Plasma membrane isolation from freshwater and salt-tolerant species of Chara: antibody cross-reactions and phosphohydrolase activities

Plasma membranes were isolated using the aqueous polymer two-phase partition method from the algae Chara corallina and Chara longifolia, algae which differ in their ability to grow in saline environments. Enrichment of plasma membrane and depletion of tonoplast relative to the microsomal fraction was monitored using phosphohydrolase assays and cross-reactions to antibodies raised against higher plant transporters. Antibodies to the vacuolar ATPase and pyrophosphatase cross-reacted with epitopes in the microsomal fraction, but showed little affinity for the plasma membrane fraction. Pyrophosphatase activity also declined in the plasma membrane fraction relative to the microsomal fraction. The V-type H(+)-ATPase activity, sensitive to nitrate or bafilomycin, was low in both fractions, though the cross-reaction to the antibody was reduced in the plasma membrane fraction. By contrast, the antibody recognition of a P-type H(+)-ATPase amino acid sequence from Arabidopsis did not occur strongly in the anticipated 90-100 kDa range. While there was enhanced recognition of a polypeptide at around 140 kDa in the plasma membrane fraction, salt treatment of Chara longifolia resulted in plasma membrane fractions with reduced amounts of this epitope, but no change in vanadate-sensitive ATPase activity, suggesting that it does not represent the only P-type ATPase. Microsomal membranes from salt-adapted C. longifolia have higher reactivity with the antibody to the tonoplast ATPase.     

Differential stimulation by Cl- of H+ transport and ATPase activity in purified plasma membrane vesicles from maize (Zea mays L.) roots

Maize (Zea mays L.) root plasma membranes purified by the aqueouspolymer two-phase technique have previously been shown to bevery low in tonoplast H⁺ -ATPase and H⁺ -PPase activities. Westernblots of a similar preparation showed that, compared to a microsomalfraction, there was practically no reaction with antibodiesto the tonoplast enzymes, but a strong reaction with an antibodyto the plasma membrane H⁺ -ATPase. Freeze/thaw treatment ofthe plasma membrane vesicles increased the proportion with aninsideout orientation to about 40%. This preparation was usedto demonstrate that substitution of KCl for K₂S0₄ resulted ina 14-fold stimulation of H⁺ transport, but an increase in ATPaseactivity of less than 10%. In contrast to its effect on tonoplastvesicles, Cl^– had only a small effect on the membranepotential of plasma membrane vesicles, assayed by oxonol V fluorescencequench recovery. To account for the apparent variability inthe H⁺/ATP coupling ratio, it may be necessary to devise a modelthat takes into consideration the possibility of non-linearbehaviour with respect to the membrane potential of the protonleak and/or of slip in the ATPase. Key words: ATPase, plasma membrane, anion stimulation, proton transport     

Comparison of Plasma Membrane and Tonoplast H+-Translocating ATPases in Phaseolus mungo L. Roots

Two membrane fractions were obtained from 16%/26% and 34%/40%interfaces following discontinuous sucrose density gradientcentrifugation of a 10,000–80,000xg pellet from mung bean(Phaseolus mungo L.) roots. The ATPases in the fractions differedfrom each other in their sensitivity toward various inhibitors,activation with salts, dependence of activity on pH, and K_mfor ATP.Mg²⁺. Judging from their sensitivity toward inhibitors,the ATPases in the low and high density membranes are consideredmainly of tonoplast and plasma membrane origin, respectively.Both ATPases were activated by gramicidin D and nigericin. ATP-inducedquenching of quinacrine fluorescence in both fractions requiredMg²⁺ and permeant anions such as Cl^– and quenching wascollapsed by carbonylcyanide p-trifluoromethoxyphenyl hydrazone.The sensitivities of quenching to the inhibitors were essentiallythe same as those of ATPase activity in the membranes. Thesefindings suggest the involvement of ATPases in H⁺-pumping acrossa plasma membrane and tonoplast. (Received April 12, 1985; Accepted October 11, 1985)     

