Insect Biochemistry and Molecular Biology 28 (1998) 201–211

Characterization of ATPases of apical membrane fractions fromLocusta migratoriaMalpighian tubules

Z.I.A. Al-Fifi a, S.L. Marshallb, D. Hyde b, J.H. Ansteeb, K. Bowler b,*

a Department of Biological Sciences, King Abdul Aziz University, Jeddah, Saudi Arabiab Department of Biological Sciences, University of Durham, Durham City, DH1 3LE, UK

Received 4 December 1997; received in revised form 3 March 1998; accepted 3 March 1998

Abstract

Apical and basal membrane fractions fromLocustaMalpighian tubules were prepared and were characterized by marker enzymeanalysis. The apical membranes contained an azide- and orthovanadate-insensitive ATPase activity that was inhibited by bafilomycinA1 (IC50 = 0.44 nM) and NEM (IC50 = 2.15 mM), and thus was characterized as a putative V-type ATPase. The enzyme wasstimulated by a variety of monovalent cations (Tris > K= Na > choline > Li= Rb) maximal stimulation occurring at 30-40 mM.It was also stimulated by a variety of monovalent anions (maximal activation 30-40 mM), but was strongly inhibited by nitrateand thiocyanate. SDS-PAGE separation of proteins present in the various membrane fractions was carried out. The apical membranefraction alone contained a 28 kDa protein band that bound a monoclonal antibody specific for a 28 kDa peptide which was acomponent of the V-type ATPase from midgut ofManduca sextaand, in native gels, possessed ATPase activity which was alsosensitive to both bafilomycin and NEM but not to azide or orthovanadate. Binding of the fluorescent monoclonal antibody waslocated at the apical boundary of the tubule cells. It was concluded that a V-type ATPase is present at the apical surface ofLocustaMalpighian tubule cells and that it is involved in their secretory functioning. 1998 Elsevier Science Ltd. All rights reserved.

Keywords:Insect; Locust; Malpighian tubules; Membrane; V-type ATPase

1. Introduction

The Malpighian tubules ofLocusta, in common withthose of a number of other insects which have been stud-ied (e.g.Calliphora, Berridge, 1968;Tipula paludosa,Coast, 1969; andSchistocerca gregaria, Maddrell andKlunsuwan, 1973), transport K+ against a chemicalgradient over a wide range of external K+ concentrations.In addition, K+ are transported in preference to Na+ evenwhen present at much lower concentrations in the bath-ing medium (Anstee et al., 1979). Measurements ofpotential difference across the tubule wall indicate thatthe lumen is positive with respect to the bathing medium(Anstee et al., 1980; Fathpour et al., 1983; Morgan andMordue, 1983; Baldrick et al., 1988). On these grounds,it has been proposed that the transport of K+ is an active

* Corresponding author. Fax:+ 44 0191 374 2417; E-mail: ken-neth.bowler@durham.ac.uk

0965-1748/98/$19.00 1998 Elsevier Science Ltd. All rights reserved.PII: S0965-1748 (98)00025-3

process. Nevertheless, both Na+ and K+ are necessary formaximal fluid secretion (Morgan and Mordue, 1981).

A number of models have been proposed to explainion and water translocation across insect Malpighiantubules (see reviews by Phillips, 1981; Maddrell andO’Donnell, 1992; Nicolson, 1993; Dow, 1994; van Ker-khove, 1994; Beyenbach, 1995; Pannabecker, 1995).Almost all require that active transport occurs across thebasal and apical cell membranes and, in particular, thatK+ and/or Na+ are transported into the lumen by an api-cal electrogenic cation pump. Harvey et al. (1983) pro-posed that the apical pump may represent a K+-activatedATPase. More recently, studies on larval midgut ofM.sexta have reported the presence of V-type ATPaseactivity associated with isolated apical membrane ves-icles from goblet cells (Wieczorek et al., 1989, 1991).It was suggested that in these cells, the apical electrog-enic K+ pump consisted of a V-type ATPase workingin parallel with a cation/proton antiporter. Subsequentphysiological studies on insect Malpighian tubules haveproduced evidence for K+ secretion via anH+/K+ anti-

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porter driven by the proton motive force produced by aV-type ATPase present in the membrane (Bertram, 1989;Bertram et al., 1991; Klein et al., 1991; Maddrell andO’Donnell, 1992; Weltens et al., 1992; Dow, 1994;Zhang et al., 1994). There is a growing body of evidencethat supports the localization of a V-type ATPase on theapical border of insect Malpighian tubule cells, (Kleinet al., 1991,M. sexta; Russell et al., 1992,Manduca;Pietrantonio and Gill, 1995,Heliothes; Garayoa et al.,1995,Formica).

