Similarities of omega gliadins from Triticum urartu to those encoded on chromosome 1A of hexaploid wheat and evidence for their post-translational processing.

The omega-gliadins encoded on chromosome 1 of the A genome were purified from Triticum aestivum L. (2n=6 x=42, AABBDD) cv. Butte86, nullisomic 1D-tetrasomic 1A of cv. Chinese Spring (CS N1DT1A), and the diploid T. urartu (2n=2 x=14, AA ). Reverse-phase high-performance liquid chromatography combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis of gliadin extracts from CS nullisomic-tetrasomic (NT) lines confirmed the assignment to chromosome 1A. The purified omega-gliadins were characterized by mass spectrometry and N-terminal sequencing. The 1A-encoded omega-gliadins were smaller than 1B- or 1D-encoded omega-gliadins. The N-terminal amino acid sequences for 1A omega-gliadin mature peptides were nearly identical to those for the T. urartu omega-gliadins and were more similar to 1D omega-gliadin sequences than to sequences for T. monococum omega-gliadins, barley C-hordeins, or rye omega-secalins. They diverged greatly from the N-terminal sequences for the 1B omega-gliadins. The data suggest that T. urartu is the A-genome donor, and that post-translational cleavage by an asparaginyl endoprotease produces those omega-gliadins with N-terminal sequences beginning with KEL.

Subunit composition and Ca(2+)-ATPase activity of the vacuolar ATPase from barley roots.

The vacuolar ATPase was purified from a tonoplast-enriched membrane fraction from barley (Hordeum vulgare cv CM72) roots. The membranes were solubilized with Triton X-100 and the membrane proteins were separated by chromatography on Sephacryl S-400 followed by fast protein liquid chromatography on a Mono-Q column. The purified vacuolar ATPase was inhibited up to 90% by KNO3 or 80% by dicyclohexylcarbodiimide (DCCI). The ATPase was resolved into polypeptides of 115, 68, 53, 45, 42, 34, 32, 17, 13, and 12 kDa. An additional purification step of centrifugation on a glycerol gradient did not result in loss of any polypeptide bands or increased specific activity of the ATPase. Antibodies against the purified holoenzyme inhibited proton transport by the native ATPase. Two peaks of solubilized Ca(2+)-ATPase were obtained from the Sephacryl S-400 column. A peak of Ca(2+)-ATPase copurified with the vacuolar ATPase during all of the purification steps and was inhibited by NO3- and DCCI. It is proposed that this Ca(2+)-ATPase is a partial reaction of the plant vacuolar ATPase. The second Ca(2+)-ATPase was greatly retarded on the Sephacryl S-400 column and eluted after the main protein peak. It was not inhibited significantly by NO3- or DCCI. The second Ca(2+)-ATPase is a major component of ATP hydrolysis by the native membranes.

In vitro and in vivo phosphorylation of polypeptides in plasma membrane and tonoplast-enriched fractions from barley roots.

Phosphorylation of polypeptides in membrane fractions from barley (Hordeum vulgare L. cv CM 72) roots was compared in in vitro and in vivo assays to assess the potential role of protein kinases in modification of membrane transport. Membrane fractions enriched in endoplasmic reticulum, tonoplast, and plasma membrane were isolated using sucrose gradients and the membrane polypeptides separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis. When the membrane fractions were incubated with gamma-[(32)P]ATP, phosphorylation occurred almost exclusively in the plasma membrane fraction. Phosphorylation of a band at 38 kilodaltons increased as the concentration of Mg(2+) was decreased from millimolar to micromolar levels. Phosphorylation of bands at 125, 86, 58, 46, and 28 kilodaltons required millimolar Mg(2+) concentrations and was greatly enhanced by Ca(2+). When roots of intact plants were labeled with [(32)P]orthophosphate, polypeptides at approximately 135, 116, 90, 46 to 53, 32, 28, and 19 kilodaltons were labeled in the plasma membrane fraction and polypeptides at approximately 73, 66, and 48 kilodaltons were labeled in the tonoplast fraction. Treatment of the roots of intact plants with 150 millimolar NaCl resulted in increased phosphorylation of some polypeptides while treatment with 100 mm NaCl had no effect.

Calcium and proton transport in membrane vesicles from barley roots.

