Calcium Fluxes across the Plasma Membrane of Commelina communis L. Assayed in a Cell-Free System

The inside-out fraction of plasma membrane-rich vesicles prepared from leaves of Commelina communis L. by aqueous twophase partitioning was loaded with ⁴⁵Ca²⁺ through the action of the plasma membrane Ca²⁺-ATPase. While the Ca²⁺-loaded vesicles were tightly sealed, trifluoperazine (TFP) (effective concentration giving 50% of maximum effect [EC₅₀] = 70 micromolar) and W-7 (EC₅₀ = 100 micromolar), but to a much lesser extent, W-5 (EC₅₀ = 500 micromolar) led to a rapid efflux of ⁴⁵Ca²⁺ from the vesicles. This efflux could be blocked efficiently with low (<1 millimolar) concentrations of La³⁺, but it remained unaffected by the addition of calmodulin (CM). Further experiments with vesicles incubated in ⁴⁵Ca²⁺ in the absence of ATP, as well as experiments performed with control liposomes and nonloaded as well as Ca²⁺-loaded plasma membrane vesicles using the indicator dye arsenazo III showed, that TFP and W-7 and, again to a lesser extent, W-5 mobilized a pool of membrane-bound Ca²⁺ from the vesicles. No indications for a detergent effect of TFP and W-7 were obtained. The EC₅₀-values of these compounds for mobilizing membrane-associated Ca²⁺ (TFP = 100 micromolar, W-7 = 100 micromolar, W-5 = 500 micromolar) or for the triggering of Ca²⁺ release from Ca²⁺-loaded vesicles (see above) were very similar, suggesting a common basis of antagonist action on both processes. Our results suggest the presence of a Ca²⁺ channel in the plasma membrane of C. communis. The channel is obtained in a Ca²⁺-inactivated state after preparation and Ca²⁺-loading of the vesicles. The inactivation is removed by TFP or W-7, presumably due to the Ca²⁺-mobilizing effect of these compounds. The activated Ca²⁺ channel is La³⁺ sensitive and, in the cell, would allow for passage of Ca²⁺ into the cell. The possibility that TFP or W-7 act independent of CM, or through CM tightly associated with the plasma membrane, is discussed. The system described allows a cell free analysis of Ca²⁺ influx, displaying channel properties, in a higher plant.     

鸭跖草花部维管束系统解剖学研究

鸭跖草花梗项部的维管束分布在中央的基本组织内。自花梗顶部至子永恒基部,维管束系统发生复杂的变化。6枚向外偏斜的维管束发生内外或左右分支,其中3枚维管束发生内外分支,其外侧的3个分支进入萼片成为萼片给管束系统,内侧3个分支进入3枚外轮雄蕊而成为外轮雄蕊维管束;另3枚维管束先发生内外分支,接着外侧3分支发生进一步的左右分支,各形成3－5个小分支,最后进入花瓣成为花瓣维管束系统,而内侧的分支则不再细分,最后伸入3枚内轮雄蕊,成为轮雄蕊维管束。另6枚近圆束形的维管束一直在中央向上延伸,进入子房屋区后,其中3格言 进入子房壁,成为3束心皮背束,最后3束心皮背束进入花柱成为花柱维管史,另3枚聚向中央,成为胎座维管束,胎座维管束至子房屋顶部时消失。文中对跖草及其有关类群的花部维管束系统的来源及演变进行了比较、讨论。     

Separation and Purification of Protoplast Types from Commelina communis L. Leaf Epidermis

Guard cell and epidermal/subsidiary cell protoplasts obtainedby enzymic digestion of peeled Commelina communis leaf epidermiswere separated and purified by discontinuous density gradientccntrifugation with media based on Percoll (Pharmacia Fine ChemicalsAB, Uppsala, Sweden). The cell types were recovered over 99.9%pure at yields exceeding 50% efficiency, and mesophyll contaminationcould be virtually eliminated when desired. Osmotic characteristicsof the protoplast types were evaluated and compared to in vivovalues, and the viability of the protoplasts, assessed usinga range of criteria, was found to be high. Purified Commelinaguard cell protoplasts were able to evolve O₂ when illuminated,and this was substantially reduced in the presence of the inhibitorDCMU, indicating that they possess photosystem II activity.Specific advantages of this method of protoplast purification,and the potential uses of separate suspensions of guard cellsand epidermal/subsidiary cells in experiments on stomatal physiologyare discussed. Key words: Commelina communis, Protoplasts, Epidermis     

