An electron paramagnetic resonance study of Mn2+ uptake by the chick chorioallantoic membrane

Mn²⁺ uptake in the chick chorioallantoic membrane, an embryonic epithelial tissue which transports Ca²⁺ in vivo was studied using electron paramagnetic resonance (EPR). Mn²⁺ was used as a paramagnetic analog for Ca²⁺, since there is evidence that Mn²⁺ is accumulated by the Ca²⁺ transport mechanism.After 1.5 h of uptake the EPR spectrum of the Mn²⁺ in the membrane indicated that 89 % of the Mn²⁺ was in a spin-exchange form, indicating close packing of Mn²⁺. The Mn²⁺ spacing was estimated from the line width to be about 4.7 Å. The remaining Mn²⁺ was very likely Mn²⁺ hexahydrate.At pH 7.4 the spin-exchange spectrum tended to broaden when uptake was inhibited, while at pH 5.0 the spin-exchange spectrum was completely abolished in the presence of inhibitors.The EPR spectrum of Mn²⁺ in the chorioallantoic membrane had a broader line width than that of Mn²⁺ in isolated mitochondria, suggesting that in this tissue mitochondria are not directly involved in divalent cation transport. These EPR studies support the concept that divalent cations are sequestered in high concentrations from the rest of the cell contents during transcellular active transport.     

Energy-dependent Mn2+ and Ca2+ uptake by the embryonic chick chorioallantoic membrane.

The chick chorioallantoic membrane is an epithelial tissue which actively transports large amounts of Ca2+ during embryonic development. In this paper Mn2+ uptake by the tissue was studied and compared to Ca2+ uptake in parallel experiments. The purpose of these experiments was to determine if Mn2+ could be used to gain more information about the Ca2+ transport system. It was found that Mn2+ uptake was reduced significantly under conditions that reduced Ca2+ uptake and that Mn2+, like Ca2+, was taken up preferentially by the ectodermal side of the tissue. Mn2+ uptake showed saturation kinetics with a Km of 0.33 MM. Mn2+ uptake was also competitively inhibited by Ca2+, and Ca2+ uptake inhibited by Mn2+. Electron microprobe studies showed that Mn2+ was localized in the ectoderm of the tissue in the same way as Ca2+. It was concluded from these studies that significant amounts of Mn2+ were accumulated by the active Ca2+ transport mechanism and that Mn2+ could be useful paramagnetic probe of divalent cation transport in this tissue.     

Evidence for more than one Ca2+ transport mechanism in mitochondria.

The active transport and internal binding of the Ca2+ analogue Mn2+ by rat liver mitochondria were monitored with electron paramagnetic resonance. The binding of transported Mn2+ depended strongly on internal pH over the range 7.7-8.9. Gradients of free Mn2+ were compared with K+ gradients measured on valinomycin-treated samples. In the steady state, the electrochemical Mn2+ activity was larger outside than inside the mitochondria. The observed gradients of free Mn2+ and of H+ could not be explained by a single "passive" uniport or antiport mechanism of divalent cation transport. This conclusion was further substantiated by observed changes in steady-state Ca2+ and Mn2+ distributions induced by La3+ and ruthenium red. Ruthenium red reduced total Ca2+ or Mn2+ uptake, and both inhibitors caused release of divalent cation from preloaded mitochondria. A model is proposed in which divalent cations are transported by at least two mechanisms: (1) a passive uniport and (2) and active pump, cation antiport or anion symport. The former is more sensitive to La3+ and ruthenium red. Under energized steady-state conditions, the net flux of Ca2+ or Mn2+ is inward over (1) and outward over (2). The need for more than one transport system inregulating cytoplasmic Ca2+ is discussed.     

