-bound closed state revealed that one of these sites, equivalent to the Na2 site in related transporters, is located between transmembrane helices 1 and 8 of the FIRL-fold; here, we confirm the location of this site by other means. Based on the pseudosymmetry of this fold, we hypothesized that the second site is located between the equivalent helices 6 and 3. Molecular dynamics simulations of the closed-state structure suggest this second sodium site involves two threonine sidechains and a backbone carbonyl from helix 3, a phenylalanine from helix 6, and a water molecule. Mutating the residues proposed to form the two binding sites increased the apparent K(m) and K(d) for sodium, as measured by betaine uptake, tryptophan fluorescence, and (22)Na(+) binding, and also diminished the transient currents measured in proteoliposomes using solid supported membrane-based electrophysiology. Taken together, these results provide strong evidence for the identity of the residues forming the sodium-binding sites in BetP.

Identification of cation-binding sites on actin that drive polymerization and modulate bending stiffness

The assembly of actin monomers into filaments and networks plays vital roles throughout eukaryotic biology, including intracellular transport, cell motility, cell division, determining cellular shape, and providing cells with mechanical strength. The regulation of actin assembly and modulation of filament mechanical properties are critical for proper actin function. It is well established that physiological salt concentrations promote actin assembly and alter the overall bending mechanics of assembled filaments and networks. However, the molecular origins of these salt-dependent effects, particularly if they involve nonspecific ionic strength effects or specific ion-binding interactions, are unknown. Here, we demonstrate that specific cation binding at two discrete sites situated between adjacent subunits along the long-pitch helix drive actin polymerization and determine the filament bending rigidity. We classify the two sites as "polymerization" and "stiffness" sites based on the effects that mutations at the sites have on salt-dependent filament assembly and bending mechanics, respectively. These results establish the existence and location of the cation-binding sites that confer salt dependence to the assembly and mechanics of actin filaments.

Zinc inhibits magnesium-dependent migration of human breast cancer MDA-MB-231 cells on fibronectin

Metastasis is the major cause of breast cancer mortality. The strength of cell adhesion to extracellular matrix is critical to cancer cell migration. Integrins, the primary mediators of cell to extra-cellular matrix adhesion, contain distinct divalent cation-binding sites. Binding of manganese and magnesium is vital to integrin-mediated cancer cell adhesion and migration. We hypothesized that zinc, a divalent cation, can modulate breast cancer metastasis through interfering with these divalent cation-dependent integrin-mediated cancer cell adhesion and migration. MDA-MB-231 cells were cultured in a zinc-depleted medium supplemented with 0 (control), 2.5, 5, 10, 25 and 50 μM of zinc to mimic severe zinc-deficiency, moderate zinc-deficiency, adequate zinc and three levels of zinc-supplementation: low-, moderate- and high-levels of zinc-supplementation, respectively. Zinc treatments had no effect on cellular zinc concentration, cell number and cell viability. Zinc at 5–50 μM reduced migration distance of MDA-MB-231 cells on fibronectin by 43–86% and migration rate on fibronectin by 72–90%. Zinc induced a dose-dependent inhibition of cell adhesion to fibronectin (R2=−0.98). Zinc at 10–50 μM reduced magnesium-facilitated cell adhesion to fibronectin in a dose-dependent manner (R2=−0.90). However, zinc had no effect on manganese-facilitated cell adhesion to fibronectin. Zinc at 5–50 μM caused rounding of the normally elongated, irregular-shaped MDA-MB-231 cells and disappearance of F-actin. Anti-integrin α5- and β1-subunit blocking antibodies inhibited magnesium-facilitated cell adhesion to fibronectin by 95 and 99%, respectively. In summary, zinc inhibited MDA-MB-231 cell migration on fibronectin by interfering with magnesium-dependent integrin-, likely integrin α5/β1-, mediated adhesion.

Post‐Synthetic Modification of Porphyrin‐Encapsulating Metal–Organic Materials by Cooperative Addition of Inorganic Salts to Enhance CO2/CH4 Selectivity

Keeping MOM: Reaction of biphenyl-3,4',5-tricarboxylate and Cd(NO3)2 in the presence of meso-tetra(N-methyl-4-pyridyl)porphine tetratosylate afforded porph@MOM-11, a microporous metal–organic material (MOM) that encapsulates cationic porphyrins and solvent in alternating open channels. Porph@MOM-11 has cation and anion binding sites that facilitate cooperative addition of inorganic salts (such as M+Cl-) in a stoichiometric fashion.