Nitrate-induced polypeptides in membranes from corn seedling roots

The polypeptide composition of the membranes from corn (Zeamays L.) seedling roots upon nitrate induction was determinedby two-dimensional gel electrophoresis and silver-staining.The synthesis of five polypeptides (49, 48, 35, 33, and 32 kDa)in the tono-plast fraction and four polypeptides (50, 49, 38,and 33 kDa) in the plasma membrane fraction was induced by both2.5 mM Ca(NO₃)₂ and 5 mM KNO₃. Extensive washing of the membraneswith salt and NaOH demonstrated that three induced polypeptides(49, 48, and 35 kDa) in the tonoplast fraction and two inducedpolypeptides (49 and 33 kDa) in the plasma membrane fractionwere integral proteins. After incubation of seedlings in N-freemedium for 4 d, the 49 and 32 kDa polypeptides in the tonoplastfraction had disappeared. By the sixth day in N-free medium,the 35 kDa polypeptide had disappeared from the tonoplast fraction.The 50 kDa polypeptide of the plasma membrane fraction was nolonger detectable in seedlings incubated for 6 d in N-free medium.The size of the spots corresponding to the 33 kDa polypeptidesof both membrane fractions and to the 49 kDa polypeptide ofthe plasma membrane fraction was reduced following incubationof seedlings in N-free medium. The changes in nitrate-inducedpolypeptides in both membrane fractions following transfer toN-free medium correlated with a reduced capacity to take upnitrate in the treated seedlings. The results support the conclusionthat the nitrate-induced polypeptides may be involved in nitratetransport across the tonoplast and plasma membrane. Key words: Nitrate transport, induction, membrane peptides     

Na+ fluxes in Chara under salt stress

The influx and efflux of Na⁺ across the plasma membrane of Characorallina and Chara longifolia were examined under mild saltstress conditions. Na⁺ influx was found to be rapid in bothspecies with the freely exchangeable cytoplasmic Na⁺ cominginto isotopic equilibrium with external ²²Na⁺ within 1 h ofexposure to isotope. Cytoplasmlc Na⁺ concentration and Na⁺ influxwere greater in C. corallina than in C. longifolla under thesame conditions. Na⁺ influx across the tonoplast was much lowerthan the flux across the plasma membrane. Na⁺ efflux was stimulatedat pH 5 relative to pH 7 by 218% in C. coralllna and 320% inC. longifolia. In both species externally applied Li⁺ inhibitedNa⁺ efflux at pH 5 but not at pH 7. Na⁺ etflux was not significantlyinhibited by amiloride. Key words: Na⁺ influx, Na⁺ efflux, Na⁺/H⁺ antiport, Chara     

Characterization of ATPases Associated with Various Cellular Membranes Isolated from Etiolated Hypocotyls of Vigna radiata (L.) Wilczek

Cellular membrane fractions, including endoplasmic reticum (ER),Golgi-enriched membrane, plasma membrane and tonoplasts, wereisolated from Vigna radiata seedlings. Each of these membranefractions was associated with specific ATPases which were highlydependent on Mg²⁺. ATPases of ER, Golgi-enriched membrane andplasma membrane were sensitive to vanadate but the tonoplastATPase was not. ATPases were mostly dependent on Cl¹, but aslight stimulation by K⁺ was observed in the case of ATPasesof Golgi-enriched membrane and plasma membrane. KNO₃ inhibitedtonoplast ATPase but stimulated the other ATPases. ER ATPasecan be distinguished from other ATPases by the following characteristics:specific inhibition by KNO₂ and Triton X-100, stimulation bylow concentrations of diethylstilbestrol and 4,4'-diisothiocyanostilbene-2,2'-disulfonicacid, and high sensitivity to heat. The ATPases showed typicalMichaelis-Menten kinetics and had K_m values of 0.5 to 0.6 ITIMMg²⁺-ATP for ER, Golgienriched-membrane and tonoplast ATPases,and 2.27 msi Mg²⁺-ATP for plasma membrane ATPase. ATPases ofGolgi-enriched membranes and plasma membranes had similar properties,but they were still distinguishable by the differences in theirK_m values and their responses to Triton X-100. Based on theseresults, it is postulated that each cellular membrane is associatedwith a specific ATPase in cells of V. radiata. ¹Contribution No. 3171 from the Institute of Low TemperatureScience. (Received April 22, 1988; Accepted September 28, 1988)     

Effects of Gamma-Irradiation on the Activity of Tonoplast H+-ATPase from Potato Tubers (Solanum tuberosum L.)