The nature of the electron spin resonance signal during aerobic uptake of Mn2+ in mitochondria from rat liver

Rat liver mitochondria take up aerobically large amounts of divalent cations in the absence of exogenous phosphate. The electron spin resonance (ESR) spectrum of matrix Mn2+ reveals the presence of two components: one, a sextet signal, corresponding to hydrated Mn2+; another, a spin exchange signal, attributed either to Mn2+ binding to specific high-energy membrane sites or to complexes of Mn2+ with inorganic phosphate. Identification of the spin exchange signal with a Mn-Pi complex is favoured by the evidence that the spin exchange signal is observed at pH 7.5 but not at pH 6.5 in the absence of exogenous Pi, but at both pH 7.5 and 6.5 in the presence of exogenous Pi. On the other hand both in the absence or presence of exogenous Pi inhibition by N-ethylmaleimide of Pi transport, abolishes the spin exchange signal. This signal is again observed when Pi is generated in the matrix, in the presence of N-ethylmaleimide, by ATP hydrolysis, and again abolished by oligomycin. Finally, addition of uncouplers results in a very slow disappearance of the signal. The amount of Mn2+ participating in the spin exchange signal has been calculated to be in the range of 50-60 nmol X mg protein-1. This amount is compatible with the amount of endogenous Pi present or generated in average mitochondrial preparations. The ESR spectrum obtained by superimposing the spectra of Mn3(PO4)2 precipitate and hydrated Mn2+, in appropriate concentrations and ratios, resembles closely the ESR spectrum during aerobic Mn2+ uptake in mitochondria. The band width of the spin exchange signal of Mn3(PO4)2 is not constant and varies between 40 and 22 mT depending on the state of aggregation of the complex. The kinetics of aggregation can be followed in solution as a function of the concentration of Mn2+, Pi and of pH. Similar kinetics can also be followed during aerobic Mn2+ uptake by controlling the rate of Mn2+ influx. The present data support the previous proposal [Pozzan et al. (1976) Eur. J. Biochem. 71, 93-99] that the spin exchange signal is essentially due to a Mn3(PO4)2 precipitate in the mitochondrial matrix.     

Manganous Ion as a Spin Label in Studies of Mitochondrial Uptake of Manganese

Manganous ion (Mn²⁺) has been used as a spin label for studies of divalent cation uptake by rat liver mitochondria. Spin exchange, observed in the electron paramagnetic resonance (EPR) spectrum of a fraction of the transported Mn²⁺, shows that this fraction is bound in regions of high local concentration within the mitochondria. The average separation of manganese ions in that fraction is estimated to be 4.0 ±0.6 A at the time of greatest concentration.     

Calcium transport into the vacuole of oat roots. Characterization of H+/Ca2+ exchange activity

Calcium (Ca2+) is sequestered into vacuoles of oat root cells through a H+/Ca2+ antiport system that is driven by the proton-motive force of the tonoplast H+-translocating ATPase. The antiport has been characterized directly by imposing a pH gradient in tonoplast-enriched vesicles. The pH gradient was imposed by diluting K+-loaded vesicles into a K+-free medium. Nigericin induced a K+/H+ exchange resulting in a pH gradient of 2 (acid inside). The pH gradient was capable of driving 45Ca2+ accumulation. Ca2+ uptake was tightly coupled to H+ loss as increasing Ca2+ levels progressively dissipated the steady state pH gradient. Ca2+ uptake displayed saturation kinetics with a Km(app) for Ca2+ of 10 microM. The relative affinity of the antiporter for transport of divalent cations was Ca2+ greater than Sr2+ greater than Ba2+ greater than Mg2+. La3+ or Mn2+ blocked Ca2+ uptake possibly by occupying the Ca2+-binding site. Ruthenium red (I50 = 40 microM) and N,N'-dicyclohexylcarbodiimide (I50 = 3 microM) specifically inhibited the H+/Ca2+ antiporter. When driven by pH jumps, the H+/Ca2+ exchange generated a membrane potential, interior positive, as shown by [14C]SCN accumulation. Furthermore, Ca2+ uptake was stimulated by an imposed negative membrane potential. The results support a simple model of one Ca2+ taken up per H+ lost. The exchange transport can be reversed, as a Ca2+ gradient (Ca2+in greater than Ca2+out) was effective in forming a pH gradient (acid inside). We suggest that the H+/Ca2+ exchange normally transports Ca2+ into the vacuole; however, under certain conditions, Ca2+ may be released into the cytoplasm via this antiporter.     

