Vanadate inhibition of active Ca2+ transport across human red cell membranes

(1) Vanadate (pentavalent vanadium) inhibits with high affinity ($\text{K}{}_{0.5}\text{= 3 μ}\text{M}$) the ATP-dependent Ca²⁺ efflux in reconstituted ghosts from human red cells. (2) To inhibit Ca²⁺ efflux vanadate has to have access to the inner surface of the cell membrane. (3) The inhibitory effect of vanadate is potentiated by intracellular Mg²⁺ and by intracellular K⁺. (4) Ca²⁺ in the external medium antagonizes the inhibitory effect of vanadate.     

The effects of vanadate on calcium transport in dialyzed squid axons Sidedness of vanadate-cation interactions

(1) Vanadate (VO₃^?) fully inhibits the ATP-dependent uncoupled Ca efflux (Ca pump) in dialyzed squid axons. (2) Vanadate inhibits with high affinity. The mean apparent affinity ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) obtained was 7 μM. (3) Inhibition by vanadate is dependent on Ca_o. External Ca lead to a release of the inhibitory effect. ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 3}\text{mM}$). This antagonic effect can be reverted by increasing the vanadate concentration. Internal K⁺ increases the affinity of the intracellular vanadate binding site. External K⁺ has no effect on the inhibition. (4) Vanadate has no effect on the Na_o-dependent Ca efflux component (forward Na-Ca exchange) in the absence of ATP. In axons containing ATP vanadate modified this component.     

Cyclic AMP transport in human erythrocyte ghosts

10^?5 M cyclic AMP has high permeability in human erythrocyte ghosts ($\text{p = 0.061 · 10}{}^{\mathrm{?6}}\text{cm}\text{·}\text{s}{}^{\mathrm{?1}}$). Saturation of influx and efflux occurs. $\text{K}{}_{\mathrm{<mtext>z</mtext><mtext>t</mtext>}}{}^{\mathrm{<mtext>oi</mtext>}}\text{= 4.43}\text{mM}$. $\text{V}{}_{\mathrm{zt}}{}^{\mathrm{oi}}\text{= 259.6 μ}\text{M · min}{}^{\mathrm{?1}}$. $\text{K}{}_{\mathrm{<mtext>z</mtext><mtext>t</mtext>}}{}^{\mathrm{<mtext>io</mtext>}}\text{= 0.475 μ}\text{M}$. $\text{V}{}_{\mathrm{<mtext>z</mtext><mtext>t</mtext>}}{}^{\mathrm{<mtext>io</mtext>}}\text{= 28.3 μ}\text{M · min}{}^{\mathrm{?1}}$ at 30°C. Equilibrium exchange entry of cyclic AMP has similar kinetics to zero trans influx, though the system does show counterflow. Cythochalasin B is an apparent competitive inhibitor of cyclic AMP exit. ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 3.9 · 10}{}^{\mathrm{?7}}\text{M}$).Control experiments indicated that cyclic AMP remains intact during incubation with red blood cell ghosts and is contained within the intravesicular space during the transport experiments.     

Kinetic characterization and substrate requirement for the Ca2+ uptake system in platelet membrane

An ATP-dependent mechanism for Ca²⁺ uptake in human platelet membrane fractions has been identified and characterized. Ca²⁺ uptake into a membrane fraction is shown to be stimulated at low concentrations of ATP and Ca²⁺ and to require magnesium ions. Initial rate kinetics, using Eadie-Scatchard analysis, indicated a single class of calcium uptake sites in the presence of ATP, with a $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ for free [Ca²⁺] of 0.145 μM. Ca²⁺ uptake in the presence of several ATP concentrations demonstrates that ATP binds to at least two sites, representing high and low affinities of 3.21 and 80.1 μM, respectively. The neuroleptic drug fluphenazine inhibited ATP-stimulated calcium uptake ($\text{IC}{}_{50}\text{= 55 μ}\text{M}$), suggesting this ATP-dependent Ca²⁺ uptake system may provide a useful ion-transport model with which to study neuroleptic therapy in humans.     

