THE UPTAKE OF LOW CONCENTRATIONS OF LITHIUM IONS INTO RAT CEREBRAL CORTEX SLICES AND ITS DEPENDENCE ON CATIONS

—Rat cerebral slices were incubated in oxygenated Krebs-Ringer bicarbonate glucose saline, and the uptake of Li⁺ was measured after periods of 15 s to 5 min. Saturation was not seen within the concentrations of Li⁺ employed (0·5-2·0 mm ). The half-time of the uptake was 7·9 min. At steady state, after 1 h incubation, the concentration of Li⁺ in the tissue was linearly related to that of the medium (0·5-1·5 mm Li⁺) with a concentration ratio of 1·29–1·66. The concentrations of K⁺ and Na⁺ in the slices incubated without Li⁺ were found to be (μmol/g incubated wt, mean ±s.d .) 63·8 ± 9·6 and 96·2 ± 7·8 respectively (n = 28). In the presence of media with 1·5 mm -Li⁺, the K⁺ and Na⁺ in the slices were 56·2 ± 8·8 and 101·0 ± 7·7 respectively (n = 37). The concentration of Li⁺ in the slices, after 1 h incubation, increased in a non linear way as the concentration of K⁺ in the medium was decreased within a range of 0·10 mm -K⁺. In the absence of K⁺ in the medium the uptake of Li⁺ was approx 50% higher than in the presence of 4·9 mm -K⁺. There was an inverse linear relationship between the concentration of Li⁺ in the slices and that of Ca²⁺ in the medium within the range of 0-5·2 mm (-0·13 mm -Li⁺/mm Ca²⁺). The concentration of Li⁺ in the slices increased by approx 10% when the Mg²⁺ in the medium was increased from 1·3 mm to 2·6 mm . Changes of the concentration of Na⁺ between 120 mm and 170 mm in the medium had no significant effect on the Li⁺ uptake.     

Kinetics of chloride transport in the cyanobacterium Synechococcus R-2 (Anacystis nidulans, S. leopoliensis) PCC 7942

The kinetics of the light-driven Cl^? uptake pump of Synechococcus R-2 (PCC 7942) were investigated. The kinetics of Cl^? uptake were measured in BG-11 medium (pH_o, 7·5; [K⁺]_o, 0·35 mol m^?3; [Na⁺]_o, 18 mol m^?3; [Cl^?]_o, 0·508 mol m^?3) or modified media based on the above. Net³⁶Cl^? fluxes (?Cl^?_o,i) followed Michaelis-Menten kinetics and were stimulated by Na⁺ [18 mol m^?3 Na⁺ BG-11 ?Cl^?_max= 3·29±0·60 (49) nmol m^?2 s^?1 versus Na⁺-free BG-11 ?Cl^?_max= 1·02±0·13 (54) nmol m^?2 s^?1] but the Km was not significantly different in the presence or absence of Na⁺ at pH_o 10; the Km was lower, but not affected by the presence or absence of Na⁺ [Km = 22·3±3·54 (20) mmol m^?3]. Na⁺ is a non-competitive activator of net ?Cl^?_o,i. High [K⁺]_o (18 mol m^?3) did not stimulate net ?Cl^?_o,i or change the Km in Na⁺-free medium. High [K⁺]_o (18 mol m^?3) added to Na⁺ BG-11 medium decreased net ?Cl^?_o,i [18 mol m^?3K⁺ BG-11; ?Cl^?_max= 2·50±0·32 (20) nmol m^?2 s^?1 versus BG-11 medium; ?Cl^?_max= 3·35±0·56 (20) nmol m^?2 s^?1] but did not affect the Km 55·8±8·100 (40) mmol m^?3]. Na⁺-stimulation of net ?Cl^?_o,i followed Michaelis-Menten kinetics up to 2–5 mol m^?3 [Na⁺]_o but higher concentrations were inhibitory. The Km for Na⁺-stimulation of net ?Cl^?_o,i [K_1/2(Na⁺)] was different at 47 mmol m^?3 [Cl^?]_o (K_1/2[Na⁺] = 123±27 (37) mmol m^?3]. Li⁺ was only about one-third as effective as Na⁺ in stimulating Cl^? uptake but the activation constant was similar [K_1/2(Li⁺) = 88±46 (16) mmol m^?3]. Br^? was a competitive inhibitor of Cl^? uptake. The inhibition constant (Ki) was not significantly different in the presence and absence of Na⁺. The overall Ki was 297±23 (45) mmol m^?3. The discrimination ratio of Cl^? over Br^? (δCl^?/δBr^?) was 6·38±0·92 (df = 147). Synechococcus has a single Na⁺-stimulated Cl^? pump because the Km of the Cl^? transporter and its discrimination between Cl^? and Br^? are not significantly different in the presence and absence of Na⁺. The Cl^? pump is probably driven by ATP.     