The effect of endogenous phosphate on the H+/Mn2+ ratio and the state of Mn2+ in the mitochondrial matrix.

1. Kinetics and stoichiometry of H+ extrusion and reuptake and of Mn2+ uptake and release have been measured in respiring liver mitochondria in the absence of external added Pi. H+ and Mn2+ fluxes are parallel during aerobic cation uptake but not during uncoupler induced cation release. The H+/Mn2+ is 1.24. Addition of SH reagents, in concentrations inhibiting the Pi carrier, modifies the kinetics of H+ extrusion and of Mn2+ uptake and release. The slow phase of uncoupler induced Mn2+ release is diminished. The H+/Mn2+ is increased to 1.72. Addition of SH reagents, after the phase of aerobic uptake is completed, results in a significant reduction of the extent of uncoupler-induced Mn2+ release. The extent of reuptake of endogenous Pi during aerobic uptake of Mn2+ is about 8 nmol x mg protein-1. 2. Aerobic uptake of Mn2+ in the absence of external Pi results in an electron spin resonance spectrum which is the sum of two components. One, denoted as S, corresponds to Mn(H2O)2+(6). Another denoted as E, reflects spin exchange narrowing. In contrast to previous claims the following evidence suggests that the spin exchange component is due to Mn3(PO4)2 precipitate: (a) the dimension of the spin exchange spectrum is markedly reduced by abolition of Pi transport; (b) the spin exchange spectrum is released very slowly by addition of uncouplers under conditions where uncouplers cause a rapid deenergization of mitochondria, reuptake of H+ and release of cations; (c) the free matrix Mn2+ is released slowly after addition of uncoupler if there is a large spin exchange signal; howeover the free matrix Mn2+ is abolished rapidly by uncoupler when formation of the spin exchange signal is prevented by pretreatment with Ca2+; (d) the band width of the spin exchange fraction is independent of the Mn2+/protein ratio either under kinetic or steady state conditions; (e) the experimental spectrum recalls closely that obtained by computer simulation by assuming it as a combination of Mn(H2O)2+(6) and Mn3(PO4)2. 3. It is concluded that endogenous Pi affects the process of aerobic divalent cation uptake. A part of Mn2+ uptake in the absence of externally added anions, consists of a Mn3(PO4)2 precipitate. This accounts for a H+/Mn2+ ratio lower than 2.     

Kinetics of Ca2+ and Sr2+ uptake by yeast. Effects of pH, cations and phosphate.

The uptake of Ca2+ and Sr2+ by the yeast Saccharomyces cerevisiae is energy dependent, and shows a deviation from simple Michaelis-Menten kinetics. A model is discussed that takes into account the effect of the surface potential and the membrane potential on uptake kinetics. The rate of Ca2+ and Sr2+ uptake is influenced by the cell pH and by the medium pH. The inhibition of uptake at low concentration of Ca2+ and Sr2+ at low pH may be explained by a decrease of the surface potential. The inhibition of Ca2+ and Sr2+ uptake by monovalent cations is independent of the divalent cation concentration. The inhibition shows saturation kinetics, and the concentration of monovalent cation at which half-maximal inhibition is observed, is equal to the affinity constant of this ion for the monovalent cation transport system. The inhibition of divalent cation uptake by monovalent cations appears to be related to depolarization of the cell membrane. Phosphate exerts a dual effect on uptake of divalent cations: and initial inhibition and a secondary stimulation. The inhibition shows saturation kinetics, and the inhibition constant is equal to the affinity constant of phosphate for its transport mechanism. The secondary stimulation can only partly be explained by a decrease of the cell pH, suggesting interaction of intracellular phosphate, or a phosphorylated compound, with the translocation mechanism.     

