Effect Surface Modifiers on an Ectoenzyme: Granulocyte 5

INFECTION AND IMMUNITY, May 1980, p. 475-485 Vol. 28, No. 20019-9567/80/05-0475/11$02.00/0

Effect of Surface Modifiers on an Ectoenzyme: Granulocyte 5'-Nucleotidase

JAMES E. SMOLENt AND MANFRED L. KARNOVSKY*Department ofBiological Chemistry, Harvard Medical School, Boston, Massachusetts 02115

Several agents that react with plasma membranes, namely the native lectinsconcanavalin A, Ricinus communes agglutinin, and wheat germ agglutinin, themodified lectin succinyl concanavalin A, and sodium meta-periodate, inhibitedthe ecto-5'-nucleotidase of intact guinea pig granulocytes. Stimulation of theenzyme was not observed at any lectin concentration. Inhibition by native lectinscould be blocked or reversed by appropriate competing hapten sugars. In the caseof concanavalin A, reversal could be achieved at 370C, but not at 5VC. Whenlectins were used in combination with each other, the effects were found to belargely independent. However, when concanavalin A and R. communes agglutininwere applied together, complications arose because the former lectin binds to thelatter as well as to the cell surface. To avoid some of the complexities inherent instudying intact cell 5'-nucleotidase and to gain additional information about thesystem, two broken cell enzyme preparations were also examined. The enzyme ofplasma membrane-enriched fractions was inhibited by all five agents mentionedabove. 5'-Nucleotidase solubilized in sodium deoxycholate was inhibited by thefour lectins but stimulated by periodate. The effects of the surface modifiers onkinetic data for all three enzyme preparations are consistent with the hypothesisthat direct interactions with the enzyme molecule give rise to changes in Vm..;interactions at membrane sites other than 5'-nucleotidase itself could causeincreases in apparent Km values. Effects of interactions of ectoenzymes with plantlectins may serve as models for phenomena that result from cell-cell interactionsor from interactions of animal cells with lectin-like components of the cellularenvironment.

The fact that high-molecular-weight nonpen-etrating molecules, such as lectins, can modifyenzymatic activities on cell surfaces is a rela-tively recent discovery (21, 25). Such modulationof ectoenzyme activities could conceivably serveas a trigger for dramatic changes in cell metab-olism or behavior, as exemplified by mitogenesis(21) and instigation of metabolic events similarto those characteristic of phagocytosis (26). Fur-thermore, lectins could provide tools for explor-ing the question of whether an enzyme is anectoenzyme, providing that entry of the lectininto the cell can be monitored (16,33). Publishedaccounts of the effects of surface modifiers onmembrane enzymes have dealt mainly with con-canavalin A (ConA) (2, 3, 25, 27-29, 32). Themost commonly investigated enzyme of the cellmembrane in this context is 5'-nucleotidase,which has been examined in rat livers (25, 27),rat mammary glands (2, 3), and C6 glioma cells(28). Both activation and inhibition of ectoen-zymes have been observed after incubations ofintact cells with lectins (3, 28).

t Present address: Department of Medicine, New YorkUniversity Medical Center, New York, NY 10016.

Although it is tempting to postulate directspecific interactions between lectins and mem-brane enzymes on the basis of such observations,further experimental criteria must be met. Forexample, direct interactions should be observedimmediately, not after long preincubations (24),during which cellular processes triggered by alectin could conceivably change the amount ordisposition of an ectoenzyme or could produceenzyme activators or inhibitors. Competingsugars should be used to block or reverse theeffect of the lectin in order to evaluate thespecificity of the effects (2, 3, 27-29, 32). It ispossible to minimize the complications inherentin the use of whole cells by conducting studiesof cell-free systems, such as microsomes orplasma membrane preparations (2, 3, 25, 32).Some account could than be taken of indirectinteractions, for example those transmitted inwhole cells by cytoskeletal structures, such asmicrotubules and microfilaments. However,even this approach would not eliminate the pos-sibility that lectin interactions at sites on themembrane other than at the target enzyme itselfcould be responsible for the phenomena ob-

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476 SMOLEN AND KARNOVSKY

served in whole cells. The use of a purifiedenzyme preparation is required for absoluteproof of direct interaction of an enzyme with alectin (27). Unfortunately, it is often very diffi-cult to purify membrane-bound enzymes to ahigh degree. As a minimum, systems with un-purified enzymes solubilized in detergents (3, 25)should be employed to circumvent some com-plications inherent in the use ofmembrane prep-arations, where indirect interactions that affectenzymatic activity might still occur. Finally, thekinetic properties of lectin-modified ectoen-zymes, which have been described in some re-ports (3, 25, 28, 32), could be revealing.