Effect of inotropic agents on the calcium binding to isolated cardiac sarcolemma

Ca²⁺ binding to fragmented sarcolemma isolated from canine heart was measured by an ultracentrifugation technique. Two classes of binding site with dissociation constants of 2.0 · 10^?5 and 1.2 · 10^?3 M were identified. The capacities of the high- and low-affinity sites were 15 and 452 nmol/mg, respectively. These sites were not affected by treatment with neuraminidase. The effects of various cations and drugs on Ca²⁺ binding were studied. All cations tested inhibited Ca²⁺ binding with the following order of potency: trivalent > divalent > monovalent cations. The order of potency for the monovalent ions was: Na⁺ > K⁺ > Li⁺ ? Cs⁺ and for the divalent and trivalent ions: La³⁺ ? Mn²⁺ > Sr²⁺ ? Ba²⁺ > Mg²⁺. 1 · 10^?3 M caffeine and 1 · 10^?8 M ouabain increased the capacity of the low-affinity sites to 1531 and 837 nmol/mg, respectively. 1 · 10^?7 M verapamil, acidosis (pH 6.4), 1^?10^?5 M Mn²⁺ and 1 · 10^?4 M ouabain depressed the capacity of the low-affinity sites to a range of 154–291 nmol/mg. The dissociation constants of the high- and low-affinity sites and the capacity of the high-affinity sites were not affected by these agents.     

Zinc and salinity effects on membrane transport in Chara connivens

Pressure-probe measurements showed that the pressure relaxation of internodal cells of the freshwater alga Chara connivens slowed considerably when 1–5 mol m^?3 Zn²⁺, or more especially Zn²⁺ and 75 mol m^?3 NaCl, were present in the medium for periods of 1 h or longer. These results indicate that the water permeability of the Chara membrane is decreased by Zn²⁺, and that this effect is enhanced by 75 mol m^?3 NaCl. Specific values taken after 375 min exposure were: 5 mol m^?3 Zn²⁺ and 75 mol m^?3 NaCl caused the half-time for bulk water movement to increase from 7·8±2·3 to 79·5±5·4s, corresponding to a decrease in the hydraulic conductivity (Lp) from (13·0±3·3) × 10^?7 m s^?1 mPa^?1 to (1·25±0·23) × 10^?7 m s^?1 MPa^?1 (mean±S.D., n= 10). These changes are not seen in the presence of NaCl alone, and to a reduced extent in the presence of 5 mol m^?3Zn²⁺ alone (after 375 min, Lp was (2·4±0·1) × 10^?7 m s^?1 MPa^?1, mean±S.D., n = 6). Ca²⁺ cannot substitute for Zn²⁺, but seems to competitively inhibit Zn²⁺. There was another, kinetically distinct effect of Zn²⁺: the ingress of Na⁺ within 15 min of exposure to 75 mol m^?3 NaCl is halved by the presence of 1–5 mol m^?3 Zn²⁺, although internal osmolality is little changed by Zn²⁺. In spite of this, Zn²⁺ does not exert the long-term protection against NaCl that has been reported for Ca²⁺. Depending on the concentration of Zn²⁺ and the duration of the exposure, the effects on water permeability were fully or partly reversible within 24–48 h. The mechanism of these changes is difficult to identify. One possibility is a zinc-induced restriction of trans-membrane channels to give single-file channels which can be blocked by salt.     

Calcium transport by pigeon erythrocyte membrane vesicles

Membrane vesicles from pigeon erythrocytes show a rapid, ATP-dependent accumulation of ⁴⁵Ca²⁺. Ca²⁺ accumulation ratios greater than or approximately equal to 10⁴ are readily attained. For ATP-dependent Ca²⁺ uptake, V is 1.5 mmol · 1^?1 · min^?1 at 27°C (approx. 0.9 nmol · mg^?1 protein · min^?1), [Ca²⁺]_{$\text{1}\text{2}$} is 0.18 μM, [ATP]_{$\text{1}\text{2}$} is 30–60 μM, the Ca²⁺ uptake rate depends on [Ca²⁺]² and the dependence of uptake rate on ATP concentration implies strong ATP-ATP cooperativity. The Arrhenius activation energy is 19.1 ± 1.4 kcal/mol and the pH optimum is approx. 6.9.     