Mitochondrial Ca2+ transport in lean and genetically obese (ob/ob) mice.

Isolated mitochondria from liver or brown adipose tissue of obese ob/ob mice demonstrated increased rates of Ca2+ uptake and release compared with those of lean mice. This enhanced transport activity was not found in mitochondria from kidney or skeletal muscle. Respiration-induced membrane potential was the same in mitochondria from lean and ob/ob mice. It is therefore concluded that the increased Ca2+ uptake rates reflect an activation of the Ca2+ uniporter rather than a change in the electrophoretic driving force. As mitochondria from pre-obese ob/ob mice did not show elevated rates of Ca2+ transport, the activated transport in the obese animals was thus a consequence of the state of obesity rather than being a direct effect of the ob/ob genotype. It is suggested that the enhanced activity of the Ca2+-transport pathways in liver and brown adipose tissue may alter metabolic functions in these tissues by modifying cytoplasmic or intramitochondrial Ca2+ concentrations.     

Unique binding site for Mn2+ ion responsible for reducing an oxidized YZ tyrosine in manganese-depleted photosystem II membranes.

Binding of Mn2+ to manganese-depleted photosystem II and electron donation from the bound Mn2+ to an oxidized YZ tyrosine were studied under the same equilibrium conditions. Mn2+ associated with the depleted membranes in a nonsaturating manner when added alone, but only one Mn2+ ion per photosystem II (PS II) was bound to the membranes in the presence of other divalent cations including Ca2+ and Mg2+. Mn2+-dependent electron donation to photosystem II studied by monitoring the decay kinetics of chlorophyll fluorescence and the electron paramagnetic resonance (EPR) signal of an oxidized YZ tyrosine (YZ+) after a single-turnover flash indicated that the binding of only one Mn2+ ion to the manganese-depleted PS II is sufficient for the complete reduction of YZ+ induced by flash excitation. The results indicate that the manganese-depleted membranes have only one unique binding site, which has higher affinity and higher specificity for Mn2+ compared with Mg2+ and Ca2+, and that Mn2+ bound to this unique site can deliver an electron to YZ+ with high efficiency. The dissociation constant for Mn2+ of this site largely depended on pH, suggesting that a single amino acid residue with a pKa value around neutral pH is implicated in the binding of Mn2+. The results are discussed in relation to the photoactivation mechanism that forms the active manganese cluster.     

Modulation of Na+/alanine cotransport in liver sinusoidal membrane vesicles by internal divalent cations

Rat liver basolateral plasma membrane (blLPM) vesicles resuspended in 5 mM Mg2(+)-, Ca2(+)-, Mn2(+)- or Co2(+)-containing media exhibited a markedly lower rate of Na(+)-stimulated L-alanine transport. Divalent cation inhibition of L-alanine uptake was dose dependent, and was observed only when the vesicles were pre-loaded with the divalent cations. The presence or absence of the metal ions in the extravesicular incubation media had no effect on L-alanine transport. Conversely, pretreatment of the vesicles with 0.2 mM of either EGTA or EDTA resulted in higher initial rates of L-alanine transport. This stimulation was overcome by addition of excess divalent cation to the vesicle suspension solution. Since these blLPM vesicles are primarily oriented right-side-out, the divalent cation inhibition of L-alanine transport appears to be a result of their interaction with cytosolic components of the cell membrane. Total Na+ flux as measured with 22Na+ was not affected by intravesicular 5 mM Mg2+ or Ca2+, indicating that the inhibition was not due to dissipation of the Na+ gradient. These observations suggest that intracellular divalent cations may serve to modulate L-alanine transport across the liver cell plasma membrane.     

