Active Glucose Transport and Proton Pumping in Tonoplast Membrane of Zea mays L. Coleoptiles Are Inhibited by Anti-H-ATPase Antibodies

A tonoplast enriched fraction was obtained from Zea mays L. coleoptiles by isopycnic centrifugation of microsomal membranes in a sucrose step gradient. At the 18/26% interface chloride-stimulated and nitrate-inhibited proton pumping activity coincided with a Mg²⁺-ATP dependent accumulation of 3-O-methyl-d-glucose (OMG) as determined by a membrane filtration technique using ¹⁴C-labeled substrate. OMG transport showed an apparently saturable component with a K_m of 110 micromolar, and was completely inhibited by 10 micromolar carbonyl cyanide m-chlorophenylhydrazone. Polyclonal antibodies against solubilized native tonoplast H⁺-ATPase and its 62 and 72 kilodalton subunits were assayed for their ability to inhibit proton pumping and OMG accumulation. Antibodies against both the native enzyme and the putative catalytic subunit (72 kilodalton) strongly inhibited proton pumping and OMG transport whereas antibodies against the 62 kilodalton subunit had only a slight effect on both processes.     

Growth cycle stage-dependent NaCl induction of plasma membrane H+-ATPase mRNA accumulation in de-adapted tobacco cells

A cDNA clone encoding an isoform of the plasma membrane H⁺-ATPase was isolated from Nicotiana tabacum. The steady-state plasma membrane H⁺-ATPase message levels were the same in unadapted tobacco cells and tobacco cells adapted to 428 mol m⁻³ NaCl. When cells adapted to 428 mol m⁻³ NaCl maintained in the absence of NaCl (deadapted) for an excess of 100 passages were exposed to 400 mol m⁻³ NaCl for 24 h, there was an increased accumulation of plasma membrane H⁺-ATPase message. The NaCl responsiveness of the deadapted cells was dependent upon the growth cycle stage. Alterations in the levels of plasma membrane FT-ATPase message during the growth cycle support a role for the H⁺-ATPase in cell growth. These results document the induction by NaCl of plasma membrane FT-ATPase message accumulation in tobacco cells, and suggest that enhanced expression of the plasma membrane FT-ATPase has a role in the short term response of cells of NaCl, but is not necessarily involved in long-term adaptation.     

Immunocytochemical Localization of the Vacuolar H-ATPase in Maize Root Tip Cells

The vacuolar H⁺-ATPase of maize (Zea mays L.) root tip cells has been localized at the EM level using rabbit polyclonal antibodies to the 69 kilodalton subunit and protein A-colloidal gold. Intracellular gold particles were detected mainly on the tonoplast and Golgi membranes. Only about 27% of the vacuoles were labeled above background. The absence of gold particles on the majority of vacuoles suggests either that the tonoplast H⁺-ATPase is degraded during tissue preparation or that the small vacuoles of root tip cells are specialized with respect to H⁺-ATP ase activity. The pattern of gold particles on the labeled vacuoles ranged from uniform to patchy. Virtually all of the Golgi bodies were labeled by the antibody, but the particle densities were too low to determine whether the H⁺-ATPase was associated with specific regions, such as the trans-face. Cell wall-labeling was also observed which could be partially prevented by the inclusion of gelatin as a blocking agent. The immunocytochemical results confirm previous biochemical studies with isolated membrane fractions (A Chanson, L Taiz 1985 Plant Physiol 78: 232-240).     

