ATP Synthase of Chloroplast Thylakoid Membranes: A More in Depth Characterization of Its ATPase Activity

In contrast to everted mitochondrial inner membrane vesicles and eubacterial plasma membrane vesicles, the ATPase activity of chloroplast ATP synthase in thylakoid membranes is extremely low. Several treatments of thylakoids that unmask ATPase activity are known. Illumination of thylakoids that contain reduced ATP synthase (reduced thylakoids) promotes the hydrolysis of ATP in the dark. Incubation of thylakoids with trypsin can also elicit higher rates of ATPase activity. In this paper the properties of the ATPase activity of the ATP synthase in thylakoids treated with trypsin are compared with those of the ATPase activity in reduced thylakoids. The trypsin-treated membranes have significant ATPase activity in the presence of Ca²⁺, whereas the Ca²⁺-ATPase activity of reduced thylakoids is very low. The Mg²⁺-ATPase activity of the trypsinized thylakoids was only partially inhibited by the uncouplers, at concentrations that fully inhibit the ATPase activity of reduced membranes. Incubation of reduced thylakoids with ADP in Tris buffer prior to assay abolishes Mg²⁺-ATPase activity. The Mg²⁺-ATPase activity of trypsin-treated thylakoids was unaffected by incubation with ADP. Trypsin-treated membranes can make ATP at rates that are 75–80% of those of untreated thylakoids. The Mg²⁺-ATPase activity of trypsin-treated thylakoids is coupled to inward proton translocation and 10 mM sulfite stimulates both proton uptake and ATP hydrolysis. It is concluded that cleavage of the γ subunit of the ATP synthase by trypsin prevents inhibition of ATPase activity by the ε subunit, but only partially overcomes inhibition by Mg²⁺ and ADP during assay.     

Anion-sensitive ATPase activity and proton transport in isolated vacuoles of species of the CAM genus Kalanchoë

Vacuoles were isolated from leaves of Kalanchoë daigremontiana Hamet et Perrier de la Bathie, and the ionic sensitivity of the vacuolar ATPase was studied in vacuole homogenates desalted on Sephadex G-25. The ATPase activity was dependent on the presence of divalent cations (Mg²⁺≥ Mn²⁺≥ Ca²⁺, Co²⁺; Zn²⁺ had no effect). Mg²⁺-dependent ATPase activity was stimulated by anions (Cl^? > malate²⁺, HCO^?₃), with maximal stimulation at concentrations above 50 mM. Mg²⁺-Dependent activity was inhibited by NO^?₃ above 2 mM, but no saturation was observed up to 100 mM. No stimulation by K⁺ or Na⁺ was detected; stimulation by NH⁺₄ was abolished by 0.01% (w/v) Triton X-100, suggesting that the NH⁺₄ effect was due to the permeability of vacuolar membrane vesicles to NH₃. Trans-tonoplast electrical potentials (Δψ) and intra-vacuolar pH were measured with glass microelectrodes and antimony covered glass micro-pH-electrodes, respectively. Free vacuofes isolated from Kalanchoë tubiflora (Harv.) Hamet were slightly positive with respect to the suspension medium. This Δψ was insensitive to the protonophore FCCP and depolarized by about 4 mV on addition of 50 mM KCl, still remaining about +5 mV. Upon addition of 7 mM Mg-ATP, vacuoles showed an FCCP-sensitive increase of Δψ from +9.2 ± 2.8 (13) to +17.8 ± 3.7 (12) mV [given as x?± sd (n)] and an internal acidification from pH 5.4 ± 0.2 (11) to pH 4.3 ± 0.4 (12). Mg-ADP and ATP without Mg²⁺ had no effect on Δψ. It is concluded that the H4 pumping at the tonoplast is due to the functioning of the anion-sensitive vacuolar ATPase and that this is an essential part of the mechanism of nocturnal acid accumulation in CAM.     

