Ca(2+)-blockable, poorly selective cation channels in the apical membrane of amphibian epithelia. UO2(2+) reveals two channel types

This study deals with the effect of mucosal UO2(2+) on the Ca(2+)- blockable, poorly selective cation channels in the apical membrane of frog skin and toad urinary bladder. Our data show that UO2(2+) inhibits the Na+ currents through the amiloride-insensitive cation pathway and confirm a previously described stimulatory effect on the amiloride- blockade Na+ transport. Noise analysis of the Ca(2+)-blockable current demonstrates that the divalent also depresses the low-frequency Lorentzian (fc = 11.7 Hz) in the power density spectrum (PDS) and reveals the presence of high-frequency relaxation noise (fc = 58.5 Hz). The action of UO2(2+) is not reversed upon washout and is not accompanied by noise, typically induced by reversible blockers. The divalent merely depresses the plateau of the low-frequency Lorentzian, demonstrating a decrease in the number of conductive cation channels. Similarly, with mucosal K+ and Rb+, UO2(2+) also unmasks the high- frequency Lorentzian by depressing the noise from the slowly fluctuating cation channels (type S). In all experiments with mucosal Cs+, the PDS contains high-frequency relaxation noise (fc = 75.1 Hz in Rana temporaria, and 65.4 Hz in Rana ridibunda). An effect of UO2(2+) on the Cs+ currents and Lorentzian plateaus could not be demonstrated, suggesting that this monovalent cation does not pass through type S channels. Experiments with the urinary bladder revealed only a UO2(2+)- insensitive pathway permeable for Na+, K+, Rb+, and Cs+. We submit that in frog skin two cation-selective channels occur, distinguished by their spontaneous gating kinetics, their sensitivity to UO2(2+), and their permeability for Cs+. In toad urinary bladder, only one kind of cation-selective channel is observed, which resembles the UO2(2+)- insensitive channel in frog skin, with fast open-closed kinetics (type F).     

Divalent cation competition with [3H]saxitoxin binding to tetrodotoxin- resistant and -sensitive sodium channels. A two-site structural model of ion/toxin interaction

Monovalent and divalent cations competitively displace tetrodotoxin and saxitoxin (STX) from their binding sites on nerve and skeletal muscle Na channels. Recent studies of cloned cardiac (toxin-resistant) and brain (toxin-sensitive) Na channels suggest important structural differences in their toxin and divalent cation binding sites. We used a partially purified preparation of sheep cardiac Na channels to compare monovalent and divalent cation competition and pH dependence of binding of [3H]STX between these toxin-resistant channels and toxin-sensitive channels in membranes prepared from rat brain. The effects of several chemical modifiers of amino acid groups were also compared. Toxin competition curves for Na+ in heart and Cd2+ in brain yielded similar KD values to measurements of equilibrium binding curves. The monovalent cation sequence for effectiveness of [3H]STX competition is the same for cardiac and brain Na channels, with similar KI values for each ion and slopes of -1. The effectiveness sequence corresponds to unhydrated ion radii. For seven divalent cations tested (Ca2+, Mg2+, Mn2+, Co2+, Ni2+, Cd2+, and Zn2+) the sequence for [3H]STX competition was also similar. However, whereas all ions displaced [3H]STX from cardiac Na channels at lower concentrations, Cd2+ and Zn2+ did so at much lower concentrations. In addition, and by way of explication, the divalent ion competition curves for both brain and cardiac channels (except for Cd2+ and Zn2+ in heart and Zn2+ in brain) had slopes of less than -1, consistent with more than one interaction site. Two-site curves had statistically better fits than one-site curves. The derived values of KI for the higher affinity sites were similar between the channel types, but the lower affinity KI's were larger for heart. On the other hand, the slopes of competition curves for Cd2+ and Zn2+ were close to - 1, as if the cardiac Na channel had one dominant site of interaction or more than one site with similar values for KI. pH titration of [3H]STX binding to cardiac channels showed a pKa of 5.5 and a slope of 0.6-0.9, compared with a pKa of 5.1 and slope of 1 for brain channels. Tetramethyloxonium (TMO) treatment abolished [3H]STX binding to cardiac and brain channels and STX protected channels, but the TMO effect was less dramatic for cardiac channels. Trinitrobenzene sulfonate preferentially abolished [3H]STX binding to brain channels by action at an STX protected site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Latrotoxin channels. Permeability for divalent cations]