Previous studies (Anstee and Bell, 1975, 1978; Ansteeand Fathpour, 1979, 1981; Fogg et al., 1991) haverevealed the presence of Mg2 + -dependent, Na+/K+-acti-vated ATPase activity in microsomal preparations fromMalpighian tubules ofLocusta, and that this activity ismainly associated with the basal plasma membranes.Fogg et al. (1991) suggested that an anion-stimulatedATPase, also present, might be a characteristic of a V-type ATPase. The acknowledged dependency of fluidtransport by Malpighian tubules on membrane transportproteins suggested that a combined biochemical andimmunocytochemical study be carried out to determinewhether a V-type ATPase is present in Malpighiantubules ofLocusta; to determine its subcellular localiz-ation and to characterize the enzyme activity present.Studies onLocustamake significant contributions to theunderstanding of the mechanisms and control of Mal-pighian tubule cell function in insects. The results of thispresent study add to this existing body of knowledge,largely derived from structural, physiological, and bio-chemical work carried out in our laboratory.

2. Materials and methods

Mature adult locusts,Locusta migratoriaL., wereused and these were taken from a population maintainedunder crowded conditions at 28± 0.5°C and 60% rela-tive humidity. The photoperiod was 12 h light: 12 h dark.

2.1. Rate of fluid secretion

In vitro measurements of the rate of fluid secretionwere carried out following the method of Maddrell andKlunsuwan (1973), modified later by Anstee and Bell(1975).

2.2. Preparation of different cell fractions

Animals were killed by decapitation and their Mal-pighian tubules quickly dissected out and placed in 10ml of an ice-cold homogenization medium consisting of250 mM sucrose in 5 mM imidazole buffer (pH 7.5).Homogenization was carried out in a glass homogenizerwith a Teflon pestle (clearance 0.1–0.15 mm) with 20passes of the plunger at 100 rev/min; the homogenization

tube was surrounded by ice throughout this procedure.The resulting homogenate was centrifuged to separatethe different membrane components using a methodbased on that described by Rodriguez and Edelman(1979) and Fogg et al. (1991). Initially, the homogenatewas centrifuged at 600g for 10 min at 0°C. The pelletfrom this spin was discarded the supernatant (S1)retained and centrifuged at 15 000g for 20 min at 0°C.The resulting mitochondrial-rich pellet (P2) was storedon ice and the associated supernatant (S2) was re-centri-fuged at 135 000g to yield a pellet (P3) which was thenresuspended by homogenization in 10 ml of homogeniz-ation medium containing 10 mM MgCl2. This was thenleft to stand on ice for 20 min prior to centrifugation at10 000g for 15 min to yield pellet P4, the basolateralmembrane fraction. The resulting supernatant (S4) wasthen centrifuged at 55 000g for 30 min. The pellet (P5)resulting from this spin represented the apical-mem-brane-rich fraction (Fogg et al., 1991). The latter pelletwas resuspended by homogenization in a known volumeof deionised water, at 4°C.

2.3. Assay of ATPase activities

Each incubation medium, consisting of 100ml of anappropriate ionic medium (see below) and 50ml ofmembrane preparation, was thermoequilibrated for 15min at 35°C in a water bath prior to starting the reactionby the addition of 50ml of 12 mM ATP (Tris salt). Incu-bations were run for 30 min at 35°C and reactions werestopped by adding 400ml of a 1:1 mixture of 1% Lubroland 1% ammonium molybdate in 0.9 M sulphuric acid(Atkinson et al., 1973). The tubes were then kept at roomtemperature for 10 min to allow the yellow colour, whichwas proportional to the amount of inorganic phosphatereleased, to develop. Absorbancy was measured at 390nm. Enzyme activity was measured by determining theamount of inorganic phosphate released.