Pressure Effects on Membrane Potentials of Mesophyll Cell Protoplasts and Epidermal Cell Protoplasts of Commelina communis L.

Pantoja, O. and Willmer, C. M. 1986. Pressure effects on membranepotentials of mesophyll cell protoplasts and epidermal cellprotoplasts of Commelina communis L.—J. exp. Bot. 37:315–320. Membrane potentials of epidermal cell protoplasts and mesophyllcell protopiasts of Comnelina communis were measured when theprotoplasts were immobilized in a suction micropipette. Whenzero suction was employed, membrane potentials of both protoplasttypes were near to zero. As suction pressure was increased,membrane potentials became increasingly more negative with gradientsof 14·3 mV/kPa and 10·5 mV/kPa for mesophyll cellprotoplasts and epidermal cell protoplasts, respectively. Theplasma membrane is stretched when suction pressure is appliedto protoplasts and it is considered that this simulates cellturgor pressure which is associated with negative membrane potentialsof intact cells. The results help to explain why some investigatorsobtain positive membrane potentials for protoplasts while othersobtain negative values. The results also indicate that considerablecaution is needed in the interpretation of ion flux data whenprotoplasts are used. Key words: Commelina communis, membrane potentials, pressure, protoplasts     

Effects of Fusicoccin in 'Isolated' Guard Cells of Commelina communis L

The effects of 3 ? 10^–2 mol m^–3 FC on rubidium fluxesand contents in isolated guard cells of Commelina communis L.have been studied using ⁸⁶RbCl. Fusicoccin causes a marked stimulationof influx and an immediate, apparently irreversible, decreasein efflux of ⁸⁶Rb. The effect on influx is usually more importantin determining the new net flux of Rb. Observed fluxes differmarkedly from those predicted by the Goldman-Hodgkin-Katz equation,suggesting that FC does not act solely via an effect upon theplasmalemma potential. Fusicoccin appears to have a more directeffect upon the ion movements associated with changes in stomatalaperture than either ABA or transfer to the dark. Observed changesin Rb content cannot account fully for the osmotic changes associatedwith aperture increase. Key words: Fusicoccin, Guard cells, Ion fluxes, Commelina communis     

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

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

Ion Fluxes in 'Isolated' Guard Cells of Commelina communis L.

Ion fluxes have been measured in ‘isolated’ guardcells of Commelina communis L. using ⁸⁶RbCl and K⁸²Br, in epidermalstrips in which all cells other than guard cells have been killedby treatment at low pH. To avoid problems of slow free spaceexchange most fluxes have been measured at pH 3.9, at whichstomata open well in K(Rb) Cl(Br) and are stable for many hours.At pH 3.9 the intracellular ⁸⁶Rb exchanged as a single compartmentwith a half-time of 2–3 h, independent of external concentration(C_o). The influx of ⁸⁶Rb rose with concentration, to a V_maxof about 23 pmol mm^–2 h^–1. The efflux curve of ⁸²Brcould be well fitted by two exponential terms, with half-timesof 38 min (independent of C_o), and 5–35 h (falling withincreasing C_o). Bromide contents of cytoplasm and vacuole (Q_cand Q_v), and fluxes at plasmalemma and tonoplast, were calculatedfrom the efflux kinetics. Over C_o 20–60 mM, as the apertureincreased from 7 µm to 17 µm, the tonoplast flux(0.5–11.5 pmol mm^–2h^–1) was always much lessthan the plasmalemma flux (7–77 pmol mm^–2 h^–1).Q_c and Q_v both increased with aperture. The increase in Q_c of10.3 pmol mm^–2 µm^–1 is adequate to accountfor the osmotic changes required to change the aperture, aspreviously estimated. However, the change in vacuolar contentof only 5.9 pmol mm^–2 µm^–1 is much too smallto account for the osmotic changes required, or to balance thecytoplasmic changes. It appears therefore that increasing KBroutside not only increases the cytoplasmic salt content, andthe Br flux at the tonoplast, but also stimulates the vacuolaraccumulation of some other solute.     