Ca(2+) uptake by membrane fractions from barley (Hordeum vulgare L. cv CM72) roots was characterized. Uptake of (45)Ca(2+) was measured in membrane vesicles obtained from continuous and discontinuous sucrose gradients. A single, large peak of Ca(2+) uptake coincided with the peak of proton transport by the tonoplast H(+)-ATPase. Depending on the concentration of Ca(2+) in the assay, Ca(2+) uptake was inhibited 50 to 75% by those combinations of ionophores and solutes that eliminated the pH gradient and membrane potential. However, 25 to 50% of the Ca(2+) uptake in the tonoplast-enriched fraction was not sensitive to ionophores but was inhibited by vanadate. The results suggest that (45)Ca uptake was driven by the low affinity, high capacity tonoplast Ca(2+)/nH(+) antiporter and also by a high affinity, lower capacity Ca(2+)-ATPase. The Ca(2+)-ATPase may be associated with tonoplast, Golgi or contaminating vesicles of unknown origin. No Ca(2+) transport was specifically associated with the distinct peak of endoplasmic reticulum that was identified by NADH cytochrome c reductase, choline phosphotransferase, and dolichol-P-man-nosyl synthase activities. A small shoulder of Ca(2+) uptake in the plasma membrane region of the gradient was inhibited by vanadate and erythrosin B and may represent the activity of a separate plasma membrane Ca(2+)-ATPase. Vesicle volumes were estimated using electron spin resonance techniques, and intravesicular Ca(2+) concentrations were estimated to be as high as 5 millimolar. ATP-driven uptake of Ca(2+) created 800- to 2000-fold concentration gradients within minutes. Problems in interpreting the effects of Ca(2+) on ATP-generated pH gradients are discussed and the suggestion is made that Ca(2+) dissipates pH gradients by a different mechanism than is responsible for Ca(2+) uptake into tonoplast vesicles.

Lipid Composition of Plasma Membranes and Endomembranes Prepared from Roots of Barley (Hordeum vulgare L.) : Effects of Salt.

Membrane fractions enriched in endoplasmic reticulum (ER), tonoplast and Golgi membranes (TG) and plasma membranes (PM) were prepared from barley (Hordeum vulgare L. cv CM 72) roots and the lipid compositions of the three fractions were analyzed and compared. Plants were grown in an aerated nutrient solution with or without 100 millimolar NaCl. Each membrane fraction had a characteristic lipid composition. The mole per cent of the individual phospholipids, glycolipids, and sterols in each fraction was not altered when roots were grown in 100 millimolar NaCl. The ER had the highest percentages of phosphatidylinositol and phosphatidylcholine of the three fractions (7 and 45 mole per cent, respectively, of the total lipid). The TG contained the highest percentage of glycosylceramide (13 mole per cent). The PM had the highest percentage of phosphatidylserine (3 mole per cent) and nearly equal percentages of phosphatidylethanolamine (15 mole per cent and phosphatidylcholine (18 mole per cent). The most abundant sterols in membranes prepared from barley roots were stigmasterol (10 mole per cent), sitosterol (50 mole per cent), and 24zeta-methylcholesterol (40 mole per cent of the total sterol). Salt-treated plants contained a slightly higher percentage of stigmasterol than controls. The percentage of stigmasterol increased with age and a simple cause and effect relationship between salt treatment and sterol composition was not observed.

Effect of temperature on the plasma membrane and tonoplast ATPases of barley roots : comparison of results obtained with acridine orange and quinacrine.

The effect of temperature on the rate of proton transport and ATP hydrolysis by plasma membrane (PM) and tonoplast (TN) ATPases from barley (Hordeum vulgare L. cv CM 72) roots were compared. Rates of proton transport were estimated using the fluorescent amine dyes quinacrine and acridine orange. The ratio between rate of transport and ATP hydrolysis was found to depend on the dye, the temperature, and the type of membrane. The PM ATPase had an estimated Arrhenius energy of activation (Ea) of approximately 18 kilocalories per mole for ATP hydrolysis, and the Ea for proton transport was best estimated with acridine orange, which gave an Ea of 19 kilocalories per mole. The TN ATPase had an Ea for ATP hydrolysis of approximately 10 kilocalories per mole and the Ea for proton transport was best estimated with quinacrine, which gave an Ea of 10 kilocalories per mole. Acridine orange did not give an accurate estimate of Ea for the TN ATPase, nor did quinacrine for the PM ATPase. Reasons for the differences are discussed. Because it was suggested (AJ Pope, RA Leigh [1988] Plant Physiol 86: 1315-1322) that acridine orange interacts with anions to dissipate the pH gradient in TN vesicles, the complex effects of NO(3) (-) on the TN ATPase were also examined using acridine orange and quinacrine and membranes from oats and barley. Fluorescent amine dyes can be used to evaluate the effects of ions, substrates, inhibitors, and temperature on transport but caution is required in using rates of quench to make quantitative estimates of proton fluxes.

