Auxin regulation of a proton translocating ATPase in pea root plasma membrane vesicles

Pea root microsomal vesicles have been fractionated on a Dextran step gradient to give three fractions, each of which carries out ATP-dependent proton accumulation as measured by fluorescence quenching of quinacrine. The fraction at the 4/6% Dextran interface is enriched in plasma membrane, as determined by UDPG sterol glucosyltransferase and vanadate-inhibited ATPase. The vanadate-sensitive phosphohydrolase is not specific for ATP, has a K_m of about 0.23 millimolar for MgATP, is only slightly affected by K⁺ or Cl⁻ and is insensitive to auxin. Proton transport, on the other hand, is more specific for ATP, enhanced by anions (NO₃⁻ > Cl⁻) and has a K_m of about 0.7 millimolar. Auxins decrease the K_m to about 0.35 millimolar, with no significant effect on the V_max, while antiauxins or weak acids have no such effect. It appears that auxin has the ability to alter the efficiency of the ATP-driven proton transport.     

Characterization of a proton-translocating ATPase in microsomal vesicles from corn roots

Sealed microsomal vesicles were prepared from corn (Zea mays, Crow Single Cross Hybrid WF9-Mo17) roots by centrifugation of a 10,000 to 80,000g microsomal fraction onto a 10% dextran T-70 cushion. The Mg²⁺-ATPase activity of the sealed vesicles was stimulated by Cl⁻ and NH₄⁺ and by ionophores and protonophores such as 2 micromolar gramicidin or 10 micromolar carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP). The ionophore-stimulated ATPase activity had a broad pH optimum with a maximum at pH 6.5. The ATPase was inhibited by NO₃⁻, was insensitive to K⁺, and was not inhibited by 100 micromolar vanadate or by 1 millimolar azide.

Quenching of quinacrine fluorescence was used to measure ATP-dependent acidification of the intravesicular volume. Quenching required Mg²⁺, was stimulated by Cl⁻, inhibited by NO₃⁻, was insensitive to monovalent cations, was unaffected by 200 micromolar vanadate, and was abolished by 2 micromolar gramicidin or 10 micromolar FCCP. Activity was highly specific for ATP. The ionophore-stimulated ATPase and ATP-dependent fluorescence quench both required a divalent cation (Mg²⁺ ≥ Mn²⁺ > Co²⁺) and were inhibited by high concentrations of Ca²⁺. The similarity of the ionophore-stimulated ATPase and quinacrine quench and the responses of the two to ions suggest that both represent the activity of the same ATP-dependent proton pump. The characteristics of the proton-translocating ATPase differed from those of the mitochondrial F₁F₀-ATPase and from those of the K⁺-stimulated ATPase of corn root plasma membranes, and resembled those of the tonoplast ATPase.

     

Proton-translocating cytochrome c oxidase in artificial phospholipid vesicles

The proton translocating properties of cytochrome c oxidase have been studied in artificial phospholipid vesicles into the membranes of which the isolated and purified enzyme was incorporated.Initiation of oxidation of ferrocytochrome c by addition of the cytochrome, or by addition of oxygen to an anaerobic vesicle suspension, leads to ejection of H⁺ from the vesicles provided that charge compensation is permitted by the presence of valinomycin and K⁺. Proton ejection is not observed if the membranes have been specifically rendered permeable to protons.The proton ejection is the result of true translocation of H⁺ across the membrane as indicated by its dependence on the intravesicular buffering power relative to the number of particles (electrons and protons) transferred by the system, and since it can be shown not to be due to a net formation of acid in the system.Comparison of the initial rates of proton ejection and oxidation of cytochrome c yields a $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ quotient close to 1.0 both in cytochrome c and oxygen pulse experiments. An approach towards the same stoichiometry is found by comparison of the extents of proton ejection and electron transfer under appropriate experimental conditions.It is concluded that cytochrome c oxidase is a proton pump, which conserves redox energy by converting it into an electrochemical proton gradient through electrogenic translocation of H⁺.     