The dependence of Ca2+ transport in synaptosomes along the channels formed by alpha-latrotoxin on [Ca2+]in and the feasibility of transport along these channels of other bivalent cations were studied. It was found that the concentration dependence of Ca2+ influx is nonlinear and is described by the Michaelis-Menten kinetics (Km = 1.07 +/- 0.19 mM). Mg2+, Ba2+, Sr2+, Mn2+ and Co2+ competitively inhibited the Ca2+ influx via latrotoxin channels. Studies with the use of the fluorescent Ca2+ probes, Quin-2 and Fura-2, revealed that these cations can also penetrate inside synaptosomes via latrotoxin channels. The bivalent cation influx via latrotoxin channels caused a decrease of the membrane potential of synaptosomes. The similarity of properties of latrotoxin and endogenous Ca2+ channels is discussed.     

Evidence that the ATP binding site of sarcoplasmic reticulum CaATPase has a Mg(2+) ion binding sub-site

The CaATPase of skeletal muscle sarcoplasmic reticulum was specifically labeled in the ATP binding site with fluorescein isothiocyanate under gentle conditions (pH 7 X 5). Fluorescence energy transfer from the attached fluorescein to Nd3+ indicated that a cation binding site was about 1 X 0 nm away from the fluorescein. Thus it appears that the ATP site includes a cation binding site. At 25 degrees C in 0 X 5 M KCl, the association constants for Nd3+, Ca2+ and Mg2+ were 3 X 3 X 10(5) M-1, 84 M-1 and 35 M-1, respectively, making it possible that, in vivo, the site binds Mg2+.     

Cation selectivity characteristics of the reconstituted voltage-dependent sodium channel purified from rat skeletal muscle sarcolemma

In this report, the alkali metal cation selectivity of the purified, voltage-dependent sodium channel from rat skeletal muscle is described. Isolated sodium channel protein (980-2840 pmol of saxitoxin binding/mg of protein) was reconstituted into egg phosphatidylcholine vesicles, and channels were subsequently activated by either batrachotoxin (5 X 10(-6) M) or veratridine (5 X 10(-4) M). Activation of the reconstituted sodium channel by batrachotoxin permitted rapid specific influx of cations into channel-containing vesicles. Quenched flow kinetic techniques were adapted to allow resolution of the kinetics of cation movement. Uptake rates for 42K+, 86Rb+, and 137Cs+ were measured directly and half-times for equilibration at 18 degrees C were determined to be 350 ms, 2.5 s, and 10 s, respectively, in this vesicle population. 22Na+ equilibration occurred within the mimimum quenching time of the apparatus (90 ms) but an upper limit of 50 ms at 18 degrees C could be assigned to its half-time. Based on this upper estimate for Na+, cation selectivity ratios of the batrachotoxin-activated channel were Na+ (1):K+ (0.14):Rb+ (0.02):Cs+ (0.005). Toxin-stimulated influx could be blocked by saxitoxin with a Ki of approximately 5 X 10(-9) M at 18 degrees C. Rates of cation movement through veratridine-activated channels were much slower, with half-times of 1.0, 1.2, 2.0, and 2.6 min at 36 degrees C for Na+, K+, Rb+, and Cs+, respectively. The temperature dependences of batrachotoxin and veratridine-stimulated cation uptake were markedly different. The activation energies for 86Rb+ and 137Cs+ movement into batrachotoxin-activated vesicles were 7.6 and 6.1 kcal/mol, respectively, while comparable measurements for these two cations in veratridine-activated vesicles yielded activation energies of 31 kcal/mol. Measurements of cation exchange with batrachotoxin-activated channels may reflect characteristics of an open sodium channel while the process of channel opening itself may be rate-limiting when veratridine is used for activation.     