Rapid induction of na/h exchange activity in barley root tonoplast.

Na(+)/H(+) exchange activity in barley (Hordeum vulgare cv CM-72) root tonoplast was induced by Na(+) even in the presence of inhibitors of protein synthesis. Induction occurred with a half-time of only 15 minutes. When salt-treated roots were transferred to a nutrient solution containing no Na(+), the activity disappeared with a similar time course. The data suggest that Na(+)/H(+) exchange was due to activation of an existing protein rather than to de novo protein synthesis.

The effects of salt stress on polypeptides in membrane fractions from barley roots.

Cell fractions enriched in endoplasmic reticulum, tonoplast, plasma membrane, and cell walls were isolated from roots of barley (Hordeum vulgare L. cv CM 72) and the effect of NaCl on polypeptide levels was examined by two-dimensional (2D) polyacrylamide gel electrophoresis. The distribution of membranes on continuous sucrose gradients was not significantly affected by growing seedlings in the presence of NaCl; step gradients were used to isolate comparable membrane fractions from roots of control and salt-grown plants. The membrane and cell wall fractions each had distinctive polypeptide patterns on 2D gels. Silver-stained gels showed that salt stress caused increases or decreases in a number of polypeptides, but no unique polypeptides were induced by salt. The most striking change was an increase in protease resistant polypeptides with isoelectric points of 6.3 and 6.5 and molecular mass of 26 and 27 kilodaltons in the endoplasmic reticulum and tonoplast fractions. Fluorographs of 2D gels of the tonoplast, plasma membrane, and cell wall fractions isolated from roots of intact plants labeled with [(35)S]methionine in vivo also showed that salt induced changes in the synthesis of a number of polypeptides. There was no obvious candidate for an integral membrane polypeptide that might correspond to a salt-induced sodium-proton anti-porter in the tonoplast membrane.

separation and Immunological Characterization of Membrane Fractions from Barley Roots.

Tonoplast and plasma membranes (PM) were isolated from barley roots (Hordeum vulgare L. cv California Mariout 72) using sucrose step gradients. The isolation procedure yielded sufficient quantities of PM and tonoplast vesicles that were sealed and of the right orientation to measure ATP-dependent proton transport in vitro. The proteins of the endoplasmic reticulum, tonoplast-plus-Golgi membrane (TG) and PM fractions were separated on sodium dodecyl sulfate gels, and immunoblots were used to test for cross-contamination between the fractions. Proteins that cross-reacted with antibodies to the PM ATPase from corn roots and Neurospora were greatly enriched in the PM fraction, as were proteins that cross-reacted with monoclonal antibodies to an arabinogalactan protein from the PM of tobacco cells. Proteins that cross-reacted with antibodies to the 58- and 72-kilodalton subunits of the tonoplast ATPase of red beet storage tissue were greatly enriched in the TG fraction. The results with immunoblots and enzyme assays indicated that there was little cross-contamination between the tonoplast and PM vesicles. The molecular weights and isoelectric points of the PM ATPase and the tonoplast ATPase subunits were also determined using immunoblots of two-dimensional gels of the PM and TG proteins.

NaCl Induces a Na/H Antiport in Tonoplast Vesicles from Barley Roots.

Evidence was found for a Na(+)/H(+) antiport in tonoplast vesicles isolated from barley (Hordeum vulgare L. cv California Mariout 72) roots. The activity of the antiport was observed only in membranes from roots that were grown in NaCl. Measurements of acridine orange fluorescence were used to estimate relative proton influx and efflux from the vesicles. Addition of MgATP to vesicles from a tonoplast-enriched fraction caused the formation of a pH gradient, interior acid, across the vesicle membranes. EDTA was added to inhibit the ATPase, by chelating Mg(2+), and the pH gradient gradually dissipated. When 50 millimolar K(+) or Na(+) was added along with the EDTA to vesicles from control roots, the salts caused a slight increase in the rate of dissipation of the pH gradient, as did the addition of 50 millimolar K(+) to vesicles from salt-grown roots. However, when 50 millimolar Na(+) was added to vesicles from salt-grown roots it caused a 7-fold increase in the proton efflux. Inclusion of 20 millimolar K(+) and 1 micromolar valinomycin in the assay buffer did not affect this rapid Na(+)/H(+) exchange. The Na(+)/H(+) exchange rate for vesicles from salt-grown roots showed saturation kinetics with respect to Na(+) concentration, with an apparent K(m) for Na(+) of 9 millimolar. The rate of Na(+)/H(+) exchange with 10 millimolar Na(+) was inhibited 97% by 0.1 millimolar dodecyltriethylammonium.