Plasma Membrane ATPase Activity following Reversible and Irreversible Freezing Injury

Plasma membrane ATPase has been proposed as a site of functional alteration during early stages of freezing injury. To test this, plasma membrane was purified from Solanum leaflets by a single step partitioning of microsomes in a dextran-polyethylene glycol two phase system. Addition of lysolecithin in the ATPase assay produced up to 10-fold increase in ATPase activity. ATPase activity was specific for ATP with a K_m around 0.4 millimolar. Presence of the ATPase enzyme was identified by immunoblotting with oat ATPase antibodies. Using the phase partitioning method, plasma membrane was isolated from Solanum commersonii leaflets which had four different degrees of freezing damage, namely, slight (reversible), partial (partially reversible), substantial and total (irreversible). With slight (reversible) damage the plasma membrane ATPase specific activity increased 1.5- to 2-fold and its K_m was decreased by about 3-fold, whereas the specific activity of cytochrome c reductase and cytochrome c oxidase in the microsomes were not different from the control. However, with substantial (lethal, irreversible) damage, there was a loss of membrane protein, decrease in plasma membrane ATPase specific activity and decrease in K_m, while cytochrome c oxidase and cytochrome c reductase were unaffected. These results support the hypothesis that plasma membrane ATPase is altered by slight freeze-thaw stress.     

Density gradient localization of plasma membrane and tonoplast from storage tissue of growing and dormant red beet : characterization of proton-transport and ATPase in tonoplast vesicles

Membranes from homogenates of growing and of dormant storage roots of red beet (Beta vulgaris L.) were centrifuged on linear sucrose gradients. Vanadate-sensitive ATPase activity, a marker for plasma membrane, peaked at 38% to 40% sucrose (1.165-1.175 grams per cubic centimeter) in the case of growing material but moved to as low as 30% sucrose (1.127 grams per cubic centimeter) during dormancy.

A band of nitrate-sensitive ATPase was found at sucrose concentrations of 25% to 28% or less (around 1.10 grams per cubic centimeter) for both growing and dormant material. This band showed proton transport into membrane vesicles, as measured by the quenching of fluorescence of acridine orange in the presence of ATP and Mg²⁺. The vesicles were collected on a 10/23% sucrose step gradient. The phosphate hydrolyzing activity was Mg dependent, relatively substrate specific for ATP (ATP > GTP > UTP > CTP = 0) and increased up to 4-fold by ionophores. The ATPase activity showed a high but variable pH optimum, was stimulated by Cl⁻, but was unaffected by monovalent cations. It was inhibited about 50% by 10 nanomolar mersalyl, 20 micromolar N,N′-dicyclohexylcarbodiimide, 80 micromolar diethylstilbestrol, or 20 millimolar NO₃⁻; but was insensitive to molybdate, vanadate, oligomycin, and azide. Proton transport into vesicles from the 10/23% sucrose interface was stimulated by Cl⁻, inhibited by NO₃⁻, and showed a high pH optimum and a substrate specificity similar to the ATPase, including some proton transport driven by GTP and UTP.

The low density of the vesicles (1.10 grams per cubic centimeter) plus the properties of H⁺ transport and ATPase activity are similar to the reported properties of intact vacuoles of red beet and other materials. We conclude that the low density, H⁺-pumping ATPase of red beets originated from the tonoplast. Tonoplast H⁺-ATPases with similar properties appear to be widely distributed in higher plants and fungi.