Tonoplast vesicles were prepared from potato tubers (Solariumtuberosum L.) on a step gradient (0% and 6%, w/w) of dextranT-70 to clarify the mechanism by which the tonoplast H⁺-ATPaseis inactivated by gamma-irradiation. H⁺-ATPase activity andH⁺ -pumping were examined after irradiation of tubers (in vivoirradiation) and of isolated tonoplast vesicles (in vitro irradiation)at doses up to 1.0 kGy. Both in vivo irradiation and in vitroirradiation resulted in significant decreases in ATPase andH⁺-pumping activities. The ATPase and H⁺-pumping activities12 h after irradiation were much lower than those 2 h afterirradiation. Solubilized H⁺-ATPase was inactivated, in a dose-dependentmanner, by irradiation (enzyme irradiation) to a greater extentthan was observed after in vitro irradiation or in vivo irradiation.The activity of ATPase 12 h after enzyme irradiation was almostthe same as it was 2 h after enzyme irradiation. The free fattyacid content of vacuolar membranes was increased by in vivoirradiation and by in vitro irradiation with an accompanyingdecrease in tonoplast H⁺-ATPase activity. Lipids from irradiatedtonoplasts had a considerable inhibitory effect on the activityof solubilized H⁺-ATPase. This result suggests that the directinactivation of H⁺-ATPase in potato tonoplast by gamma-irradiationis augmented by the effects of deterioration of membrane lipidsthat is induced by the irradiation. (Received December 21, 1994; Accepted May 16, 1994)     

Reconstitution of Tonoplast H+-ATPase from Mung Bean (Vigna radiata L.) Hypocotyls in Liposomes

Reconstituted proteoliposomes of tonoplast ATPase are formedon solubilization of tonoplast membranes from mung bean (Vignaradiata L.) with deoxycholate (DOC) in the presence of a mixtureof soybean phospholipids (asolectin), after removal of DOC bypassage through a PD-10 column (Pharmacia). This method is idealbecause of its simplicity and rapidity. Selective insertionof sets of tonoplast H⁺-ATPase polypeptides (68 kDa, 60 kDa,16 kDa and several minor polypeptides) into liposomes usingthis method was confirmed by SDS-PAGE and immuno-blotting withantibodies raised against 68-kDa and 60-kDa polypeptides. Pumping of protons across the membranes of the proteoliposomeswas demonstrated by quinacrine-fluorescence quenching in thepresence of ATP-Mg²⁺. ATP-Mg²⁺ was shown to be the preferredsubstrate in both reconstituted and native tonoplast vesicles,and its optimum concentration was 0.75 to 3.0 mM. Quenchingwas completely abolished by a channel-forming ionophore, gramicidinD, and an inhibitor of tonoplast H⁺-ATPase, KNO₃. Antibodiesto 68-kDa and 60-kDa peptides partially inhibited the pumpingof protons. The rate of pumping of protons increased with thenumber of proteoliposomes, the maximal concentration of whichwas equivalent to 250 µg of protein per reaction mixture.The optimum pH for pumping was 6.5 when inside of proteoliposomeswere loaded pH at 7.2. The rate of pumping of protons was reducedwhen proteoliposomes were made using asolectin and cholesterolat 3 : 1 (w/w), as compared with those made with asolectin alone. The ATPase activity in reconstituted proteoliposomes was inhibitedby KNO₃, with half-maximal inhibition at approximately 7 mM.The enzyme actively hydrolyzed ATP in preference to GTP, CTP,UTP, and ADP, but it did not hydrolyze pNPP or AMP. Antibodiesagainst the 60-kDa polypeptide strongly inhibited ATPase activityas compared to antibodies against the 68-kDa polypeptide. Theresults obtained in this study demonstrate directly that functionaltonoplast H⁺-ATPase can be inserted selectively into liposomes. (Received August 31, 1990; Accepted April 18, 1991)     

Separation of K+-and Cl --selective ion channels from rye roots on a continuous sucrose density gradient