Reconstitution and Characterization of H+-Translocating ATPase from the Plasma Membrane of Phaseolus mungo L. Roots

Plasma membrane H⁺-translocating ATPase was partially purifiedfrom mung bean (Phaseolus mungo L.) roots and reconstitutedinto soybean phospholipid (asolectin) liposomes by the n-octylglucosidedilution method. The resulting proteoliposomes were mainly unilamellarvesicles ranging in size from 0.05 to 0.2 µm. The existenceof ATP-drived H⁺-pumping across the proteoliposomes was demonstratedby the quenching of quinacrine fluorescence in the presenceof Mg²⁺. The quenching could be abolished by an uncoupler, FCCP,and an inhibitor of H⁺-translocating ATPase, vanadate. The reconstitutedATPase consisted of three major polypeptides of 105 KDa, 67KDa and 57 KDa. Its pH optimum, divalent cation stimulationand vanadate sensitivity were similar to those of partiallypurified ATPase. However, the specificity toward ATP was muchgreater following reconstitution. Also reconstitution reducedthe degree of inhibition by DCCD. Local anesthetics (e.g. dibucaine)had no effect on H⁺-pumping activity but increased the ATPaseactivity when proteoliposomes were reconstituted in their presence. (Received May 2, 1986; Accepted October 17, 1986)     

Some Biochemical Characteristics of Guard Cell and Mesophyll Cell Protoplasts from Commelina communis L.

Guard cell and mesophyll cell protoplasts of Commelina communisL., were isolated and used to investigate their various biochemicalcharacteristics. Contamination of the samples by other celltypes was very low and viability of the protoplasts, assessedby the use of neutral red, Evans blue and fluorescein diacetate,was high (89–98%). Mesophyll cell protoplasts containedmore chlorophyll (x 47), more soluble protein (x 10), more totalN (x 36) and more DNA (x 9) than guard cell protoplasts. Theabsorption spectra of protoplast extracts were similar for bothcell types except that below 400 nm there was a large increasein absorption by the guard cell protoplast extract. In guardcell protoplast extracts, high levels of activity of phosphoenolpyruvatecarboxylase (E.C. 4.1.1.31 [EC] ), NAD malate dehydrogenase (E.C.1.1,1.37), NADP malic enzyme (E.C. 1.1.1.40 [EC] ) and carbonic anhydrase(E.C. 4.2.1.1 [EC] ) were detected while only low levels of pyruvate-orthophosphatedikinase (E.C. 2.7.9.1 [EC] ) activity were detected. Glycollate oxidase(E.C. 1.1.3.1 [EC] ), ribulose-l,5-bisphosphate carboxylase (E.C 4.1.1.39 [EC] ),NADP malate dehydrogenase (E.C. 1.1.1.82 [EC] ) and NAD malic enzyme(E.C. 1.1.1.39 [EC] ) were not detected in guard cell protoplast extracts.High levels of ribulose-1, 5-bisphosphate carboxylase, glycollateoxidase, NAD malate dehydrogenase and carbonic anhydrase weredetected in mesophyll cell protoplast extracts which is typicalof C₃ plants. A pathway of carbon flow during stomatal openingand closing is proposed. Key words: Carbon metabolism, Commelina communis, guard cell protoplasts, mesophyll cell protoplasts, stomata     

Effects of ABA in 'Isolated' Guard Cells of Commelina communis L.

