Proton-pumping activities of soybean (Glycine max L.) root microsomes: Localization and sensitivity to nitrate and vanadate

Plant Science Letters, 36 (1984) 187--193 Elsevier Scientific Publishers Ireland Ltd.

187

PROTON-PUMPING ACTIVITIES OF SOYBEAN (GLYCINE M A X L.) ROOT MICROSOMES: LOCALIZATION AND SENSITIVITY TO NITRATE AND VANADATE*

ROGER

R. L E W and R O G E R

M. S P A N S W I C K

Section of Plant Biology, Division of Biological Sciences, Plant Science Building, Cornell University, Ithaca, N Y 14853 (U.S.A.) (Received February 22nd, 1984) (Revision received June 18th, 1984) (Accepted June 25th, 1984) Soybean (Glycine max L. cv. Williams '79) root microsomal suspensions exhibit two proton-pumping activities. The largest proportion o f activity, localized at a low density on sucrose gradients and inhibited by nitrate, does not correspond to markers for Golgi, endoplasmic reticulum, plasma membrane, or mitochondria and presumably originates from the tonoplast. There is a small shoulder of proton-pumping activity, which is sensitive to vanadate, localized at a higher density on gradients (37%, w/w). This activity corresponds fairly well with vanadate-sensitive ATPase activity (39%, w/w) and a shoulder of glucan synthetase II activity (39%, w/w). The main peak of glucan synthetase II activity co~quilibriates with the mitochondrial marker cytochrome c oxidase at 43% (w/w). It is likely that this activity originates from the plasma membrane.

Key words: soybean root; microsomes; proton-pumping; tonoplast; plasma membrane

Introduction A number of recent reports have documented the presence of vanadate-sensitive, ATP-dependent proton-pumping activity in microsomal fractions of a variety of higher plant tissues which is separable from nitratesensitive activity on the basis of density [1--3 ]. These activities may represent electrogenic proton pumps which have been postulated to exist at both the plasma membrane and the tonoplast of plant cells, and which may generate a proton electrochemical gradient which 'drives' the transport of ions *This research was supported b y NSF grant PCM 8111007 to R.M.S. and a NSF graduate fellowship to R.R.L. Abbreviations: BTP, 1,3-bis [ tris(hydroxymethyl )m e t h y l a m i n o ] p r o p a n e ; EGTA, ethyleneglycol-bis(2-aminoethylether)N,N,N',N'-tetraacetic acid; FCCP, carbonyl cyanide p-trifluoro methoxyphenylhydrazone; lyso-PE, lyso-phosphatidylethanolamine; MES, 2(N-morpholino)ethanesulfonic acid; TLC, thin-layer chromatography.

and small molecules [4]. We have previously studied the electrogenicity of soybean roots, examining the effect of various inhibitors and determining the ATP
Microsomal suspension preparation Soybean seeds (Glycine max L. cv. Williams '79) were surface sterilized by treatment for 2 min in 0.05% (w/v) sodium hypochlorite containing about 1% (v/v) ethanol. They were rinsed thoroughly in distilled deionized water, placed between two sheets of well-moistened germination paper (Anchor Paper, S t . Paul, MN), in trays covered with aluminum foil

0304-4211/84/$03,00 © 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

188 and incubated in the dark at 25°C. Three
medium was used. This was homogenized for 15 s at setting 8 with the polytron. The filtered homogenate was underlain with 5 ml each of 20, 34, and 42% (w/w) sucrose in 2 SW 27 tubes, and centrifuged for 75 min at 85 000 × g. The 20/34 and 34/42% (w/w) sucrose interfaces were removed with a bent tip pasteur pipette, diluted with 20 ml of the grinding medium and centrifuged at 85 000 × g for 20 min in a Beckman 50.2 Ti fixed angle rotor. The pellets were resuspended in 0.5 ml o f grinding medium (a protein concentration o f about 10 mg/ml).

Total lipid extraction and thin-layer chromatography (TLC) Total lipid extracts were prepared by homogenizing the microsomal pellets in 5 ml of chloroform/methanol ( 2 : 1 ) using a 10-ml capacity tissue homogenizer with a teflon pestle, t h e n following standard procedures [7]. One notable modification was the use of 0.73% (w/v) NaC1 which was acidified with HC1 (to allow better recovery of acidic phospholipids [8] ) for the phase separation. The lipid extract was dissolved in benzene and stored at - 2 0 ° C until TLC analysis. Two-dimensional chromatography was done with LK-6 linear K silica gel plates with a preabsorbent area (Whatman Chemical Separation Inc., Clifton, NJ 07014). About 2 mg of the total lipid extract was applied to the plate. The first development used chloroform/ methanol/water (65 : 2 5 : 4 ) (v/v/v); the second development used chloroform/acetone/ methanol/acetic acid/water (100 : 40 : 20 : 20 : 10) (v/v/v/v/v). Identification ofphospholipids was based on the use of m o l y b d e n u m blue reagent spray for phosphorus [9], ninhydrin for amines [9], and 40% (v/v) sulfuric acid for all lipids, and on comparison of Rf~values with standards obtained from Sigma Chemical Co. (St. Louis, MO 63178) or Avanti Polar Lipids Inc. (Birmingham, AL 35216). Phospholipid composition was determined using sulfuric acid digestion of the phospholipid spots scraped from the TLC plates [9] following the procedure o f Bartlett [10].