The permeability of the lysosomal membrane to small ions

The permeability of the lysosomal membrane to small anions and cations was studied at 37°C and pH 7.0 in a lysosomal-mitochondrial fraction isolated from the liver of untreated rats. The extent of osmotic lysis following ion influx was used as a measure of ion permeancy. In order to preserve electroneutrality, anion influx was coupled to an influx of K⁺ in the presence of valinomycin, and cation influx was coupled to an efflux of H⁺ using the protonophore $\text{3-tert-}\text{butyl-5,2′-dichloro-4′-nitrosalicilylanilide}$. Lysosomal lysis was monitored by observing the loss of latency of two lysosomal hydrolases.The order of permeability of the lysosomal membrane to anions was found to be $\text{SCN}{}^{\mathrm{?}}\text{> I}{}^{\mathrm{?}}\text{> CH}{}_3\text{COO}{}^{\mathrm{?}}\text{> Cl}{}^{\mathrm{?}}\text{≈ HCO}{}^{\mathrm{?}}{}_3\text{≈ P}{}_{\mathrm{i}}\text{> SO}{}_4{}^{\mathrm{2?}}$ and that to cations $\text{Cs}{}^{\mathrm{+}}\text{> K}{}^{\mathrm{+}}\text{> Na}{}^{\mathrm{+}}\text{> H}{}^{\mathrm{+}}$. These orders are largely in agreement with the lyotropic series of anions and cations.The implications of these findings for the mechanism by means of which a low intralysosomal pH is produced and maintained are discussed.     

Asymmetric or symmetric? Cytosolic modulation of human erythrocyte hexose transfer

(1) The Michaelis-Menten parameters for hexose transfer in erythroctes, erythrocyte ghosts and inside-out vesicles at 20°C were determined using the light scattering method of Sen and Widdas ((1962) J. Physiol. 160, 392–403). (2) The external $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for infinite-cis exit of d-glucose in cells and ghosts is $\text{3.6 ± 0.5}\text{mM}$. (3) Dilution of cellular solute (up to × 90 dilution) by lysing and resealing cells in varying volumes of lysate is without effect on the $\text{V}{}_{\mathrm{<mtext>m</mtext>}}$ for net d-glucose exit. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for net exit, however, falls from $\text{32.4 ± 3.7}\text{mM}$ in intact cells to $\text{12.9 ± 2.3}\text{mM}$ in ghosts. This effect is reversible. (4) Infinite-cis net d-glucose uptake measurements in cells and ghosts reveal the presence of a low $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$, high affinity internal site of $\text{5.9 ± 0.8}\text{mM}$. The $\text{V}{}_{\mathrm{<mtext>m</mtext>}}$ for net glucose entry increases from $\text{23.2 ± 3.7}\text{mmol/l per min}$ in intact cells to $\text{55.4 ± 6.3}\text{mmol/l per min}$ in ghosts. (5) The external $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for infinite-cisd-glucose exit in inside-out vesicles is $\text{6.8 ± 2.7}\text{mM}$. The kinetics of zero-transd-glucose exit from inside-out vesicles are changed markedly when cellular solute (obtained by lysis of intact cells) is applied to either surface of inside-out vesicles. When solute is present externally, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ and $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ for zero-trans exit are decreased by up to 10-fold. When solute is present at the interior of inside-out vesicles, $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ for zero-trans exit is reduced; $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for exit is unaffected. In the nominal absence of cell solute, transfer is symmetric in inside-out vesicles. The orientation of transporter in the bilayer is unaffected by the vesiculation procedure. (6) External application of cellular solute to ghosts reduces $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ for d-glucose exit but is without effect on the external $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for infinite-cis exit. (7) The inhibitory potency of cell lysate on hexose transfer is lost following dialysis indicating that the factors responsible for transfer modulation are low molecular weight species. (8) We consider the hexose transfer in human erythrocytes is intrinsically symmetric and that asymmetry of transfer is conferred by interaction of the system with low molecular weight cytosolic factors.     

Relationship between calcium ion transport and (Ca2+ + Mg2+)-ATPase activity in adipocyte endoplasmic reticulum