Separation of tonoplast and plasma membrane H+-ATPase from Zucchini hypocotyls by consecutive sucrose and glycerol gradient centrifugation

Summary In order to isolate tonoplast and plasma membrane vesicles involved in ATP-dependent proton transport we devised a preparative procedure with two consecutive centrifugations. Three fractions were obtained on a sucrose step gradient: light microsomes, heavy microsomes, and a mitochondria-rich fraction. The light and heavy microsomal fractions were each recentrifuged on an isopycnic glycerol density gradient. Recentrifugation of light microsomes resulted in two fractions with H⁺-ATPase activity, one equilibrating at a density less than 1.11 g/cm³ and one equilibrating at a density of about 1.17g/cm³. Comparison with marker enzyme activities suggests that the upper fraction was enriched in tonoplast, and the dense fraction with plasma membrane. In addition to marker enzyme content, H⁺ transport in the H⁺-ATPase-containing fractions was further characterized with respect to pH dependence, cation and anion dependence, and uncouplers and inhibitors. H⁺ transport in all fractions was strongly dependent on the presence of halides but no specific stimulation by potassium or any other monovalent cation was found. Of the anions tested, malate and fumarate preferentially stimulated H⁺ transport in the tonoplast-enriched fraction. It is suggested that a Ca²⁺/H⁺ antiporter is present in all fractions. Only H⁺-ATPase in the plasma membrane-enriched fractions was sensitive to nystatin, an uncoupler, and to orthovanadate, an inhibitor. The tonoplast fraction was more sensitive to nitrate than the plasma membrane-enriched fraction, and all fractions showed some sensitivity to high concentrations of oligomycin. Oligomycin sensitivity was not due to the presence of mitochondria.     

Role of Tonoplast Proton Pumps and Na+/H+ Antiport System in Salt Tolerance of Populus euphratica Oliv.

Populus euphratica has been used as a plant model to study resistance against salt and osmotic stresses, with recent studies having characterized the tonoplast and the plasma membrane ATPases, and two Na⁺/H⁺ antiporters, homologs of the Arabidopsis tonoplast AtNHX1, were published in databases. In the present work we show that P. euphratica suspension-cultured cells are highly tolerant to high salinity, being able to grow with up to 150 mM NaCl in the culture medium without substantial modification of the final population size when compared to the control cells in the absence of salt. At a salt concentration of 300 mM, cells were unable to grow but remained highly viable up to 17 days after subculture. The addition of a 1-M-NaCl pulse to unadapted cells did not promote a significant loss in cell viability within 48 h. In tonoplast vesicles purified from cells cultivated in the absence of salt and from salt-stressed cells, vacuolar H⁺-pyrophosphatase (V-H⁺-PPase) seemed to be the primary tonoplast proton pump; however, there appears to be a decrease in V-H⁺-PPase activity with exposure to NaCl, in contrast to the sodium-induced increase in the activity of vacuolar H⁺-ATPase (V-H⁺-ATPase). Despite reports that in P. euphratica there is no significant difference in the concentration of Na⁺ in the different cell compartments under NaCl stress, in the present study, confocal and epifluorescence microscopic observations using a Na⁺-sensitive probe showed that suspension-cultured cells subject to a salt pulse accumulated Na⁺ in the vacuole when compared with control cells. Concordantly, a tonoplast Na⁺/H⁺ exchange system is described whose activity is upregulated by salt and, indirectly, by a salt-mediated increase of V-H⁺-ATPase activity.     

Purification of an h-translocating inorganic pyrophosphatase from vacuole membranes of red beet

An H⁺-translocating inorganic pyrophosphatase (PPase) was isolated and purified from red beet (Beta vulgaris L.) tonoplast. One major polypeptide of molecular weight 67 kilodalton copurified with fluoride-inhibitable PPase activity when subjected to one-dimensional polyacrylamide gel electrophoresis. Overall, a 150-fold purification of the PPase was obtained, from the tonoplast fraction, through anion exchange chromatography of the detergent-solubilized membranes followed by ammonium sulfate precipitation and gel filtration chromatography. The purified polypeptide showed no cross-reactivity with antibodies raised against the 67 kilodalton subunit of the tonoplast ATPase.     