189 were t hen washed three times with 4 ml of 70% (v/v) ethanol. T he filters were placed in vials containing 9 ml o f scintillation cocktail (Liquiscint, National Diagnostics, NJ 08876) and c o u n t e d for radioactivity using a Beckman LS-100 liquid scintillation counter. T he fluorescent p r o b e quinacrine was used to measure p r o t o n - p u m p i n g activity. An aliquot o f vesicles was added to an assay buffer containing 250 mM sorbitol, 25 mM BTP/MES (pH 6:5), 5 mM MgSO4, 0.01 mM quinacrine, and either 50 mM KC1 or KNO3 (final volume 1.5 ml). After t e m p e r a t u r e equilibration at 30°C in a Perkin-Elmer Model 650-10S fluorescence s p e c t r o p h o t o m e t e r , a 15-pl aliquot o f 0.5 M Na2ATP was added. T he initial rate of the decrease in fluorescence was used as a measure o f p r o t o n - p u m p i n g activity [ 15 ]. Protein was d e t e r m i n e d according t o Bradford [ 16 ].

Assays

ATPase assays were p e r f o r m e d as previously described [5]. L a t e n t UDPase (a biochemical marker for Golgi) was assayed essentially as in R e f 11. Aliquots (5 ul) o f the gradient fractions were pre-incubated for 20 min at r o o m temperature in t h e reaction m i x t u r e (containing 3 mM MnSO4, 60 mM Tris--MES, pH 6.5) with or w i t h o u t T r i t o n X-100 (0.03%) (w/v), t h e n 3 mM Tris--UDP (prepared using Dowex) was added, incubated at 30°C f or 20 min, and t h e reaction stopped by adding Ames reagent [12] containing sodium lauryl sulfate. As20 nm was measured 20 rain later. C y t o c h r o m e c oxidase and c y t o c h r o m e c reductase were assayed as in Ref. 13 with modifications as in Ref. 14. Glucan synthetase II was assayed as in Ref. 14. Briefly, a 50~al aliquot f r om t h e linear gradient fractions was m i xed with 80 pl o f 24 mM Tris (pH 8.0), 210 nCi [glucose-~4C (U)] UDP (New England Nuclear, Boston, MA 02118), and 0.77 mM UDP-glucose. This m i x t u r e was incubated for 15 min at 25°C with gentle shaking. T he reaction was s t oppe d b y heating th e reaction m i x t u r e t o 100°C, 4 ml of 70% (v/v) ethanol was added, and the m i x t u r e was stored at 4°C f or 12 h. T h e reaction mixtures were t h e n filtered using Gelman t y p e A/E glass fiber filters which

Results and discussion

Preparation o f microsomal suspensions which exhibit p r o t o n - p u m p i n g activity is difficult since t he microsomal vesicles must be intact and must be oriented in the p r o p e r direction. In soybeans, certain factors appeared to be i m p o r t a n t for t he preparation o f intact

Table I. Effect of grinding medium on proton-pumping activity and phospholipid composition of soybean root microsomes. The grinding medium consisted of 0.25 M sucrose, 25 mM MES, 2.5 mM dithiothreitoi, 1% (w/v) bovine serum albumin pH adjusted to 7 with KOH, plus the additions noted below (numbers indicate mM or % (v/v) in the case of glycerol and methanol). Microsomal pellets from the same preparation were either resuspended in suspension medium and assayed for proton-pumping or a total lipid extract prepared for phospholipid composition determination as described in the methods. Grinding medium

% quenching min-' (0.1 mg protein)-'