Cooperative binding of calcium to glycerinated skeletal muscle fibers

The binding of ⁴⁵Ca²⁺ to glycerinated rabbit psoas fibers was measured by means of a double isotope technique. With 5 mM Mg²⁺ (no ATP) binding was half-maximal at 1.4 · 10^?6M Ca²⁺ and the maximal amount bound was 1.6 μmol/g protein. At < 50% saturation, the Scatchard plot had a positive slope and the Hill coefficient was 2.2. At greater than 50% saturation, the Scatchard plot was linear with a negative slope (K′ = 0.8 · 10⁶ M^?1) and the Hill coefficient was 1.0. In the absence of Mg²⁺, binding was half-maximal at 3 · 10^?7 M Ca²⁺ and the maximal amount bound was 2.9 μmol/g protein. The Scatchard plot indicated two classes of sites with K′ values of about 2 · 10⁷ and 2 · 10⁶ M^?1. The Hill coefficient in the mid-saturation range was approx. 0.6. The data indicate that in the presence of Mg²⁺ binding to about half of the total Ca²⁺ binding sites is suppressed and there is a strong positive cooperativity involving half of the remaining sites.     

Interaction of hemoglobin with salts: Effects on the functional properties of human hemoglobin

Oxygen isotherms of human hemoglobin measured in distilled water and in solutions of different inorganic salts in the concentration range from below 10^?3 m to above 1·5 m at neutral pH indicate that the oxygen affinity decreases with increasing salt concentration in the lower range of ionic strength; above the physiological range, there is in most cases a further decrease in oxygen affinity, but this varies with the nature of the salt and, in some instances, the affinity goes through a maximum.The effect of cations, which is opposite to that of anions, operates primarily in the higher concentration range; i.e. above 0·1 m. This effect is especially large for Li⁺, Ca²⁺ and Mg²⁺.The alkaline Bohr effect depends strongly on anion concentration, being displaced towards higher pH values and being reduced in magnitude as chloride concentration is increased. On the other hand, the acid Bohr effect, observed below pH 6, appears to be independent of chloride concentration from 6 × 10^?2 m to 2 m.The overall heat of oxygenation has been determined for the isoionic protein as well as at different concentrations of chloride and phosphate. The average intrinsic heat of reaction of hemoglobin with oxygen in solution is found to be ?14·6 kcal/mol of O₂.     

Studies on the luminescent response of the Ca2+-activated photoprotein,obelin

1. The kinetics and stoicheiometry of the Ca²⁺-activated luminescent reaction of the photoprotein obelin were studied at different temperatures and in the presence of various substances, including the physiologically occurring cations K⁺, Na⁺, Ca²⁺, Mg²⁺ and H⁺. 2. The results suggest Ca²⁺-independent rates of rise and fall in obelin luminescence following sudden changes in [Ca²⁺] and indicate that changes in [Ca²⁺] over the range 1 · 10^?6?3 · 10^?4 M are followed significantly faster by the obelin response (approx. 3 ms delay at 20°C) than by the aequorin response (approx. 10 ms delay at 20°C). 3. Obelin was found to emit low-intensity light (less than 10^?6 of the maximum Ca²⁺-activated response), which was independent of Ca²⁺ at concentrations below about 10^?7 M. The level of this Ca²⁺-independent light emission is sensitive to temperature and the ionic composition of the solution. 4. The log-log plot of light intensity against ionized Ca indicates a maximum slope of 2.5, suggesting the involvement of three Ca ions in the luminescent reaction. 5. Increases in the concentration of K⁺, Na⁺, Mg²⁺ and H⁺ generally shift the Ca²⁺ activation curve for obelin toward higher Ca²⁺ concentrations. These cations can also affect the maximum rate of obelin utilization at more extreme concentrations. 6. The maximal rate of obelin utilization was also affected to varying degrees by the presence of uncharged substances such as glucose, sucrose and polyvinylpyrrolidone. However, neither the sensitivity of obelin to Ca²⁺ nor the quantum yield were modified by these substances. 7. Caffeine (less than 20 mM), procaine (less than 20 mM) and sodium dantrolene (saturated solution), substances known to modify cellular Ca²⁺ movements, had little effect on the Ca²⁺-induced luminescent reaction. The general anaesthetics chlorpromazine and halothane appeared to lower greatly the quantum yield without, however, modifying the maximum rate of obelin utilization. 8. A scheme of reaction for obelin activation by Ca²⁺ is presented which adequately explains the experimental observations and allows one to make accurate predictions regarding the relative obelin respones under a variety of ionic conditions at room temperature.     