In determining different ATPase activities, a varietyof different ionic media having the following ionic com-positions were used (final concentrations):

2.3.1. Na+ /K + -ATPase activity(1) 4 mM MgCl2; (2) 4 mM MgCl2, 100 mM NaCl

and 20 mM KCl; (3) 4 mM MgCl2, 100 mM NaCl, 20mM KCl plus 1 mM ouabain. Each medium contained20 mM imidazole/HCl (pH 7.2). Na+/K+-ATPase activitywas determined as the difference in the amount of inor-ganic phosphate liberated in the presence of ionicmedium 2 and ionic medium 3.

2.3.2. V-type ATPase activityThis was assayed as azide- and orthovanadate-insensi-

tive ATPase activity as described by Schweikl et al.(1989): 1 mM MgCl2, 20 mM KCl, 0.5 mM NaN3, 0.1

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mM Na3VO4, 0.1 mM EGTA and 0.3 mg BSA ml−1 in50 mM Tris–MOPS buffer (pH 7.5).

Appropriate controls were run to determine the extentof non-enzymatic hydrolysis of ATP. All ATPase activi-ties are expressed in nmoles Pi liberated mg protein−1

min−1.

2.4. Marker enzyme assays for membrane fractionidentification

Basal membrane fractions were identified by enrich-ment of Na+/K+-ATPase (EC 3.6.1.3) activity. Succinatedehydrogenase (EC 1.3.99.1) activity was determinedaccording to the method described by King (1967) as amarker for mitochondrial membranes. Alkaline phospha-tase (EC 3.1.3.1) activity was determined using themethod of Bowers and McComb (1966) to identify api-cal membrane fractions.

2.5. Determination of protein content

Protein determinations were carried out using theCoomassie Brilliant Blue binding method of Bradford(1976), using bovine serum albumin fraction V as stan-dard.

2.6. SDS–polyacrylamide gel electrophoresis (SDS–PAGE)

The method used was essentially that described byLaemmli (1970) for discontinuous gels. Membrane frac-tions (of< 15 mg protein) in sample buffer were loadedonto a 12.5% (w/v) polyacrylamide gel via a 3.9% (w/v)stacking gel. The gels were run at a constant voltage (40mV) and then fixed and stained for protein.

2.7. Western blotting

Proteins were blotted from acrylamide gels to nitrocel-lulose using the semi-dry blotting method of Kyhse-Anderson (1984). Transfer was carried out on a Bio–RadTrans blot, semi-dry transfer cell at 25 V and 220 mA(5.5 mA.cm−2) for 30 min. After transfer, the nitrocellu-lose sheet was blocked (PBS, 5% Marvel and 1%Tween-20) for 1 h at room temperature. It was then incu-bated with primary antibody in anti-sera buffer (PBS,5% Marvel and 0.1% Tween-20) overnight at 4°C, withshaking. Best results were obtained using 1:4000dilution. Following 3 × 5 min washes with anti-serabuffer, the nitrocellulose sheet was incubated with thesecondary antibody, goat anti-mouse IgG (H+ L) horser-adish peroxidase conjugate (Bio–Rad cat. no. 170-6516),at 1:5000 dilution, for 2 h at room temperature. Afterthorough washing in 0.1% Tween-20 in PBS and rinsingwith distilled water, antibody binding was visualized

using an Enhanced Chemiluminescence Detection (ECL)system (Amersham Life Science).

2.8. Native microgradient PAGE

Continuous microgradient acrylamide gels were pro-duced by controlled mixing of 3 and 15% buffered acryl-amide solutions (Schweikl et al., 1989; Hames, 1990).

Apical membrane-enriched pellets (P5), produced asdescribed previously, were resuspended and solubilizedin a 1% solution of TritonX-100 in 10 mM Tris/HCl (pH7.5) containing 0.32 mM EDTA and 5% glycerol. Thismixture was stirred on ice for 10 min prior to centrifug-ation at 100 000g for 1 h, using a fixed angle rotor. Thesupernatant was then mixed with an equal volume of asample buffer consisting of 0.0625 M Tris/HCl (pH 6.8),10% (v/v) glycerol and 5% (v/v) 2-mercaptoethanol(modified from Laemmli, 1970). The resulting mixturewas loaded onto the stacking gel and electrophoresis car-ried out at 20 mA constant current for 4–5 h at| 4°C.