Stimulation of Bacillus subtilis membrane adenosine triphosphatase by cationic bactericidal agents

The environmental Mg²⁺ used in preparation of Bacillus subtilis membranes was found to influence the responses of the associated ATPase to cetyltrimethylammonium bromide (CTAB). Membranes prepared using fluids containing higher Mg²⁺ levels exhibited lower control activity than was seen with low Mg²⁺ membranes. Increased environmental Mg²⁺ resulted in higher stimulations with lower doses of the agent. ATPase of all three membrane types was stimulated in two concentration ranges, but in the doses tested, CTAB inhibited the ATPase of only those membranes obtained using fluids containing high Mg²⁺ for every stage of the isolation. Sonication of membranes for 25 s at half maximum output yielded three fractions, consisting of a soluble form which was sensitive to CTAB stimulation at 25 μg/ml of assay mixture; small, 95–110 nm, vesicles, which were resistant to CTAB at 25, 75, and 150 μg/ml, and large vesicles, similar to untreated membranes both in morphology and responses to detergent. Combinations of detergent and protein (β-lysin or arginine-rich histone) produced activity appearing to be additive when the protein level was present in a high concentration and the detergent was present at levels corresponding to the minimum influence. Mixtures of a maximally stimulating dose (75 or 100 μg/ml) of detergent and a small amount of protein produced activities that were at least 92% or more of the expected sums of individual stimulations. Interference occurred with the following mixtures: high amounts of detergent and protein; high protein and 10 or 15 μg/ml CTAB; and β-lysin and arginine-rich histone, both at high levels. These data are consistent with a hypothesis that the two peaks in CTAB stimulation reflect two adjacent ATPase sites, one of which is also susceptible to stimulation by cationic protein.     

Ca-Mg-ATPase activity in brain coated microvesicles purified on immunosorbents

Coated microvesicle fractions isolated from ox forebrain cortex by the ultracentrifugation procedure of Pearse (1) and by the modified, less time consuming method of Keen et al (2) had comparable Ca²⁺+Mg²⁺ dependent ATPase activities (about 9 μmol/h per mg protein). The Na⁺+K⁺+Mg²⁺ dependent ATPase activity was 3.2 μmol/h per mg (±1.0, S.D., n=3) when microvesicles were prepared according to (1) and 1.5 μmol/h per mg (±1.0, S.D., n=3) when prepared according to (2).Oligomycin, ruthenium red, and trifluoperazine, inhibitors of Ca²⁺ transport in mitochondria and erythrocyte membranes had no effect on Ca²⁺+Mg²⁺ dependent ATPase from any of the preparations.As demonstrated both by ATPase assays and electron microscopy, coated microvesicles could be bound to immunosorbents prepared with poly-specific antibodies against a coated microvesicle fraction obtained by the method of Pearse (1). The binding could be inhibited by dissolved coat protein using partially purified clathrin. The fraction of coated vesicles eluted from the immunosorbent was purified relative to the starting material as judged by electron microscopy.The Ca²⁺+Mg²⁺ ATPase activity and calmodulin content was copurified with the coated microvesicles and the specific activity of Na⁺+K⁺+Mg²⁺ ATPase was decreased.Na⁺+K⁺+Mg²⁺ dependent ATPase activity in the coated microvesicle fraction could be ascribed to membranes with the appearance of microsomes. These membranes were also bound to the immunosorbents, but the binding was not influenced by clathrin. The capacity of the immunosorbents for these membranes was less than for the coated microvesicles, resulting in a decrease of Na⁺+K⁺+Mg²⁺ dependent ATPase activity in the eluted coated microvescile fraction.It was concluded that Ca²⁺+Mg²⁺ ATPase activity is not a contamination from plasma membrane vesicles or mitochondrial membranes but seems to be an integral part of the coated vesicle membrane.     

Role of Mg2+ ions in the subunit structure and membrane binding properties of bacterial energy transducing ATPase.

ATPase extracted from $\text{Streptococcus faecalis}$ membranes was purified by preparative slab gel electrophoresis in the presence of Mg⁺⁺ (plus Mg²⁺ ATPase) and without Mg²⁺ (minus Mg²⁺ ATPase). The subunit composition and membrane binding capacity of both preparations was then examined. The plus Mg²⁺ ATPase had 5 types of subunits (αβγδ?) and reattached normally to depleted membranes. The minus Mg²⁺ ATPase had the αβγ and ? chains, but no δ chain, and failed to reattach to membranes. These data indicate that Mg²⁺ or a similar cationic ligand anchors the δ chain to the core enzyme complex and that the δ chain in turn is needed for membrane attachment. For the plus Mg²⁺ ATPase the data are consistent with the subunit stoichiometry and arrangement, (α₃β₃ γ ?)-Mg²⁺)_n?(δ).     

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.

