Proc. Nati. Acad. Sci. USAVol. 87, pp. 1456-1460, February 1990Biochemistry

Intestinal brush border membrane Na+/glucose cotransporterfunctions in situ as a homotetramer

(intestine/sodium-glucose symporter/radiation inactivation)

BRUCE R. STEVENS*t, ALARICO FERNANDEZ*, BRUCE HIRAYAMAt, ERNEST M. WRIGHTt,AND ELLIS S. KEMPNER§*Department of Physiology, College of Medicine, University of Florida, Gainesville, FL 32610-0274; tDepartment of Physiology, University ofCalifornia-Los Angeles Medical Center, Los Angeles, CA 90024-1751; and §National Institutes of Health, Bethesda, MD 20892

Communicated by Bernhard Witkop, November 29, 1989 (received for review October 4, 1989)

ABSTRACT The functional unit molecular size of theintestinal brush border membrane-bound Na+/glucosecotransporter was determined by radiation inactivation. Puri-fied brush border membrane vesicles preserved in cryopro-tectant buffer were irradiated (-1350C) with high-energyelectrons from a 13-MeV (1 eV = 1.602 x 10-'9 J) linearaccelerator at doses from 0 to 70 Mrad (1 rad = 0.01 Gy). Aftereach dose, the cotransporter was investigated with respect to (i)Na'-dependent transport activity and (ii) immunologic blotanalysis with antibodies against the cloned rabbit intestinalcotransporter. Increasing radiation decreased the maximalNa'-dependent cotransporter activity JfaX without affectingapparent Km. The size of the transporting functional unit was290 ± 5 kDa. Immunologic blot analysis of brush bordermembranes gave a single band ofMr 70,000, which decreasedin intensity with increased radiation dose and gave a target sizeof 66 ± 11 kDa. We conclude that activity of the intestinalNa+/glucose cotransporter in situ in the brush border mem-brane requires the simultaneous presence of four intact, inde-pendent, identical subunits arranged as a homotetramer.

The intestinal brush border membrane Na+/glucose cotrans-porter is among the most thoroughly studied eukaryoticsystems demonstrating secondary active (ion-coupled) trans-port. Biochemical and immunological experiments have iden-tified the cotransporter as a polypeptide ofMr 70,000-75,000on SDS/PAGE reducing gels (1-3), and the cloned cotrans-porter has a predicted molecular mass of 73 kDa (5, 6).However, information regarding structure-function relation-ships of the cotransporter in situ in the native membrane islacking. In this study we used high-energy electron radiationinactivation to investigate the membrane-bound in situ size ofthe cotransporter. Our results indicate that the cotransporterfunctions in the membrane as a 290-kDa homotetramercomprised of four independent 73-kDa subunits.

METHODS AND MATERIALSBrush border membrane vesicles were prepared from maleNew Zealand White rabbit small intestines by a 10 mM MgCl2precipitation procedure (7, 8). The membrane vesicles wereenriched =15-fold in the brush border membrane markerenzymes alkaline phosphatase, leucine aminopeptidase, su-crase, and 'y-glutamyltranspeptidase. Vesicles (20 ,ug of pro-tein per ,l) were equilibrated in a cryoprotectant buffer (9,10) that contained 14% (vol/vol) glycerol, 1.4% (wt/vol)D-sorbitol, 150 mM KCI, and 5 mM Hepes-Tris (pH 7.5).Beliveau et al. (9, 10) have established that this bufferpreserves glucose and phosphate transport, and Na+/H+

antiport in kidney brush border membrane vesicles. Aliquotsof the vesicle suspension were frozen in 2-ml glass ampules(type 12012; Kimble, Toledo, OH). The sealed ampules werethen kept at -80'C and transported on dry ice. The mem-branes were irradiated at -1350C with a beam of 13 MeV (1eV = 1.602 x 10-1' J) electrons produced by a linearaccelerator, as described elsewhere (11). After irradiation theampules were held at -800C until the contents were assayed.The irradiated frozen ampules were opened and purged

