Characterization of native and reconstituted plasma membrane H+-ATPase from the plasma membrane ofBeta vulgaris

Summary Characteristics of the native and reconstituted H⁺-ATPase from the plasma membrane of red beet (Beta vulgaris L.) were examined. The partially purified, reconstituted H⁺-ATPase retained characteristics similar to those of the native plasma membrane H⁺-ATPase following reconstitution into proteoliposomes. ATPase activity and H⁺ transport of both enzymes were inhibited by vanadate, DCCD, DES and mersalyl. Slight inhibition of ATPase activity associated with native plasma membranes by oligomycin, azide, molybdate or NO ₃ ^– was eliminated during solubilization and reconstitution, indicating the loss of contaminating ATPase activities. Both native and reconstituted ATPase activities and H⁺ transport showed a pH optimum of 6.5, required a divalent cation (Co²⁺>Mg²⁺>Mn²⁺>Zn²⁺>Ca²⁺), and preferred ATP as substrate. The Mg:ATP kinetics of the two ATPase activities were similar, showing simple Michaelis-Menten kinetics. Saturation occurred between 3 and 5mM Mg: ATP, with aK _m of 0.33 and 0.46mM Mg: ATP for the native and reconstituted enzymes, respectively. The temperature optimum for the ATPase was shifted from 45 to 35°C following reconstitution. Both native and reconstituted H⁺-ATPases were stimulated by monovalent ions. Native plasma membrane H⁺-ATPase showed an order of cation preference of K⁺>NH ₄ ⁺ >Rb⁺>Na⁺>Cs⁺>Li⁺>choline⁺. This basic order was unchanged following reconstitution, with K⁺, NH ₄ ⁺ , Rb⁺ and Cs⁺ being the preferred cations. Both enzymes were also stimulated by anions although to a lesser degree. The order of anion preference differed between the two enzymes. Salt stimulation of ATPase activity was enhanced greatly following reconstitution. Stimulation by KCl was 26% for native ATPase activity, increasing to 228% for reconstituted ATPase activity. In terms of H⁺ transport, both enzymes required a cation such as K⁺ for maximal transport activity, but were stimulated preferentially by Cl^– even in the presence of valinomycin. This suggests that the stimulatory effect of anions on enzyme activity is not simply as a permeant anion, dissipating a positive interior membrane potential, but may involve a direct anion activation of the plasma membrane H⁺-ATPase.     

Characteristics of non-specific permeability and H+-ATPase inhibition induced in the plasma membrane of Nitella flexilis by excessive Cu2+

Effects of Cu²⁺ on a non-specific conductance and H⁺-ATPase activity in the plasma membrane of the freshwater alga Nitella flexilis L. Agardh was studied using a conventional microelectrode voltage-clamp technique. We show that a Cu²⁺-induced increase in the non-specific conductance is related to the formation of pores in the plasma membrane. Pore formation is the result of unidentified chemical reactions, since the Q₁₀ for the rate of increase of conductance over time was about 3. Various oxidants and antioxidants (10 mmol/l H₂O₂, 10 mmol/l ascorbate, 100 μg/ml superoxide dismutase, and 100 μg/ml catalase) did not alter Cu²⁺-induced changes in the plasma membrane conductance, suggesting that the effect of Cu²⁺ was unrelated to peroxidation of plasma-membrane lipids. In contrast, organic and inorganic Ca²⁺-channel antagonists (nifedipine, Zn²⁺, Cd²⁺, Fe²⁺, Ni²⁺) inhibited the Cu²⁺-induced non-specific conductance increase. This suggests that changes in Ca²⁺ influx underlie this effect of Cu²⁺. Decreasing the pH or the ionic strength of external solutions also inhibited the Cu²⁺-induced plasma-membrane conductance increase. Copper was also found to inhibit plasma-membrane H⁺-ATPase activity with half-maximal inhibition occurring at about 5–20 μmol/l and full inhibition at about 100–300 μmol/l. The Hill coefficient of Cu²⁺ inhibition of the H⁺-ATPase was close to two. Received: 8 December 1999 / Accepted: 16 August 2000     