Calcium-Activated K+ Channels and Calcium-Induced Calcium Release by Slow Vacuolar Ion Channels in Guard Cell Vacuoles Implicated in the Control of Stomatal Closure

Stomatal closing requires the efflux of K+ from the large vacuolar organelle into the cytosol and across the plasma membrane of guard cells. More than 90% of the K+ released from guard cells during stomatal closure originates from the guard cell vacuole. However, the corresponding molecular mechanisms for the release of K+ from guard cell vacuoles have remained unknown. Rises in the cytoplasmic Ca2+ concentration have been shown to trigger ion efflux from guard cells, resulting in stomatal closure. Here, we report a novel type of largely voltage-independent K+-selective ion channel in the vacuolar membrane of guard cells that is activated by physiological increases in the cytoplasmic Ca2+ concentration. These vacuolar K+ (VK) channels had a single channel conductance of 70 pS with 100 mM KCI on both sides of the membrane and were highly selective for K+ over NH4+ and Rb+. Na+, Li+, and Cs+ were not measurably permeant. The Ca2+, voltage, and pH dependences, high selectivity for K+, and high density of VK channels in the vacuolar membrane of guard cells suggest a central role for these K+ channels in the initiation and control of K+ release from the vacuole to the cytoplasm required for stomatal closure. The activation of K+-selective VK channels can shift the vacuolar membrane to more positive potentials on the cytoplasmic side, sufficient to activate previously described slow vacuolar cation channels (SV-type). Analysis of the ionic selectivity of SV channels demonstrated a Ca2+ over K+ selectivity (permeability ratio for Ca2+ to K+ of ~3:1) of these channels in broad bean guard cells and red beet vacuoles, suggesting that SV channels play an important role in Ca2+-induced Ca2+ release from the vacuole during stomatal closure. A model is presented suggesting that the interaction of VK and SV channel activities is crucial in regulating vacuolar K+ and Ca2+ release during stomatal closure. Furthermore, the possibility that the ubiquitous SV channels may represent a general mechanism for Ca2+-induced Ca2+ release from higher plant vacuoles is discussed.     

Purification and characterization of the FokI restriction endonuclease

The restriction endonuclease FokI from Flavobacterium okeanokoites was purified to homogeneity. Based on gel filtration, sedimentation and sodium dodecyl sulfate-polyacrylamide-gel electrophoresis, the following properties of the enzyme were determined: FokI exists in one active monomeric form, and has an Mr of 64-65.4 x 10(3).FokI is a strongly basic protein with an isoelectric point of 9.4. The enzyme exhibits restriction activity in the pH range 5.0 to 10.5 (maximum level at pH 7.0-8.5) and its divalent cation requirement is satisfied not only by Mg2+, but also by Co2+, Mn2+, Ni2+, Cd2+, Zn2+ and Fe2+.     

Reconstitution of sarcoplasmic reticulum Ca2+-ATPase vesicles lacking ion channels and demonstration of electrogenicity of Ca2+-pump

Sarcoplasmic reticulum (SR) vesicles were reconstituted by the salting out method in the presence of excess phospholipids: the lipid-to-protein ratio ranged from 10 to 100. It was found that the reconstituted vesicles could be separated by KC1 density gradient centrifugation into four types: those having both cation and anion channels (CASR), those having only cation channels (CSR), those having only anion channels (ASR), and those having no ion channels (PSR). From the yield of these vesicles, it was estimated that one native SR vesicle contains 19 cation channels and 1.4 anion channels on average; the amount of cation channels is 14 times larger than that of anion channels. Although all vesicles thus prepared are considered to contain the Ca2+-ATPase protein, the PSR vesicles alone did not take up Ca2+, but they did do so in the presence of valinomycin. This result indicates that the Ca2+-ATPase takes up Ca2+ in an electrogenic manner. The electromotive force was estimated to be about 50 mV.     

Binding constants of Li+, K+, and Tl+ in the gramicidin channel determined from water permeability measurements.

In an open circuit there can be no net cation flux through membranes containing only cation-selective channels, because electroneutrality must be maintained. If the channels are so narrow that water and cations cannot pass by each other, then the net water flux through those "single-file" channels that contain a cation is zero. It is therefore possible to determine the cation binding constants from the decrease in the average water permeability per channel as the cation concentration in the solution is increased. Three different methods were used to determine the osmotic water permeability of gramicidin channels in lipid bilayer membranes. The osmotic water permeability coefficient per gramicidin channel in the absence of cations was found to be 6 x 10(-14) cm3/s. As the cation concentration was raised, the water permeability decreased and a binding constant was determined from a quantitative fit to the data. When the data were fitted assuming a maximum of one ion per channel, the dissociation constant was 115 mM for Li+, 69 mM for K+, and 2 mM for Tl+.     