Following electrophoresis, ATPase activity was ident-ified and localized using the procedure described bySchweikl et al. (1989). Initially, gels were preincubatedin a medium consisting of 5 mM MgSO4 and 100 mMTris/HCl (pH 7.5) for 15 min at 35°C in a water bath.ATP (5 mM, final concentration) orb-glycerophosphate(control) was then added and the gel incubated for afurther 1 h. At the end of this time, the gel was washedin double distilled water and placed in 2.5 mM lead acet-ate in 80 mM Tris-maleate buffer (pH 7.0) for 30 min,at room temperature. The gel was then thoroughlywashed in deionised water and 2% ammonium sulphideadded. Sites of ATPase or alkaline phosphatase wererevealed as dark brown/black bands on the gel.

2.9. Immunocytochemistry

Malpighian tubules from adultLocustawere dissectedout and placed in ice-cold fixative consisting of 2% par-aformaldehyde, 0.075% lysine, 0.01 M sodium periodatein 0.1 M sodium phosphate buffer (pH 7.3) for 2 h(McLean and Nakane, 1974). The tissues were washed inphosphate buffer and transferred to 30% sucrose buffer,overnight, before embedding in Tissue Tek and frozenin liquid nitrogen. Cryostat sections were collected onglass slides coated with 0.01% aqueous poly-l-lysine.Immunofluorescent staining was carried out according toKlein et al. (1991).

2.10. Reagents

All solutions were prepared in glass-distilled deion-ised water. All inorganic salts were AnalaR grade or thepurest commercially available. Bafilomycin A1 was pur-chased from Professor Dr K. Altendorf, Universita¨tOsnabru¨ck, Germany. The concentration was estimated

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photometrically (Bowman et al., 1988) using anabsorbance maximum of 245 nm. Bafilomycin A1 wasdissolved in DMSO before adding it to the appropriatesolution and the final concentration of DMSO in theexperimental solutions was, 1%, the same concen-tration of the solvent was included in the controls.

Monoclonal antibodies 230-3, 221-67, 224-3 and 90-7, directed to the V-type ATPase from insect plasmamembrane (midgut of larvalM. sexta), were a gift fromProfessor Dr Ulla Klein, Laboratory Wieczorek,Zoological Institute of the University of Munich, Ger-many.

3. Results

3.1. Rate of fluid secretion

The effect of bafilomycin A1 on the rate of fluidsecretion is shown in Fig. 1(A). Comparison betweennormalized control and experimental values establishedthat 5 mM bafilomycin A1 caused a 73% inhibition inthe rate of fluid secretion (P , 0.001), the IC50 was cal-culated to be 0.82mM. The effect of NEM on the rateof fluid secretion is shown in Fig. 1(B), significant inhi-bition was observed over the concentration range 10−7

to 10−3M, with an IC50 of 9.05 mM.

3.2. Marker enzyme activities of cell fractions

Table 1 shows the subcellular distribution of Na+/K+-ATPase (basolateral marker), succinate dehydrogenase(mitochondrial marker) and alkaline phosphatase (anapical membrane marker) activities in the various cellfractions studied. Pellets P3 and especially P4 exhibitedthe highest specific levels of Na+/K+-ATPase while alka-line phosphatase activity was largely associated with P5.Previous cytochemical studies (Fogg et al., 1991) haveshown that alkaline phosphatase is confined to the apicalcell surface in Malpighian tubules ofLocusta. These dif-ferential distributions of Na+/K+-ATPase and alkalinephosphatase are consistent with P4 and P5 representingbasal and apical membrane enriched fractions, respect-ively. Table 1 also shows that SDH activity was maximalin pellet P2 with minimal levels of activity beingobserved in P5 suggesting low mitochondrial contami-nation of the apical membrane enriched fraction.