with air, and the contents were thawed at room temperature.The vesicles were resuspended by drawing through a 27-gauge syringe needle and then were placed on ice. Glucosetransport activity was measured during a 10-sec influx periodwith and without NaCi, using a rapid-mix/rapid-filtrationtechnique (7). The characteristic transport properties ofbrush border membrane vesicles (7, 8, 12) were preserved byusing the cryoprotectant buffer at temperatures ranging from-80 to -196TC, as also verified by Beliveau et al. (9, 10). TheNa'-dependent cotransporter activity (J) at each radiationdose was the difference in total transport measured in NaCl(JtotaI) minus transport measured in choline chloride buffer(JNa-indePendent). J at each radiation dose was assessed relativeto control unirradiated transporter activity (J0). Data werefitted by a least-squares analysis of the relation ln(J/JO) =-kD, where D is the radiation dose in Mrad (106 rad; 1 rad= 0.01 Gy), and k is the slope of the inactivation curve. Thecomputer regression program was constrained such that J/Jo= 1 when D = 0. The molecular mass of the functional unit,or target size, was subsequently determined (13-15) from therelationship:

target mass = 17.9 x 105 k, [1]

which includes the temperature correction factor for irradi-ations performed at -135°C. Dosimetry was performed asdescribed elsewhere (16).

Anti-Peptide Antibodies. Polyclonal antibodies AbE andAbC (B.H., H. C. Wong, M. A. Hediger, and E.M.W.,unpublished work) were made against selected regions of thecloned rabbit intestinal Na+/glucose cotransporter (5). AbEwas against nonadecapeptide from the major extracellularhydrophobic loop (residues 402-420), and AbC was against adodecapeptide from the major intracellular hydrophobic loop(residues 604-615). These antibodies were raised in rabbitsaccording to Walsh and Wong (17) and were the IgG fractionspurified using protein A-Sepharose (Pharmacia).Immunologic (Western) Blots. Immunodetection of the

brush border Na+/glucose cotransporter on Western blotswas carried out using goat anti-rabbit IgG conjugated tohorseradish peroxidase. Briefly, the desired amount of brushborder membrane protein was solubilized in SDS samplebuffer [2% SDS/62.5 mM Tris-HCI, pH 6.8/1 mM dithio-

tTo whom reprint requests should be addressed.

1456

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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threitol/20% (vol/vol) glycerol] immediately after the samplehad thawed and was boiled for 3 min before loading onto thegel. Protein was electrophoresed in a minigel format (Mini-Protean; Bio-Rad) and then electrophoretically transferred tonitrocellulose paper (BA85; Schleicher & Schuell). The re-maining nonspecific protein-binding sites on the nitrocellu-lose were blocked by preincubation with 0.05% (vol/vol)Tween-20 and 0.5% (wt/vol) nonfat dried milk powder (Car-nation), for 1 hr at 220C. The blots were then incubated in theantibodies (1:1000 dilution) for 2 hr. The bound antibodieswere detected with goat anti-rabbit IgG conjugated to horse-radish peroxidase (Calbiochem) and developed in diami-nobenzidine (4, 17). Care was taken to develop the blot suchthat the intensity of staining was directly proportional to theamount of antigen.

Identical blots were run under peptide-blocked conditionsto visualize the nonspecific binding (background) staining. Inthis case, the antibody dilution was preincubated with 0.5 ,gof peptide per ml of the relevant peptide at 370C for 1 hr. Thebackground in the area of interest was minimal comparedwith an empty lane.

In all experiments a standard serial dilution of controlrabbit brush border protein (40-2.5 Ag) was included to verifythat the samples gave density readings in the linear range.The blots were scanned on a densitometer (GS300; Hoefer)in the reflectance mode, and the data were analyzed bycomputer with the companion software. The nonspecificbinding was subtracted from the total binding to give thespecific immunoreactivity. We observed an increase in thepeptide-blockable immunostaining over the entire molecularweight range with increased radiation dose, which was re-lated to a generalized increase in background staining (18).This immunostaining was regarded as background and wassubtracted from the specific immunostaining by continuingthe profile of the background through the peak area (A) todetermine the amount of the area over this background. Theremaining relative area of the peak (A/AO) at each radiationdose, D, was then employed in the relation In(A/AO) = -kDto calculate the inactivation slope k. The target size wassubsequently determined using Eq. 1.