Measurement of plasma membrane potentials of yeast cells with glass microelectrodes

Glass microelectrodes were used to measure the electrical potential difference (Δψ) across plasma membrane of the yeast Pichia humboldtii. The cells were captured in the neck of a glass microfunnel and impaled with a glass microelectrode. The measurements were reproducible and stable for several minutes. The highest Δψ values were obtained in cells metabolizing glucose at pH 6. Δψ in cells deenergized by uncouplers or in dead cells was reduced to about one third of the maximal value. This residual Δψ probably represented Donnan potential. Δψ also was reduced by increasing concentrations of K⁺ in the medium. Other monovalent cations were distinctly less effective: Li⁺ ⪡ Na⁺ < K⁺, and Ca²⁺ was without effect. These experiments prove the applicability of the electrophysiological technique on yeast cells and thus open the way for direct determination of the electrical component of the plasma membrane electrochemical proton gradient.     

Cadmium impairs ion homeostasis by altering K+ and Ca2+ channel activities in rice root hair cells

Cadmium (Cd²⁺) interferes with the uptake, transport and utilization of several macro‐ and micronutrients, which accounts, at least in part, for Cd²⁺ toxicity in plants. However, the mechanisms underlying Cd²⁺ interference of ionic homeostasis is not understood. Using biophysical techniques including membrane potential measurements, scanning ion‐selective electrode technique for non‐invasive ion flux assays and patch clamp, we monitored the effect of Cd²⁺ on calcium (Ca²⁺) and potassium (K⁺) transport in root hair cells of rice. Our results showed that K⁺ and Ca²⁺ contents in both roots and shoots were significantly reduced when treated with exogenous Cd²⁺. Further studies revealed that three cellular processes may be affected by Cd²⁺, leading to changes in ionic homeostasis. First, Cd²⁺‐induced depolarization of the membrane potential was observed in root hair cells, attenuating the driving force for cation uptake. Second, the inward conductance of Ca²⁺ and K⁺ was partially blocked by Cd²⁺, decreasing uptake of K⁺ and Ca²⁺. Third, the outward K⁺ conductance was Cd²⁺‐inducible, decreasing the net content of K⁺ in roots. These results provide direct evidence that Cd²⁺ impairs uptake of Ca²⁺ and K⁺, thereby disturbing ion homeostasis in plants.     

Mechanism for Cd2+ inhibition of (K++ Mg2+)ATPase activity and K+ (86Rb+) uptake join roots of sugar beet (Beta vulgaris)

Steady state kinetics were used to examine the influence of Cd²⁺ both on K⁺ stimulation of a membrane-bound ATPase from sugar beet roots (Beta vulgaris L. cv. Monohill) and on K⁺(⁸⁶Rb⁺) uptake in intact or excised beet roots. The in vitro effect of Cd²⁺ was studied both on a 12000–25000 g root fraction of the (Na⁺+K⁺+Mg²⁺)ATPase and on the ATPase when further purified by an aqueous polymer two-phase system. The observed data can be summarized as follows: 1) Cd²⁺ at high concentrations (>100 μM) inhibits the MgATPase activity in a competitive way, probably by forming a complex with ATP. 2) Cd²⁺ at concentrations <100 μM inhibits the specific K⁺ activation at both high and low affinity sites for K⁺. The inhibition pattern appears to be the same in the two ATPase preparations of different purity. In the presence of the substrate MgATP, and at K⁺ <5 mM, the inhibition by Cd²⁺ with respect to K⁺ is uncompetitive. In the presence of MgATP and K⁺ >10 μM, the inhibition by Cd²⁺ is competitive. 3) At the low concentrations of K⁺, Cd²⁺ also inhibits the 2,4-dinitrophenol(DNP)-sensitive (metabolic) K⁺(⁸⁶Rb⁺) uptake uncompetitively both in excised roots and in roots of intact plants. 4) The DNP-insensitive (non metabolic) K⁺(⁸⁶Rb⁺) uptake is little influenced by Cd²⁺. As Cd²⁺ inhibits the metabolic uptake of K⁺(⁸⁶Rb⁺) and the K⁺ activation of the ATPase in the same way at low concentrations of K⁺, the same binding site is probably involved. Therefore, under field conditions, when the concentration of K⁺ is low, the presence of Cd²⁺ could be disadvantageous.     