3.3. Effect of NEM and bafilomycin A1 on azide- andorthovanadate-insensitive ATPase (V-type ATPase)activity

Pellet P5 also contained significant levels of ATPaseactivity that was insensitive to both azide and orthovan-adate, these reagents inhibit F-type and P-type ATPases,respectively. This ATPase activity, in the apical mem-

brane-enriched fraction, was very sensitive to bafilomy-cin A1; all concentrations used effecting a significantinhibition (Fig. 1(C)) with an IC50 of 0.44 nM. NEM alsoinhibited this ATPase activity of the apical membranefraction over the concentration range 0.5 to 100mM,with an IC50 of 2.15 mM (Fig. 1(D)). These results areconsistent with this ATPase activity being a V-type inits properties (Al-Awqati, 1986; Bowman et al., 1988;Mattsson et al., 1991).

3.4. Effects of pH and the requirements for ATP andMg2 + for azide and orthovanadate-insensitive ATPaseactivity (V-type ATPase)

Fig. 2(A) shows the effect of pH on the V-typeATPase activity, maximal activity was determined at pH7.5. Fig. 2(B and C) show that maximal activity of theenzyme was also obtained at equimolar (4 mM) concen-trations of Mg2 and ATP.

3.5. Effect of various ions on azide- andorthovanadate-insensitive ATPase (V-type ATPase)activity

The effect of various ions on V-type ATPase activitywere determined in the presence of 1 mM MgCl2, 5 mMTris–HCl (pH 7.5), 0.1 mM EDTA, 0.5 mM NaN3, 0.1mM Na3VO4 and 0.05% Triton X-100 (Schweikl et al.,1989). The effect of altering the concentrations of anumber of monovalent (K+, Na+, Li+, Rb+, choline andTris) chlorides on V-type ATPase activity was determ-ined. Maximal activation occurred in all cases between30 and 40 mM, higher concentrations tended to beinhibitory. The maximal activation achieved variedbetween 251% for Tris to 141% for Rb+, the stimulationobtained with K+ was 191%. Cations were stimulatoryin the following sequence Tris > K= Na > choline > Li= Rb. The effect of different anion salts of K+ (SO4

2 − ,Fl−, Br−, gluconate and N03−) on V-type ATPase activitywas also determined. Once again activation was maximalbetween 30 and 40 mM. Gluconate and SO4

2 − gaveabout 50% stimulation whereas Br− and F− produced asmaller stimulation of activity of about 25–30%. It issignificant that NO3− caused progressive inhibition withan IC50 of about 9 mM; 10 mM NaSCN also stronglyinhibited the V-type ATPase reducing its activity to 24.2± 5.7% of control values. What is clear is that both theanion and cation can affect enzyme activity, with HCO3

−

being the most effective stimulating anion (252.4±8.4%), with bromide, fluoride and sulphate being lesseffective than chloride.

3.6. SDS–PAGE

The different membrane fractions isolated from Mal-pighian tubules ofLocusta and a mixture of standard

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Fig. 1. The effect of varying concentrations of bafilomycin A1 and NEM on fluid production and V-type ATPase activity of Malpighian tubulecells of Locusta. Fluid production was measured at 30°C for 20 min in control saline (rate 1) and then for a further 20 min in control salinecontaining a stated concentration of bafilomycin (A) or NEM (B) to give rate 2. The percentage inhibition was determined by comparisons of rates1 and 2. These percentages were transformed into probits, the IC50 values shown were determined by regression analysis (Finney, 1952). V-typeATPase activity associated with the apical rich membrane fractions was determined in the presence and absence of different concentrations ofbafilomycin (C) or NEM (D). The percentage inhibition was determined by comparison of the enzyme activity in the presence and absence of theinhibitor, and the percentages were transformed into probits. (Each point represents mean± SEM, n = 5).

molecular weight markers were subjected to analysis bySDS–PAGE, under denaturing conditions (Fig. 3(i) lanesA–D). Western blots of these protein bands were probedwith monoclonal antibodies 230-3, 221-67, 224-3 and90-7, which recognized various subunits of V-typeATPase in crude homogenates of midgut ofM. sexta

(Klein et al., 1991). Only monoclonal antibody 230-3showed immunoreactivity with preparations fromLocustaand then only to one of the protein bands in theapical membrane-rich fraction (Fig. 3(ii), lanes A andB). This corresponded to the protein band with an esti-mated mass of 28 kDa.