Spin-labeling of Escherichia coli membrane by enzymatic synthesis of phosphatidylglycerol and divalent cation-induced interaction of phosphatidylglycerol with membrane proteins.

Escherichia coli membrane particulate fraction has been spin-labeled by incubating with sn-glycerol-3-phosphate, CTP, palmitoyl CoA and 12-nitroxide stearoyl CoA. Incorporation of the spin-labeled acyl chain into phosphatidyl-glycerol was confirmed. ESR spectrum of the spin-labeled phosphatidylglycerol in E. coli membrane consisted at least of two components; one due to the labels undergoing rapid anisotropic motions and the other due to strongly immobilized labels (the overall splitting value, approx. 58 G). The relative intensity of the two components was dependent on the concentration of divalent cations. The immobilized component decreased on treatment of the membrane with EDTA and increased on addition of Mg2+ or Ca2+. The spectrum at 1 mM Mg2+ or Ca2+ consisted almost only of the immobilized component. Spin-labeled phosphatidylglycerol in total lipid membrane showed ESR spectrum due to mobile labels and the spectrum was not affected appreciably by the divalent cations. The results suggest the divalent cation-mediated interaction of phosphatidylglycerol with proteins in E. coli membrane. Phosphoenolpyruvate-dependent uptake of methyl-alpha-D-glucoside was markedly accelerated by Mg2+. Ca2+ was not effective for the enhancement. The divalent cation-induced interaction of phosphatidylglycerol with proteins was discussed in relation to the sugar transport.     

Transport of Ca and Mn by mitochondria from rat liver, heart and brain

The energy-dependent, respiration-supported uptake and the uncoupler- or Na+-induced release of Ca2+ and Mn2+ by mitochondria from rat liver, heart and brain were investigated, using as indicators radioisotopes (45Ca and 54Mn), proton ejection, oxygen consumption, nicotinamide nucleotide oxidation-reduction and, in the case of Ca2+, the metallochromic dye Arsenazo III. Ca2+ uptake in the presence of Pi was rapid in mitochondria from liver and brain, and less rapid in those from heart. Mn2+ uptake was much slower than that of Ca2+ in liver and heart, but only slightly slower in brain. When added together, Ca2+ accelerated the uptake of Mn2+, and Mn2+ retarded the uptake of Ca2+, by mitochondria from all three tissues. When Mn2+ was present during Ca2+ uptake, its own uptake remained accelerated even after Ca2+ uptake was terminated. Mg2+, which was not taken up, inhibited Ca2+ uptake by mitochondria from all three tissues, and, when present during Ca2+ uptake, accelerated the subsequent uptake of Mn2+. The uncoupler CCCP induced a release of both Ca2+ and Mn2+ from all three sources of mitochondria; yet, release of Mn2+ took place only in the absence of Pi. The release followed the same pattern as the uptake, i.e., Ca2+ accelerated the release of Mn2+ and Mn2+ retarded the release of Ca2+. Na+ induced a release of both Ca2+ and Mn2+ from heart and brain but not from liver mitochondria; again, Mn2+ release occurred only in the absence of Pi. The Na+-induced release of Ca2+ was inhibited by Mn2+, but the Na+-induced release of Mn2+ was not accelerated by Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional involvement of carbonic anhydrase in calcium transport of the chick chorioallantoic membrane.

Carbonic anhydrase activity was demonstrated in the chick-embryonic chorioallantoic membrane and was correlated with the Ca2+-transport activity of the membrane. It is inhibited by sulphonamides and is expressed in the chorioallantoic membrane in an age-dependent fashion during embryonic development. Ca2+ uptake by the chorioallantoic membrane in vivo also increases in a similar age-dependent manner. The temporal increase in these activities is coincident with calcium deposition in the embryonic skeleton. Incubation of the chorioallantoic membrane in ovo with sulphonamides specifically inhibits both the carbonic anhydrase and the Ca2+ uptake activities of the membrane in vivo. Enzyme histochemistry revealed the carbonic anhydrase activity is localized in the Ca2+-transporting ectodermal cells of the chorioallantoic membrane. These results, taken together, indicate that carbonic anhydrase may be functionally important in the Ca2+-transport activity of the chorioallantoic membrane.     