Na+/H+ antiport activity in tonoplast vesicles isolated from sunflower roots induced by NaCl stress

Na⁺ transport across the tonoplast and its accumulation in the vacuoles is of crucial importance for plant adaptation to salinity. Mild and severe salt stress increased both ATP- and PP_i-dependent H⁺ transport in tonoplast vesicles from sunflower seedling roots, suggesting the possibility that a Na⁺/H⁺ antiport system could be operating in such vesicles under salt conditions (E. Ballesteros et al. 1996. Physiol. Plant. 97: 259–268). During a mild salt stress, Na⁺ was mainly accumulated in the roots. Under a more severe salt treatment, Na⁺ was equally distributed in shoots and roots. In contrast to what was observed with Na⁺, all the salt treatments reduced the shoot K⁺ content. Dissipation by Na⁺ of the H⁺ gradient generated by the tonoplast H⁺-ATPase, monitored as fluorescence quenching of acridine orange, was used to measure Na⁺/H⁺ exchange across tonoplast-enriched vesicles isolated by sucrose gradient centrifugation from sunflower (Helianthus annuus L.) roots treated for 3 days with different NaCl regimes. Salt treatments induced a Na⁺/H⁺ exchange activity, which displayed saturation kinetics for Na⁺ added to the assay medium. This activity was partially inhibited by 125 μM amiloride, a competitive inhibitor of Na⁺/H⁺ antiports. No Na⁺/H⁺ exchange was detected in vesicles from control roots. The activity was specific for Na⁺. since K⁺ added to the assay medium slightly dissipated H⁺ gradients and displayed non-saturating kinetics for all salt treatments. Apparent K_m for Na⁺/H⁺ exchange in tonoplast vesicles from 150 mM NaCl-treated roots was lower than that of 75 mM NaCl-treated roots, V_max remaining unchanged. The results suggest that the existence of a specific Na⁺/H⁺ exchange activity in tonoplast-enriched vesicle fractions, induced by salt stress, could represent an adaptative response in sunflower plants, moderately tolerant to salinity.     

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.     

Enhanced Net K Uptake Capacity of NaCl-Adapted Cells

Maintenance of intracellular K⁺ concentrations that are not growth-limiting, in an environment of high Na⁺, is characteristic of NaCl-adapted cells of the glycophyte, tobacco (Nicotiana tabacum/gossii). These cells exhibited a substantially greater uptake of ⁸⁶Rb⁺ (i.e. an indicator of K⁺) relative to unadapted cells. Potassium uptake into NaCl-adapted cells was 1.5-fold greater than unadapted cells at 0 NaCl and 3.5-fold greater when cells were exposed to 160 millimolar NaCl. The difference in net K⁺ uptake between unadapted and NaCl-adapted cells was due primarily to higher rates of entry rather than to reduced K⁺ leakage. Presumably, enhanced K⁺ uptake into adapted cells is a result of electrophoretic flux, and a component of uptake may be linked to vanadate-sensitive H⁺ extrusion.     

Characterization of tonoplast polypeptides isolated from corn seedling roots

Tonoplast vesicles were isolated by discontinuous sucrose gradient centrifugation in the presence of Mg²⁺ from 5 day old corn (Zea mays L., Golden Cross Bantam) seedling roots. Marker enzyme assays indicated only a low degree of cross-contamination of tonoplast vesicles at the 10/23% (weight/weight) interface by other membrane components. Severalfold enrichment of tonoplast ATPase and pyrophosphatase was indicated in tonoplast fractions by dot blot studies with antibodies against an oat tonoplast ATPase and a mung bean tonoplast pyrophosphatase. Comparison of two-dimensional electrophoretic gels of tonoplast and microsomal membrane polypeptides revealed approximately 68 polypeptides to be specific to tonoplast by silver staining. Immunoblot analysis with antibodies against a tonoplast holoenzyme ATPase from oat roots revealed the presence of the 72, 60, and 41 kilodalton polypeptides in isolated tonoplast vesicles from corn roots. Affinity blotting with concanavalin A and secondary antibodies indicated the degree of glycosylation of tonoplast polypeptides, where 21 of 68 tonoplast-specific polypeptides contained detectable carbohydrate moieties. Salt and NaOH washes removed 38 of the tonoplast-specific polypeptides, indicating a peripheral association with the membrane. Thirteen of the peripheral polypeptides and eight of the integral polypeptides were identified as glycoproteins. This information on the polypeptide composition of the tonoplast of root cells will aid in gaining insight into the role of this membrane in controlling vacuolar functions.     