     

Characterization of Mg2+- and (Ca2+ + Mg2+)-ATPase activity in adipocyte endoplasmic reticulum

The presence of an energy-dependent calcium uptake system in adipocyte endoplasmic reticulum (D. E. Bruns, J. M. McDonald, and L. Jarett, 1976, J. Biol. Chem.251, 7191–7197) suggested that this organelle might possess a calcium-stimulated transport ATPase. This report describes two types of ATPase activity in isolated microsomal vesicles: a nonspecific, divalent cation-stimulated ATPase (Mg²⁺-ATPase) of high specific activity, and a specific, calcium-dependent ATPase (Ca²⁺ + Mg²⁺-ATPase) of relatively low activity. Mg²⁺-ATPase activity was present in preparations of mitochondria and plasma membranes as well as microsomes, whereas the (Ca²⁺ + Mg²⁺)-ATPase activity appeared to be localized in the endoplasmic reticulum component of the microsomal fraction. Characterization of microsomal Mg²⁺-ATPase activity revealed apparent K_m values of 115 μm for ATP, 333 μm for magnesium, and 200 μm for calcium. Maximum Mg²⁺-ATPase activity was obtained with no added calcium and 1 mm magnesium. Potassium was found to inhibit Mg²⁺-ATPase activity at concentrations greater than 100 mm. The energy of activation was calculated from Arrhenius plots to be 8.6 kcal/mol. Maximum activity of microsomal (Ca²⁺ + Mg²⁺)-ATPase was 13.7 nmol ³²P/mg/min, which represented only 7% of the total ATPase activity. The enzyme was partially purified by treatment of the microsomes with 0.09% deoxycholic acid in 0.15 m KCl which increased the specific activity to 37.7 nmol ³²P/mg/min. Characterization of (Ca²⁺ + Mg²⁺)-ATPase activity in this preparation revealed a biphasic dependence on ATP with a Hill coefficient of 0.80. The apparent K_m^s for magnesium and calcium were 125 and 0.6–1.2 μm, respectively. (Ca²⁺ + Mg²⁺)-ATPase activity was stimulated by potassium with an apparent K_m of 10 mm and maximum activity reached at 100 mm potassium. The energy of activation was 21.5 kcal/mol. The kinetics and ionic requirements of (Ca²⁺ + Mg²⁺)-ATPase are similar to those of the (Ca²⁺ + Mg²⁺)-ATPase in sarcoplasmic reticulum. These results suggest that the (Ca²⁺ + Mg²⁺)-ATPase of adipocyte endoplasmic reticulum functions as a calcium transport enzyme.     

Isolation and properties of ion-stimulated ATPase activity associated with cauliflower plasma membranes

The association of K⁺-stimulated, Mg²⁺-dependent ATPase activity with plasma membranes from higher plants has been used as a marker for the isolation and purification of a plasma membrane-enriched fraction from cauliflower (Brassica oleraceae L.) buds. Plasma membranes were isolated by differential centrifugation followed by density gradient centrifugation of the microsomal fraction. The degree of purity of plasma membranes was determined by increased sensitivity of Mg²⁺-ATPase activity to stimulation by K⁺ and by assay of approximate marker enzymes. In the purified plasma membrane fraction, Mg²⁺-ATPase activity was stimulated up to 700% by addition of K⁺. Other monovalent cations also markedly stimulated the enzyme, but only in the presence of the divalent cation Mg²⁺. Ca²⁺ was inhibitory to enzyme activity. ATPase was the preferred substrate for hydrolysis, there being little hydrolysis in the presence of ADP, GTP, or p-nitrophenylphosphate. Monovalent cation-stimulated activity was optimum at alkaline pH. Enzyme activity was inhibited nearly 100% by AgNO₃ and about 40% by diethylstilbestrol.     

A comparison of the properties of ATPase associated with wheat and cauliflower plasma membranes

Plasma membrane-associated ATPase obtained from cauliflower (Brassica oleraceae L.) florets isolated and assayed by several different procedures was stimulated 150 to 400% by K⁺. In contrast, winter wheat (Triticum aestivum L. cv. Kharkov 22 MC) shoot and root ATPase obtained by the same methods exhibited only 10 to 25% stimulation by K⁺. The level of K⁺-stimulation of the wheat enzyme was not significantly increased by purifying the crude microsomal membrane fraction using sucrose density gradients. ATPase associated with density gradient-purified cauliflower membranes was inhibited by Ca²⁺, high ATP concentration in the presence of low Mg²⁺, and by several metabolic inhibitors. In contrast, the wheat enzyme was largely unaffected by all of these treatments. The plasma membranes of intact wheat and cauliflower cells gave a positive reaction with the plasma membrane-specific, phosphotungstic acid-chromic acid stain (PACP). A high proportion of the cauliflower membrane vesicles in the putative plasma membrane-enriched fraction stained with PACP, whereas only a small proportion of the wheat membrane vesicles reacted positively with PACP. These results indicate that a plasma membrane-enriched fraction has been isolated successfully from cauliflower floret tissue, but that none of the procedures used effectively separate plasma membranes from homogenates of wheat shoots and roots.     