The subcellular localization of neutral sphingomyelinase in rat liver.

The subcellular distribution of neutral sphingomyelinase activity has been determined in rat liver. Neutral sphingomyelinase is present in the plasma membrane. This enzyme requires either Mg2+ or Mn2+ for full activity; these cations cannot be replaced by Co2+ or Ca2+. The plasma membrane sphingomyelinase is strongly inhibited by Hg2+. A small amount of neutral spingomyelinase activity appears to be present in microsomes. No neutral sphingomyelinase activity is present in liver mitochondria or bytosol. Lysosomal sphingomyelinase is fully active at pH 4.4--4.8 without added divalent cations. However, between pH 5.0 and 7.5 lysosomal sphingomyelinase activity is stimulated by Mg2+, Mn2+, Co2+, and Ca2+. Below pH 4.8, Mg2+ inhibits the reaction. In contrast to the results obtained with the neutral sphingomyelinase activity of plasma membranes and microsomes, lysosomal sphingomyelinase is unaffected by sulfhydryl inhibitors.     

Regulation of 22Na+ transport by calcium in an established kidney epithelial cell line.

The role of calcium in regulating the Na+ channel in an established kidney epithelial cell line has been examined. Extracellular calcium was inhibitory to Na+ uptake, and a Dixon plot of the initial Na+ uptake rate in the presence of Ca2+ was nonlinear, suggesting a mixed pattern of inhibition. Similar patterns of inhibition were also observed for other divalent cations, including Ba2+, Mg2+, and Mn2+. In contrast elevated concentrations of intracellular calcium resulted in a stimulation of Na+ entry. This intracellular effect was specific to calcium, with Mg2+ and Mn2+ appearing much less effective. Lineweaver-Burk plots of Na+ influx in calcium-loaded and unloaded cells were linear, suggesting that under both conditions a single system transported Na+. Although Na+ entry was stimulated by intracellular Ca2+, the cells did not exhibit other counter transport phenomena reported with cell types in which a Na+/Ca2+ exchange system is operative. Thus, the results indicate that calcium acts as an allosteric regulator of Na+ transport by the Na+ channel.     

Interaction of bacteriophage lambda protein phosphatase with Mn(II): evidence for the formation of a [Mn(II)]2 cluster.

The interaction of bacteriophage lambda protein phosphatase with Mn2+ was studied using biochemical techniques and electron paramagnetic resonance spectrometry. Reconstitution of bacteriophage lambda protein phosphatase in the presence of excess MnCl2 followed by rapid desalting over a gel filtration column resulted in the retention of approximately 1 equiv of Mn2+ ion bound to the protein. This was determined by metal analyses and low-temperature EPR spectrometry, the latter of which provided evidence of a mononuclear high-spin Mn2+ ion in a ligand environment of oxygen and nitrogen atoms. The Mn2+-reconstituted enzyme exhibited negligible phosphatase activity in the absence of added MnCl2. The EPR spectrum of the mononuclear species disappeared upon the addition of a second equivalent of Mn2+ and was replaced by a spectrum attributed to an exchange-coupled (Mn2+)2 cluster. EPR spectra of the dinuclear (Mn2+)2 cluster were characterized by the presence of multiline features with a hyperfine splitting of 39 G. Temperature-dependent studies indicated that these features arose from an excited state. Titrations of the apoprotein with MnCl2 provided evidence of one Mn2+ binding site with a micromolar affinity and at least one additional Mn2+ site with a 100-fold lower affinity. The dependence of the phosphatase activity on Mn2+ concentration indicates that full enzyme activity probably requires occupation of both Mn2+ sites. These results are discussed in the context of divalent metal ion activation of this enzyme and possible roles for Mn2+ activation of other serine/threonine protein phosphatases.     