Localization of neutral,magnesium-stimulated sphingomyelinase in plasma membrane of cultured neuroblastoma cells

A parallel is shown between the distribution of neutral sphingomyelinase and plasma membrane enzymes (5′-nucleotidase and (Na⁺ + K⁺)-activated ATPase) in cultured neuroblastoma cells. In contrast there is no evidence of localization in lysosomes (β-hexosaminidase and acid sphingomyelinase), mitochondria (carnitine palmitoyltransferase), or cytosol. Activity in the microsomal fraction is attributed primarily to plasma membrane contamination.     

A novel role of Ca2+ and Zn2+: Protection of cells against membrane damage

Certain cytotoxic agents damage cells by the induction of pores across their plasma membrane. Ca²⁺ and Zn²⁺ protect against such damage by promoting pore closure. Zn²⁺ may play a beneficial role in this regard in certain disease states.     

Effects of plasma membrane oxidoreductases on Ca2+ mobilization and protein phosphorylation in rat brain synaptosomes

We have investigated the possible role of plasma membrane oxidoreductases in the Ca²⁺ export mechanisms in rat brain synaptic membranes. Ca²⁺ efflux in nerve terminals is controlled both by a high-affinity/low capacity Mg-dependent ATP-stimulated Ca²⁺ pump and by a low affinity/high capacity ATP-independent Na⁺-Ca²⁺ exchanger. Both Ca²⁺ efflux mechanisms were strongly inhibited by pyridine nucleotides, in the order NADP>NAD>NADPH>NADH with IC₅₀ values of ca. 10 mM for NADP and ca. 3 mM for the other agents in the case of the ATP-driven Ca²⁺ pump and with IC₅₀ values between 8 and 10 mM for the Na⁺-Ca²⁺ exchanger. Oxidizing agents such as DCIP and ferricyanide inhibited the ATP-driven Ca²⁺ efflux mechanism but not the Na⁺-Ca²⁺ exchanger. In addition, full activation of plasma membrane oxidoreductases requires both an acceptor and an electron donor; therefore the combined effects of both substrates added together were also studied. When plasma membrane oxidoreductases of the synaptic plasma membrane were activated in the presence of both NADH (or NADPH) and DCIP or ferricyanide, the inhibition of the ATP-driven Ca²⁺ pump was optimal; by contrast, the pyridine nucleotide-mediated inhibition of the Na⁺-Ca²⁺ exchanger was partially released when both substrates of the plasma membrane oxidoreductases were present together. Furthermore, the activation of plasma membrane oxidoreductases also strongly inhibited intracellular protein phosphorylation in intact synaptosomes, mediated by eithercAMP-dependent protein kinase, Ca²⁺ calmodulin-dependent protein kinases, or protein kinase C.Abbreviations Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid - SDS sodium dodecyl sulfate - EGTA ethylenglycol-bis(-aminoethylether)-N,N,N,N-tetraacetic acid - DCIP dichlorophenol-indophenol     

Effects of magnesium availability on the activity of plasma membrane ion transporters and light-induced responses from broad bean leaf mesophyll