The cyanobacterium Synechococcus R-2 (Anacystis nidulans, S. leopoliensis) PCC 7942 has a sodium-dependent chloride transporter

Synechococcus R-2 (PCC 7942) actively accumulated Cl^? in the light and dark, under control conditions (BG-11 media: pH_o, 7·5; [Na⁺]_o, 18 mol m^?3; [Cl^?]_o, 0·508 molm^?3). In BG-11 medium [Cl^?], was 17·2±0·848 mol m^?3 (light), electrochemical potential of Cl^? (ΔμCl^?_i,o) =+211±2mV; [Cl^?]_i= 1·24±0·11 mol m^?3(dark), ΔμCl^?_i,o=+133±4mV. Cl^? fluxes, but not permeabilities, were much higher in the light: ?Cl^?_i,o= 4·01±5·4 nmol m^?2 s^?1, ^PCl^?_i,o= 47±5pm s^?1 (light); ?Cl^?_i,o= 0·395±0·071 nmol m^?2 s^?1, _PCl^?_i,o= 69±14 pm s^?1 (dark). Chloride fluxes are inhibited by acid pH_o (pH_o 5; ?Cl^?_i,o= 0·14±0·04 nmol m^?2 s^?1); optimal at pH_o 7·5 and not strongly inhibited by alkaline pH_o (pH_o 10; ?Cl^?1_i,o= 1·7±0·14 nmol m^?2 s^?1). A Cl^?_in/2H⁺_in coporter could not account for the accumulation of Cl^? alkaline pH_o. Permeability of Cl^? is very low, below 100pm s^?1 under all conditions used, and appears to be maximal at pH_o 7·5 (50–70 pm s^?1) and minimal in acid pH_o (20pm s^?1). DCCD (dicyclohexyl-carbodiimide) inhibited ?Cl^?_i,o in the light about 75% and [Cl^?]_i fell to 2·2±0·26 (4) mol m^?3. Valinomycin had no effect but monensin severely inhibited Cl^? uptake ([Cl^?]_i= 1·02±0·32 mol m^?3; ?Cl^?_i,o= 0·20±0·1 nmol m^?2 s^?1). Vanadate (200 mmol m^?3) accelerated the Cl^? flux (?Cl^?_i,o= 5·28±0·64 nmol m^?2 s^?1) but slightly decreased accumulation of Cl^? ([Cl^?], = 13·9±1·3 mol m^?3) in BG-11 medium but had no significant effect in Na⁺-free media. DCMU (dichlorophenyldimethylurea) did not reduce [Cl^?], or ?Cl^?_i,o to that found in the dark ([Cl^?]_i= 8·41±0·76 mol m^?3; ?Cl^?_i,o= 2·06±0·36 nmol m^?2 s^?1). Synechococcus also actively accumulated Cl^? in Na⁺-free media, [Cl^?]_i was lower but ΔΨ_i,o hyperpolarized in Na⁺-free media and so the ΔμCl^?_i,o was little changed ([Cl^?]_i= 7·98±0·698 mol m^?3; ΔμCl^?_i,o=+203±3 mV). Net Cl^? uptake was stimulated by Na⁺; Li⁺ acted as a partial analogue for Na⁺. Synechococcus has a Na⁺ activated Cl^? transporter which is probably a primary 2Cl^?/ATP pump. The Cl^? pump is voltage sensitive. ΔμCl^?_i,o is directly proportional to ΔΨ_i,o(P»0·01%): ΔμCl^?_i,o= -1·487 (±0·102) ×ΔΨ_i,o, r= -0·983, n= 31. The ΔμCl^?_i,o increased (more positive) as the Δμ_i,o became more negative. The ΔμCl^?_i,o has no known function, but might provide a driving force for the uptake of micronutrients.     