Actomyosin-like ATPase extracted from plain synaptic vesicle fractions

Ca²⁺-sensitive Mg²⁺-dependent ATP phosphohydrolase (EC 3.6.1.3, ATPase) was extracted from the plain synaptic vesicle fractions that were virtually devoid of contamination. The protein pattern of the ATPase preparation on SDS polyacrylamide gel electrophoresis closely resembled that of actomyosin from skeletal muscle. The finding suggests that the main components of the ATPase are actin- and myosin-like proteins of the brain (stenin and neurin). Microsome and synaptosomal plasmalemma fractions were extracted under the same conditions to examine the possibility that the ATPase extracted derived from contaminating particulates. An entirely different ATPase was extracted from microsomes, and no protein from plasma membranes. Although Ca²⁺-sensitive Mg²⁺-dependent ATPase was extracted from coated vesicle fraction, the electrophoretic pattern was dissimilar to that of the ATPase from plain synaptic vesicle fractions. It may be inferred that the whole complex of neurostenin is located in plain synaptic vesicles from the brain.     

Freeze-thaw damage in isolated lobster sarcoplasmic reticulum membranes: A model system for membrane damage

Sarcoplasmic reticulum membrane vesicles (SRV), isolated from the abdominal muscle of Maine lobsters, were put through a freeze-thaw cycle in order to study membrane freezing damage on a molecular basis, The major membrane protein in SRV is a (Ca²⁺ − Mg²⁺) ATPase capable of accumulating Ca²⁺ with the concomitant hydrolysis of ATP, After being frozen and thawed in the presence of NaCl, the SRV showed an increased ATPase activity and a decreased ability to accumulate Ca²⁺. The degree of increased ATPase activity and decreased Ca²⁺ accumulation was dependent upon the NaCl concentration (damage increased with increased NaCl concentration) and cooling rate (damage was only observed at slow cooling rates, i.e., less than 10 °C/min). Slow thawing rates also increased the amount of damage.The freeze-thaw damage of the SRV membranes is probably not due to osmotic shock, since the vesicles are quite resistant to osmotic stress and are highly permeable to small molecules and monovalent ions. Incubation of the SRV in 2 NaCl at 22 °C has no effect on Ca²⁺ accumulation whereas freezing in 0.25 NaCl totally abolishes their ability to take up Ca²⁺. Thus, a combination of salt and low temperature is necessary for damage. The freeze-thaw damage can be largely prevented by the addition of DMSO, glycerol, or PVP. The factors above have implications for the storage of tissue or membranes for subsequent analysis of membrane-bound enzymes. The SRV mimic the behavior of cells in their response to cooling and thawing rates, salts and cryoprotectants.     

The Ca-Transport ATPase of Plant Plasma Membrane Catalyzes a nH/Ca Exchange

Microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings accumulate Ca²⁺ upon addition of MgATP. MgATP-dependent Ca²⁺ uptake co-migrates with the plasma membrane H⁺-ATPase on a sucrose gradient. Ca²⁺ uptake is insensitive to oligomycin, inhibited by vanadate (IC₅₀ 40 micromolar) and erythrosin B (IC₅₀ 0.2 micromolar) and displays a pH optimum between pH 6.6 and 6.9. MgATP-dependent Ca²⁺ uptake is insensitive to protonophores. These results indicate that Ca²⁺ transport in these microsomal vesicles is catalyzed by a Mg²⁺-dependent ATPase localized on the plasma membrane. Ca²⁺ strongly reduces ΔpH generation by the plasma membrane H⁺-ATPase and increases MgATP-dependent membrane potential difference (Δψ) generation. These effects of Ca²⁺ on ΔpH and Δψ generation are drastically reduced by micromolar erythrosin B, indicating that they are primarily a consequence of Ca²⁺ uptake into plasma membrane vesicles. The Ca²⁺-induced increase of Δψ is collapsed by permeant anions, which do not affect Ca²⁺-induced decrease of ΔpH generation by the plasma membrane H⁺-ATPase. The rate of decay of MgATP-dependent ΔpH, upon inhibition of the plasma membrane H⁺-ATPase, is accelerated by MgATP-dependent Ca²⁺ uptake, indicating that the decrease of ΔpH generation induced by Ca²⁺ reflects the efflux of H⁺ coupled to Ca²⁺ uptake into plasma membrane vesicles. It is therefore proposed that Ca²⁺ transport at the plasma membrane is mediated by a Mg²⁺-dependent ATPase which catalyzes a nH⁺/Ca²⁺ exchange.     