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Table 1Distribution of enzyme activities in the various fractions derived from the differential centrifugation of a homogenate made from Malpighian tubulesof Locusta

Membrane fraction Mg2 + -ATPase Na+K+-ATPase Alkaline phosphatase Succinic dehydrogenase

Crude 163.4± 8.0 66.8± 4.6 325.4± 31.0 123.0± 6.0P1 196.7± 12.2 55.4± 9.0 181.6± 12.1 115.4± 12.3P2 279.4± 21.1 71.4± 6.9 466.3± 19.4 287.2± 29.6P3 440.7± 27.9 131.9± 11.2 1017.7± 38.5 103.2± 15.0P4 446.5± 11.7 331.9± 21.9 530.1± 45.5 133.1± 23.4P5 597.4± 38.1 26.7± 5.6 3603.3± 145.5 10.0± 1.5S1 192.3± 8.6 65.1± 7.7 467.4± 30.7 115.8± 13.2S2 169.6± 8.7 48.2± 5.5 402.4± 93.2 69.4± 15.3S3 21.8± 1.8 1.1± 0.5 36.4± 4.1 16.5± 2.9S5 37.5± 6.2 2.4± 0.6 182.0± 10.8 0

ATPase activities are expressed as nmoles Pi liberated mg protein−1 min−1. Alkaline phosphatase activity is expressed as nmoles p-nitrophenolliberated mg protein−1 min−1. Succinic dehydrogenase activity is expressed as nmoles succinate oxidized mg protein−1 min−1. Mean value± SEMfor three independent experiments.

Fig. 2. The effect of pH (A), Mg2 + (B) and ATP (C) concentrations of V-type ATPase activity of an apical membrane rich fraction fromLocustaMalpighian tubules. Enzyme activity expressed as nmoles Pi liberated mg protein−1 min−1. Mean± SEM from five experiments.

3.7. Native microgradient PAGE

Apical membrane enriched fractions of tubules ofLocusta, were subjected to native microgradient PAGE.Two protein bands showing ATPase activity, as revealedby hydrolysis of ATP, were identified (Fig. 3(iii)). Band1 was specific for ATP whereas band 2 also effectedhydrolysis of b-glycerophosphate (lane F), suggestingthat the latter was a non-specific alkaline phosphatase.The ATPase activity was particularly sensitive to inhi-bition by bafilomycin (lane B) and NEM (lane C) butwas not sensitive to the presence of either azide or ortho-vanadate (lanes D and E). In a parallel study, unstainedprotein bands were transferred to nitrocellulose and pro-bed with antibody 230-3. Fig. 3(iv) (lane B) shows that

only the protein corresponding to band 1 (i.e. that show-ing ATPase activity) was recognized by the V-typeATPase specific antibody fromM. sexta. In a controlexperiment a sample of a crude homogenate ofLocustaMalpighian tubules and a mitchondrial fraction were alsoprobed with antibody 230-3 and neither preparationshowed cross-reactivity with the antibody.

3.8. Immunocytochemistry

Incubations with antibodies 221-67, 224-3 and 90-7showed no significant binding to the cells of Malpighiantubule cells ofLocusta. In contrast, examination Fig. 4,shows that antibody 230-3 bound specifically to the api-cal microvillar surface of the tubule cells indicating the

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Fig. 3. Characterization and separation of proteins present in membrane fractions ofLocustaMalipghian tubules. (i) Microgradient SDS–PAGEgel. Lane A, mitochondrial fraction (P2); lane B basal membrane fraction (P4); lane C apical membrane fraction (P5); lane D molecular weightmarkers. Coomassie blue staining. (ii) Western blot of an SDS–PAGE microgradient gel prepared identically to (i) above. The blot was probedwith a monoclonal antibody 230-3 to the 28 kDa subunit of the midgut V-type ATPase fromManduca sexta(lane A). Molecular weight markersare shown in lane B. (iii) Native micrograrient PAGE of the apical membrane fraction ofLocustaMalipghian tubules. The gels were stained forATPase activity (lanes A–E) orb-glycerophosphatase activity (lane F). The ATPase activity was demonstrated in control media (lane A); in thepresence of 5mM bafiloycin A1 (lane B); 10 mM NEM (lane C); 100 mM NaN3 (lane D); or 1 mM Na3VO4 (lane E). Phosphate release wasvisualized by lead acetate plus ammonium sulphite precipitation. (iv) Microgradient gels prepared as described in (i) above. Lane A is stained forATPase activity and lane B was probed with antibody 230-3.