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

The effects of NO(3) (-) 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 KNO(3) when assayed at 24 degrees 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 KNO(3) 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 KNO(3) inhibited ATP hydrolysis by the tonoplast ATPase at 12 degrees C and the initial rate of proton transport was stimulated by 50 millimolar KNO(3). The time course for fluorescence quench indicated that addition of ATP in the presence of KNO(3) caused a pH gradient to form that subsequently collapsed. This biphasic time course for proton transport in the presence of KNO(3) was explained by the temperature-dependent delay of the inhibition by KNO(3). The plasma membrane H(+)-ATPase maintained a pH gradient in the presence of KNO(3) for up to 30 minutes at 24 degrees C.

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/cm(3)). Activities of NADH cytochrome (Cyt) c reductase, Ca(2+)-ATPase, and Mg(2+)-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 Ca(2+)-phosphatase was at the top of the gradient, NADH Cyt c reductase at 1.08 g/cm(3), a peak of ATP-dependent proton transport at 1.09 to 1.12 g/cm(3), a peak of nitrate-inhibited ATPase at 1.09 to 1.12 g/cm(3), and of vanadate-inhibited ATPase at 1.16 g/cm(3). The Ca(2+)-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 Ca(2+)-phosphatase was removed from the microsomes by washing with 150 millimolar KCl. The vanadate-sensitive ATPase required Mg(2+), 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.

Effect of Chilling Temperatures upon Cell Cultures of Tomato.

The effect of chilling temperatures upon cell cultures of tomato (Lycopersicon esculentum Mill cv ;VF36,' and cv ;VFNT Cherry,' and L. hirsutum Humb. & Bonpl.) was tested. Doubling times for L. esculentum were 2 to 3 days at 28 degrees C, and 3 to 8 days at 12 degrees C. No growth was observed at 8 degrees C, indicating an abrupt limit to growth between 8 and 12 degrees C. Fluorescein diacetate staining indicated that 80 to 90% of the cells were alive when cells were maintained at 8 degrees C for up to 2 weeks. When cultures kept at 8 degrees C for up to 30 days were transferred to 28 degrees C, growth resumed quickly, and at a rate virtually identical to that for unchilled cells. Similar results were found for cells maintained at 0 degrees C, and for cells of ;VFNT Cherry' and of L. hirsutum. Under certain conditions, cultures slowly doubled in fresh weight and cell volume at 8 or 9 degrees C but additional growth at 8 degrees C did not occur, nor could growth be maintained by subculture at 8 or 9 degrees C. The results are contrary to reports that cell cultures of tomato die when exposed to temperatures below 10 degrees C for 1 or 2 weeks. Our observations indicate that chilling temperatures quickly inhibit growth of tomato cells, but do not kill them.

Preparation of Membrane Vesicles Enriched in ATP-Dependent Proton Transport from Suspension Cultures of Tomato Cells.

Membranes enriched in ATP-dependent proton transport were prepared from suspension cultures of tomato cells (Lycopersicon esculentum Mill cv VF36). Suspension cultures were a source of large quantities of membranes from rapidly growing, undifferentiated cells. Proton transport activity was assayed as quench of acridine orange fluorescence. The activity of the proton translocating ATPase and of several other membrane enzymes was measured as a function of the cell culture cycle. The relative distribution of the enzymes between the 3,000, 10,000, and 100,000g pellets remained the same throughout the cell culture cycle, but yield of total activity and activity per gram fresh weight with time had a unique profile for each enzyme tested. Maximal yield of the proton translocating ATPase activity was obtained from cells in the middle logarithmic phase of growth, and from 50 to 90% of the activity was found in the 10,000g pellet. The proton translocating ATPase activity was separable from NADPH cytochrome c reductase and cytochrome c oxidase on a sucrose gradient. Proton transport activity had a broad pH optimum (7.0-8.0), was stimulated by KCl with a K(m) of 5 to 10 millimolar, stimulation being due to the anion, Cl(-), and not the cation, K(+), and was not inhibited by vanadate, but was inhibited by NO(3) (-). The activity is tentatively identified as the tonoplast ATPase.

Acclimation to low temperature by microsomal membranes from tomato cell cultures.