Visualization by Freeze-Fracture Electron Microscopy of Intramembraneous Particles corresponding to the Tonoplast H+-Pyrophosphatase and H+-ATPase of Kalanchoë daigremontiana Hamet et Perrier de la Bâthie*

The H⁺-PPase and the H⁺-ATPase of the vacuolar membrane were separated during purification of tonoplast proteins of Kalanchoë daigremontiana Hamet et Perrier de la Bǎthie. Three membrane protein fractions prepared contained firstly, the H⁺-PPase protein without any subunits of the H⁺-ATPase, secondly, the H⁺-PPase protein with only minute traces of the intramembraneous 16 kDa c-subunit of the H⁺-ATPase, and thirdly, the H⁺-ATPase subunits without H⁺-PPase peptides as verified by SDS-PAGE. These three preparations were reconstituted into soybean (Glycine max L.)-phospholipid vesicles, and compared with proteoliposomes obtained by reconstitution of total solubilized tonoplast proteins as well as with native tonoplast vesicles. Analysis of freeze-fracture replicas prepared from these five different types of vesicles showed that there are two populations of intramembraneous particles, one with a diameter of 6.7-7.2 nm corresponding to the H⁺-PPase, and one with an average diameter of 9.1 nm belonging to the H⁺-ATPase. Thus, freeze-fracture electron microscopy allows one to visualize H⁺-PPase particles in addition to H⁺-ATPase particles in the tonoplast of Kalanchoë daigremontiana.     

Sucrose transport in tonoplast vesicles of red beet roots is linked to ATP hydrolysis

Sucrose uptake into tonoplast vesicles, which were prepared from red beet (Beta vulgaris L.) vacuoles isolated by two different methods, was stimulated by MgATP. Using the same medium as for osmotic disruption of vacuoles, membrane vesicles were prepared from tissue homogenates of dormant red beet roots and separated by high-speed centrifugation through a discontinuous dextran gradient. A low-density microsomal fraction highly enriched in tonoplast vesicles could be further purified from contaminating ER vesicles by inclusion of 5 mM MgCl₂ in the homogenization medium. These vesicles were able to transport sucrose in an ATP-dependent manner against a concentration gradient, whereas vesicles from regions of other densities lacked this feature, indicating that ATP stimulation of sucrose uptake took place only at the tonoplast membrane. Sucrose uptake was optimal at pH 7 in the presence of MgATP and could be stimulated by superimposed pH gradients (vesicle interior acidic) in the absence of MgATP, which is consistent with the operation of a sucrose/H⁺-antiporter at the tonoplast. Tonoplast vesicles, obtained in high yield from tissue homogenates of red beet roots, exhibited sugar-uptake characteristics comparable to those of intact vacuoles; these characteristics included similarities in K _m (1.7 mM), sensitivity to inhibitors and specificity for sucrose.Many experiments were carried out at the Experiment Station of the HSPA, Aiea, Hawaii and financed by an NSF grant to Dr. Maretzki and Mrs. M. Thom.     