The effects of 2 x 10^–3 M ABA on ion fluxes in isolatedguard cells of Commelina communis L. have been studied, using⁸⁶RbCl and K⁸²Br, in epidermal strips in which all cells otherthan guard cells have been killed by treatment at low pH. Theeffect of ABA on influx is small, if present, and the majoreffect is a marked transient stimulation of the efflux of both⁸⁶Rb and ⁸²Br at the plasmalemma; there is also an increasein the flux of ⁸²Br from vacuole to cytoplasm. The stimulationis transient, and the cells do not simply become more leaky.The results are not consistent with previous speculations onthe mechanism by which ABA reduces aperture.     

Role of Ca and EGTA on Stomatal Movements in Commelina communis L

Ca²⁺ (0.1-1.0 millimolar) accelerated dark-induced stomatal closure and reduced stomatal apertures in the light in epidermal peels of Commelina communis L. In contrast, ethyleneglycol-bis-(β-aminoethyl ether) N,N′tetraacetic acid (EGTA) (2 millimolar), a Ca²⁺ chelator, prevented closure in the dark and accelerated opening in the light. EGTA did not promote significant opening in the dark. It is therefore concluded that EGTA does not increase ion uptake into guard cells, but rather prevents ion efflux. Addition of EGTA to incubating solutions with 10 millimolar KCl resulted in steady state apertures of 15.6 micrometers, whereas in the absence of EGTA similar apertures required 55 millimolar KCl and 150 millimolar KCl was needed in the presence of 1 millimolar CaCl₂. The results demonstrate the importance of Ca²⁺ in the regulation of stomatal closure and point to a role of Ca²⁺ in the regulation of K⁺ efflux from stomatal guard cells.     

Responses of Commelina communis L. Guard Cell Protoplasts to Abscisic Acid

Fitzsimons, P. J. and Weyers, J. D. B. 1987. Responses of Commelinacommunis L. guard cell protoplasts to abscisic acid.—J.exp. Bot. 38: 992–1001. Guard cell protoplasts (GCPs) isolated from the leaf epidermisof Commelina communis L. responded to abscisic acid (ABA) ina manner which was qualitatively and quantitatively similarto that of intact stomata. ABA inhibited swelling of GCPs underlow-CO₂ conditions and swollen GCPs responded to the hormoneby shrinking. Both the absolute volume decrease and the initialrate of shrinking were commensurate with the extent and ratesof solute loss computed for ABA-treated intact, open stomata.This indicates that GCPs represent a suitable experimental systemfor studies of ABA-mediated solute fluxes. A radiotracer equilibrationmethod was developed for the rapid estimation of GCP osmoticvolume changes. Using this technique it was found that, on average,82% of the reduction in solute content caused by ABA treatmentwas due to the loss of K⁺. It is envisaged that electroneutralitymight be maintained during ABA-induced shrinkage of GCPs bynet inward proton movement leading to acidification of the vacuole. Key words: Abscisic acid, Commelina communis L., guard cells, protoplasts     

Effect of total alkaloids from Commelina communis L. on lung damage by influenza virus infection

Whether administration of total alkaloids from Commelina communis L. (TAC) reduces lung damage in influenza virus-infected mice was investigated. Compared with untreated mice, significantly less severe damage was found in the lungs of mice administered TAC at 8 mg/kg per day for 6 days. TAC significantly decreased viral loads in the lungs. The concentrations of IFN-γ in the serum of TAC-treated mice were significantly lower than those of virus control mice at 4 and 6 days post-infection. The results indicate that TAC imparted partial protection to the mice by reducing pulmonary viral loads and limiting lesions in the lungs.     

Profiles of Chloride Concentration and PD in the Root of Commelina communis L.

Chloride concentrations in longitudinal files of cells acrossthe root of Commelina communis have been determined. Vacuolarsap was taken from the root using a microsampling techniqueand chloride concentration determined on nanolitre samples byelectrometric microtitration. No radial gradient in vacuolar chloride was observed, eitherfor roots grown in a nutrient solution containing a low level,or for those grown in a solution containing a high level, ofchloride. Vacuolar electrical potentials were also determined,on attached and excised roots. The PD was found not to varysignificantly across the root from epidermis to pericycle despitethe PD in attached roots being 50 mV more negative than thatin excised roots. These results are discussed in relation tothe mechanism of ion transport across the root.     