Plasma membranes from candida tropicalis grown on glucose or hexadecane. I. Isolation,identification and purification

Plasma membranes from Candida tropicalis grown on glucose or hexadecane were isolated using a method based on the difference in surface charge of mitochondria and plasma membranes.After mechanical disruption of the cells, a fraction consisting of mitochondrial and plasma membrane vesicles was obtained by differential centrifugation.Subsequently the mitochondria were separated from the plasma membrane vesicles by aggregation of the mitochondria at a pH corresponding to their isoelectric point. Additional purification of the isolated plasma membrane vesicles was achieved by osmolysis. Surface charge densities of mitochondria and plasma membranes were determined and showed substrate-dependent differences.The isolated plasma membranes were morphologically characterized by electron microscopy and, as a marker enzyme, the activity of Mg²⁺-dependant ATPase was determined.By checking for three mitochondrial marker enzymes the plasma membrane fractions were estimated to be 94% pure with regard to mitochondrial contamination.     

Substrate requirements and subcellular distribution of calcium transport activities in brain membranes

Ca²⁺ transport activity in synaptosomal membranes has been identified as having two major components: Ca²⁺-stimulated ATP hydrolysis and ATP-dependent CA²⁺ uptake. Both processes exhibit similar affinities for Ca²⁺ and operate maximally under identical buffer conditions. Subcellular fractionation studies revealed the Ca²⁺/Mg²⁺ ATPase and ATP-dependent CA²⁺ uptake activities to be highest in synaptic plasma membrane fractions 1 and 2, with lesser activity in synaptic vesicles and mitochondria. Progressive treatment with Triton X-100 activated, then decreased Ca²⁺/Mg²⁺ ATPase, Mg²⁺ ATPase and Ca²⁺ ATPase. ATP-dependent Ca²⁺ uptake was progressively decreased by similar treatment with Triton X-100. These studies illustrate that Ca²⁺ ATPase and ATP-dependent Ca²⁺ uptake may provide two important mechanisms for buffering of cytosolic Ca²⁺ at the nerve terminal. These systems may function to rapidly sequester cytosolic Ca²⁺ following a rise during depolarization and then extrude Ca²⁺ from the terminal against a concentration gradient. This regulation of cytosolic Ca²⁺, represented by two processes of the type seen in other plasma membranes, may play critical roles in calcium homeostasis in nerve cells.Footnote: Portions of this research were submitted by K. M. Garrett in partial fulfillment of requirements for the Doctor of Philosophy Degree in Pharmacology at the University of Texas Health Science Center.     

Highly purified sarcoplasmic reticulum vesicles are devoid of Ca2+-independent (‘basal’) ATPase activity

On solubilization with Triton X-100 of sarcoplasmic reticulum vesicles isolated by differential centrifugation, the Ca²⁺-ATPase is selectively extracted while approximately half of the initial Mg²⁺-, or ‘basal’, ATPase remains in the Triton X-100 insoluble residue. The insoluble fraction, which does not contain the 100 000 dalton polypeptide of the Ca²⁺-ATPase, contains high levels of cytochrome c oxidase. Furthermore, its Mg²⁺-ATPase activity is inhibited by specific inhibitors of mitochondrial ATPase, indicating that the ‘basal’ ATPase separated from the Ca²⁺-ATPase by detergent extraction originates from mitochondrial contaminants.To minimize mitochondrial contamination, sarcoplasmic reticulum vesicles were fractionated by sedimentation in discontinuous sucrose density gradients into four fractions: heavy, intermediate and light, comprising among them 90–95% of the initial sarcoplasmic reticulum protein, and a very light fraction, which contains high levels of Mg²⁺-ATPase. Only the heavy, intermediate and light fractions originate from sarcoplasmic reticulum; the very light fraction is of surface membrane origin. Each fraction of sarcoplasmic reticulum origin was incubated with calcium phosphate in the presence of ATP and the loaded fractions were separated from the unloaded fractions by sedimentation in discontinuous sucrose density gradients. It was found that vesicles from the intermediate fraction had, after loading, minimal amounts of mitochondrial and surface membrane contamination, and displayed little or no Ca²⁺-independent basal ATPase activity. This shows conclusively that the basal ATPase is not an intrinsic enzymatic activity of the sarcoplasmic reticulum membrane, but probably originates from variable amounts of mitochondrial and surface membrane contamination in sarcoplasmic reticulum preparations isolated by conventional procedures.     