Membrane transport in isolated vesicles from sugarbeet taproot : I. Isolation and characterization of energy-dependent, h-transporting vesicles

Sealed membrane vesicles were isolated from homogenates of sugarbeet (Beta vulgaris L.) taproot by a combination of differential centrifugation, extraction with KI, and dextran gradient centrifugation. Relative to the KI-extracted microsomes, the content of plasma membranes, mitochondrial membranes, and Golgi membranes was much reduced in the final vesicle fraction. A component of ATPase activity that was inhibited by nitrate co-enriched with the capacity of the vesicles to form a steady state pH gradient during the purification procedure. This suggests that the nitrate-sensitive ATPase may be involved in driving H⁺-transport, and this is consistent with the observation that H⁺-transport, in the final vesicle fraction was inhibited by nitrate. Proton transport in the sugarbeet vesicles was substrate specific for ATP, insensitive to sodium vanadate and oligomycin but was inhibited by diethylstilbestrol and N,N′-dicyclohexylcarbodiimide. The formation of a pH gradient in the vesicles was enhanced by halide ions in the sequence I⁻ > Br⁻ > Cl⁻ while F⁻ was inhibitory. These stimulatory effects occur from both a direct stimulation of the ATPase by anions and a reduction in the vesicle membrane potential. In the presence of Cl⁻, alkali cations reduce the pH gradient relative to that observed with bis-tris-propane, possibly by H⁺/alkali cation exchange. Based upon the properties of the H⁺-transporting vesicles, it is proposed that they are most likely derived from the tonoplast so that this vesicle preparation would represent a convenient system for studying the mechanism of transport at this membrane boundary.     

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 [³⁵S]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.     

Extracellular polysaccharides and proteins of tobacco cell cultures and changes in composition associated with growth-limiting adaptation to water and saline stress

The chemical composition of extracellular polymers released by cells of tobacco (Nicotiana tabacum L. cv W38) adapted to a medium containing 30% polyethylene glycol 8000 (−28 bar) or 428 millimolar NaCl (−23 bar) was compared to the composition of those released by unadapted cells. Unadapted cells released uronic acid-rich material of high molecular weight, arabinogalactan-proteins, low molecular weight fragments of hemicellulosic polysaccharides, and a small amount of protein. Cells adapted to grow in medium containing NaCl released arabinogalactan and large amounts of protein but not the uronic acid-rich material, and cells adapted to grow in polyethylene glycol released only small amounts of an arabinogalactan of much lower molecular weight and some protein. Secretion of all material was nearly blocked by polyethylene glycol, but when cells were transferred to a medium containing iso-osmolar mannitol, they again released extracellular polymers at rates similar to those of unadapted cells. Like cells adapted to NaCl, however, these cells released arabinogalactan and large amounts of protein but only small amounts of the uronic acid-rich material. Media of NaCl-adapted cells were enriched in 40, 29, and 11 kilodalton polypeptides. CaCl₂ extracted the 40 and 11 kilodalton polypeptides from walls of unadapted cells, but the 29 kilodalton polypeptide was found only in the medium of the NaCl-adapted cells. Accumulation of low molecular weight polysaccharide fragments in the medium was also substantially reduced in both NaCl- and polyethylene glycol-adapted cells, and specifically, the material was composed of lower proportions of xyloglucan fragments. Our results indicate that adaptation to saline or water stress results in inhibition of both the hydrolysis of hemicellulosic xyloglucan and release of uronic acid-rich material into the culture medium.     

Ionophorous functions of phosphatidic acid in the plant cell

Effects of phosphatidic acid (PA), a product of phospholipase D activity, on Ca²⁺ and H⁺ transport were investigated in membrane vesicles obtained from roots and coleoptiles of maize (Zea mays L.). Calcium flows were measured with fluorescent probes indo-1 and chlorotetracycline loaded into the vesicles and added to the incubation medium, respectively. Phosphatidic acid (50–500 μM) was found to induce downhill flow of Ca²⁺ along the concentration gradient into the plasma membrane vesicles and endomembrane vesicles (tonoplast and endoplasmic reticulum). Protonophorous functions of PA were probed with acridine orange. First, the ionic H⁺ gradient was created on the tonoplast vesicles by means of H⁺-ATPase activation with Mg-ATP addition. Then, the vesicles were treated with 25–100 μM PA, which induced the release of protons from tonoplast vesicles and dissipation of the proton gradient. Thus, PA could function as an ionophore and was able to transfer Ca²⁺ and H⁺ across plant cell membranes along concentration gradients of these ions. The role of PA in mechanisms of intracellular signaling in plants is discussed.     