Electrophoresis-grade glycine, Tris, SDS, and the proteinassay kit were purchased from Bio-Rad. All other reagents

U(I,0)

E"1-5

E

0

CL

0

U_j

200

150-

100-

50-

were obtained from Sigma, unless otherwise specified andwere of the highest grade available. D-[6-3H]glucose (30Ci/mmol; 1 Ci = 37 GBq) was purchased from New EnglandNuclear. Data points are presented as means ± SEM (n 2 3assays). Results were confirmed for three independent radi-ation experiments using five separate brush border vesiclepreparations from a total of 10 rabbit intestines. Data wereanalyzed using linear and nonlinear regression computerprograms.

RESULTSTransport Activity Functional Unit Size. After membrane

vesicles were irradiated with high-energy electrons, glucoseuptake was measured as a function of radiation dose, asshown in Fig. 1. In NaCl buffer, total glucose uptake de-creased with radiation dose to an asymptote value equivalentto 5% of the control. The Na+-independent glucose uptakewas a constant value unaffected by radiation dose; thisdemonstrated that the vesicles were not leaky and retainedtheir integrity in the cryoprotectant buffer even up to thehighest radiation dose. Fig. 2 shows that Na+/glucosecotransporter activity decayed as a single exponential func-tion of radiation dose. Based on three independent experi-mental data sets, the mean target size for function was 290 ±50 kDa.The data of Fig. 1 demonstrated a radiation-resistant

component ofJt tai, which was equal to jNa-indePendent. There-fore, the constant value ofJtotal determined at radiation doses>30 Mrads could be used in place of jNa-indePendent measuredin the presence of choline. This analysis yielded an averagetarget size of 290 ± 5 kDa (n = 3).

Effects of Radiation on Cotransporter Kinetics. Na+/glucose cotransporter uptake kinetics were determined inmembranes irradiated at doses of 0, 3, and 6 Mrad. Nonlinearregression analysis of the kinetic data gave the same Km =303 ± 26 ,uM glucose, regardless of radiation dose; this valuewas similar to the previously published value of 340 ,M forthe intestinal brush border membrane major Na+-dependentglucose cotransporter (8) measured under similar conditions.The jmax decreased with dose, however. Based on jmaxvalues, the target size was 284 ± 28 kDa (n = 3).

Total in Noa

0

Total incholine 1 _ @ 0 __a____

.

1 2 24 36 48 60 72Dose (Mrad)

FIG. 1. Glucose (2 ,M) initial rate of transport activity as function of radiation dose. Transport was initiated by adding 10,l of the irradiatedprotein suspension to 40 ,ul of uptake buffer containing 5 mM Hepes'Tris (pH 7.5), 14% glycerol, 1.4% D-sorbitol, 2 ,uM D-[6-3H]glucose, and150 mM NaCI or 150 mM choline chloride. Uptake was arrested after 10 sec by using an ice-cold stop solution containing 500 p.M phlorizin,5 mM Hepes-Tris (pH 7.5), 14% glycerol, 1.4% D-sorbitol, and 150 mM NaCI. Increasing the radiation dose decreased JtotaI (o) measured inthe presence of NaCI to an asymptote value equal to the constant jNa-indcpendent (0) measured in the presence ofcholine chloride. Points representmeans ± SEM (n = 4 replicates) from a radiation experiment using a single batch of membranes and is typical of three independent experiments.

Biochemistry: Stevens et al.

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1 .00

c)0

u

aL.