Ca2+ - and Mg2+-dependent ATPase activities in the deoxycholate-treated rat heart sarcolemma

• 1.1. Isolated rat heart sarcolemma was treated with different concentrations of an ionic detergent, deoxycholate (DOC) and ATP hydrolysis in the presence of Ca²⁺ or Mg²⁺ was determined.
• 2.2. Both Ca²⁺-dependent ATPase and Mg²⁺-dependent ATPase activities were decreased in the DOC-treated membranes; however, the depression of Mg²⁺-dependent ATPase activity was greater than that of Ca²⁺-dependent ATPase.
• 3.3. The differential changes in Ca²⁺-dependent ATPase and Mg²⁺-dependent ATPase activities were apparent when incubations with DOC were carried out for different time intervals and at different temperatures.
• 4.4. In DOC-treated preparations, the K_m value for Ca²⁺-dependent ATPase was decreased whereas that for Mg²⁺-dependent ATPase was increased. The half maximal velocities of the Ca²⁺-dependent ATPase and Mg²⁺-dependent ATPase enzyme reactions in the treated preparations were obtained at a DOC: membrane protein ratio of 3.0 and 0.6, respectively.
• 5.5. In the DOC-treated membranes exhibiting the half maximal velocities of enzyme reactions, the K_i value for Ca²⁺-dependent ATPase was drastically reduced but remained unchanged for Mg²⁺-dependent ATPase.
• 6.6. The DOC treatment was associated with a loss of protein as well as phospholipids and resulted in changes in the ultrastructural integrity of the membrane.
• 7.7. Varying degrees of decreases in the activities of sarcolemmal adenylate cyclase. (Na⁻-K⁺)-ATPase. 5'-nucleotidase and calcium binding were seen upon DOC treatment.
• 8.8. The extent of reduction in Ca²⁺-dependent ATPase and Mg²⁺-dependent ATPase activities were also different when the membrane was treated with a non-ionic detergent, Lubrol PX.
• 9.9. These data suggest that Ca²⁺-dependent ATPase in heart sarcolemma is more resistant than Mg²⁺-dependent ATPase to detergent treatments and further indicate some differences in the properties of these enzymes.

     

Separation of the Mg-ATPases from the Ca-Phosphatase Activity of Microsomal Membranes Prepared from Barley Roots

Two methods for preparing membrane fractions from barley (Hordeum vulgare cv California Mariout 72) roots were compared in order to resolve reported differences between the characteristics of the plasma membrane ATPase of barley and that of other species. When microsomal membranes were prepared by a published procedure and applied to a continuous sucrose gradient, the membranes sedimented as a single broad band with a peak density of 1.16 grams per cubic centimeter (g/cm³). Activities of NADH cytochrome (Cyt) c reductase, Ca²⁺-ATPase, and Mg²⁺-ATPase were coincident and there was little ATP-dependent proton transport anywhere on the gradient. When the homogenization procedure was modified by increasing the pH of the buffer and the ratio of buffer to roots, the microsomal membranes separated as several components on a continuous sucrose gradient. A Ca²⁺-phosphatase was at the top of the gradient, NADH Cyt c reductase at 1.08 g/cm³, a peak of ATP-dependent proton transport at 1.09 to 1.12 g/cm³, a peak of nitrate-inhibited ATPase at 1.09 to 1.12 g/cm³, and of vanadate-inhibited ATPase at 1.16 g/cm³. The Ca²⁺-phosphatase had no preference for ATP over other nucleoside di- and tri-phosphates and was separated from the vanadate-inhibited ATPase on a sucrose gradient; approximately 70% of the Ca²⁺-phosphatase was removed from the microsomes by washing with 150 millimolar KCl. The vanadate-sensitive ATPase required Mg²⁺, was highly specific for ATP, and was not affected by the KCl wash. These results show that barley roots have a plasma membrane ATPase similar to that of other plant species.     