Calcium uptake by adipocyte endoplasmic reticulum was studied in a rapidly obtained microsomal fraction. The kinetics and ionic requirements of Ca²⁺ transport in this preparation were characterized and compared to those of (Ca²⁺ + Mg²⁺)-ATPase activity. The time course of Ca²⁺ uptake in the presence of 5 mM oxalate was nonlinear, approaching a steady-state level of 10.8–11.5 nmol Ca²⁺/mg protein after 3–4 min of incubation. The rate of Ca²⁺ transport was increased by higher oxalate concentrations with a near linear rate of uptake at 20 mM oxalate. The calculated initial rate of calcium uptake was 18.5 nmol Ca²⁺/mg protein per min. The double reciprocal plot of ATP concentration against transport rate was nonlinear, with apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values of 100 μM and 7 μM for ATP concentration ranges above and below 50 μM, respectively. The apparent $\text{K}\text{m}$ values for Mg²⁺ and Ca²⁺ were 132 μM and 0.36–0.67 μM, respectively. The energy of activation was 23.4 kcal/mol. These kinetic properties were strikingly similar to those of the microsomal ($\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}$)-ATPase. The presence of potassium was required for maximum Ca²⁺ transport activity. The order of effectiveness of monovalent cations in stimulating both Ca²⁺ transport and ($\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{-}\text{ATPase}$ activity was $\text{K}{}^{\mathrm{+}}\text{>}\text{Na}{}^{\mathrm{+}}\text{=}\text{NH}{}_4{}^{\mathrm{+}}\text{>}\text{Li}{}^{\mathrm{+}}\text{.}\text{Ca}{}^{\mathrm{2+}}$ transport and ($\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ activity were both inhibited 10–20% by 6 mM procaine and less than 10% by 10 mM sodium azide. Both processes were completely inhibited by 3 mM dibucaine or 50 μM $\text{p-}\text{chloromercuribenzene}$ sulfonate. The results indicate that Ca²⁺ transport in adipocyte endoplasmic reticulum is mediated by a $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ and suggest an important role for endoplasmic reticulum in control of intracellular Ca²⁺ distribution.     

Regulatory interaction between calmodulin and ATP on the red cell Ca2+ pump

The interactions between calmodulin, ATP and Ca²⁺ on the red cell Ca²⁺ pump have been studied in membranes stripped of native calmodulin or rebound with purified red cell calmodulin. Calmodulin stimulates the maximal rate of $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ by 5–10-fold and the rate of Ca²⁺-dependent phosphorylation by at least 10-fold. In calmodulin-bound membranes ATP activates $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ along a biphasic concentration curve ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>1</mtext>}}\text{≈ 1.4 μ}\text{M}\text{, K}{}_{\mathrm{<mtext>m</mtext><mtext>2</mtext>}}\text{≈ 330 μ}\text{M}$), but in stripped membranes the curve is essentially hyperbolic ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{≈ 7 μ}\text{M}$). In calmodulin-bound membranes Ca²⁺ activates $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ at low concentrations ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{< 0.28 μ}\text{M}$) in stripped membranes the apparent Ca²⁺ affinities are at least 10-fold lower.The results suggest that calmodulin (and perhaps ATP) affect a conformational equilibrium between E₂ and E₁ forms of the Ca²⁺ pump protein.     

Stimulating effects of monovalent cations on activator-dissociated and activator-associated states of Ca2+-ATPase in human erythrocytes

The additional activation by monovalent cations of the $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-dependent}$ ATPase (ATP phosphohydrolase, EC 3.6.1.3) in human erythrocyte membranes was studied.The Ca²⁺-ATPase occurs in two different states. In the A-state the enzyme is virtually free of protein activator and the kinetics of Ca²⁺ activation is characterized by low apparent Ca²⁺ affinity and low maximum activity. In the B-state the enzyme is associated with activator and the kinetics is characterized by high Ca²⁺ affinity and high maximum activity.At optimum concentrations of Ca²⁺ the additional activation of the B-state by K⁺, NH₄⁺, Na⁺ and Rb⁺ exceeded the corresponding activations of the A-state, and half-maximum activations by K⁺, NH₄⁺, and Na⁺ were achieved at lower concentrations in the B-state than in the A-state. Li⁺ and Cs⁺ activated the two states almost equally but maximum activation was obtained at lower cation concentrations in the B-state than in the A-state.The activation of the B-state by the various cations decreased in the order $\text{K}{}^{\mathrm{+}}\text{> NH}{}_4{}^{\mathrm{+}}\text{> Na}{}^{\mathrm{+}}\text{= Rb}{}^{\mathrm{+}}\text{> Li}{}^{\mathrm{+}}\text{= Cs}{}^{\mathrm{+}}$. The A-state was activated almost equally by K⁺, Na⁺, NH₄⁺, and Rb⁺ and to a smaller extent by Li⁺ and Cs⁺.At sub-optimum concentrations of Ca²⁺ high concentrations of monovalent cations (100 mM) activated the Ca²⁺-ATPase equally in the A-state and the B-state. In the absence of Ca²⁺ the monovalent cations inhibited the Mg²⁺-dependent ATPase in both types of membranes. This dependence on Ca²⁺ indicates that the monovalent cations interact with the Ca²⁺ sites in the B-state.The results suggest that K⁺ or Na⁺, or both, contribute to the regulation of the Ca²⁺ pump in erythrocytes.     