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Fig. 4. Fluorescent labeling of Malpighian tubule cells ofLocusta.Frozen sections were labeled with the primary monoclonal antibody230-3 to the midgut V-type ATPase fromManduca sexta. FITC-conju-gated sheep-antimouse antibody was used to locate the bound pri-mary antibody.

recognition of a specific V-type ATPase epitope. Fluor-escence labeling of the basal cell surface and cytoplasmwas not significant, being similar to that seen in controls.

4. Discussion

As described previously by Fogg et al. (1991), differ-ential separation of the apical cell membrane markerenzyme alkaline phosphatase (Ernst and Mills, 1980) andthe basolateral membrane marker, Na+/K+-ATPase, wasobserved at the 10 000g spin. Pellet P4 contained rela-tively high specific Na+/K+-ATPase activity with littleapical membrane contamination, reflected by the rela-tively low alkaline phosphatase activity. The high spe-cific activity reported of 331 nmoles Pi liberated mgprotein−1 min−1 was greater than that reported for thesame tissue using a deoxycholate/NaI extraction pro-cedure, which gave specific activities of 220–290(Anstee and Bowler, 1984); however the latter prep-arations contained appreciably less Mg2+ -ATPaseactivity (Table 1). The high specific activity for Na+/K+-ATPase confirms that P4 is basolateral-membrane rich.The specific activity of alkaline phosphatase was greatest

in pellet P5, which, in turn, exhibited relatively lowNa+/K+-ATPase activity. Mitochondrial contaminationof P5 fraction, as indicated by succinate dehydrogenaseactivity, was extremely low.

Subsequent studies on the apical membrane-rich frac-tion (P5) revealed the presence of azide- and orthovanad-ate insensitive ATPase activity similar to that reported inlepidopteran midgut goblet cells (Schweikl et al., 1989;Wieczorek et al., 1989). This enzyme activity was sub-stantially inhibited by NEM (IC50 | 2 mM) in agreementwith the reported NEM sensitivity for the enzyme ofM.sexta (Schweikl et al., 1989). This, together with thehigh bafilomycin sensitivity of the enzyme activity, sug-gested that a substantial proportion of this azide- andorthovanadate-insensitive activity present was due to aV-type ATPase (Schweikl et al., 1989); however, neitherbafilomycin or NEM totally eradicated apical ATPaseactivity.

Monoclonal antibodies which had been raised to dif-ferent subunits of the V-type ATPase from the gut ofM.sextawere used to probe different enriched membranefractions of the Malpighian tubules ofL. migratoria. Ofthese antibodies, only one, antibody 230-3 (specific forthe 28 kDa subunit inM. sexta), recognized a proteinpresent in the Malpighian tubules ofLocustaand thisprotein was confined to the apical membrane rich frac-tion. Support for the apical locatization of a V-typeATPase was provided by the immunocytochemicalstudy, where once again, antibody 230-3 bound specifi-cally to the cell’s apical surface. This is supported bythe fact that ATPase activity was confined to one proteinband separated by native electrophoresis, and this bandreacted with antibody 230-3 rather than the band whichshowed alkaline phosphatase activity. Furthermore, thisATPase activity was sensitive to bafilomycin A1 andNEM. The fact that only one of theManducamono-clonal antibodies cross-reacted with a subunit of theLocusta V-type ATPase suggests that this antibodyrecognized a conserved epitope between the species.There is therefore clear evidence that a V-type ATPaseis present in Malpighian tubule cells ofLocusta, and thatthis activity is associated with their apical surface.Vacuolar-type ATPases were originally considered to belocalized in the endomembrane systems of eukaryoticcells, e.g. lysosomes and the Golgi complex, although,more recently, they have also been reported in theplasma membranes of vertebrate urinary epithelia (Gluckand Caldwell, 1987; see also Forgac, 1989).