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32

190

wesicles. Roots excised into distilled deionized water containing CaC12 did not exhibit protonpumping activity. Using a molar ratio of chelate (either EDTA or EGTA) to divalent cation (MgSO4) of 1 in the grinding medium gave much greater activity than a ratio of 2 (Table I). This was true whether 10 mM chelate/10 mM Mg2÷ or 5 mM concentrations of each were used. Glycerol and methanol [17] were added based on the premise that they 'protect' the membranes during isolation. In fact, activities were greater when they were present (Table I). The glycerol- and methanolinduced increases in proton-pumping activity are additive in other plant tissue (we have not examined this using soybean roots). Protonpumping activity survived a freeze-thaw cycle when membrane vesicles are isolated as described. One possible cause of variation in protonpumping activity is the production of lyotropic membrane components during homogenization and isolation. Soybean root microsomes contain relatively high levels of the lyotropic phospholipid lyso-phosphatidylethanolamine (I0--13% of phospholipid phosphate) [cf. 18], so we examined changes in phospholipid composition with the different homogenizing media described above. There were no quantitative differences in phospholipid composition (Table I). Nor were there qualitative differences in sulfuric acid charred Table II. Effect of lyso-PE on proton-pumping activity o f soybean root microsomes. Aliquots of 5 mg/ml lyso-PE in chloroform/methanol ( 2 : 1 ) w e r e added to test tubes, then the test tubes were placed under vacuum for 30 rain. Microsomes (200 ug) were added to the test tubes which were then vortex-mixed for 15 s. The tubes were kept on ice and were assayed within 2 h. Proton-pumping was assayed using 100 ug of the microsomes. Lyso-PE concentration

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Fig. 1. The effect of inhibitors on ATP-dependent quenching of quinaerine fluorescence in microsoma] suspensions. Inhibitors were added about 5 rain prior to the addition of ATP except for FCCP which was added at the upward pointing arrow. Coneentrations were: vanadate (0.1 raM), oligomycin (5 ~g/m]), FCCP (6 /~M). (A) Assay in the presence of 250 mM sorbito|, 25 mM BTP/MES, 5 mM MgSO,, 0.01 mM quinacrine, with 50 mM KC] except where KNO~ replaces KC] as marked. ATP (5 mM final) was added at the downward pointing arrow. (B) Assay with KNO 3 replacing KC]. Note the larger amount of microsomai suspension used.

191

ATP concentration required for half-maximal quenching (with 5 mM MgSO~ present) is about 0.3 mM when 50 mM KC1 is present and about 1.5 mM when KNO~ replaces KC1 (Fig. 2). The activity is partially inhibited by nitrate, oligomycin and vanadate, with most of the activity being inhibited by nitrate (Fig. 1). Nitrate inhibits even in the presence of KC1. Usually, vanadate~sensitive activity was apparent only in assays where KNO~ was present instead of KC1 (Fig. 1B); it was a very small component of the total proton-pumping activity. Half-maximal inhibition of protonpumping by vanadate occurred at about 20 ~M. This is slightly higher than the concentration required to inhibit microsomal ATPase half-maximally (about 10 ~M). Oligomycin sensitivity was leas in the presence of KNO~ compared to its effect in the presence of KCI (Fig. 1, taking into account the difference in protein concentrations used for Fig. 1A and 1B), suggesting that it was inhibiting mostly nitrate-sensitive activity. Although this inhibition may be due to the presence of submitochondrial particles, oligomycin may act directly on nitrate- and vanadate-sensitive activities [20]. Sucrose density gradient fractionation was used to determine the location of proton-

pumping activity relative to biochemical markers for sub~ellular organelles. It was necessary to use two linear gradients to yield enough material for all assays. The A~s0- ~0 nm traces and sucrose densities in each fraction for these gradients were nearly identical so the results were combined in Fig. 3. ATPdependent fluorescence quenching (using 0.1ml aliquots per assay), cytochrome c reductase and oxidase were assayed using one linear gradient. Latent UDPase, ATPase (using 5-~1 aliquots and a 1-h incubation period), and

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ATP (raM) Fig. 2. ATP dependence o f quenching in the presence o f KCI o r KNOs. ATP dependence was measured w i t h the MgSO, concentration constant at 5 raM, otherwise the reaction m i x is as described in Fig. 1. Open symbols show the dependence f o r microsomes (100 /~g) in the presence o f 50 m M KC1. Closed symbols show the dependence for microsomes (400 /~g) w i t h KCI replaced by KNO~. T w o separate

experiments for each treatment are shown.

Linear sucrose density gradient of microsomal suspension. The upper panel shows ATPdependent quenching of quinacrine fluorescence and percent inhibition when KNO~ replaces KCI and in the presence of 0.1 mM vanadate (with KCI present). The next panel shows Mg-KCI-ATPase and nitrate(50 mM KNO~ replacing KCI) and vanadate- (0.1 mM in the presence of 50 mM KCi) inhibited activities. These were calculated by subtracting activities in the presence of inhibitor from the control activity. The 3rd panel shows latent UDPase, glucan synthetase II, cytochrome c reductase and oxidase. The lowermost panel shows sucrose density and protein.