     

Calcium transport in tonoplast and endoplasmic reticulum vesicles isolated from cultured carrot cells

Two active calcium (Ca²⁺) transport systems have been identified and partially characterized in membrane vesicles isolated from cultured carrot cells (Daucus carota Danvers). Both transport systems required MgATP for activity and were enhanced by 10 millimolar oxalate. Ca²⁺ transport in membrane vesicles derived from isolated vacuoles equilibrated at 1.10 grams per cubic centimeter and comigrated with Cl⁻-stimulated, NO₃⁻-inhibited ATPase activity on sucrose density gradients. Ca²⁺ transport in this system was insensitive to vanadate, but was inhibited by nitrate, carbonyl cyanide-m-chlorophenylhydrazone (CCCP), N,N′-dicyclohexylcarbodiimide (DCCD), and 4,4-diisothiocyano-2,2′-stilbene disulfonic acid (DIDS). The K_m for MgATP and Ca²⁺ were 0.1 mm and 21 micromolar, respectively. The predominant Ca²⁺ transport system detectable in microsomal membrane preparations equilibrated at a density of 1.13 grams per cubic centimeter and comigrated with the endoplasmic reticulum (ER) marker, antimycin A-insensitive NADH-dependent cytochrome c reductase. Ca²⁺ transport activity and the ER marker also shifted in parallel in ER shifting experiments. This transport system was inhibited by vanadate (I₅₀ = 12 micromolar) and was insensitive to nitrate, CCCP, DCCD, and DIDS. Transport exhibited cooperative MgATP dependent kinetics. Ca²⁺ dependent kinetics were complex with an apparent K_m ranging from 0.7 to 2 micromolar. We conclude that the vacuolar-derived system is a Ca²⁺/H⁺ antiport located on the tonoplast and that the microsomal transport system is a Ca,Mg-ATPase enriched on the ER. These two Ca²⁺ transport systems are proposed to restore and maintain cytoplasmic Ca²⁺ homeostasis under changing cellular and environmental conditions.     

Phosphate absorption rates and adenosine 5'-triphosphate concentrations in corn root tissue

The correlations between ATP concentration in corn (Zea mays) root tissue and the rate of phosphate absorption by the tissue have been examined. Experimental variation was secured with 2,4-dinitrophenol, oligomycin, mersalyl, l-ethionine, 2-deoxyglucose, N₂ gassing and inhibition of protein synthesis. It is concluded that ATP could be the energy source for potassium phosphate absorption, but only if the transport mechanism possesses certain properties: oligomycin-sensitivity; creation of a proton gradient susceptible to collapse by uncouplers; phosphate transport via a mersalyl-sensitive Pi⁻-OH⁻ transporter; good activity at energy charge as low as 0.4; short enzymatic half-life for the ATPase or phosphate transporter; a linked mechanism for K⁺-H⁺ exchange transport, possibly electrogenic.     

Variable Effects of Nitrate on ATP-Dependent Proton Transport by Barley Root Membranes

The effects of NO₃⁻ and assay temperature on proton translocating ATPases in membranes of barley (Hordeum vulgare L. cv California Mariout 72) roots were examined. The membranes were fractionated on continuous and discontinuous sucrose gradients and proton transport was assayed by monitoring the fluorescence of acridine orange. A peak of H⁺-ATPase at 1.11 grams per cubic centimeter was inhibited by 50 millimolar KNO₃ when assayed at 24°C or above and was tentatively identified as the tonoplast H⁺-ATPase. A smaller peak of H⁺-ATPase at 1.16 grams per cubic centimeter, which was not inhibited by KNO₃ and was partially inhibited by vanadate, was tentatively identified as the plasma membrane H⁺-ATPase. A step gradient gave three fractions enriched, respectively, in endoplasmic reticulum, tonoplast ATPase, and plasma membrane ATPase. There was a delay before 50 millimolar KNO₃ inhibited ATP hydrolysis by the tonoplast ATPase at 12°C and the initial rate of proton transport was stimulated by 50 millimolar KNO₃. The time course for fluorescence quench indicated that addition of ATP in the presence of KNO₃ caused a pH gradient to form that subsequently collapsed. This biphasic time course for proton transport in the presence of KNO₃ was explained by the temperature-dependent delay of the inhibition by KNO₃. The plasma membrane H⁺-ATPase maintained a pH gradient in the presence of KNO₃ for up to 30 minutes at 24°C.     