Considering the physiological significance of Mg homeostasis in plants, surprisingly little is known about the molecular and ionic mechanisms mediating Mg transport across the plasma membrane and the impact of Mg availability on transport processes at the plasmalemma. In this study, a non-invasive ion-selective microelectrode technique (MIFE) was used to characterize the effects of Mg availability on the activity of plasma membrane H⁺, K⁺, Ca²⁺, and Mg²⁺ transporters in the mesophyll cells of broad bean (Vicia faba L.) plants. Based on the stoichiometry of ion-flux changes and results of pharmacological experiments, we suggest that at least two mechanisms are involved in Mg²⁺ uptake across the plasma membrane of bean mesophyll cells. One of them is a non-selective cation channel, also permeable to K⁺ and Ca²⁺. The other mechanism, operating at concentrations below 30 M, was speculated to be an H⁺/Mg⁺ exchanger. Experiments performed on leaves grown at different levels of Mg availability (from deficient to excessive) showed that Mg availability has a significant impact on the activity of plasma-membrane transporters for Ca²⁺, K⁺, and H⁺. We discuss the physiological significance of Mg-induced changes in leaf electrophysiological responses to light and the ionic mechanisms underlying this process.     

Assessing the efficacy of vesicle fusion with planar membrane arrays using a mitochondrial porin as reporter

The gene for a putative cation calcium exchanger (CCX) from Arabidopsis thaliana, AtCCX5, was cloned and its function was analyzed in yeast. Green fluorescent protein-tagged AtCCX5 expressed in yeast was localized in the plasma membrane and nuclear periphery. The yeast transformants expressing AtCCX5 were created and their growth in the presence of various cations (K⁺, Na⁺, Ca²⁺, Mg²⁺, Fe²⁺, Cu²⁺, Co²⁺, Cd²⁺, Mn²⁺, Ba²⁺, Ni²⁺, Zn²⁺, and Li⁺) were analyzed. AtCCX5 expression was found to affect the response to K⁺ and Na⁺ in yeast. The AtCCX5 transformant also showed a little better growth to Zn²⁺. The yeast mutant 9.3 expressing AtCCX5 restored growth of the mutant on medium with low K⁺ (0.5 mM), and also suppressed its Na⁺ sensitivity. Ion uptake experiments showed that AtCCX5 mediated relatively high-affinity K⁺ uptake and was also involved in Na⁺ transport in yeast. Taken together, these findings suggest that the AtCCX5 is a novel transport protein involves in mediating high-affinity K⁺ uptake and Na⁺ transport in yeast.     

Metal ion inhibition of corn root plasma membrane atpase

In the presence of K⁺, the hydrolysis of ATP catalysed by the ATPase of corn plasma membrane showed negative cooperative kinetics. When the complexes of ATP and Mg²⁺, Mn²⁺, Ca²⁺ or Cd²⁺ were used as substrates, the catalysed hydrolysis changed to follow simple Michaelis-Menten kinetics. However, this change was not observed with Zn²⁺-ATP as the substrate. A substantial enhancement of the hydrolysis was observed only when the complexes of Mg²⁺ and Mn²⁺ were used. Kinetic parameter determination indicated that the enzyme exhibited a similar binding property but a different catalytic efficiency to Mg²⁺, Mn²⁺ and Ca²⁺-ATP. The enzyme formed a more stable but less reactive complex with Cd²⁺-ATP. The presence of aluminium ions competitively inhibited the membrane-catalysed hydrolysis of Mg²⁺-ATP, but showed no effect when free ATP was the substrate. This finding suggested that aluminium might bind in the vicinity of the Mg²⁺ of Mg²⁺-ATP in the active site of the enzyme. On the basis of these observed inhibitory effects, possible origins of metal ion toxicity to root plasma membrane ATPase activity are discussed.     