Ion channel formation by Alzheimer's disease amyloid beta-peptide (Abeta40) in unilamellar liposomes is determined by anionic phospholipids

Incorporation of Alzheimer's disease amyloid beta-proteins (AbetaPs) across natural and artificial bilayer membranes leads to the formation of cation-selective channels. To study the peptide-membrane interactions involved in channel formation, we used cation reporter dyes to measure AbetaP-induced influx of Na+, Ca2+, and K+ into liposomes formed from phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylcholine (PC). We found that Abeta40, but not Abeta40-1 or Abeta28, caused a dose-dependent increase in the concentration of each cation in the lumen of liposomes formed from the acidic phospholipids PS and PI. The Abeta40-induced changes in cation concentration, which we attribute to ion entry through Abeta40 channels, were not observed when using liposomes formed from the neutral phospholipid PC. Using mixtures of phospholipids, the magnitude of the AbetaP40-induced ion entry increased with the acidic phospholipid content of the liposomes, with entry being observed with as little as 5% PS or PI. Thus, while negatively charged phospholipids are required for formation of cation-permeable channels by Abeta40, a small amount is sufficient to support the process. These results have implications for the mechanisms of AbetaP cytotoxicity, suggesting that even a small amount of externalized negative charge could render cells susceptible to the deleterious effects of unregulated ion influx through AbetaP channels.     

Lanthanide ions and Cd2+ are able to substitute for Ca2+ in regulating phosphorylase kinase

Trivalent lanthanide ions and Cd2+ were found to mimic effectively the stimulatory action of Ca2+ on rabbit muscle phosphorylase kinase. In the range of concentrations tested, Cd2+ and lanthanides (Tb3+, Gd3+, Pr3+, Ce3+) could substitute for Ca2+ in activating the enzyme to about 60% and 70% respectively of the maximal level seen with Ca2+, at pH 8.2. The effect induced by Cd2+ was biphasic (stimulation followed by inhibition with increasing metal cation concentration). Similar results were obtained at pH 6.8. Cd2+ and Tb3+ were also able to replace Ca2+ required for the stimulation of phosphorylase kinase activity at pH 8.2 by exogenous calmodulin. Maximal stimulation induced by calmodulin in presence of Cd2+ was significantly higher than that in presence of Ca2+ or Tb3+.     

Extracellular polyvalent cation block of slow Na+ channels in Xenopus laevis oocytes

Sustained depolarization of the Xenopus oocyte membrane elicits a slowly activating Na+ current, thought to be due to the opening of sodium selective channels. These channels are induced to become voltage gated by the depolarization. They show unconventional gating properties and are insensitive to tetrodotoxin (TTX). The present study was undertaken to evaluate the effect of extracellular multivalent cations (Ca2+, Co2+, Cd2+, La3+, Mg2+, Mn2+, Ni2+, Sr2+ and Zn2+) on these TTX-resistant channels, either on membrane potential responses or on current responses. Our data show that all the polyvalent cations used blocked Na+ channels in a concentration-dependent manner. The order of potency of the most efficient cations was Co2+ < Ni2+ < Cd2+ < Zn2+, the respective concentration required to cause a 50% decrease of Na+ current was 0.9+/-0.29; 0.47+/-0.15; 0.36+/-0.09 and 0.06+/-0.02 mmol/l. The comparison of the activation curves from controls and after treatment with the cations indicated a shift towards more positive voltages. These results can be interpreted as due to the screening effect of divalent cations together with an alteration of the Na+ channel gating properties. We also show that divalent cations blocked Na+ channels in an open state without interfering with the induction mechanism of the channels. The possibility that cation block was due to a possible interaction between cations and SH-groups was investigated, but a sulphydryl alkylating reagent failed to abolish Zn2+ block.     