The Nitric-oxide Reductase from Paracoccus denitrificans Uses a Single Specific Proton Pathway

The NO reductase from Paracoccus denitrificans reduces NO to N₂O (2NO + 2H⁺ + 2e⁻ → N₂O + H₂O) with electrons donated by periplasmic cytochrome c (cytochrome c-dependent NO reductase; cNOR). cNORs are members of the heme-copper oxidase superfamily of integral membrane proteins, comprising the O₂-reducing, proton-pumping respiratory enzymes. In contrast, although NO reduction is as exergonic as O₂ reduction, there are no protons pumped in cNOR, and in addition, protons needed for NO reduction are derived from the periplasmic solution (no contribution to the electrochemical gradient is made). cNOR thus only needs to transport protons from the periplasm into the active site without the requirement to control the timing of opening and closing (gating) of proton pathways as is needed in a proton pump. Based on the crystal structure of a closely related cNOR and molecular dynamics simulations, several proton transfer pathways were suggested, and in principle, these could all be functional. In this work, we show that residues in one of the suggested pathways (denoted pathway 1) are sensitive to site-directed mutation, whereas residues in the other proposed pathways (pathways 2 and 3) could be exchanged without severe effects on turnover activity with either NO or O₂. We further show that electron transfer during single-turnover reduction of O₂ is limited by proton transfer and can thus be used to study alterations in proton transfer rates. The exchange of residues along pathway 1 showed specific slowing of this proton-coupled electron transfer as well as changes in its pH dependence. Our results indicate that only pathway 1 is used to transfer protons in cNOR.     

Further Characterization on the Transport Property of Plasmalemma NADH Oxidation System in Isolated Corn Root Protoplasts

Recent experiments show that exogenous NADH increases the O₂ consumption and uptake of inorganic ions into isolated corn (Zea mays L. Pioneer Hybrid 3320) root protoplasts (Lin 1982, Proc Natl Acad Sci USA 79: 3773-3776). A mild treatment of protoplasts with trypsin released most of the NADH oxidation system from the plasmalemma (Lin 1982 Plant Physiol 70: 326-328). Further studies on this system showed that exogenous NADH (1.5 millimolar) tripled the proton efflux from the protoplasts thus generating a greater electrochemical proton gradient across the plasmalemma. Trypsin also released ubiquinone (11.95 nanomoles per milligrams protein) but not flavin or cytochrome from the system. Kinetic analyses showed that 1.5 millimolar NADH quadrupled V_max of the mechanism I (saturable) component of K⁺ uptake, while K_m was not affected. Diethylstibestrol and vanadate inhibited basal (ATPase-mediated) K⁺ influx and H⁺ efflux, while NADH-stimulated K⁺ uptake was not or only slightly inhibited. p-Chloromercuribenzene-sulfonic acid, N,N′-dicyclohexylcarbodiimide, ethidium bromide, and oligomycin inhibited both ATPase- and NADH-mediated H⁺ and K⁺ fluxes. A combination of 10 millimolar fusicoccin and 1.5 millimolar NADH gave an 11-fold increase of K⁺ influx and a more than 3-fold increase of H⁺ efflux. It is concluded that a plasmalemma ATPase is not involved in the NADH-mediated ion transport mechanism. NADH oxidase is a -SH containing enzyme (protein) and the proton channel is an important element in this transport system. Fusicoccin synergistically stimulates the effect of NADH on K⁺ uptake.     

Proton Transport Activity of the Purified Vacuolar H-ATPase from Oats : Direct Stimulation by Cl