Ca2+-cardiolipin interaction in a model system Selectivity and apparent high affinity

The interaction of cardiolipin with Ca²⁺ was assessed by measuring the cardiolipin-mediated extraction of ⁴⁵Ca²⁺ from an aqueous to an organic (methylene chloride) phase. Cardiolipin binds Ca²⁺ with high affinity [$\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{(}\text{apparent}\text{)=0.70±0.17 μ}\text{M (S.D.)}$]. Cation-cardiolipin interactions are selective. Interaction of cardiolipin with Ca²⁺ is insensitive to Na⁺, but is inhibited by divalent cations with $\text{Mn}{}^{\mathrm{2+}}\text{>}\text{Zn}{}^{\mathrm{2+}}\text{>}\text{Mg}{}^{\mathrm{2+}}$. In addition La³⁺ and Ruthenium red are particularly potent inhibitors of Ca²⁺ binding by cardiolipin. Cardiolipin-mediated extraction of Ca²⁺ into an aqueous phase is also inhibited by phosphatidylcholine. Inhibition of Ca²⁺-cardiolipin interaction by phosphatidylcholine (a phospholipid known to stabilize the bilayer conformation) may implicate inverted, non-bilayer lipid structures in the binding.     

Pyruvate flux into resealed ghosts from human erythrocytes

The kinetics of pyruvate transport across the isolated red blood cell membrane were studied by a simple and precise spectrophotometric method: following the oxidation of NADH via lactate dehydrogenase trapped within resealed ghosts. The initial rate of pyruvate entry was linear. Influx was limited by saturation at high pyruvate concentration. Pyruvate influx was greatly stimulated by increasing ionic strength in the outer but not the inner aqueous compartment. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ ranged from 15.0 mM at $\text{μ = 0.05}$ to 3.7 mM at $\text{μ = 0.01}$, while the $\text{V}$ went from $\text{0.611 · 10}{}^{-15}$ to $\text{0.137 · 10}{}^{-15}\text{mol}\text{·}\text{min}{}^{-1}\text{·}\text{ghost}{}^{-1}$. Ionic strength was shown to affect the translocation step and not pyruvate binding. The energy of activation of pyruvate flux into resealed ghosts was 25 kcal/mol, similar to that found in intact red blood cells. Inhibitors of pyruvate influx included such anions as thiocyanate, chloride, bicarbonate, α-cyanocinnamate, salicylate and ketomalonate (but not acetate); noncompetitive inhibitors were phloretin, 1-fluoro-2,4-dinitrobenzene, 4-acetamido-4′-isothiocyanate-stilbene-2,2′-disulfonic acid and o-phenanthroline/CuSO₄ mixtures. The last reagent, known to induce disulfide links in certain membrane proteins, blocked the ionic strength stimulation of pyruvate influx in this study.     

Asymmetric binding of cytochrome b5 to the membrane of human erythrocyte ghosts

The intact, amphipatic form of cytochrome $\text{b}{}_5$ could bind to unsealed ghosts, but not to resealed ghosts, suggesting that the cytochrome could bind only to the inner (cytoplasmic) surface of the ghost membrane. This was further confirmed by the finding that the cytochrome could bind to closed, inside-out vesicles prepared from the ghosts. This asymmetric binding was not due to the exclusive localization of sialic acid and sugar chains on the outer surface of the ghosts membrane, because the cytochrome could not bind to ghosts even after enzymatic removal of these components. Although liposomes consisting of phosphatidylcholine or both phosphatidylcholine and sphingomyelin could effectively bind the cytochrome, this binding capacity was progressively decreased as increasing amount of cholesterol was included in the composition of phosphatidylcholine liposomes. Removal of cholesterol from resealed ghosts by incubation with egg phosphatidylcholine liposomes resulted in the binding of cytochrome $\text{b}{}_5$ to the outer surface of the treated ghosts. The possibility is discussed that the asymmetric binding is due to preferential localization of cholesterol in the outer leaflet of the lipid bilayer that constitutes the ghost membrane.     