鼎湖山主要森林类型土壤交换性阳离子含量及其季节动态特征

研究鼎湖山自然保护区马尾松林、马尾松荷木混交林和季风常绿阔叶林三种代表性森林类型表层土壤(0~20cm)交换性阳离子含量及其季节动态。结果表明:土壤交换性阳离子含量因元素种类、森林类型和季节不同而异。三种森林土壤交换性阳离子含量都表现为:Al3+>H+>K+>Ca2+、Mg2+、Na+。几乎所有调查的阳离子含量在阔叶林显著高于马尾松林和混交林,但后两者之间大多数阳离子含量差异不显著。鼎湖山森林土壤可交换性阳离子含量虽然较高,但盐基饱和度却很低。马尾松林、混交林和阔叶林土壤可交换性阳离子含量在1997年6月份分别为:58.3、84.5和118.7mmolc/kg,盐基饱和度分别为:5.5%、3.2%和4.5%。三种森林土壤交换性Ca2+、Mg2+、K+和H+含量季节差异极显著(P<0.001),但交换性Al3+含量只在马尾松林土壤存在极显著的季节性差异(P<0.001)。同一元素季节变化大小程度趋向马尾松林>混交林>阔叶林。森林土壤交换性Ca2+、Na+和H+含量与土壤pH值相关关系不明显,但交换性Mg2+、K+和Al3+与土壤pH值间呈极显著负相关。     

Na+, K+ and Ca2+ antagonize the glutamate- and glycine-induced decrease of [3H]MK-801 binding observed in the presence of Mg2+ at low pH.

NMDA receptors are glutamate-regulated ion channels that are of great importance for many physiological and pathophysiological conditions in the mammalian central nervous system. We have previously shown that, at low pH, glutamate decreases binding of the open-channel blocker [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten, 5,10-imine ([3H]MK-801) to NMDA receptors in the presence of 1 mM Mg2+ but not in Krebs buffer. Here, we investigated which cations that block the glutamate-induced decrease in Krebs buffer, using [3H]MK-801 binding assays in membrane preparations from the rat cerebral cortex. At pH 6.0, Na+, K+, and Ca2+ antagonized the glutamate-induced decrease with cross-over values, which is a measure of the antagonist potencies of the cations, of 81, 71, and 26 mM, respectively, in the absence of added glycine. Thus, in Krebs buffer only the concentration of Na+ (126 mM) is sufficiently high to block the glutamate-induced decrease observed at low pH. In the presence of 1 mM Mg2+ and 10 mM Ca2+ at pH 7.4, the cross-over values for Na+, K+, and Ca2+ were 264, 139, and 122 mM, respectively, in the absence of added glycine. This is the same rank order of potency as observed at pH 6.0, suggesting that the less H+-sensitive and the less Ca2+-sensitive, glutamate-induced decreases in [3H]MK-801 binding represent the same entity. The glycine site antagonists 7-chlorokynurenate (10 microM) and 7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2(H)-quinoline (L-701,324; 1 microM) antagonized the glutamate-induced decrease in [3H]MK-801 binding observed in presence of Mg2+ at pH 6.0, suggesting that glycine is required together with glutamate to induce the decrease observed at low pH. These results suggest that in addition to a previously described high-affinity binding site for H+ and Ca2+ there exist a low-affinity binding site for H+, Ca2+, Na+, and K+ on NMDA receptors. The latter site may under physiological conditions be blocked by Na+ or K+, depending on the extra/intracellular localization of the modulatory site. Both the high-affinity and low-affinity cation sites mediate antagonistic effects on the glutamate- and glycine-induced decrease of the affinity of the [3H]MK-801 binding site, which may correspond to similar changes in the affinity of the voltage-sensitive Mg2+-block site inside the NMDA receptor channel pore, which in turn may affect current and Ca2+ influx through activated NMDA receptor channels.     