Sealed vesicles were prepared from microsomal membranes from cell suspension cultures of tomato (Lycopersicon esculentum Mill cv VF36). ATP-dependent proton transport activity by the vesicles was measured as quenching of fluorescence of acridine orange. Measurements of proton transport were correlated with the activity of a nitrate-inhibitable ATPase. The initial rate of proton influx into the vesicles was strongly temperature dependent with a Q(10) of 2 and a maximum rate near 35 degrees C. The data suggest that passive permeability did not increase at chilling temperatures but did increase rapidly with temperatures above 30 degrees C. A comparison was made between membranes from cell cultures grown at 28 degrees C and 9 degrees C. The temperature optimum for proton transport broadened and shifted to a lower temperature range in membranes from cells maintained at 9 degrees C.

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 Mg(2+)-ATPase activity of the sealed vesicles was stimulated by Cl(-) and NH(4) (+) 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 NO(3) (-), 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 Mg(2+), was stimulated by Cl(-), inhibited by NO(3) (-), 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 (Mg(2+) >/= Mn(2+) > Co(2+)) and were inhibited by high concentrations of Ca(2+). 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 F(1)F(0)-ATPase and from those of the K(+)-stimulated ATPase of corn root plasma membranes, and resembled those of the tonoplast ATPase.

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 beta-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.

Characterization of a partially purified adenosine triphosphatase from a corn root plasma membrane fraction.

The (K(+),Mg(2+))-ATPase was partially purified from a plasma membrane fraction from corn roots (WF9 x Mol7) and stored in liquid N(2) without loss of activity. Specific activity was increased 4-fold over that of the plasma membrane fraction. ATPase activity resembled that of the plasma membrane fraction with certain alterations in cation sensitivity. The enzyme required a divalent cation for activity (Co(2+) > Mg(2+) > Mn(2+) > Zn(2+) > Ca(2+)) when assayed at 3 millimolar ATP and 3 millimolar divalent cation at pH 6.3. When assayed in the presence of 3 millimolar Mg(2+), the enzyme was further activated by monovalent cations (K(+), NH(4) (+), Rb(+) >> Na(+), Cs(+), Li(+)). The pH optima were 6.5 and 6.3 in the absence and presence of 50 millimolar KCl, respectively. The enzyme showed simple Michaelis-Menten kinetics for the substrate ATP-Mg, with a K(m) of 1.3 millimolar in the absence and 0.7 millimolar in the presence of 50 millimolar KCl. Stimulation by K(+) approached simple Michaelis-Menten kinetics, with a K(m) of approximately 4 millimolar KCl. ATPase activity was inhibited by sodium orthovanadate. Half-maximal inhibition was at 150 and 35 micromolar in the absence and presence of 50 millimolar KCl. The enzyme required the substrate ATP. The rate of hydrolysis of other substrates, except UDP, IDP, and GDP, was less than 20% of ATP hydrolysis. Nucleoside diphosphatase activity was less than 30% of ATPase activity, was not inhibited by vanadate, was not stimulated by K(+), and preferred Mn(2+) to Mg(2+). The results demonstrate that the (K(+),Mg(2+))-ATPase can be clearly distinguished from nonspecific phosphohydrolase and nucleoside diphosphatase activities of plasma membrane fractions prepared from corn roots.

Solubilization and partial purification of the adenosine triphosphatase from a corn root plasma membrane fraction.

The K(+)-stimulated ATPase was partially purified from a plasma membrane fraction from corn roots (WF9 x Mo 17) by solubilization with 30 millimolar octyl-beta-d-glucopyranoside followed by precipitation with dilute ammonium sulfate. The specific activity of the enzyme was increased about five times by this procedure. The molecular weight of the detergent-extracted ATPase complex was estimated to be at least 500,000 daltons by chromatography on a Bio-Gel A-5m column. Negative staining electron microscopy indicated that the detergent-extracted material consisted of amorphous particles, while the ammonium sulfate precipitate was composed of uniform vesicles with an average diameter of 100 nanometers. The protein composition of the ammonium sulfate precipitate was significantly different from that of the plasma membrane fraction when compared by sodium dodecyl sulfate gel electrophoresis. The characteristics of the partially purified ATPase resembled those of the plasma membrane associated enzyme. The ATPase required Mg(2+), was further stimulated by K(+), was almost completely inhibited by 0.1 millimolar diethylstilbestrol, and was not affected by 5.0 micrograms per milliliter oligomycin. Although the detergents sodium cholate, deoxycholate, Triton X-100 and Lubrol WX also solubilized some membrane protein, none solubilized the K(+)-stimulated ATPase activity. Low concentrations of each detergent, including octyl-beta-d-glucopyranoside, activated the ATPase and higher concentrations inactivated the enzyme. These results suggest that the plasma membrane ATPase is a large, integral membrane protein or protein complex that requires lipids to maintain its activity.