Microsomal membranes from rye (Secale cereale L.) roots wereseparated by isopycnic sucrose density gradient centrifugation.The ion channels present in gradient fractions were assayedby reconstitution into planar 1-palmitoyl-2-oleoyl phosphatidylethanolaminebilayers (PLB) and the distributions of ion channel activitieswere compared with membrane markerenzyme activities. A numberof ion channel activities were observed and could be distinguishedon the combined bases of their conductance, selectivity, kineticsand pharmacology. A voltage-dependent maxi (498 pS) cation-channel,a voltage-dependent 199-pS cationchannel, 48-pS and 18-pS K⁺channels, and a 148-pS Cl^– channel (all unitary conductancesdetermined in asymmetrical cis trans 325:100mM KCl) colocalizedwith the plasma membrane marker-enzyme, vanadatesensitive ATPase.A weakly K ⁺-selective (108 pS) channel, a 1249-pS cation-channeland a 98-pS K ⁺ channel colocalized with the tonoplast markerenzyme,nitrate-sensitive ATPase. A 706-pS K⁺ channel colocalized withthe expected distribution of intact plastids and a 38-pS Cl^–channel colocalized with either plastid or ER membranes. Themembrane location of several other channels including a hypervoltage-sensitivemaxi (497 pS) cation-channel, a 270-pS K⁺ channel, an 8-pS K⁺channel and a 4-pS K⁺ channel was equivocal, but they were tentativelyassigned to the Golgi. Thus, the plasma membrane and tonoplastorigin of ion channels previously characterized following theincorporation of plasma membrane prepared by aqueous-polymertwo-phase partitioning or tonoplast derived from isolated vacuolesinto PLB was confirmed and the ion channel complement of previouslyunassayed membranes was defined. This demonstrates the usefulnessof PLB in identifying and characterizing ion channels from plantcell membranes, in particular, those of membranes which areinaccessible to patch-clamp electrodes. Key words: Chloride (Cl^–) channel, potassium (K⁺) channel, planar lipid bilayer, root, rye, Secale cerealeL.     

A Comparison Between the ATPase and Proton Pumping Activities of Plasma Membranes Isolated from the Stele and Cortex of Zea mays Roots

Plasma membrane vesicles of high purity, determined by markerenzyme assays, were obtained by phase partitioning microsomalfractions from stelar and cortical tissues of Zea mays (cv.LG11) roots. ATP hydrolytic activities in both of the plasmamembrane fractions were inhibited by vanadate, SW26 and erythrosinB, but were insensitive to nitrate. Activity in both fractionsexhibited a marked pH optimum of 6·5 and displayed typicalMichaelis-Menten kinetics. A high substrate specificity wasapparent in both the stele and cortex plasma membrane fractions,while the lower fractions, after phase partitioning, showedlower specificity for nucleotide substrates. Specific activitiesof the stele (67·8 µmol Pi mg^–1 h^–1)and cortex (78·4 µmol Pi mg^–1 h–¹)plasma membrane H⁺ -ATPases were very similar. Proton pumping activities in microsomal membrane fractions fromstele and cortex were inhibited by nitrate and insensitive tovanadate. Homogenization of stele and cortex tissue in the presenceof 250 mol m^–3 KI resulted in microsomal fractions exhibitingvanadate-sensitive, nitrate-insensitive proton pumping activity,suggesting a plasma membrane origin for this activity. SW26was also an effective inhibitor of proton pumping activity,although results indicated an interaction between SW26 and thefluorescent probes quinacrine and acridine orange. The results are discussed in relation to models for the transportof ions into the stele and are consistent with a role for theH⁺ -ATPase activity in this process. Key words: ATPase, cortex, plasma membrane, stele, Zea mays     

Subcellular Distribution of Basal and Cation-sensitive ATPases in Roots of Phaseolus vulgaris

A smooth microsomal fraction isolated from homogenates of Pbaseolus vulgaris root tissue has been found to possesss a highly active basal ATPase (measured in the absence of added cations). The microsomal membranes also feature a cation-sensitive ATPase which responds to Mg²⁺, Na⁺ and K⁺, but in a manner that is highly variable with pH. In contrast, membrane fragments prepared by a technique designed to yield purified plasma membrane were capable of little or no hydrolysis of ATP either in the presence or absence of added cations. This suggests that the microsomal activity is a reflection of membrane-bound ATPase which has been derived from cytoplasmic membranes, possibly the tonoplast, rather than plasma membrane.     