Inhibition of Cation Channels in Human Erythrocytes by Spermine

In erythrocytes, spermine concentration decreases gradually with age, which is paralleled by increases of cytosolic Ca2+ concentration, with subsequent cell shrinkage and cell membrane scrambling. Cytosolic Ca2+ was estimated from fluo-3 fluorescence, cell volume from forward scatter, cell membrane scrambling from annexin V binding and cation channel activity with whole-cell patch-clamp in human erythrocytes. Extracellular spermine exerted a dual effect on erythrocyte survival. At 200 μM spermine blunted the increase of intracellular Ca2+, cell shrinkage and annexin V binding following 48 h exposure of cells at +37 °C. In contrast, short exposure (10-30 min) of cells to 2 mM spermine was accompanied by increased cytosolic Ca2+ and annexin binding. Intracellular addition of spermine at subphysiological concentration (0.2 μM) significantly decreased the conductance of monovalent cations (Na+, K+, NMDG+) and of Ca2+. Moreover, spermine (0.2 μM) blunted the stimulation of voltage-independent cation channels by Cl? removal. Spermine (0.2 and 200 μM) added to the extracellular bath solution similarly inhibited the cation conductance in Cl?-containing bath solution. The effect of 0.2 μM spermine, but not the effect of 200 μM, was rapidly reversible. Acute addition (250 μM) of a naphthyl acetyl derivative of spermine (200 μM) again significantly decreased basal cation conductance in NaCl bath solution and inhibited voltage-independent cation channels. Spermine is a powerful regulator of erythrocyte cation channel cytosolic Ca2+ activity and, thus, cell survival.     

Ion selectivity of porcine skeletal muscle Ca2+ release channels is unaffected by the Arg615 to Cys615 mutation.

The Arg615 to Cys615 mutation of the sarcoplasmic reticulum (SR) Ca2+ release channel of malignant hyperthermia susceptible (MHS) pigs results in a decreased sensitivity of the channel to inhibitory Ca2+ concentrations. To investigate whether this mutation also affects the ion selectivity filter of the channel, the monovalent cation conductances and ion permeability ratios of single Ca2+ release channels incorporated into planar lipid bilayers were compared. Monovalent cation conductances in symmetrical solutions were: Li+, 183 pS +/- 3 (n = 21); Na+, 474 pS +/- 6 (n = 29); K+, 771 pS +/- 7 (n = 29); Rb+, 502 pS +/- 10 (n = 22); and Cs+, 527 pS +/- 5 (n = 16). The single-channel conductances of MHS and normal Ca2+ release channel were not significantly different for any of the monovalent cations tested. Permeability ratios measured under biionic conditions had the permeability sequence Ca2+ >> Li+ > Na+ > K+ > or Rb+ > Cs+, with no significant difference noted between MHS and normal channels. This systematic examination of the conduction properties of the pig skeletal muscle Ca2+ release channel indicated a higher Ca2+ selectivity (PCa2+:Pk+ approximately 15.5) than the sixfold Ca2+ selectivity previously reported for rabbit skeletal (Smith et al., 1988) or sheep cardiac muscle (Tinker et al., 1992) Ca2+ release channels. These results also indicate that although Ca2+ regulation of Ca2+ release channel activity is altered, the Arg615 to Cys615 mutation of the porcine Ca2+ release channel does not affect the conductance or ion selectivity properties of the channel.     

A fluorescence lifetime study of virginiamycin S using multifrequency phase fluorometry

Using multifrequency phase fluorometry, fluorescence lifetimes have been assigned to the different protolytic forms of the antibiotic virginiamycin S. These lifetimes are 0.476 +/- 0.005 ns for the uncharged form, 1.28 +/- 0.2 and 7.4 +/- 0.2 ns for the zwitterionic form, 1.19 +/- 0.01 ns for the negatively charged form, and 1.9 +/- 0.1 ns for the double negatively charged form. The assignments are based on lifetime measurements as a function of pH, volume percent ethanol, and excitation wavelength. Excited-state proton transfer is taken into account. It is complete at pH values lower than 1, and no fluorescence of the fully protonated charged form is observed. At pH 8, an excited-state pK* increase is calculated, but proton association is too slow to cause excited-state proton transfer. The addition of divalent cations, at pH 9.4, increases the lifetime of the negatively charged form to a value dependent upon the specific nature of the cation (7.58 +/- 0.06 ns for Mg2+, 6.54 +/- 0.02 ns for Ca2+, and 3.74 +/- 0.05 ns for Ba2+). Monovalent cations do not influence the lifetimes, indicating that their binding to the macrocycle does not influence the fluorescent moiety. The model compound 3-hydroxypicolinamide shows an analogous behavior, but the retrieved lifetime can differ significantly.     