Calcium transport and Ca2+-ATPase activity in ram spermatozoa plasma membrane vesicles

Plasma membrane vesicles, isolated from ejaculated ram sperm, were found to contain Ca²⁺-activated Mg²⁺-ATPase and Ca²⁺ transport activities. Membrane vesicles that were exposed to oxalate as a Ca²⁺-trapping agent accumulated Ca²⁺ in the presence of Mg²⁺ and ATP. The $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ for Ca²⁺ uptake was 33 nmol/mg protein per h, and the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for Ca²⁺ and ATP were 2.5 μM and 45 μM, respectively. 1 μM of the Ca²⁺ ionophore A23187, added initially, completely inhibited net Ca²⁺ uptake and, if added later, caused the release of Ca²⁺ previously accumulated. A Ca²⁺-activated ATPase was present in the same membrane vesicles which had a $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of 1.5 μmol/mg protein per h at free Ca²⁺ concentration of 10 μM. This Ca²⁺-ATPase had $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values of 4.5 μM and 110 μM for Ca²⁺ and ATP, respectively. This kinetic parameter was similar to that observed for uptake of Ca²⁺ by the vesicles. The Ca²⁺-ATPase activity was insensitive to ouabain. Both Ca²⁺ transport and Ca²⁺-ATPase activity were inhibited by the flavonoid quercetin. Thus, ram spermatozoa plasma membranes have both a Ca²⁺ transport activity and a Ca²⁺-stimulated ATPase activity with similar substrate affinities and specificities and similar sensitivity to quercetin.     

Regulation of the Ca2+-pump by calmodulin in intact cells

ATP-enriched human red cells display high rates of Ca²⁺-dependent ATP hydrolysis (16 mmol·litre cells^?1·h^?1) with a high Ca²⁺ affinity ($\text{K}{}_{0.5}\text{～0.2 μ}\text{M}$). The finding suggests a mechanism for regulation of cell Ca²⁺ levels, involving highly-cooperative stimulation of active Ca²⁺ extrusion following binding of calmodulin to the $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$.     

Na+-driven Ca2+ transport in alkalophilic Bacillus

Ca²⁺ transport was studied in membrane vesicles of alkalophilic Bacillus. When Na⁺-loaded membrane vesicles were suspended in KHCO₃/KOH buffer (pH 10) containing Ca²⁺, rapid uptake of Ca²⁺ was observed. The apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for Ca²⁺ measured at pH 10 was about 7 μM, and the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value shifted to 24 μM when measured at pH 7.4. The efflux of Ca²⁺ was studied with Ca²⁺-loaded vesicles. Ca²⁺ was released when Ca²⁺-loaded vesicles were suspended in medium containing 0.4 M Na⁺.Ca²⁺ was also transported in membrane vesicles driven by an artificial pH gradient and by a membrane potential generated by K⁺-valinomycin in the presence of Na⁺.These results indicate the presence of Ca²⁺/Na⁺ and H⁺/Na⁺ antiporters in the alkalophilic Bacillus A-007.     

The relative effectiveness of .OH,H2O2,O2−, and reducing free radicals in causing damage to biomembranes: A study of radiation damage to erythrocyte ghosts using selective free radical scavengers

The relative effectiveness of oxidizing (^.OH, H₂O₂), ambivalent (O₂^?) and reducing free radicals (e^? and CO₂^?) in causing damage to membranes and membrane-bound glyceraldehyde-3-phosphate dehydrogenase of resealed erythrocyte ghosts has been determined. The rates of damage to membranebound glyceraldehyde-3-phosphate dehydrogenase ($\text{R}$(enz)) were measured and the rates of damage to membranes ($\text{R}$(mb)) were assessed by measuring changes in permeability of the resealed ghosts to the relatively low molecular weight substrates of glyceraldehyde-3-phosphate dehydrogenase. Each radical was selectively isolated from the mixture produced during gamma-irradiation, using appropriate mixtures of scavengers such as catalase, superoxide dismutase and formate. ^.OH, O₂^? and H₂ O₂ were approximately equally effective in inactivating membrane-bound glyceraldehyde-3-phosphate dehydrogenase, while e^? and CO₂^? were the least effective. $\text{R}$(enz) values of O₂^? and H₂O₂ were 10-times and of ^.OH 15-times that of e^?. $\text{R}$(mb) values were quite similar for e^? and H₂O₂ (about twice that of O₂^?), while that of ^.OH was 3-times that of O₂^?. Hence, with respect to $\text{R}$(mb): ${}^{\mathrm{.}}\text{OH}\text{>}\text{e}{}^{\mathrm{?}}\text{=}\text{H}{}_2\text{O}{}_2\text{>}\text{O}{}_2{}^{\mathrm{?}}$ , and with respect to $\text{R}$(enz): ${}^{\mathrm{.}}\text{OH}\text{>}\text{O}{}_2{}^{\mathrm{?}}\text{=}\text{H}{}_2\text{O}{}_2\text{>}\text{e}{}^{\mathrm{?}}$. The difference between the effectiveness of the most damaging and the least damaging free radicals was more than 10-fold greater in damage to the enzyme than to the membranes. Comparison between H₂O₂ added as a chemical reagent and H₂O₂ formed by irradiation showed that membranes and membrane-bound glyceraldehyde-3-phosphate dehydrogenase were relatively inert to reagent H₂O₂ but markedly susceptible to the latter.     