Actions of cadmium on basolateral plasma membrane proteins involved in calcium uptake by fish intestine

Summary The inhibition of Ca^2–-ATPase, (Na⁺+K⁺)-ATPase and Na⁺/Ca²⁺ exchange by Cd²⁺ was studied in fish intestinal basolateral plasma membrane preparations. ATP driven ⁴⁵Ca²⁺ uptake into inside-out membrane vesicles displayed a K _m for Ca²⁺ of 88±17 nm, and was extremely sensitive to Cd²⁺ with an IC₅₀ of 8.2±3.0 pM Cd²⁺, indicating an inhibition via the Ca²⁺ site. (Na⁺+K⁺)-ATPase activity was half-maximally inhibited by micromolar amounts of Cd²⁺, displaying an IC₅₀ of 2.6±0.6 m Cd²⁺. Cd²⁺ ions apparently compete for the Mg²⁺ site of the (Na^– +K⁺)-ATPase. The Na⁺/Ca²⁺ exchanger was inhibited by Cd²⁺ with an IC₅₀ of 73±11 nm. Cd²⁺ is a competitive inhibitor of the exchanger via an interaction with the Ca²⁺ site (K _i = 11 nm). Bepridil, a Na⁺ site specific inhibitor of Na⁺/Ca²⁺ exchange, induced an additional inhibition, but did not change the K _i of Cd²⁺. Also, Cd²⁺ is exchanged against Ca²⁺, albeit to a lesser extent than Ca²⁺. The exchanger is only partly blocked by the binding of Cd²⁺. In vivo cadmium that has entered the enterocyte may be shuttled across the basolateral plasma membrane by the Na⁺/Ca²⁺ exchanger. We conclude that intracellular Cd²⁺ ions will inhibit plasma membrane proteins predominantly via a specific interaction with divalent metal ion sites.We would like to thank Dr. D. Fackre (University of Alberta, Canada) for stimulating discussions and Mr. F.A.T. Spanings (University of Nijmegen, The Netherlands) for excellent fish husbandry. The fura-2 measurements of intracellular Ca²⁺ concentrations in tilapia enterocytes were carried out in the Department of Physiology, School of Medicine, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Th.J.M. Schoenmakers and G. Flik were supported by travel grants from the Foundation for Fundamental Biological Research (BION) and the Netherlands Organization for Scientific Research (NWO).     

Amino acid-dependent sodium transport in plasma membrane vesicles from rat liver

Summary Thel-alanine-dependent transport of sodium ions across the plasma membrane of rat-liver parenchymal cells was studied using isolated plasma membrane vesicles. Sodium uptake is stimulated specifically by thel-isomer of alanine and other amino acids, whose transport is sodium-dependent in rat-liver plasma membrane vesicles. Thel-alanine-dependent sodium flux across the membrane is inhibited by an excess of Li⁺ ions, but not by K⁺ or choline ions. Sodium transport is sensitive to-SH reagents and ionophores, and is an electrogenic process: a membrane potential (negative inside) can enhancel-alanine-dependent sodium accumulation. The data presented provide further evidence for a sodium-alanine cotransport mechanism.     

Cd2+ effects on transmembrane electrical potential difference,respiration and membrane permeability of rice (Oryza sativa L) roots.

Among other detrimental effects of the heavy metal Cd²⁺, a decrease in the plant content of essential mineral nutrients is known. In this study, the effect of Cd²⁺ on different physiological activities of rice roots involved in nutrient acquisition has been studied. Upon addition of 0.1 or 1 mM Cd²⁺ to the experimental solution, root cell membranes depolarized in few minutes, reaching very low Em values. This effect was transient and the initial membrane potential recovered totally within 6–8 h. Only the highest concentration used had an inhibitory effect on root respiration. Significant respiratory inhibition appeared after 2 h of exposure to Cd²⁺ and lasted for at least 4 h. In turn, membrane permeability increased in the presence of Cd²⁺ for at least 8 h, inducing K⁺ efflux from the roots. The relationship between these parameters and their possible involvement in lowered nutrient content in Cd²⁺-treated plants is discussed.     