Effect of calcium ions on electron transfer between hemes a and a 3 in cytochrome c oxidase

Kinetics of the reduction of the hemes in cytochrome c oxidase in the presence of high concentration of ruthenium(III)hexaammine chloride was examined using a stopped-flow spectrophotometer. Upon mixing of the oxidized enzyme with dithionite and Ru(NH(3))(6)(3+), three well-resolved phases were observed: heme a reduction reaching completion within a few milliseconds is followed by two slow phases of heme a(3) reduction. The difference spectrum of heme a(3) reduction in the visible region is characterized by a maximum at ~612 nm, rather than at 603 nm as was believed earlier. It is shown that in the case of bovine heart cytochrome c oxidase containing a special cation-binding site in which reversible binding of calcium ion occurs, heme a(3) reduction is slowed down by low concentrations of Ca(2+). The effect is absent in the case of the bacterial cytochrome oxidase in which the cation-binding site contains a tightly bound Ca(2+) ion. The data corroborate the inhibition of the cytochrome oxidase enzymatic activity by Ca(2+) ions discovered earlier and indicate that the cation affects intramolecular electron transfer.

Opticin Exerts Its Anti-angiogenic Activity by Regulating Extracellular Matrix Adhesiveness

Opticin is an extracellular matrix glycoprotein that we identified associated with the collagen network of the vitreous humor of the eye. Recently, we discovered that opticin possesses anti-angiogenic activity using a murine oxygen-induced retinopathy model: here, we investigate the underlying mechanism. Using an ex vivo chick chorioallantoic membrane assay, we show that opticin inhibits angiogenesis when stimulated by a range of growth factors. We show that it suppresses capillary morphogenesis, inhibits endothelial invasion, and promotes capillary network regression in three-dimensional matrices of collagen and Matrigel(TM). We then show that opticin binds to collagen and thereby competitively inhibits endothelial cell interactions with collagen via α(1)β(1) and α(2)β(1) integrins, thereby preventing the strong adhesion that is required for proangiogenic signaling via these integrins.

Charge Compensation Mechanism of a Na+-coupled, Secondary Active Glutamate Transporter

Forward glutamate transport by the excitatory amino acid carrier EAAC1 is coupled to the inward movement of three Na(+) and one proton and the subsequent outward movement of one K(+) in a separate step. Based on indirect evidence, it was speculated that the cation binding sites bear a negative charge. However, little is known about the electrostatics of the transport process. Valences calculated using the Poisson-Boltzmann equation indicate that negative charge is transferred across the membrane when only one cation is bound. Consistently, transient currents were observed in response to voltage jumps when K(+) was the only cation on both sides of the membrane. Furthermore, rapid extracellular K(+) application to EAAC1 under single turnover conditions (K(+) inside) resulted in outward transient current. We propose a charge compensation mechanism, in which the C-terminal transport domain bears an overall negative charge of -1.23. Charge compensation, together with distribution of charge movement over many steps in the transport cycle, as well as defocusing of the membrane electric field, may be combined strategies used by Na(+)-coupled transporters to avoid prohibitive activation barriers for charge translocation.

Evidence for a Non-Catalytic Ion-Binding Site in Multiple RNA-Dependent RNA Polymerases

A high-affinity divalent cation-binding site located proximal to the catalytic center has been identified in several RNA-dependent RNA polymerases (RdRps), but the characteristics of such a site have not been systematically studied. Here, all available polymerase structures that follow the hand-like structural motif were screened for the presence of a divalent cation close to the catalytic site but distinct from catalytic metal ions. Such non-catalytic ions were found in all RNA virus families for which there were high-resolution RdRp structures available. Bound ions were always located in structurally similar locations at an approximate 6-Å distance from the catalytic site. Furthermore, the second aspartate residue in the highly conserved GDD sequence was found to be involved in the coordination of the bound ion in all viral RdRps studied. These results suggest that a non-catalytic ion-binding site is conserved across positive-sense, single-stranded, and double-stranded RNA viruses. Interestingly, a non-catalytic ion was also observed in a similar position in the reverse transcriptase of the human immunodeficiency virus. Moreover, two members of the DNA-dependent DNA polymerase B family displayed an ion at a comparable distance from the catalytic site, but the position was clearly distinct from the non-catalytic ion-binding sites of RdRps.