Calcium Inhibits Ion-Stimulated Stomatal Opening in Epidermal Strips of Commelina communis L.

Inoue, H. and Katoh, Y. 1987. Calcium inhibitsion-stimulatedstomatal opening in epidermal strips of Commelina communis L.—J.exp. Bot. 38: 142–149. Ca²⁺ suppressed both the ion-stimulated stomatal opening andH⁺ extrusion of pre-illuminated epidermal strips isolated fromCommelina communis L. In the absence of Ca²⁺, the rate of H⁺release was 18 nmol H⁺ cm^–2 h^–1 per epidermal stripunit area in 150 mol m^–3 KCL at pH 7?4. Half-maximum inhibitionof stomatal opening was observed with 220 mmol m^–3 ofCa²⁺. The hexavalent dye, ruthenium red, showed concentration-dependentprevention of the inhibition by Ca²⁺ of the ion-stimulated stomatalopening. The effect of ruthenium red was non-competitive, andthe K₁ for the calcium inhibition was found to be 3?6 mmol m^–3.The calcium inhibition of H⁺ extrusion was also prevented byruthenium red. These results suggest that Ca²⁺ inhibits theactivity of electrogenic H⁺ translocating ATPase of the guardcell plasma membrane and leads to the suppression of stomatalopening. Key words: Calcium, Commelina communis, ruthenium red, stomata     

Responses of Commelina communis Stomata In Vitro

Weyers, J. D. B. and Paterson, N. W. 1987. Responses of Commelinacommunis stomata in vitro.— J. exp. Bot. 38: 631–641. Analysis of the kinetics of movements of Commelina communisL. stomata in vitro revealed a sequence of opening and closingphases dependent on the incubation medium used and the physiologicalstate of the plant material. In buffer containing 50 mol m ^–3KC1 the sequence of aperture changes appeared to be influencedby equilibration of cell water potentials with that of the mediumand by solute fluxes (dependent and independent on metabolicactivity). The results indicate that the stomatal aperture afterseveral hours of incubation may not always provide a reliablequantitative estimate of the ability of the stomata to operate.As a consequence, modifications are suggested to the ways inwhich experiments using epidermal strips are carried out andreported. Key words: Epidermal strips, guard cells, hydroactive, hydropassive, kinetics, potassium chloride, mannitol, osmotic effects, solute fluxes.     

Functional Reconstitution of Plasma Membrane H+-ATPase from Mung Bean (Vigna radiata L.) Hypocotyls in Liposomes Prepared with Various Molecular Species of Phospholipids

The activity of solubilized plasma membrane ATPase is affectedby the nature of exogenously added molecular species of phospholipids.To examine the role of the polar head group and of the molecularspecies of phospholipids in H⁺-pumping, the ATPase solubilizedfrom plasma membranes of mung bean (Vigna radiata L.) hypocotylswas reconstituted in liposomes prepared with a variety of phospholipids. The extent of activation of solubilized plasma membrane ATPasedue to the addition of 1-palmitoyl 2-oleoyl-phospholipids (PO-phospholipids)and asolectin decreased in the following order: POPS POPC asolectin POPG > POPE > POPA (see List of Abbreviations). H⁺-pumpinginto proteoliposomes reconstituted with asolectin and plasmamembrane ATPase was demonstrated by quinacrine fluorescencequenching in the presence of ATP-MgSO₄. H⁺-pumping was inhibitedby VO₄ and gramicidin D. When plasma membrane ATPase was reconstitutedin liposomes prepared with various PO-phospholipids, the abilityof PO-phospholipids to support H⁺-pumping into the proteoliposomesdecreased in the following order: POPG POPS > asolectin POPC. POPE and POPA failed to support any H⁺-pumping. A remarkablyhigh rate of H⁺-pumping was observed in proteoliposomes preparedwith 1-saturated 2-unsaturated fatty acids, such as POPC, butH⁺-pumping could hardly be detected in proteoliposomes preparedwith 1-, 2-unsaturated or 1-, 2-saturated fatty acids, suchas PSPC or DLPC. ATPase activity in proteoliposomes was dependenton the species of PO-phospholipids used for reconstitution anddecreased in the following order: POPS > POPG > POPC asolectin > POPA > POPE. DLPC (see List of Abbreviations)which includes a 1-, 2-unsaturated fatty acid supported onlymarkedly depressed activity. Both H⁺-pumping and the hydrolysis of ATP by the plasma membraneATPase are strongly affected by the polar head group and compositionof the fatty acyl chain of phospholipids used to prepare liposomesfor reconstitution of the ATPase. (Received May 31, 1991; Accepted September 18, 1991)     