Generation of plasma membrane potential by the Na+-pump coupled to proton extrusion

Lettré cells maintain a plasma membrane potential near — 60mV, yet are scarcely depolarized by 80 mM Rb⁺ and are relatively impermeable to ⁸⁶Rb⁺. They are depolarized by ouabain without a concomitant change in intracellular cation content. Addition of K⁺ to cells suspended in a K⁺ free medium, or of Na⁺ to cells in a Na⁺ free medium, hyperpolarizes the cells. They contain electroneutral transport mechanisms for Na⁺, K⁺ and H⁺ which can function as Na⁺:K⁺ and Na⁺:H⁺ exchanges. It is concluded that plasma membrane potential of Lettré cells, in steady-state for Na⁺ and K⁺, is produced by an electrogenic Na⁺ pump sustained by electroneutral exchanges, and restricted by anion leakage.     

Activation of the H+-ATPase in the plasma membrane of cells of Saccharomyces cerevisiae grown under mild copper stress

Cells of Saccharomyces cerevisiae exibited a more active plasma membrane H⁺-ATPase during growth in media supplemented with CuSO₄ concentrations equal to or below 1 mM than did cells cultivated in the absence of copper stress. Maximal specific activities were found with 0.5 mM CuSO₄. ATPase activity declined when cells were grown with higher concentrations up to 1.5 mM (the maximal concentration that allowed growth), probably due to severe disorganization of plasma membrane. Cu²⁺-induced maximal activation was reflected in an increase of V _max (approximately threefold) and in the slight decrease of the K _m for MgATP (from 0.93 ± 0.13 to 0.65 ± 0.16 mM). The expression of the gene encoding the essential plasma membrane ATPase (PMA1) was reduced with a dose-dependent pattern in cells grown with inhibitory concentrations of copper, while the weakly expressed PMA2 gene promoter was moderately more efficient in cells cultivated under mild copper stress (1.5-fold maximal activation). ATPase was activated by copper despite the slightly lower content of ATPase protein in the plasma membrane of Cu²⁺-grown cells and the powerful inhibitory effect of Cu²⁺ in vitro. Received: 6 May 1998 / Accepted: 14 September 1998     

Ca2+/H+ exchange in the plasma membrane of Arabidopsis thaliana leaves

A large number of plant Ca²⁺/H⁺ exchangers have been identified in endomembranes, but far fewer have been studied for Ca²⁺/H⁺ exchange in plasma membrane so far. To investigate the Ca²⁺/H⁺ exchange in plasma membrane here, inside-out plasma membrane vesicles were isolated from Arabidopsis thaliana leaves using aqueous two-phase partitioning method. Ca²⁺/H⁺ exchange in plasma membrane vesicles was measured by Ca²⁺-dependent dissipation of a pre-established pH gradient. The results showed that transport mediated by the Ca²⁺/H⁺ exchange was optimal at pH 7.0, and displayed transport specificity for Ca²⁺ with saturation kinetics at K _m = 47 μM. Sulfate and vanadate inhibited pH gradient across vesicles and decreased the Ca²⁺-dependent transport of H⁺ out of vesicles significantly. When the electrical potential across plasma membrane was dissipated with valinomycin and potassium, the rate of Ca²⁺/H⁺ exchange increased comparing to control without valinomycin effect, suggesting that the Ca²⁺/H⁺ exchange generated a membrane potential (interior negative), i.e. that the stoichiometric ratio for the exchange is greater than 2H⁺:Ca²⁺. Eosin Y, a Ca²⁺-ATPase inhibitor, drastically inhibited Ca²⁺/H⁺ exchange in plasma membrane as it does for the purified Ca²⁺-ATPase in proteoliposomes, indicating that measured Ca²⁺/H⁺ exchange activity is mainly due to a plasma membrane Ca²⁺ pump. These suggest that calcium (Ca²⁺) is transported out of Arabidopsis cells mainly through a Ca²⁺-ATPase-mediated Ca²⁺/H⁺ exchange system that is driven by the proton-motive force from the plasma membrane H⁺-ATPase.     