Conformational changes in NhaA Na+/H+antiporter

Na(+)/H(+) antiporters play a primary role in Na(+)/H(+) homeostasis in cells and many organelles and have long been drug targets. The X-ray structure of NhaA, the main antiporter of Escherichia coli, provided structural insights into the antiport mechanism and its pH regulation and revealed a novel fold; six of the 12 TMs (Trans membrane segments) are organized in two topologically inverted repeats, each with one TM interrupted by an extended chain creating a unique electrostatic environment in the middle of the membrane at the cation binding site. Remarkably, inverted repeats containing interrupted helices with similar functional implications have since been observed in structures of other bacterial secondary transporters with almost no sequence homology. Finally, the structure reveals that NhaA is organized into two functional regions: a 'pH sensor' - a cluster of amino acyl side chains that are involved in pH regulation; and a catalytic region that is 9 Å removed from the pH sensor. Alternative accessibility of the binding site to either side of the membrane, i.e., functional-dynamics, is the essence of secondary transport mechanism. Because NhaA is tightly pH regulated, structures of the pH-activated and ligand-activated NhaA conformations are needed to identify its functional-dynamics. However, as these are static snapshots of a dynamic protein, the dynamics of the protein both in vitro and in situ in the membrane are also required as reviewed here in detail. The results reveal two different conformational changes characterizing NhaA: One is pH-induced for NhaA activation; the other is ligand-induced for antiport activity.

Role of K+ binding residues in stabilization of heme spin state of Leishmania major peroxidase

The endogenous cation in peroxidases may contribute to the type of heme coordination. Here a series of ferric and ferrous derivatives of wild-type Leishmania major peroxidase (LmP) and of engineered K+ site mutants of LmP, lacking potassium cation binding site, has been examined by electronic absorption spectroscopy at 25°C. Using UV–visible spectrophotometry, we show that the removal of K+ binding site causes substantial changes in spin states of both the ferric and ferrous forms. The spectral changes are interpreted to be, most likely, due to the formation of a bis-histidine coordination structure in both the ferric and ferrous oxidation states at neutral pH 7.0. Stopped flow spectrophotometric techniques revealed that characteristics of Compound I were not observed in the K+ site double mutants in the presence of H2O2. Similarly electron donor oxidation rate was two orders less for the K+ site double mutants compared to the wild type. These data show that K+ functions in preserving the protein structure in the heme surroundings as well as the spin state of the heme iron, in favor of the enzymatically active form of LmP.

Interaction of Counterions with Subtilisin in Acetonitrile: Insights from Molecular Dynamics Simulations

A recent X-ray structure has enabled the location of chloride and cesium ions on the surface of subtilisin Carlsberg in acetonitrile soaked crystals. (1) To complement the previous study and analyze the system in solution, molecular dynamics (MD) simulations, in acetonitrile, were performed using this structure. Additionally, Cl(-) and Cs(+) ions were docked on the protein surface and this system was also simulated. Our results indicate that chloride ions tend to stay close to the protein, whereas cesium ions frequently migrate to the solvent. The distribution of the ions around the enzyme surface is not strongly biased by their initial locations. Replacing cesium by sodium ions showed that the distribution of the two cations is similar, indicating that Cs(+) can be used to find the binding sites of cations like Na(+) and K(+), which, unlike Cs(+), have physiological and biotechnological roles. The Na(+)Cl(-) is more stable than the Cs(+)Cl(-) ion pair, decreasing the probability of interaction between Cl(-) and subtilisin. The comparison of water and acetonitrile simulations indicates that the solvent influences the distribution of the ions. This work provides an extensive theoretical analysis of the interaction between ions and the model enzyme subtilisin in a nonaqueous medium.

Phototriggered DNA Complexation and Compaction Using Poly(vinyl alcohol) Carrying a Malachite Green Moiety

Photoinduced DNA compaction was performed using the interaction of DNA with a photoresponsive random copolymer of poly(vinyl alcohol) carrying a malachite green moiety (PVAMG). Although PVAMG does not have any affinity for DNA under dark conditions, it undergoes photoionization upon exposure to UV light, consequently resulting in a cationic binding site for DNA. Electrophoresis results demonstrated that irradiation of PVAMG retarded the DNA bands due to their complexation, whereas the bands remained unchanged under dark conditions. The binding of PVAMG to DNA occurs at a cationic site/DNA phosphate ratio of approximately 0.036. Single-molecule observations of DNA by fluorescence microscopy revealed that irradiation of PVAMG induced a coil-globule transition in the DNA molecule. Complete compaction of DNA has been accomplished at a cationic site/DNA phosphate ratio >8.0, indicating that PVAMG offers an effective system to photochemically trigger DNA compaction.