To determine whether the detergent-solubilized and purified vacuolar H⁺-ATPase from plants was active in H⁺ transport, we reconstituted the purified vacuolar ATPase from oat roots (Avena sativa var Lang). Triton-solubilized ATPase activity was purified by gel filtration and ion exchange chromatography. Incorporation of the vacuolar ATPase into liposomes formed from Escherichia coli phospholipids was accomplished by removing Triton X-100 with SM-2 Bio-beads. ATP hydrolysis activity of the reconstituted ATPase was stimulated twofold by gramicidin, suggesting that the enzyme was incorporated into sealed proteoliposomes. Acidification of K⁺-loaded proteoliposomes, monitored by the quenching of acridine orange fluorescence, was stimulated by valinomycin. Because the presence of K⁺ and valinomycin dissipates a transmembrane electrical potential, the results indicate that ATP-dependent H⁺ pumping was electrogenic. Both H⁺ pumping and ATP hydrolysis activity of reconstituted preparations were completely inhibited by <50 nanomolar bafilomycin A₁, a specific vacuolar type ATPase inhibitor. The reconstituted H⁺ pump was also inhibited by N,N′-dicyclohexylcarbodiimide or NO₃⁻ but not by azide or vanadate. Chloride stimulated both ATP hydrolysis by the purified ATPase and H⁺ pumping by the reconstituted ATPase in the presence of K⁺ and valinomycin. Hence, our results support the idea that the vacuolar H⁺-pumping ATPase from oat, unlike some animal vacuolar ATPases, could be regulated directly by cytoplasmic Cl⁻ concentration. The purified and reconstituted H⁺-ATPase was composed of 10 polypeptides of 70, 60, 44, 42, 36, 32, 29, 16, 13, and 12 kilodaltons. These results demonstrate conclusively that the purified vacuolar ATPase is a functional electrogenic H⁺ pump and that a set of 10 polypeptides is sufficient for coupled ATP hydrolysis and H⁺ translocation.     

Anion-Sensitive, H-Pumping ATPase of Oat Roots : Direct Effects of Cl, NO(3), and a Disulfonic Stilbene

To understand the mechanism and molecular properties of the tonoplast-type H⁺-translocating ATPase, we have studied the effect of Cl⁻, NO₃⁻, and 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid (DIDS) on the activity of the electrogenic H⁺-ATPase associated with low-density microsomal vesicles from oat roots (Avena sativa cv Lang). The H⁺-pumping ATPase generates a membrane potential (Δψ) and a pH gradient (ΔpH) that make up two interconvertible components of the proton electrochemical gradient (μh⁺). A permeant anion (e.g. Cl⁻), unlike an impermeant anion (e.g. iminodiacetate), dissipated the membrane potential ([¹⁴C]thiocyanate distribution) and stimulated formation of a pH gradient ([¹⁴C]methylamine distribution). However, Cl⁻-stimulated ATPase activity was about 75% caused by a direct stimulation of the ATPase by Cl⁻ independent of the proton electrochemical gradient. Unlike the plasma membrane H⁺-ATPase, the Cl⁻-stimulated ATPase was inhibited by NO₃⁻ (a permeant anion) and by DIDS. In the absence of Cl⁻, NO₃⁻ decreased membrane potential formation and did not stimulate pH gradient formation. The inhibition by NO₃⁻ of Cl⁻-stimulated pH gradient formation and Cl⁻-stimulated ATPase activity was noncompetitive. In the absence of Cl⁻, DIDS inhibited the basal Mg,ATPase activity and membrane potential formation. DIDS also inhibited the Cl⁻-stimulated ATPase activity and pH gradient formation. Direct inhibition of the electrogenic H⁺-ATPase by NO₃⁻ or DIDS suggest that the vanadate-insensitive H⁺-pumping ATPase has anion-sensitive site(s) that regulate the catalytic and vectorial activity. Whether the anion-sensitive H⁺-ATPase has channels that conduct anions is yet to be established.     

ATPase in ripening strawberries

ATPase was found in 1000g, 13 000g and 80 00Og fractions from strawberry fruits. The optima pH for ATPase was the same (i.e. 6) for the 3 fractions, which also showed similar substrate specificity. However, the enzyme associated with the 80 000 g fraction showed the highest affinity for ATP and the maximum V_max/K_m value. As the fruit ripened, from the green to dark-red stage, ATPase activity in the 80 000 g fraction increased more than three times. The ATP content of the fruit pulp, which was high at the green stage, decreased as the fruit matured and ripened. Na⁺ and K⁺ slightly stimulated enzyme activity associated with the 1000 g,80 000 g and soluble fractions, whereas, Ca²⁺ and Mg²⁺ inhibited the enzyme activity in all fractions. However, the extent of inhibition due to divalent cations lessened as the fruit ripened.     