192

glucan synthetase II were assayed using the other gradient. To obtain proton-pumping activity in the denser regions of the gradient, it ~vas necessary to heavily overload the gradients. This overloading and the short length of the gradients were the probable cause of the broad distribution of some of the markers (notably latent UDPase and cytochrome c reductase). On these gradients, Goigi were located at 29%--33% (w/w) sucrose density (fractions 4--6), endoplasmic reticulum at 33%--38% (w/w) (fractions 6--8), glucan synthetase II at 43% (w/w) (fraction 10) with a 36%--40% (w/w) shoulder (fractions 8--9), and mitochondria at 43% (w/w) (fraction 10). The peak of proton-pumping activity was at 26% (w/w) (fraction 3) with activity tapering off to about 40% (w/w). Nitrate-inhibited protonpumping activity was located at 31% (w/w) (fraction 5), while vanadate-inhibited activity was located at a much higher density (37% (w/w), fractions 7--8). Oligomycin-inhibited activity was examined in one experiment and did not correspond to the distribution of the mitochondrial marker but was distributed between 30% and 40% (w/w) sucrose density. It may arise from a population of submitochondrial particles having a density lower than that of mitochondria. The high density location of endoplasmic reticulum was due to the high levels of Mg2+ in the grinding medium. When lower levels are used, it is located at 20--25% (w/w) [5]. Although KCl~stimulated ATPase is often used as a marker for the plasma membrane, we find the greatest stimulation (about 30% stimulation) at lower densities; it is correlated with nitrate~sensitive ATPase. We assume this stimulation is due to C1--stimulation of the nitrate~sensitive ATPase. The lack of stimulation at high densities is presumably due to the relatively low incubation temperature used for the ATPase reaction in this and previous studies [ 5 ]. The nitrate-inhibited proton-pumping activity is closely correlated with the distribution of nitrate-inhibited ATPase activity. This

nitrate-inhibited ATPase is a very small component of total ATPase; it is barely measurable in microsomal suspensions (data not shown), yet it appears to account for the majority of proton.pumping activity in this system. Nitrate-inhibited proton-pumping activity and ATPase are not correlated with markers for endoplasmic reticulum, mitochondria or plasma membrane. They match the location of Golgi to some extent but the distribution of Golgi is broader, extending deeper into the gradient, so we assume they originate from the tonoplast as reported by others [21,22]. Vanadate-inhibited proton-pumping is localized at a slightly lower density than vanadate-inhibited ATPase and glucan synthetase II; perhaps due to loss of vesicle intactness at higher sucrose densities. The two markers for plasma membrane, vanadate-inhibited ATPase and glucan synthetase II, are not localized at the same density. Rather, glucan synthetase II occurs as a shoulder with the same density as vanadate-inhibited ATPase activity, and a peak at the same location as the mitochondrial marker cytochrome c oxidase. Similar results have been reported by Hendriks [23] for maize coleoptiles. Thus we are unable to identify plasma membrane unequivocally with this marker. The density of vanadate-inhibited proton-pumping and ATPase activity differs from densities for soybean plasma membrane reported using radioactive labeling of protoplast plasma membrane (32% compared to 37%--39% (w/w)) [24]. It is similar to the densities reported using phosphotungstic acid~hromic acid staining and concanavalin A binding as plasma membrane markers (34%--45% w/w) [25,26]. The density location of vanadateinhibited proton-pumping activity and its association with vanadate-inhibited ATPase and a shoulder of glucan synthetase II suggests that it m a y originate from the plasma m e m brane. It is possible to separate nitrate- and vanadate~sensitive proton-pumping activity by using step gradient preparations (Fig. 4). The

193

,[

[ 21,22 ]. The vanadate-inhibited activity may originate from the plasma membrane.

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Fig. 4. Proton-pumping activity of 20/34 and 34/ 42% (w/w) sucrose interfaces. Inhibitors were added about 5 min prior to the addition o f ATP. Concentrations were the same as in Fig. 1 except oligomycin which was used at 10 ,g/rnl. Percentage o f quenching min -I is shown in parentheses. (A) 20/34% (w/w) i interface. One-hundred micrograms o f protein were used. (B) 34/42% (w/w) interface. Five hundred micrograms of protein were used.

vanadate-sensitive activity is quite low, similar to the results using the linear gradient, but is clearly enriched on the 34/42% (w/w) interface and separable from the nitrate-sensitive activity. In summary, the proton-pumping activity of soybean root microsomes is primarily located at a low density on gradients and is inhibited to a great extent by nitrate. There is a small amount of vanadate-inhibited activity which is located at a high density on gradients. The distribution of this vanadate-inhibited activity is fairly well correlated with that of vanadate-inhibited ATPase. It is not correlated with the major peak of the plasma membrane marker glucan synthetase II, but is correlated with a 39% (w/w) shoulder of glucan synthetase II activity. Nitrate-inhibited activity probably originates from the tonoplast

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