Identification of a Divalent Metal Cation Binding Site in Herpes Simplex Virus 1 (HSV-1) ICP8 Required for HSV Replication

Herpes simplex virus 1 (HSV-1) ICP8 is a single-stranded DNA-binding protein that is necessary for viral DNA replication and exhibits recombinase activity in vitro. Alignment of the HSV-1 ICP8 amino acid sequence with ICP8 homologs from other herpesviruses revealed conserved aspartic acid (D) and glutamic acid (E) residues. Amino acid residue D1087 was conserved in every ICP8 homolog analyzed, indicating that it is likely critical for ICP8 function. We took a genetic approach to investigate the functions of the conserved ICP8 D and E residues in HSV-1 replication. The E1086A D1087A mutant form of ICP8 failed to support the replication of an ICP8 mutant virus in a complementation assay. E1086A D1087A mutant ICP8 bound DNA, albeit with reduced affinity, demonstrating that the protein is not globally misfolded. This mutant form of ICP8 was also recognized by a conformation-specific antibody, further indicating that its overall structure was intact. A recombinant virus expressing E1086A D1087A mutant ICP8 was defective in viral replication, viral DNA synthesis, and late gene expression in Vero cells. A class of enzymes called DDE recombinases utilize conserved D and E residues to coordinate divalent metal cations in their active sites. We investigated whether the conserved D and E residues in ICP8 were also required for binding metal cations and found that the E1086A D1087A mutant form of ICP8 exhibited altered divalent metal binding in an in vitro iron-induced cleavage assay. These results identify a novel divalent metal cation-binding site in ICP8 that is required for ICP8 functions during viral replication.

Kinetic Analysis of Gill (Na+,K+)-ATPase Activity in Selected Ontogenetic Stages of the Amazon River Shrimp, Macrobrachium amazonicum (Decapoda, Palaemonidae): Interactions at ATP- and Cation-Binding Sites

We investigated modulation by ATP, Mg²⁺, Na⁺, K⁺ and NH₄⁺ and inhibition by ouabain of (Na⁺,K⁺)-ATPase activity in microsomal homogenates of whole zoeae I and decapodid III (formerly zoea IX) and whole-body and gill homogenates of juvenile and adult Amazon River shrimps, Macrobrachium amazonicum. (Na⁺,K⁺)-ATPase-specific activity was increased twofold in decapodid III compared to zoea I, juveniles and adults, suggesting an important role in this ontogenetic stage. The apparent affinity for ATP (K(M) = 0.09 ± 0.01 mmol L⁻¹) of the decapodid III (Na⁺,K⁺)-ATPase, about twofold greater than the other stages, further highlights this relevance. Modulation of (Na⁺,K⁺-ATPase activity by K⁺ also revealed a threefold greater affinity for K⁺ (K₀.₅ = 0.91 ± 0.04 mmol L⁻¹) in decapodid III than in other stages; NH₄⁺ had no modulatory effect. The affinity for Na⁺ (K₀.₅ = 13.2 ± 0.6 mmol L⁻¹) of zoea I (Na⁺,K⁺)-ATPase was fourfold less than other stages. Modulation by Na⁺, Mg²⁺ and NH₄⁺ obeyed cooperative kinetics, while K⁺ modulation exhibited Michaelis-Menten behavior. Rates of maximal Mg²⁺ stimulation of ouabain-insensitive ATPase activity differed in each ontogenetic stage, suggesting that Mg²⁺-stimulated ATPases other than (Na⁺,K⁺)-ATPase are present. Ouabain inhibition suggests that, among the various ATPase activities present in the different stages, Na⁺-ATPase may be involved in the ontogeny of osmoregulation in larval M. amazonicum. The NH₄⁺-stimulated, ouabain-insensitive ATPase activity seen in zoea I and decapodid III may reflect a stage-specific means of ammonia excretion since functional gills are absent in the early larval stages.

Structural and functional roles of potassium ions on aldehyde dehydrogenases

The homotetrameric betaine aldehyde dehydrogenase from Pseudomonas aeruginosa (PaBADH) dissociates into inactive monomers at low ionic strength in the absence of K+ ions while the association state of the homodimeric enzyme from spinach is not affected, in spite of both enzymes having the same intrasubunit and intersubunit K+‐binding sites. The prevention of PaBADH dissociation by K+ ions is due to non‐specific effects of ionic strength, since the enzyme did not dissociate in the presence of tetraethylammonium (TEA) chloride. Cation‐binding sites unique to PaBADH, located in the negatively charged central cavity of this enzyme, are responsible for its dissociation in the absence of cations. In addition to their non‐specific, ionic‐strength effects, K+ ions have specific effects on enzyme structure, which result in increased affinity for NAD(P)+ as indicated by equilibrium binding studies. The latter effects probably arise from binding of the cation to the intrasubunit K+‐binding site, which is a common structural feature of aldehyde dehydrogenases. The result of pre‐steady state kinetic studies, carried out at very high protein concentration, to prevent enzyme dissociation, and at high NAD(P)+ concentration, to ensure saturation by the nucleotide, showed that the enzyme activity in non‐salt or TEACl buffers is similar to that in KCl buffer. Therefore, K+ ions are not required for the catalytic steps. Supported by CONACYT 16712 grant to RAMC.