Doxorubicin inhibits the phosphate-transport protein reconstituted in liposomes. A study on the mechanism of the inhibition

Using Thr(P)-inhibitor-1 and Ser(P)-casein as substrates, studies on the activation of calcineurin purified from bovine brain have been carried out. The phosphatase requires the synergistic action of Ca2+, calmodulin and another divalent cation (Mg2+, Mn2+, Co2+ or Ni2+, but not Zn2+) for full expression of its activity. Ca2+ and Ca2+ X calmodulin act as allosteric activators to transform the phosphatase to a relaxed conformation, while Mg2+ acts solely as a cofactor for the catalytic action of the enzyme. In addition to their function as cofactors for catalysis, transition metal ions can also substitute for Ca2+ as allosteric activators. Ca2+ and calmodulin exert their activating effects mainly by increasing the Vm of the phosphatase reaction with little effect on the Km values for the substrates or on the KA values for the divalent cation cofactors. The predominant factor in dictating the catalytic properties of calcineurin is the divalent cation cofactor. For example, with Mg2+ as a cofactor, the phosphatase exhibits an optimum around pH 8.0-8.5; while with a transition metal ion as a cofactor, the optimum is around pH 7.0-7.5, regardless of whether Thr(P)-inhibitor-1 or Ser(P)-casein serves as a substrate, in the absence or the presence of Ca2+ X calmodulin.     

Monovalent cation binding to cubic insulin crystals.

Two localized monovalent cation binding sites have been identified in cubic insulin from 2.8 A-resolution difference electron density maps comparing crystals in which the Na+ ions have been replaced by Tl+. One cation is buried in a closed cavity between insulin dimers and is stabilized by interaction with protein carbonyl dipoles in two juxtaposed alternate positions related by the crystal dyad. The second cation binding site, which also involves ligation with carbonyl dipoles, is competitively occupied by one position of two alternate His B10 side chain conformations. The cation occupancy in both sites depends on the net charge on the protein which was varied by equilibrating crystals in the pH range 7-10. Detailed structures of the cation binding sites were inferred from the refined 2-A resolution map of the sodium-insulin crystal at pH 9. At pH 9, the localized monovalent cations account for less than one of the three to four positive counterion charges necessary to neutralize the negative charge on each protein molecule. The majority of the monovalent counterions are too mobile to show up in the electron density maps calculated using data only at resolution higher than 10 A. Monovalent cations of ionic radius less than 1.5 A are required for crystal stability. Replacing Na+ with Cs+, Mg++, Ca++ or La+++ disrupts the lattice order, but crystals at pH 9 with 0.1 M Li+, K+, NH4+, Rb+ or Tl+ diffract to at least 2.8 A resolution.     

Regulation of the L-lactase dehydrogenase from Lactobacillus casei by fructose-1,6-diphosphate and metal ions.

The lactate dehydrogenase of Lactobacillus casei, like that of streptococci, requires fructose-1,6-diphosphate (FDP) for activity. The L. casei enzyme has a much more acidic pH optimum (pH 5.5) than the streptococcal lactate dehydrogenases. This is apparently due to a marked decrease in the affinity of the enzyme for the activator with increasing pH above 5.5; the concentration of FDP required for half-maximal velocity increase nearly 1,000-fold from 0.002 mM at pH 5.5 to 1.65 mM at 6.6. Manganous ions increase the pH range of activity particularly on the alkaline side of the optimum by increasing the affinity for FDP. This pH dependent metal ion activation is not specific for Mn2+. Other divalent metals, Co2+, Cu2+, Cd2+, Ni2+, Fe2+, Fe2+, and Zn2+ but not Mg2+, will effectively substitute for Mn2+, but the pH dependence of the activation differs with the metal ion used. The enzyme is inhibited by a number of commonly used buffering ions, particularly phosphate, citrate, and tris (hydroxymethyl) aminomethane-maleate buffers, even at low buffer concentrations (0.02 M). These buffers inhibit by affecting the binding of FDP.