Chymotrypsinogen · inositol phosphatide complexes and the transport of digestive enzyme across membranes

Chymotrypsinogen A was almost quantitatively extracted from aqueous solution in the presence of inositol phosphatides at relatively low concentrations of both ligands. Calcium ion facilitated the interaction at concentrations of 10^?4–10^?5 M. A water-insoluble chymotrypsinogen · Ca²⁺ · inositol phosphatide complex was formed with an apparent stochiometry of 3 mol phospholipid : 3 mol Ca²⁺ : 1 mol protein. Small changes in the structure of the protein prevented complex formation; in particular, the almost identical α-chymotrypsin, did not form complexes under the conditions studied. On the other hand, an homologous, but structurally substantially different, secretory protein, trypsinogen, did form complexes. Water-insoluble complexes were not formed with albumin, carbonic anhydrase or lactic dehydrogenase under the same circumstances. Neither phosphatidylethanolamine nor phosphatidylcholine formed complexes with chymotrypsinogen. Phosphatidylserine formed complexes, but was less effective than inositol phosphatides. Complex formation and stability was dependent upon “critical” concentrations of both Ca²⁺ and H⁺. Extraction of the protein from solution increased from neglible to complete when the calcium concentration of the medium was raised slightly from 1.0 · 10^?4 M to 1.5·10^?4 M. Conversely, dissociation was complete when H⁺ concentration was decreased slightly from pH 6.5 to 7.0. The complex is apparently formed as the result of specific electrostatic interactions between the polar head group of the inositol phosphatide and the protein, with the nonpolar alipathic fatty acid chains of the phospholipid providing a hydrophobic microenvironment for the protein. It is proposed that such complexes could account for the movement of digestive enzyme through membranes.     

Lysophosphatidylcholine-activated,vanadate-inhibited,Mg2+-ATPase from radish microsomes

An orthovanadate-inhibited, nitrate-insensitive, phospholipid-requiring Mg²⁺-ATPase has been partially purified (approx. 40-fold) from microsomal preparations from 24 h germinated radish seedlings. The specific activity obtained was 10–13 μmol P_i · min^?1 per mg protein, namely by 4- to 10-fold higher than that reported for the known similar enzyme preparations from corn and oat roots, and by 3- to 10-fold lower than that of the extensively purified plasmalemma enzymes from Neurospora and yeast. The partially purified activity was fairly specific for ATP, other nucleotide triphosphates being hydrolysed at less than 10% the rate with ATP; no activity was present towards ADP, AMP, p-nitrophenyl phosphate and other phosphate esters. The activity was strongly dependent on the presence of phospholipids with a marked preference for lysophosphatidylcholine, and showed an absolute requirement for Mg²⁺ or some other divalent cations (CO²⁺, Mn²⁺, Mg²⁺, Ni²⁺, Zn²⁺ in order of decreasing effectiveness); Ca²⁺ could not substitute for Mg²⁺ and was strongly inhibitory in its presence. K⁺, Rb⁺ and Na⁺ and also to a lesser extent NH₄⁺ and Li⁺ were significantly stimulatory, while the anions NO₃^?, H₂PO₄^?, Cl^? and SO₄^2? were ineffective. Orthovanadate, N,N′-dicyclohexylcarbodiimide, diethylstilbestrol, p-chloromercuribenzensulfonate, tetraiodofluorescein and tetrachlorotetraiodofluorescein were strongly inhibitory. The coincidence of the K_m for ATP with that for Mg²⁺ suggested that ATP-Mg is the true substrate. Accordingly, the enzyme showed a normal Michaelis-Menten kinetics for ATP-Mg with an apparent K_m of approx. 0.5 mM. The similarity of the characteristics of this enzyme with those of the plasmalemma enzymes from lower plants suggests its location at the plasma membrane, while some data ‘in vivo’ and in native sealed vesicle systems indicate its involvement in active proton transport.     

Ca2+- or Mg2+-dependent ATPase in plasma membrane of cultured endothelial cells from bovine carotid artery

ATPase was found in plasma membrane of cultured endothelial cells from bovine carotid artery. The activity of the enzyme solubilized by octaethyleneglycol mono-n-dodecyl ether was enhanced by the addition of Ca²⁺ or Mg²⁺ and was not affected by F-actin and ouabain. V_max was 2.8 and 10.0 μmol P_i/mg protein per h for Ca²⁺- and Mg²⁺-dependent activity, respectively, and the corresponding K_m was 4.8·10^?4 M and 3.2·10^?4 M. Molecular weight of the protein was estimated to be approx. 250 000, as determined by activity-staining electrophoresis with polyacrylamide gels.     