Presence of a Na+-activated ATPase in the Plasma Membrane of the Marine Raphidophycean Heterosigma akashiwo

Highly purified plasma membranes were isolated from Heterosigmaakashiwo cells, a marine raphidophycean unicellular biflagellate,by the silica microbead method, and the ATPase activity of themembranes was characterized. The ionic requirements and spectrumof effective inhibitors enable us to identify a novel Na⁺-activatedATPase in the plasma membrane of this organism. Furthermore,we detected two phosphorylated intermediate forms of ATPases,with molecular weights of 150 kDa and 95 kDa as judged by acidSDS-polyacrylamide gel electrophoresis of extracts of isolatedplasma membrane. The 150 kDa intermediate was phosphorylated in the presenceof both Mg²⁺ and Na⁺, while the 95 kDa intermediate was phosphorylatedin the presence of Mg²⁺ alone. Both were dephosphorylated inthe presence of monovalent cations. These results indicate thatthe former intermediate was a Na⁺-activated ATPase, similarto Na⁺,K⁺-ATPases from animals, and the latter was similar toH⁺,K⁺-ATPases from higher plants. The physiological significanceof the two kinds of ATPase in the plasma membrane of marinealgae. (Received March 15, 1989; Accepted June 23, 1989)     

Isolation of Smooth Endoplasmic Reticulum and Tonoplast from Mung Bean Hypocotyls (Vigna radiata [L.] Wilczek) Using a Ficoli Gradient and Two-Polymer Phase Partition

A new method for the isolation of smooth endoplasmic reticulumand tonoplast from etiolated mung bean hypocotyls (Vigna radiata[L.] Wilczek) has been developed. After centrifugation in aFicoli density gradient [5.5% (w/w) in 15% (w/w) sucrose] ofa crude microsomal membrane fraction (10,000–156,000?gpellet) which had been prepared and resuspended in buffer systemsthat contained 0.25 M sorbitol, more than 80% of the total amountsof smooth endoplasmic reticulum and tonoplast were co-bandedat the interface between the sample load and the Ficoll layer,while most of the other cellular membranes, including plasmamembrane, Golgi membranes and yellow-colored membrane materials,which were presumably the etioplast envelopes, were sedimentedthrough the Ficoli layer. Smooth endoplasmic reticulum and tonoplastwere separated from each other to a high degree of enrichmentby a subsequent two-polymer phase partitioning. The separationis based on the principle that mung bean tonoplast has a highpartition coefficient for the polyethylene glycol-enriched upperphase and the smooth endoplasmic reticulum has a high partitioncoefficient for the Dextran-enriched lower phase. Assessed interms of degree of contamination by activities of membrane markerenzymes, the isolated smooth endoplasmic reticulum and tonoplastfractions were found to be highly purified. An ATPase sensitiveto neutral detergent and vanadate was found to be specificallyassociated with the smooth endoplasmic reticulum. ¹Contribution No. 3172 from the Institute of Low TemperatureScience (Received April 22, 1988; Accepted September 28, 1988)     

The Permeability of the Guard Cell Plasma Membrane and Tonoplast

Uptake experiments and efflux compartmental analysis of planthormones, osmotica and toxins using ‘isolated’ guardcells of Valerianella locusta and guard cell protoplasts (GCP)of Vicia faba were performed in order to study the permeabilityproperties of guard cell plasma membrane and tonoplast. Theplasma membrane of guard cells exhibits a higher permeabilitythan plasma membranes of mesophyll cells for most solutes investigated.The permeability coefficients (P_s calculated for the guard cellplasma membranes are also significantly higher than the P_s valuesfor the guard cell tonoplast. This applies also for protonatedABA. We suppose that the high permeability for ABAH could bepart of the target cell properties. A Collander analysis demonstratesa linear correlation between P_s, values and the ratio K_r/M_r^1,5for both plasma membrane (r = 0.87) and for the tonoplast (r=0.93). Because of deviations from the observed correlations,the permeation of some solutes (ABA, GA, IAA through the tonoplast;methylamine through the plasma membrane) seems to be facilitatedby an additional transport mechanism. The Collander analysisof the plasma membrane of GCP shows very similar results tothe analysis of the plasma membrane of ‘isolated’guard cells, indicating that isolation of protoplasts does notalter the permeability of the guard cell plasma membrane. Key words: Permeability coefficient, guard cells, plasma membrane, tonoplast     

H-ATPase Activity from Storage Tissue of Beta vulgaris: III. Modulation of ATPase Activity by Reaction Substrates and Products