Characterization of an ATPase Associated with the Inner Envelope Membrane of Amyloplasts from Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.)

Amyloplast envelope membranes isolated from cultured, white-wild cells of sycamore (Acer pseudoplatanus L.) have been found to contain a Mg²⁺-ATPase, ranging in specific activity from 5 to 30 nanomoles per minute per milligram protein. This ATPase hydrolyzes a broad range of nucleoside triphosphates, whereas it hydrolyzes nucleoside mono- and diphosphates poorly, if at all. The ATPase activity was stimulated by several divalent cations, including Mg²⁺, Mn²⁺ and Ca²⁺, whereas it was not affected by Sr²⁺, K⁺, or Na⁺. The K_m for total ATP was 0.6 millimolar, and the activity showed a broad pH optimum between 7.5 and 8.0. The ATPase was insensitive to N,N′-dicyclohexylcarbodiimide and oligomycin, but it was inhibited by vanadate. All these characteristics are basically similar to those reported previously for the Mg²⁺-ATPase of the chloroplast inner-envelope membrane. Likewise, the amyloplast envelope enzyme was shown to be located specifically on the inner envelope membrane. The amyloplast envelope membranes were chemically modified with a series of unique affinity labeling reagents, the adenosine polyphosphopyridoxals (M Tagaya, T Fukui 1986 Biochemistry 25: 2958-2964). About 90% of the ATPase activity was lost when the envelope membranes were preincubated with 0.1 millimolar adenosine triphosphopyridoxal. Notably, the enzyme was protected completely from inactivation in the presence of its substrate, ATP. In contrast, both adenosine diphosphopyridoxal and pyridoxal phosphate caused much less of an inhibitory effect. This greater relative reactivity of the triphosphopyridoxal analog is similar to that reported previously with Escherichia coli F₁ ATPase (T Noumi et al. 1987 J Biol Chem 262: 7686-7692).     

Characterization of ATPase activity associated with corn leaf plasma membranes

A Mg²⁺-dependent, cation-stimulated ATPase was associated with plasma membranes isolated from corn leaf mesophyll protoplasts. Potassium was the preferred monovalent cation for stimulating the ATPase above the Mg²⁺-activated level. The enzyme was substrate-specific for ATP, was inhibited by N,N′-dicyclohexylcarbodiimide, diethylstilbestrol, p-chloromercuribenzoate, and orthovanadate, but was insensitive to oligomycin or sodium azide. A K_m of 0.28 millimolar Mg²⁺-ATP was determined for the K⁺-ATPase, and the principal effect of potassium was on the V_max for ATP hydrolysis. Since potassium stimulation was not saturated at high concentrations, a nonspecific role was proposed for potassium stimulation. A nonspecific phosphatase was also found to be associated with corn leaf plasma membranes. However, it could not be determined positively whether this activity represented a separate enzyme.     

Alterations in Ca2+/Mg2+ ATPase activity upon treatment of heart sarcolemma with phospholipases

In order to examine the role of phospholipids in the activation of membrane bound Ca²⁺/Mg²⁺ ATPase, the activities of Ca²⁺ ATPase and Mg²⁺ ATPase were studied in heart sarcolemma after treatments with phospholipases A, C and D. The Mg²⁺ ATPase activity was decreased upon treating the sarcolemmal membranes with phospholipases, A, C and D; phospholipase A produced the most dramatic effect. The reduction in Mg², ATPase activity by each phospholipase treatment was associated with a decrease in the V_max value without any changes in the K_a value. The depression of Mg²⁺ ATPase in the phospholipase treated preparations was not found to be due to release of fatty acids in the medium and was not restored upon reconstitution of these membranes by the addition of synthetic phospholipids such as lecithin, lysolecithin or phosphatidic acid. In contrast to the Mg²⁺ ATPase, the sarcolemmal Ca²⁺ ATPase was affected only slightly by phospholipase treatments. The greater sensitivity of Mg^- ATPase to phospholipase treatments was also apparent when deoxycholate-treated preparations were employed. These results indicate that glycerophospholipids are required for the sarcolemmal Mg²⁺ ATPase activity to a greater extent in comparison to that for the Ca²⁺ ATPase activity and the phospholipids associated with Mg²⁺ ATPase are predominantly exposed at the outer surface of the membrane.