The behaviors of metal ions in the CdTe quantum dots–H2O2 chemiluminescence reaction and its sensing application

The behaviors of 15 kinds of metal ions in the thiol‐capped CdTe quantum dots (QDs)–H₂O₂ chemiluminescence (CL) reaction were investigated in detail. The results showed that Ag⁺, Cu²⁺ and Hg²⁺ could inhibit CdTe QDs and H₂O₂ CL reaction. A novel CL method for the selective determination of Ag⁺, Cu²⁺ and Hg²⁺ was developed, based on their inhibition of the reaction of CdTe QDs and H₂O₂. Under the optimal conditions, good linear relationships were realized between the CL intensity and the logarithm of concentrations of Ag⁺, Cu²⁺ and Hg²⁺. The linear ranges were from 2.0 × 10^?6 to 5.0 × 10^?8 mol L^?1 for Ag⁺, from 5.0 × 10^?6 to 7.0 × 10^?8 mol L^?1 for Cu²⁺ and from 2.0 × 10^?5 to 1.0 × 10^?7 mol L^?1 for Hg²⁺, respectively. The limits of detection (S/N = 3) were 3.0 × 10^?8, 4.0 × 10^?8 and 6.7 × 10^?8 mol L^?1 for Ag⁺, Cu²⁺ and Hg²⁺, respectively. A possible mechanism for the inhibition of CdTe QDs and H₂O₂ CL reaction was also discussed. Copyright © 2009 John Wiley & Sons, Ltd.     

Occurrence of passive furosemide-sensitive transmembrane potassium transport in cultured cells

Furosemide ($\text{1 · 10}{}^{\mathrm{?4}}\text{M}$) inhibits a proportion of the total passive (ouabain-insensitive) K⁺ influx into primary chick heart cell cultures (85%), BC₃H1 cells (75%), MDCK cells (40%) and HeLa cells (57%). This action of furosemide upon K⁺ influx is independent of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{pump}$ inhibition since the furosemide-sensitive component of the K⁺ influx is identical in the presence and absence of ouabain ($\text{1 · 10}{}^{\mathrm{?3}}\text{M}$). For HeLa cells the passive, furosemide-sensitive component of K⁺ influx is markedly dependent upon the external K⁺, Na⁺ and Cl^? content. Acetate, iodide and nitrate are ineffective as substitutes for Cl^?, whereas Br^? is partially effective. Partial Cl^? replacement by NO₃^? gave an apparent affinity of 100 mM [Cl]. Na⁺ replacement by choline⁺ abolishes the furosemide-sensitive component, whereas Li⁺ replacement reduces this component by 48%. Partial Na⁺ replacement by choline⁺ gives an apparent affinity of 25 mM [Na⁺]. Variation in the external K⁺ content gives an affinity for the furosemide-sensitive component of approx. 1.0 mM. Furosemide inhibition of the passive K⁺ inflúx is of high affinity, half-maximal inhibition being observed at $\text{5 · 10}{}^{\mathrm{?6}}\text{M}$ furosemide. Piretanide ($\text{1 · 10}{}^{\mathrm{?4}}\text{M}$) and phloretin ($\text{1 · 10}{}^{\mathrm{?4}}\text{M}$) inhibit the same component of passive K⁺ influx as furosemide; ethacrynic acid and amiloride ($\text{both}\text{1 · 10}{}^{\mathrm{?4}}\text{M}$) partially so. The stilbene, SITS ($\text{1 · 10}{}^{\mathrm{?6}}\text{M}$), was ineffective as an inhibitor of the furosemide-sensitive component.     