Kinetic analysis of proton transport by the vanadate-sensitive ATPase from maize root microsomes

Proton transport by the nitrate-insensitive, vanadate-sensitive ATPase in Kl-washed microsomes and reconstituted vesicles from maize (Zea mays L.) roots was followed by changes in acridine orange absorbance in the presence of either KNO₃ or KCl. Data from such studies obeyed a kinetic model in which net proton transport by the pump is the difference between the rate of proton transport by the action of the ATPase and the leak of protons from the vesicles in the direction opposite from the pump. After establishing the steady state proton gradient, the rate of return of transported protons was found to obey first-order kinetics when the activity of the ATPase was completely and rapidly stopped. The rate of return of these protons varied with the quencher used. When the substrate Mg:ATP was depleted by the addition of either EDTA or hexokinase, the rate at which the proton gradient collapsed was faster than when vanadate was used as the quencher. These trends were independent of the anion accompanying the K and the transport assay used.     

Localization of a proton-translocating ATPase on sucrose gradients

Ionophore-stimulated ATPase activity and ATP-dependent quinacrine quench were enriched in parallel when microsomal vesicles were prepared from corn (Crow Single Cross Hybrid WF9-Mo17) roots and collected on a cushion of 10% dextran. Activities were highest in the apical 1.5 centimeters of the roots. Vesicles collected on the dextran cushion also contained NADH cytochrome c reductase (enriched in the apical 0.5 cm of the root) and nucleoside diphosphatase (distributed throughout the first four cm). On continuous sucrose gradients, ATP-dependent proton transport and ionophore-stimulated ATPase activity coincided in a broad band extending from 1.08 to 1.15 grams per cubic centimeter with maximum activity at 1.10 to 1.12 grams per cubic centimeter. Large portions of the proton-translocating ATPase activity and ionophore-stimulated ATPase activity were clearly separable from mitochondrial membranes containing cytochrome c oxidase activity and azide-sensitive, pH 8.5 ATPase activity and from membranes bearing β-glucan synthetase I and II. The vesicles coincided with a minor portion of the NADH-cytochrome c reductase and nucleoside diphosphatase activities. It is suggested that the vesicles are of tonoplast origin.     

A Ca/H Antiport System Driven by the Proton Electrochemical Gradient of a Tonoplast H-ATPase from Oat Roots

Two types of ATP-dependent calcium (Ca²⁺) transport systems were detected in sealed microsomal vesicles from oat roots. Approximately 80% of the total Ca²⁺ uptake was associated with vesicles of 1.11 grams per cubic centimeter and was insensitive to vanadate or azide, but inhibited by NO₃⁻. The remaining 20% was vanadate-sensitive and mostly associated with the endoplasmic reticulum, as the transport activity comigrated with an endoplasmic reticulum marker (antimycin A-insensitive NADH cytochrome c reductase), which was shifted from 1.11 to 1.20 grams per cubic centimeter by Mg²⁺.

Like the tonoplast H⁺-ATPase activity, vanadate-insensitive Ca²⁺ accumulation was stimulated by 20 millimolar Cl⁻ and inhibited by 10 micromolar 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid or 50 micromolar N,N′-dicyclohexylcarbodiimide. This Ca²⁺ transport system had an apparent K_m for Mg-ATP of 0.24 millimolar similar to the tonoplast ATPase. The vanadate-insensitive Ca²⁺ transport was abolished by compounds that eliminated a pH gradient and Ca²⁺ dissipated a pH gradient (acid inside) generated by the tonoplast-type H⁺-ATPase. These results provide compelling evidence that a pH gradient generated by the H⁺-ATPase drives Ca²⁺ accumulation into right-side-out tonoplast vesicles via a Ca²⁺/H⁺ antiport. This transport system was saturable with respect to Ca²⁺ (K_m apparent = 14 micromolar). The Ca²⁺/H⁺ antiport operated independently of the H⁺-ATPase since an artifically imposed pH gradient (acid inside) could also drive Ca²⁺ accumulation. Ca²⁺ transport by this system may be one major way in which vacuoles function in Ca²⁺ homeostasis in the cytoplasm of plant cells.