Metabolism of Abscisic Acid in Guard Cells of Vicia faba L. and Commelina communis L

Metabolism of abscisic acid (ABA) was investigated in isolated guard cells and in mesophyll tissue of Vicia faba L. and Commelina communis L. After incubation in buffer containing [G-³H]±ABA, the tissue was extracted by grinding and the metabolites separated by thin layer chromatography. Guard cells of Commelina metabolized ABA to phaseic acid (PA), dihydrophaseic acid (DPA), and alkali labile conjugates. Guard cells of Vicia formed only the conjugates. Mesophyll cells of Commelina accumulated DPA while mesophyll cells of Vicia accumulated PA. Controls showed that the observed metabolism was not due to extracellular enzyme contaminants nor to bacterial action.

Metabolism of ABA in guard cells suggests a mechanism for removal of ABA, which causes stomatal closure of both species, from the stomatal complex. Conversion to metabolites which are inactive in stomatal regulation, within the cells controlling stomatal opening, might precede detectable changes in levels of ABA in bulk leaf tissue. The differences observed between Commelina and Vicia in metabolism of ABA in guard cells, and in the accumulation product in the mesophyll, may be related to differences in stomatal sensitivity to PA which have been reported for these species.

     

Redox Processes in the Blue Light Response of Guard Cell Protoplasts of Commelina communis L

Guard cell protoplasts from Commelina communis L. illuminated with red light responded to a blue light pulse by an H⁺ extrusion which lasted for about 10 minutes. This proton extrusion was accompanied by an O₂ uptake with a 4H⁺ to O₂ ratio. The response to blue light was nil in darkness without a preillumination period of red light and increased with the duration of the red light illumination until about 40 minutes. However, acidification in response to a pulse of blue light was obtained in darkness when external NADH (1 millimolar) was added to the incubation medium, suggesting that redox equivalents necessary for the expression of the response to blue light in darkness may be supplied via red light. In accordance with this hypothesis, the photosystem II inhibitor 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (10 micromolar) decreased the acidification in response to blue light more efficiently when it was added before red light illumination than before the blue light pulse. In the presence of hexacyanoferrate, the acidification in response to a blue light pulse was partly inhibited (53% of control), suggesting a competition for reducing power between ferricyanide reduction and the response to blue light.     

Characterization and purification of the fusicoccin-binding complex from plasma membranes of Commelina communis

The fungal phytotoxin fusicoccin binds with high affinity to plasma membranes of the monocotyledonous plant, Commelina communis L. The sites bind the toxin with an apparent Kd of 5.2 nM and a pH optimum of 6.0. They occur at a level of approximately 6-8 pmol/mg plasma membrane protein. Photoaffinity labeling with the biologically active fusicoccin derivative 9'-nor-8'-(4-azido[3,5-3H]benzoyl) diaminoethylfusicoccin identified a polypeptide of 31.5 kDa on SDS/PAGE which was strongly labeled. A second 32.5-kDa band was also consistently labeled, although not to the same extent. The binding sites were solubilized in functional form and a purification scheme was developed based on affinity and ion-exchange procedures. The purified fraction contains two polypeptides of apparent molecular masses of 30.5 kDa and 31.6 kDa. A detailed molecular analysis of the fusicoccin-binding complex is now possible.