Kinetics of cation-induced aggregation of Torpedo electric organ synaptic vesicles

Synaptic vesicles from the Torpedo ray can be induced to aggregate in the presence of Ca²⁺ and K⁺ in the 4 mM and 50 mM range, respectively. The reactions are strikingly similar to those of chromaffin granule membranes reported previously (Morris, S.J., Chiu, V.C.K. and Haynes, D.H. (1979) Membrane Biochem. 2, 163–202). The Ca²⁺-induced reaction includes dimerization and higher order aggregation, and is shown to be due to electrostatic screening interactions and binding to negatively-charged groups on the membrane surface. The K⁺-induced reaction includes only dimerization and is shown to be due to screening interactions alone.The kinetics of the dimerization reactions were studied using the stopped-flow rapid mixing technique. The Ca²⁺-induced reaction has a ‘bimolecular’ rate constant of 4.77 · 10⁸ M^?1 · s^?1 while the value for the K⁺-induced reaction is 7.05 · 10⁹ M^?1 · s^?1. These values are close to the limit of diffusion control (8.03 · 10⁹ M^?1 · s^?1), indicating that no large energy barriers or structural barriers to aggregation exist. Arrhenius plots for the Ca²⁺-induced aggregation showed a break at 5°C. Above this temperature, the activation energy is low ($\text{+0.65}\text{kcal/mol}$), consistent with the above. Below this temperature, the activation energy is high, consistent with a membrane structure change increasing the energetic and structural barriers. This information, and the observation of a high stability constant of the complex, were taken as evidence for the involvement of ‘recognition sites’ on the membrane surface.     

Calcium transport by rabbit skeletal muscle microsomes (“fragmented sarcoplasmic reticulum”)

Ca²⁺ binding by skeletal muscle microsomes in 5 mM ATP exhibited saturation kinetics in the range of Ca²⁺ concentrations between 3 · 10^?8 and 10^?5 M. Approximately 140 nmoles binding sites per mg protein were found. These had a Ca²⁺ binding constant of approximately 4.5 · 10⁶ M^?1 with half saturation at 2.2 · 10^?7 M Ca²⁺. In the presence of oxalate, much larger amounts of Ca²⁺ (approx. 6 μmoles/mg protein) were taken up by the microsomes (Ca²⁺ uptake), but the rate of Ca²⁺ uptake increased linearly with [Ca²⁺] when ionized Ca²⁺ concentrations were below 3 · 10^?6 M. At Ca²⁺ concentrations above 3 · 10^?6, Ca²⁺ uptake was inhibited. Double reciprocal plots of the Ca²⁺ dependence of the initial rates of Ca²⁺ uptake in the concentration range between 3 · 10^?7 M and 10^?5 M, unlike those of Ca²⁺ binding, did not demonstrate saturation kinetics, but could be interpreted as representing a non-saturable system with inhibition at higher Ca²⁺ concentrations. In view of these differences, and because Ca²⁺-binding sites were almost fully saturated at 10^?6 M Ca²⁺, whereas Ca²⁺ uptake rate increased linearly with increasing [Ca²⁺] to approximately 3 · 10^?6 M, the Ca²⁺-binding sites are not shown kinetically to participate in oxalate-dependent Ca²⁺ uptake.     

Energy metabolism of reticulocytes: Two different sources of energy for Na+K+-ATPase activity

Total energy production in rabbit reticulocytes amounted to 136·52 ± 6·50μmol ATP h^?1ml^?1 of reticulocytes: 88·3 per cent was provided by oxidative phosphorylation, whereas only 11·7 per cent by aerobic glycolysis. Na⁺K⁺-ATPase accounted for 23 per cent, i.e. 27·65 ± 2·55μmol ATP h^?1ml^?1 of reticulocytes, in the overall energy consumption in reticulocytes of rabbits. Under basal conditions ATP for Na⁺K⁺-ATPase activity was derived exclusively from oxidative phosphorylation. However, when the activity of Na⁺K⁺-ATPase was increased due to the stimulation of adenylate cyclase by (?)-isoprenaline, the additional energy required was provided by aerobic glycolysis. These results indicate that two different compartments, one cytosolic and the other mitochondrial, provide energy for Na⁺K⁺-ATPase activity in reticulocytes.     

Equilibrium constants under physiological conditions for the reactions of the nonphosphorylated pathway of L-serine biosynthesis