Li-

0)

c

c

E

0.10

0.01

0 20 40 60DOSE (Mrads)

FIG. 2. Determination of the molecular mass of the membrane-bound Na+/glucose cotransporter. For each of three independentexperiments similar to Fig. 2, the Na'-dependent glucose cotrans-porter activity (J) was assessed at each radiation dose and expressedrelative to control unirradiated transporter activity (JO), where J =

Jtot _ jNa-indePendcnt. The logarithm of remaining activity (e) was alinear function of dose. Based on three independent experiments, theaverage target size was 290 ± 50 kDa. In irradiated membranes fromthree independent experiments, the amount of subunit polypeptidewas measured on Western blots using AbC and AbE after gelelectrophoresis, as described in Methods and Materials and Fig. 3.The fraction of polypeptide remaining is shown (o); these experi-ments yielded an average target size of 66 11 kDa.

Effects ofRadiation on the Monomer Band on Western Blots.Polyclonal antibodies were made against synthetic peptidesthat represented portions of the primary sequence of thecloned rabbit intestinal Na+/glucose cotransporter; the pep-tides were selected based on the secondary structural model(5, 6). Western blots with antibodies to the cloned rabbitintestinal Na+/glucose cotransporter gave a single, specificband Of Mr 70,000 (Fig. 3). Fig. 3 shows the effect of radiationon the Mr 70,000 band in rabbit brush border membranes byusing AbC. The area of the peak was assessed at each dose

-

Q)

Q)

cc

o Mrald

24 Mrad

3

97 66 43 x1O

Mr

FIG. 3. Effect of increasing radiation dose on Na+/glucosecotransporter monomer. In this example, brush border membraneswere irradiated at 0 and 24 Mrad and then probed by Western blotanalysis by using polyclonal anti-Na+/glucose cotransporter anti-body AbC. Shown are densitometry scans of the blots. The amountof the cotransporter was estimated from the area of the peaks (Mr70,000) after subtraction of background.

up to 72 Mrad, and the remaining fraction of peak area wasexpressed relative to the control unirradiated peak area, asshown in Fig. 2. In three independent experiments, the targetsize of the monomer peak in brush border membranes was 66± 11 kDa. Identical results were obtained with AbE.

DISCUSSIONMonomer Genetic Translation. The intestinal Nat/glucose

cotransporter has been extensively studied, and much isknown about its unique location in the brush border mem-brane of the enterocyte, sugar specificity, cation specificity,and kinetics (1-3, 5, 6, 8, 19). Biochemical experiments haveidentified the rabbit cotransporter as a polypeptide of Mr70,000-75,000 on reducing SDS/PAGE gels and providedevidence that the Na' and D-glucose binding sites are both onthe same polypeptide (20, 21). The rabbit Na+/glucosecotransporter has been cloned, sequenced, and expressed inXenopus oocytes (5, 6). The cDNA codes for a protein of 662amino acids with a predicted mass of 73 kDa. The propertiesof the clone expressed in oocytes are virtually indistinguish-able from those of the native brush border transporter (22).It may be concluded that the functional Na+/glucose cotrans-porter is a single gene product with a mature Mr of 70,000-75,000 on SDS/PAGE reducing gels.Some kinetic studies with intestinal brush border vesicles

have suggested the presence of two cotransporters (8). Onlyone has been identified, cloned, and expressed, and otherexplanations can account for the kinetic behavior in vesicles(22). It is our working model that there is only one intestinalbrush border Na+/glucose cotransporter.