Two distinct membrane fractions containing H⁺-ATPase activity were prepared from red beet. One fraction contained a H⁺-ATPase activity that was inhibited by NO₃⁻ while the other contained a H⁺-ATPase inhibited by vanadate. We have previously proposed that these H⁺-ATPases are associated with tonoplast (NO₃⁻-sensitive) and plasma membrane (vanadate-sensitive), respectively. Both ATPase were examined to determine to what extent their activity was influenced by variations in the concentration of ATPase substrates and products. The substrate for both ATPase was MgATP²⁻, and Mg²⁺ concentrations in excess of ATP had only a slight inhibitory effect on either ATPase. Both ATPases were inhibited by free ATP (i.e. ATP concentrations in excess of Mg²⁺) and ADP but not by AMP. The plasma membrane ATPase was more sensitive than the tonoplast ATPase to free ATP and the tonoplast ATPase was more sensitive than the plasma membrane ATPase to ADP.

Inhibition of both ATPases by free ATP was complex. Inhibition of the plasma membrane ATPase by ADP was competitive whereas the tonoplast ATPase demonstrated a sigmoidal dependence on MgATP²⁻ in the presence of ADP. Inorganic phosphate moderately inhibited both ATPases in a noncompetitive manner.

Calcium inhibited the plasma membrane but not the tonoplast ATPase, apparently by a direct interaction with the ATPase rather than by disrupting the MgATP²⁻ complex.

The sensitivity of both ATPases to ADP suggests that under conditions of restricted energy supply H⁺-ATPase activity may be reduced by increases in ADP levels rather than by decreases in ATP levels per se. The sensitivity of both ATPases to ADP and free ATP suggests that modulation of cytoplasmic Mg²⁺ could modulate ATPase activity at both the tonoplast and plasma membrane.

     

Proton transport and phosphorylation of tonoplast polypeptides from zucchini are stimulated by the phospholipid platelet-activating factor

The ether phospholipid platelet-activating factor and certain similar phospholipids, including lysophosphatidylcholine, are known to stimulate both H⁺ transport and protein phosphorylation in plant microsomal membranes. In the present work, several polypeptides in highly purified tonoplast membranes from zucchini (Cucurbita pepo L.) showed platelet-activating factor-dependent phosphorylation. Comparison of protein phosphorylation in different membrane fractions separated by sucrose step density gradient centrifugation indicated that some of the phosphoproteins were contaminants or were common to several membrane fractions, but platelet-activating factor-dependent phosphorylation of peptides at 30, 53, and perhaps 100 kilodaltons was tonoplast specific. The phosphoprotein of 53 kilodaltons was shown by three different approaches (one- and two-dimensional polyacrylamide gel electrophoresis, western blots, and immunoprecipitation) to cross-react with antibody raised against the B subunit of the tonoplast ATPase from red beet (Beta vulgaris L.).     

Isolation and Characterization of the Plasma Membrane by Two-Phase Partitioning from the Alkalophilic Cyanobacterium Spirulina platensis

Partition in aqueous dextran-polyethylene glycol two-phase systemwas used to isolate the plasma membranes from the alkalophiliccyanobacterium Spirulina platensis. The upper phase containeda colorless membranes obtained in relatively short time, 3–4h. This fraction had a different protein profile than that ofthe thylakoid fraction obtained in the lower phase. It did notcontain cytochrome c-oxidase activity, but retained characteristicMg²⁺-ATPase activity that is sensitive to vanadate, stimulatedby K⁺, and has a pH optimum near 8.5. These data support ourassumption that the upper phase of the gradient consist of theplasma membrane of S. platensis. (Received November 25, 1993; Accepted April 12, 1994)     

Endoplasmic reticulum Ca(2+)-ATPase inhibitors stimulate membrane guanylate cyclase in pancreatic acinar cells

In this study, we show thatparticulate guanylate cyclase (GC) is present in rat pancreatic acinarcells and is located both on plasma membrane and membranes ofendoplasmic reticulum (ER). Western blot analysis indicates that theenzyme isoform GC-A is present in the acinar cell membranes. Thespecific inhibitors of ERCa²⁺-ATPase thapsigargin,2,5-di-(t-butyl)-1,4-hydroquinone(BHQ), and cyclopiazonic acid all activated particulate GC inpancreatic acini, both in membrane fractions and intact cells. Theseinhibitors also induced dephosphorylation of GC. Dose dependencies ofCa²⁺-ATPase inhibition and GCactivation by BHQ are very similar, and those for thapsigarginpartially overlap. ER Ca²⁺-ATPaseand GC are coimmunoprecipitated both by antisera against membrane GCand by antisera against ERCa²⁺-ATPase, suggesting a physicalassociation between the two enzymes. The results suggest thatthapsigargin and the other inhibitors act through ERCa²⁺-ATPase to activate membraneGC in pancreatic acinar cells, although their direct effect on GCcannot be excluded.