     

Characterization of the Proton-Translocating Cytochrome c Oxidase Activity in the Plasma Membrane of Intact Anacystis nidulans Spheroplasts

Intact spheroplasts of the cyanobacterium (blue-green alga) Anacystis nidulans oxidized various exogenous c-type cytochromes with concomitant outward proton translocation while exogenous ferricytochrome c was not reduced. The H⁺/e⁻ stoichiometry was close to 1 with each of the cytochromes and did not depend on the actual rate of the oxidase reaction. Observed proton ejections were abolished by the uncoupler carbonyl cyanide m-chlorophenylhydrazone. Cyanide, azide, and carbon monoxide inhibited cytochrome c oxidation and proton extrusion in parallel while dicyclohexylcarbodiimide affected proton translocation more strongly than cytochrome c oxidation. The cytoplasmic membrane of A. nidulans appears to contain a proton-translocating cytochrome c oxidase similar to the one described for mitochondria.     

K+-stimulated ATPase in purified microsomes of bullfrog oxyntic cells

Differential centrifugation of oxyntic cell homogenates yielded microsomal fractions which contained large amounts of mitochondrial membrane. The presence of marker enzymes (succinate dehydrogenase and cytochrome c oxidase) indicated that mitochondrial contamination of crude microsomes ranged from 20 to 60% in different preparations. A discontinuous sucrose density gradient procedure was developed for the routine preparation of purified oxyntic cell microsomes. A K⁺-stimulated, Mg²⁺-requiring ATPase was localized in these purified membranes and coincided with the presence of a K⁺-stimulated p-nitrophenylphosphatase. Na⁺ and ouabain had no effect on the K⁺ stimulation of the microsomal ATPase. The apparent activation constant for K⁺ was approximately 1 mM at pH 7.5, the optimal pH for stimulation.An anion-sensitive ATPase has been widely studied in gastric microsomal preparations. We found that the basal microsomal ATPase (i.e. without K⁺) and the mitochondrial ATPase were inhibited by SCN^? and enhanced by HCO₃^?, however, the K⁺-stimulated component of the microsomal ATPase was virtually unaffected by these anions.     

Effects of Cr on proton extrusion, potassium uptake and transmembrane electric potential in maize root segments

Abstract Proton extrusion of maize root Zea mays segments, was inhibited by the presence of Cr (o.n. + 6; present in solution as CrO₄^2-, Cr₂O₇^2-) in the incubation medium: the minimum inhibiting concentration was 2 × 10^?3 mol m^?3 and the inhibition progressively increased with Cr concentration. Cr inhibited proton extrusion. Also, when this activity was stimulated by the presence of K⁺ or fusicoccin (FC) in the incubation medium, the K⁺ and FC stimulating effect was still present when proton extrusion was inhibited by Cr. In addition, Cr inhibited K⁺ uptake. This inhibition was higher (50%) at K⁺ concentrations up to 1 mol m^?3 lower (15%) at higher K⁺ concentrations. This result indicates that the system responsible for K⁺ uptake operating at low K⁺ concentrations is more sensitive to Cr inhibition. Cr had no effect on transmembrane electric potential (PD). The depolarizing and hyper-polarizing effect of K+ and FC, respectively, were not affected by Cr; but Cr enhances the depolarizing effect of the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCP). These results indicate that Cr inhibited the proton translocating mechanism coupled with K⁺ uptake, but did not change the net transport of charges through the plasmalemma. The Cr effect is discussed, taking into account the possibility of a direct effect of Cr at the membrane level or, alternatively, of an effect on some metabolic processes controlling membrane function.