Ion modulation of membrane permeability: Effect of cations on intact cells and on cells and phospholipid bilayers treated with pore-forming agents

Summary Leakage of ions (Na⁺, K⁺) and phosphorylated metabolites (phosphorylcholine, 2-deoxyglucose 6-phosphate) through membrane lesions in intact cells or in cells modified by pore-forming agent has been studied. Leakage from intact cells isinduced by protons and by divalent cations such as Cu²⁺, Cd²⁺ or Zn²⁺. Leakage from agent-modified cells—or across phospholipid bilayers modified by agent—isprevented by low concentrations of the same cations and by higher concentrations of Ca²⁺, Mn²⁺ or Ba²⁺; Mg²⁺, dimethonium, spermine, or spermidine are virtually ineffective. The relative efficacy of a particular cation (e.g. Ca²⁺) depends more on cell type than on the nature of the pore-forming agent. The predominant effect is on binding of cation to specific sites, not on surface charge. Surface charge, on the other hand, does affect leakage from agent-modified cells in that suspension in nonionic media reduces leakage, which can be restored by increasing the ionic strength: univalent (Na⁺, K⁺, Rb⁺, NH ₄ ⁺ ) and divalent (Mg²⁺, dimethonium) cations are equally effective; addition of protons or divalent cations such as Zn²⁺ to this system inhibits leakage. From this and other evidence here presented it is concluded that leakage across membranes is modulated by the presence of endogenous anionic components: when these are in the ionized state, leakage is favored; when unionized (as a result of protonation) or chelated (by binding to divalent cation), leakage is prevented. It is suggested that such groups are exposed at the extracellular face of the plasma membrane.     

Phosphoprotein intermediate in the ca2+-dependent atpase reaction of macrophage plasma membrane

ATPase activity and phosphorylation by [γ-³²P] ATP of isolated plasma membrane of alveolar macorphages are stimulated in a parallel fashion by physiologic concentrations of Ca²⁺, with half-maximal activating effect of this ion at (3–7) × 10^?7 M. For various membrane preparations, a direct proportionality exists between Ca²⁺-dependent ATPase activity and amount of ³²P incorporated. Labeling of membrane attains the steady-state level by 10 sec at 0°C, and is rapidly reversed by adenosine diphosphate (ADP). K⁺ decreases the amount of membrane-bound ³²P, mainly by enhancing the rate of dephosphorylation of the ³²P-intermediate. Hydroxylamine causes a release of about 90% of ³²P bound to the membrane, thus indicating that the ³²P-intermediate contains an acyl-phosphate bond. When the labeled plasma membrane is solubilized and electrophoresed on acrylamide gels in the presence of sodium dodecyl sulphate, the radioactivity appears to be largely associated with a single protein fraction of 132,500 ± 2,000 apparent molecular weight. These features of the macrophage Ca²⁺-ATPase suggest that the enzyme activity might be part of a surface-localized Ca²⁺-extrusion system, participating in the regulation of Ca²⁺-dependent activities of the macrophage.     

Isolation and characterization of plasma membrane fromAcanthamoeba culbertsoni

A rapid method for preparation of plasma membrane fromAcanthamoeba culbertsoni involving toluene treatment followed by lithium bromide extraction is described. In the plasma membrane preparation, 5′-nucleotidase, Na⁺ + K⁺ -ATPase, Mg²⁺ -ATPase and glucose-6-phosphatase activities were enriched. The membrane preparation was free from nucleic acid, cytochrome P-450 and cytochrome b5. Amino acid (¹⁴C-Ieucine) was not incorporated in the plasma membrane in 2 min. Succinic dehydrogenase was not detectable in the plasma membrane preparation. The molar ratio of cholesterol and phospholipids was 0.95 which is characteristics for plasma membranes. Under electronmicroscopy the preparation was homogenous without any other component of the cell. Plasma membrane proteins and glycoproteins were separated on acrylamide gel electrophoresis