     

Selective Effect of Salt Stress on the Activity of Two ATPases in the Cell Membrane of Nostoc muscorum

ATPase activity in the cell membrane of a salt-stressed cyanobacterium,Nostoc muscorum M-14, was examined cytochemically by three differentstaining protocols. Application of Hulstaert's method resultedin distinct precipitation of the reaction products of ATPaseinside the cell membrane exclusively. No reaction products wereformed when ATP was replaced by GTP or when dicyclohexylcarbodiimideor N-ethylmaleimide was present in the reaction mixture. Bycontrast, low levels were detectable after the reaction in thepresence of ouabain. Bafilomycin did not affect the formationof products. Mayahara's method, which is considered to demonstratethe reaction of Na⁺,K⁺-ATPase activity, revealed the presenceof a ouabain-sensitive Na⁺,K⁺-ATPase in the cell membrane, whileWachstein-Meisel's method revealed the presence of an ATPaseactivity that was resistant to ouabain. It appears, therefore,that cell membranes of Nostoc muscorum contain both ouabain-sensitiveATPase and ouabain-insensitive ATPase. Comparison of the stainingprofiles of salt-stressed cells with those of control cellssuggested that a high-salt environment activates the ouabain-sensitiveNa⁺,K⁺-ATPase, which seems likely to be involved in the effluxof Na⁺ ions. (Received February 7, 1995; Accepted August 9, 1995)     

Monoclonal antibody TOP 71 recognizes a tonoplast protein of wide distribution in the plant kingdom

Highly purified tonoplast and plasma-membrane vesicles isolated from roots of Lepidium sativum L. (garden cress) were used as a starting material for generating a monoclonal antibody against plant tonoplast. Tonoplast vesicles were isolated by discontinuous-sucrose-gradient centrifugation followed by free-flow electrophoresis. The deglycosylated tonoplast fraction was used to generate monoclonal antibodies by immunization of Balb/c-mice and by fusion of their -lymphocytes with the mouse cell line X 63 Ag 8.653. Using plasma membrane purified by two-phase partitioning and freeflow electrophoresis to define the negative signal in screening, and purified tonoplast to define the positive signal in screening, a monoclonal antibody (TOP 71) was obtained which recognized a tonoplast protein of 71 kDa by immunoblotting in cress-root membrane fractions. Two-dimensional gel electrophoresis, affinoblotting and binding to concanavalin A showed that the TOP 71-antigen was a glycosylated protein and had an isoelectric point (pI) of 4.5. The TOP 71-antigen was found in the different tissues of organs of several higher plants (Glycine max L., Curcurbita pepo L., Zea mays L.) where it did not cross-react with the purified plasma-membrane fractions of these plants. Additionally, TOP 71 recognized its antigen in microsomal fractions of two lower plants (Chara globularis Thuili., Matteucia struthiopteris Tod.).Abbreviations ELISA enzyme-linked immunosorbent assay - FFE free-flow electrophoresis - IEF isoelectric focusing - MAB monoclonal antibody - P_FFE purified plasma membrane after FFE - pI isoelectric point - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis - T_gr tonoplast-enriched fraction (gr = gradient) - T_FFE purified tonoplast after FFE We thank I. Hartmann for technical assistance, R. Görlich (Institut für Landwirtschaftliche Botanik, Universität Bonn, Bonn, FRG) for advice on hybridoma techniques, M.F. Manolson (Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pa., USA) for the gift of the anti-A subunit-ATPase antibody, and R. Liedtke, H. Geithmann, and A. Heppekausen for preparation of figures. This work was financially supported by the Deutsche Forschungsgemeinschaft and the Bundesministerium für Forschung und Technologie.