The observed equilibrium constants (K_obs) for the reactions of d-2-phosphoglycerate phosphatase, d-2-Phosphoglycerate^3? + H₂O → d-glycerate^? + HPO₄^2?; d-glycerate dehydrogenase (EC 1.1.1.29), d-Glycerate^? + NAD⁺ → NADH + hydroxypyruvate^? + H⁺; and l-serine:pyruvate aminotransferase (EC 2.6.1.51), Hydroxypyruvate^? + l-H · alanine^± → pyruvate^? + l-H · serine^±; have been determined, directly and indirectly, at 38 °C and under conditions of physiological ionic strength (0.25 m) and physiological ranges of pH and magnesium concentrations. From these observed constants and the acid dissociation and metal-binding constants of the substrates, an ionic equilibrium constant (K) also has been calculated for each reaction. The value of K for the d-2-phosphoglycerate phosphatase reaction is 4.00 × 10³m [ΔG⁰ = ?21.4 kJ/mol (?5.12 kcal/mol)]([H₂0] = 1). Values of K_obs for this reaction at 38 °C, [K⁺] = 0.2 m, I = 0.25 M, and pH 7.0 include 3.39 × 10³m (free [Mg²⁺] = 0), 3.23 × 10³m (free [Mg²⁺] = 10^?3m), and 2.32 × 10³m (free [Mg²⁺] = 10^?2m). The value of K for the d-glycerate dehydrogenase reaction has been determined to be 4.36 ± 0.13 × 10^?13m (38 °C, I = 0.25 M) [ΔG⁰ = 73.6 kJ/mol (17.6 kcal/mol)]. This constant is relatively insensitive to free magnesium concentrations but is affected by changes in temperature [ΔH⁰ = 46.9 kJ/mol (11.2 kcal/mol)]. The value of K for the serine:pyruvate aminotransferase reaction is 5.41 ± 0.11 [ΔG⁰ = ?4.37 kJ/mol (?1.04 kcal/mol)] at 38 °C (I = 0.25 M) and shows a small temperature effect [ΔH⁰ = 16.3 kJ/ mol (3.9 kcal/mol)]. The constant showed no significant effect of ionic strength (0.06–1.0 m) and a response to the hydrogen ion concentration only above pH 8.5. The value of K_obs is 5.50 ± 0.11 at pH 7.0 (38 °C, [K⁺] = 0.2 m, [Mg²⁺] = 0, I = 0.25 M). The results have also allowed the value of K for the d-glycerate kinase reaction (EC 2.7.1.31), d-Glycerate^? + ATP^4? → d-2-phosphoglycerate^3? + ADP^3? + H⁺, to be calculated to be 32.5 m (38 °C, I = 0.25 M). Values for K_obs for this reaction under these conditions and at pH 7.0 include 236 (free [Mg²⁺] = 0) and 50.8 (free [Mg²⁺] = 10^?3m).     

Exposure of colonic epithelial cells to oxidative and endoplasmic reticulum stress causes rapid potassium efflux and calcium influx

Endoplasmic reticulum (ER) stress and oxidative stress have recently been linked to the pathogenesis of inflammatory bowel diseases. Under physiological conditions, intestinal epithelial cells are exposed to ER and oxidative stress affecting the cellular ionic homeostasis. However, these altered ion flux ‘signatures’ during these stress conditions are poorly characterized. We investigated the kinetics of K⁺, Ca²⁺ and H⁺ ion fluxes during ER and oxidative stress in a colonic epithelial cell line LS174T using a non‐invasive microelectrode ion flux estimation technique. ER and oxidative stress were induced by cell exposure to tunicamycin (TM) and copper ascorbate (CuAsc), respectively, from 1 to 24 h. Dramatic K⁺ efflux was observed following acute ER stress with peak K⁺ efflux being ?30·6 and ?138·7 nmolm^?2 s^?1 for 10 and 50 µg ml^?1, respectively (p < 0·01). TM‐dependent Ca²⁺ uptake was more prolonged with peak values of 0·85 and 2·68 nmol m^?2 s^?1 for 10 and 50 µg ml^?1 TM, respectively (p < 0·02). Ion homeostasis was also affected by the duration of ER stress. Increased duration of TM treatment from 0 to 18 h led to increases in both K⁺ efflux and Ca²⁺ uptake. While K⁺ changes were significantly higher at each time point tested, Ca²⁺ uptake was significantly higher only after prolonged treatment (18 h). CuAsc also led to an increased K⁺ efflux and Ca²⁺ uptake. Functional assays to investigate the effect of inhibiting K⁺ efflux with tetraethylammonium resulted in increased cell viability. We conclude that ER/oxidative stress in colonic epithelial cells cause dramatic K⁺, Ca²⁺ and H⁺ ion flux changes, which may predispose this lineage to poor stress recovery reminiscent of that seen in inflammatory bowel diseases. Copyright © 2012 John Wiley & Sons, Ltd.