Proton Transport in Plasma Membrane and Tonoplast Vesicles from Red Beet (Beta vulgaris L.) Storage Tissue : A Comparative Study of Ion Effects on DeltapH and DeltaPsi

The proton transport properties of plasma membrane and tonoplast vesicles isolated from red beet (Beta vulgaris L.) storage tissue were examined and compared. Membrane vesicles isolated with 250 millimolar KCl in the homogenization media and recovered at low density following sucrose density gradient centrifugation displayed characteristics of proton transport (nitrate inhibition, no inhibition by orthovanadate, pH optimum of 7.75, pyrophosphate-driven proton transport) which were consistent with a tonoplast origin. When the KCl in the homogenization medium was replaced by 250 millimolar KI, sealed membrane vesicles were recovered at higher densities in sucrose gradients and displayed properties (orthovanadate sensitivity, no inhibition by nitrate, pH optimum of 6.5) consistent with a plasma membrane origin. A comparison of anion effects (potassium salts) upon ΔpH and ΔΨ revealed a direct correspondence between the relative ability of anions to stimulate proton transport and reduce ΔΨ. For tonoplast vesicles, the relative order for this effect was KI > KBr ≥ KCl > KClO₃ > K₂SO₄ while for plasma membrane vesicles, a different order KI > KNO₃ ≥ KBr ≥ KClO₃ > KCl > K₂SO₄ was observed. Proton transport in plasma membrane and tonoplast vesicles was inhibited by fluoride; however, plasma membrane vesicles appeared to be more sensitive to this anion. In order to correlate anion effects in the two vesicle fractions with anion transport, the kinetics of anion stimulation of steady-state pH gradients established in the absence of monovalent ions was examined. Anions were added as potassium salts and the total potassium concentration (100 millimolar) was maintained through the addition of K⁺/Mes. For plasma membrane vesicles, chlorate and nitrate displayed saturation kinetics while chloride displayed stimulation of proton transport which followed a linear profile. For tonoplast vesicles, the kinetics of chloride stimulation of proton transport displayed a saturable component. The results of this study indicate differences in proton transport properties of these two vesicle types and provide information on conditions where proton transport in the two fractions can be optimized.     

ATP-induced sucrose efflux from red-beet tonoplast vesicles

 Sucrose efflux from the vacuole of mobilizing red-beet (Beta vulgaris L.) hypocotyl cells was investigated using purified tonoplast vesicles. Tonoplast vesicle purity was assured by the immunoreactivity to antibodies raised against the vacuolar ATPase and by the strong inhibition exhibited by the H⁺-ATPase to bafilomycin-A and NO₃ ⁻. Inhibition of the H⁺-ATPase by vanadate and azide was negligible. Sucrose was loaded into tonoplast vesicles by using the pH-jump method of energization. Addition of ATP to sucrose-loaded vesicles in the presence of bafilomycin-A resulted in efflux of a significant amount of sucrose. During ATP-induced sucrose efflux, bafilomycin-insensitive ATPase activity increased significantly with no increase in H⁺-translocating activity. The additional bafilomycin-A insensitive ATPase activity observed in sucrose-loaded vesicles was completely inhibited by vanadate as was the efflux of sucrose. Similar to vanadate, thapsigargin was also inhibitory to sucrose efflux and to the bafilomycin-A insensitive ATPase activity. The data indicate that vacuolar sucrose can be actively mobilized by a specific ATP-dependent efflux mechanism. Received: 12 October 1999 / Accepted: 18 November 1999