Biochemical studies on the V-type ATPase revealedthat maximal ATP hydrolysis was obtained at pH 7.5, avalue similar to that reported for the enzyme from mam-malian brush border (Ait-Mohamed et al., 1986; Wangand Gluck, 1990, but lower than that reported for theenzyme fromManducamidgut (Wieczorek et al., 1986).In common with many other ATPases the enzyme wasmaximally stimulated at equimolar (4 mM) concen-

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trations of Mg2 + and ATP, which is consistent with aMgATP complex forming the substrate for the enzyme.

V-type ATPases are reported to be activated by a var-iety of both anions and monovalent cations. The fact thatthis activation occurred in the range 30–40 mM for avariety of salts, suggested that it may be non-specificand probably a response to the change in ionic strength.The enzyme seemed more specifically sensitive to anionsthan cations. It was strongly inhibited by nitrate, a fea-ture of V-type ATPases from other sources, (O’Neill etal., 1983; Lichko and Okorokov, 1984) and by thiocyan-ate, both features of the anion-activated ATPasedescribed in this tissue (Fogg et al., 1991; Fathpour andDahlman, 1994). Fogg et al. (1991) suggested that theanion-stimulated activity present in the apical mem-branes of Malpighian tubules may be due to a V-typeATPase. It is of interest that recently Dschida and Bow-man (1995) have questioned the mechanism of enzymeinhibition by NO3

− and SCN−, whether it be by chao-tropic action or by an influence of the redox state of theenzyme. The low concentration of the reagents used inour study would argue against a chaotropic action.

The physiological significance of this V-type ATPasein tubule function is suggested from the studies on tubulesecretion in vitro. NEM and bafilomycin A1 bothinhibited fluid secretion. The concentrations of bafilo-mycin A1 needed to produce a significant inhibition offluid secretion in the present study were higher thanthose reported to inhibit the V-type ATPase activity inthe apical membrane rich fractions. Nevertheless theywere lower than those that inhibit P- and F-type ATPases(Forgac, 1989). However, it is likely that the intracellularconcentration of bafilomycin in these physiologicalexperiments is much lower than that applied in themedium bathing tubules (0.5–5mM). Other workersreport similar findings e.g. bafilomycin A1 (1 mM)caused significant inhibition of fluid production bytubules of Drosophila hydeiand 10 mM completelyarrested it within 15–30 min (Bertram et al., 1991); fluidsecretion was also significantly inhibited in the tubulesof Formica polyctenausing 5 mM bafilomycin A1

(Weltens et al., 1992).A higher concentration of NEM (100mM) was also

needed to produce significant inhibition of fluid secretionthan was required to inhibit the partially purifiedATPase. The concentrations of NEM compared with thatreported by other workers. Weltens et al. (1992) workingon the tubules ofF. polyctenafound that a concentrationof 500 mM was required to inhibit in this species andBertram et al. (1991) found significant effects onsecretion of fluid byD. hydeiat concentrations of NEMgreater than 10mM with complete inhibition at 1 mMNEM being found. The significance of this inhibition offluid production by NEM is complicated because 100mM NEM is also at the lower end of the sensitivity ofP-type ATPases, and even though it is unlikely that the

intracellular concentrations would be that high, thepossibility that the inhibitory effect on fluid secretion isnot due to V-type ATPase inhibition cannot be excluded.

The biochemical properties of the enzyme reportedhere, together with the immunocytochemical evidence,strongly support the existence of a classical V-typeATPase on the apical surface of the Malpighian tubulesof Locusta. It is proposed that, in common with otherinsect secretory epithelia, this enzyme creates a protonmotive force across the apical membrane, and acts inparallel with a K+/H+ antiporter to effect the active trans-port of K+ across the apical membrane which in turn isthe driving force for fluid secretion.LocustaMalpighiantubules also secrete appreciable quantities of Na+ and itis possible that Na+ and K+ share a common antiporter,however Pivovarova et al. (1994) have provided evi-dence in support of separate exit ports for these two ions,both of which may depend on the proton motive forcegenerated by the V-type ATPase.

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