Ammonium (methylammonium) transport by Klebsiella pneumoniae

Klebsiella pneumoniae can accumulate methylammonium up to 80-fold by means of a transport system as indicated by the energy requirement, saturation kinetics and a narrow pH profile around pH 6.8. Methylammonium transport (apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 100 μ}\text{M}$, $\text{V = 40 μ}\text{mol/min}$ per g dry weight at 15°C) is competitively inhibited by ammonium (apparent $\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 7 μ}\text{M}$). The low $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ value and the finding that methylammonium cannot serve as a nitrogen source indicate that ammonium rather than methylammonium is the natural substrate. Uphill transport is driven by a component of the protonmotive force, probably the membrane potential. The transport system is under genetic control; it is partially repressed by amino acids and completely by ammonium. Analysis of mutants suggest that the synthesis of the ammonium transport system is subject to the same ‘nitrogen control’ as nitrogenase and glutamine synthetase.     

Determination of the kinetic constants of fructose transport and phosphorylation in the perfused rat liver

The kinetics of fructose uptake was determined in perfused rat liver during steady-state fructose elimination. On the basis of the corresponding values of fructose concentration in the affluent and in the effluent medium, and the fructose and ATP concentration in biopsies, the kinetics of membrane transport and intracellular phosphorylation in the intact organ was calculated according to a model system. Carrier-mediated fructose transport has a high $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ (67 mM) and $\text{V}$ (30 μmoles · min^?1 ·g^?1). The calculated kinetic constants of the intracellular phosphorylation were compared with values obtained with an acid-treated rat liver high speed supernatant (values given in parentheses). $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ with fructose 1.0 mM (0.7 mM), $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ with ATP 0.54 mM (0.37 mM), $\text{V}$ 10.3 μmoles · min^?1 · g^?1 (10.1 μmoles · min^?1 · g^?1, calculated on the basis of the highest measured rate of fructose uptake correcting the ATP concentration to saturating values). The kinetics of fructose uptake reveals that at Physiological fructose concentrations the membrane transport limits the rate of fructose uptake, thus protecting the liver from severe depletion of adenine nucleotides.     

Ouabain-insensitive Na+-stimulated ATPase activity of basolateral plasma membranes from guinea-pig kidney cortex cells: II. Effect of Ca2+

The ouabain-insensitive, Mg²⁺-dependent, Na⁺-stimulated ATPase activity present in fresh basolateral plasma membranes from guinea-pig kidney cortex cells (prepared at pH 7.2) can be increased by the addition of micromolar concentrations of Ca²⁺ to the assay medium. The Ca²⁺ involved in this effect seems to be associated with the membranes in two different ways: as a labile component, which can be quickly and easily ‘deactivated’ by reducing the free Ca²⁺ concentration of the assay medium to values lower than 1 μM; and as a stable component, which can be ‘deactivated’ by preincubating the membranes for periods of 3–4 h with 2 mM EDTA or EGTA. Both components are easily activated by micromolar concentrations of Ca²⁺. The $\text{K}{}_{\mathrm{<mtext>a</mtext>}}$ of the system for Na⁺ is the same, 8 mM, whether only the stable component or both components, stable and labile, are working. In other words, the activating effect of Ca²⁺ on the Na⁺-stimulated ATPase is on the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$, and not on the $\text{K}{}_{\mathrm{<mtext>a</mtext>}}$ of the system for Na⁺. The activating effect of Ca²⁺ may be related to some conformational change produced by the interaction of this ion with the membranes, since it can also be obtained by resuspending the membranes at pH 7.8 or by ageing the preparations. Changes in the Ca²⁺ concentration may modulate the ouabain-insensitive, Na⁺-stimulated ATPase activity. This modulation could regulate the magnitude of the extrusion of Na⁺ accompanied by Cl^? and water that these cells show, and to which the Na⁺-ATPase has been associated as being responsible for the energy supply of this mode of Na⁺ extrusion.