Functional Unit Size in Situ. The molecular organization ofthe Na+/glucose cotransporter has been suggested to be anoligomer in the brush border membrane of intestine (23) andrenal proximal tubule (9). In the present study we usedradiation inactivation in conjunction with transporter func-tion and Western blot analysis to assess the functional unitmolecular mass of the Na+/glucose cotransporter in cryo-protected native membranes. The theory and practice ofradiation inactivation is well established in confirming thefunctional unit size of a wide variety of enzymes (11). Thetechnique has also been used to determine the in situ func-tional unit size of brush border membrane-bound enzymes(24, 25) and transporters (9, 10). Specifically, the radiationtarget size of the kidney brush border membrane Na+/glucose cotransporter in situ has been measured in a varietyof species, with a target size of 288 kDa (9) and 298 kDa (26)for transporter activity. Inasmuch as the cloned intestinalNa+/glucose cotransporter is homologous to the human renalclone (27), our finding (Fig. 2) that the intestinal brush bordermembrane-bound in situ radiation target size is 290 kDa isconsistent with these renal target sizes. Parenthetically,insensitivity of the Na+-independent uptake to radiationsuggests that this transport mode is primarily not protein-mediated at 2 ,uM glucose.Homotetramer Organization. Radiation target theory as-

signs the size of the functional unit but does not assignmolecular organization of subunits (15, 28). When a high-energy electron hits a polypeptide chain, the deposited en-

ergy completely destroys the chain, resulting in loss offunction. The energy can be absorbed by a single polypeptideand not spread to adjacent chains (14); in such oligomericproteins, each subunit is destroyed as a single entity.

In the present study, we obtained two different sizes for therabbit cotransporter, depending on the postirradiation func-tional assay. In Fig. 3 the Western blot protein band at Mr70,000 gave a target size of 66 ± 11 kDa, corresponding to themature Mr 70,000-75,000 protein, whereas the Na'-depen-dent glucose transporter activity gave a target size of 290 ±5 kDa. Furthermore, only the transport Jmax, but not Km,

1458 Biochemistry: Stevens et al.

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0*

........0FIG. 4. Tentative model of the membrane-bound arrangement of the intestinal brush border Nat/glucose cotransporter. The transporting

functional unit (collectively -290 kDa) requires four intact independent identical monomer subunits (each 73 kDa). At the extracellular surfaceNa' binds to the cotransporter, which results in a conformational change that permits glucose to bind to the glucose-binding site. With bothsubstrate species bound, the cotransporter undergoes a new conformational shift, placing Na' and glucose near the inner surface of themembrane. Na' and glucose are released to the cytoplasm, thereby triggering a cotransporter conformational shift to expose the binding sitesonce again to the extracellular surface of the membrane. The cotransporter thus again assumes the optimal conformation for binding extracellularNa' and glucose. For reasons of clarity, only one Na' and one glucose are shown to interact with the cotransporter. Previous studies haveindicated that each 73-kDa polypeptide has both Na' and glucose active sites (1, 21).

varied as a function of radiation dose, giving a target size of284 + 28 kDa; this finding offers strong evidence that a singlehigh-energy electron hit completely inactivated the cotrans-porter functional unit, rather than partially affecting cotrans-porter activity by reducing substrate affinity.The target size of 290 kDa could be due to the cloned

73-kDa peptide plus other, as yet unidentified, proteins.However, because only a single protein species (5, 6) isrequired for transport activity (22), our results indicate thatthe function of the intestinal Na+/glucose cotransporter insitu in the brush border membrane requires the simultaneouspresence of four intact, independent, identical 73-kDa sub-units arranged as a homotetramer. This model is similar tothat proposed in a radiation inactivation study of the sixindependent subunits of glutamate dehydrogenase (14).There are two simple models for transport activity that

would be consistent with these results: (i) a single functionalsite is shared by the four subunits or (ii) an independent site ison each subunit, but the conformation needed for activity ismaintained only by the mutual interaction of all four subunits.Our depiction of the homotetramer arrangement in the mem-brane (Fig. 4) could accommodate either mechanism. How-ever, there is strong evidence that each monomer potentiallypossesses independent binding sites for both Na+ and glucose(20, 21), and that the in situ contransporter possesses discreteNa+ and glucose binding and translocation steps (4). There-fore, the present data suggest that the intestinal Na+/glucose

cotransporter exists as four subunits working in concert andthat an individual subunit's activity can only be manifested bythe interaction of all the other components.

We thank John Freeman for technical assistance. This work wassupported by Grants DK-38715 and DK-19567 from the NationalInstitutes of Health.

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