Paramagnetic probes in NMR and EPR studies of membrane enzymes

The applications of paramagnetic probes to problems of structure and mechanism are discussed from the point of view of the membrane enzymologist. Problems unique to membrane systems are discussed, and a variety of nuclear and paramagnetic probes are evaluated. Three membrane ATPase (kidney (Na+ + K+)-ATPase, Ca2+-ATPase from sarcoplasmic reticulum and Mg2+-ATPase from kidney) are used to describe the types of experiments which can be done, the information which can be obtained and the limitations involved. Nuclear relaxation studies employing 1H, 7Li+, 31P and 205Tl+ nuclei are described. The advantages and disadvantages of Mn2+, Gd3+ and Cr3+ as paramagnetic probes are discussed in terms of the three ATPases. The theory and interpretation of Mn2+ and Gd3+ EPR spectra are evaluated in studies with the (Na+ + K+)-ATPase and Ca2+-ATPase, respectively.     

Manganese stimulates calcium flux through the mitochondrial uniporter

Mn2+ alters the balance between the simultaneous uptake and release of Ca2+ across the mitochondrial inner membrane toward a lower external level. Addition of as little as 0.5 microM Mn2+ to energised mitochondria from rat liver, rat heart or guinea-pig brain changed the level at which they buffered Ca2+ in the medium. That extramitochondrial Mn2+ was responsible was suggested by a partial decay in the shift in Ca2+ steady state at a rate similar to the rate at which Mn2+ was accumulated by the mitochondria. The alteration of transmembrane Ca2+ distribution by Mn2+ required that both Mg2+ and Pi be present, and was almost maximal at Mg2+ and Pi levels in the physiological range. Substitution of spermine or Ni2+ for Mg2+, or acetate for Pi, abolished the effect. In contrast to Sr2+, Mn2+ did not inhibit either EGTA- or Ruthenium red-induced release of Ca2+ from the mitochondria. However, when flux through the uniporter was rate-limiting, Mn2+ accelerated Ca2+ uptake. The stimulation showed hyperbolic kinetics, with an element of competition discernible in the Mn2+-Mg2+ interaction. Thus, extramitochondrial Mn2+ at levels occurring in vivo can alter the mitochondrial 'set-point' by stimulating Ca2+ influx through the uniporter.     

Inorganic cation transport and the effects on C4 dicarboxylate transport in Bacillus subtilis

The complex interrelationships between the transport of inorganic cations and C4 dicarboxylate were examined using mutants defective in potassium transport and retention, divalent cation transport, or phosphate transport. The potassium transport system, studied using 86Rb+ as a K+ analogue, kinetically appeared as a single system (Km 200 microM for Rb+, Ki 50 microM for K+), the activity of which was only slightly reduced in K+ retention mutants. Divalent cation transport, studied using 54Mn2+, 60Co2+, and 45Ca2+, was more complex being represented by at least two systems, one with a high affinity for Mn2+ (Km 2.5 microM) and a more general one of low affinity (Km 1.3-10 mM) for Mg2+, Mn2+, Ca/2+, and Co2+. Divalent cation transport was repressed by Mg2+, derepressed in K+ retention mutants, and defective in Co2+-resistant mutants. Phosphate was required for both divalent cation and succinate transport, and phosphate transport mutants (arsenate resistant) were found to be defective in both divalent cation and succinate transport. Divalent cations, especially Mg2+ and Co2+, decreased Km for succinate transport approximately 20-fold over that achieved with K+; neither cation was required stoichiometrically for succinate transport. The loss of divalent cation transport in cobalt-resistant mutants has been correlated with the loss of a 55,000 molecular weight membrane protein. Similarly, the loss of phosphate transport in arsenate-resistant mutants has been correlated with the loss of a 35,000 molecular weight membrane component.