In accordance with these desiderata we stud-ied the effects of several surface modifiers,namely, ConA, Ricinus communes agglutinin(RCA), wheat germ agglutinin (WGA), succinylconcanavalin A (S-ConA), and sodium meta-per-iodate, on the 5'-nucleotidase of intact granulo-cytes and on membranes and detergent extractsderived from those cells. Guinea pig granulo-cytes are particularly suitable for these studiessince virtually all of the cellular 5'-nucleotidaseis an ectoenzyme (6, 7).The physiological roles ofectoenzymes are not

yet clear, and the relevance of interactions ofthese enzymes with lectins can only be surmisedat present. However, the presence of glycopro-teins on cell surfaces is well established, andthere are strong indications of what may betermed "animal lectins" (9) on cell surfaces.Phagocytic leukocytes, in particular, do makecontact with exogenous particles that could haveavailable lectin-like surface components. Cell-to-cell contacts, phagocyte-particle contacts, andcellular exposure to soluble lectins or lectin-likesubstances could all modulate the activity ofectoenzymes of leukocytes.

MATERIALS AND METHODSCells. Guinea pig polymorphonuclear leukocytes

were harvested 15 to 20 h after intraperitoneal injec-tion of a sterile sodium caseinate solution (6). Thecells were washed three times before use in Krebs-Ringer phosphate buffer (KRP), pH 7.4. Cell mono-layers were formed on plastic culture dishes by a minormodification of the method of Michell et al. (19).Plasma membrane-enriched preparations. Ho-

mogenates free of nuclei were prepared from non-phagocytizing cells by the method of DePierre andKarnovsky (5). A 5-ml amount of homogenate wasoverlaid on a discontinuous sucrose density gradientconsisting of 6.5 ml of 40% (wt/vol) sucrose and 6.5 mlof 8% (wt/vol) sucrose. The tubes were then centri-fuged at 27,000 rpm (100,000 x g) in an SW27.1 rotorfor 1 h. Material harvested from the 8%/40% interfacewas dispersed in KRP buffer pH 7.4, by gentle homog-enization. This fraction typically contained 30 to 40%of the total plasma membrane marker 5'-nucleotidase,with a 6- to 10-fold increase in specific activity com-

pared to that of the original homogenate. It also con-tained 5% of the total succinate dehydrogenase, 5% ofthe total /?-glucuronidase, and 10 to 20% of the totalalkaline phosphatase (assays were performed by themethod of Michell et al. [18]). These enzymes aremarkers for mitochondria and for structures particu-larly characteristic of granulocytes: azurophilic (pri-mary, lysosomal) granules and specific (secondary)granules, respectively. It is likely that the alkalinephosphatase activity reflects contamination of themembrane fraction with specific granules because pre-liminary electron photomicrographs showed substan-tial contamination of the membrane-enriched fractionwith particles in the size range expected for specificgranules. Furthermore, repeated freeze-thaw treat-ments of these samples followed by centrifugationremoved almost all of the alkaline phosphatase con-tamination (as well as the lesser ,B-glucuronidase con-tamination) from the particulate material (membrane-enriched fraction). Attempts to improve the quality ofthe membrane preparation have failed, as methodswhich slightly improve the specific activity of 5'-nucle-otidase produce large decreases in membrane yield.Experiments with these preparations were performedimmediately after isolation.

Detergent-solubilized 5'-nucleotidase. Cellswhich were washed in KRP were suspended in 25pellet volumes of 0.9% NaCl-10 mM tris(hydroxy-methyl)aminomethane hydrochloride, pH 7.0. A con-centrated solution of sodium deoxycholate (DOC) wasadded to the suspension to a final concentration of0.5%. The resulting gel was incubated at 37°C for 30min and then centrifuged at 25,000 x g and 4°C for 30min. Centrifugation separated the viscous gel into twophases, a clear fluid supernatant phase, which wasdecanted and saved, and a gel pellet which was dis-carded. The supernatant contained solubilized 5'-nu-cleotidase; the yield of this enzyme was 40 to 60% ofthe starting value (the remainder was in the pelletfraction). The specific activity (based on protein) wassomewhat increased (up to 50%). The DOC extractcould be stored overnight at 4°C with a loss of onlyone-third of the enzyme activity. Any gelling of theextract during storage could be eliminated by gentlerotary action at room temperature.

Triton X-100, which has been used by other inves-tigators (17), was not employed since it interfered withthe enzyme assay. Solubilization of 5'-nucleotidase byDOC was demonstrated by the fact that centrifugationat 100,000 x g for 3 h did not sediment any of theenzyme. Furthermore, the homogeneous distributionof 5'-nucleotidase in the centrifuge tube was notchanged by this procedure.General experimental procedure. (i) Cell

monolayers. All solutions of ConA (grade IV; SigmaChemical Co.), WGA (Sigma), and sodium meta-per-iodate were prepared from dry solids immediatelybefore use. Solutions ofRCA (prepared by the methodof Nicolson and Blaustein [20]) and of S-ConA (pre-pared by the method of Gunther et al. [12]) were madeup daily from concentrated frozen stocks.

In most experiments, duplicate cell monolayerswere incubated with 1 ml of KRP containing thespecified lectin (0.1 to 3,400 ,Ag/ml) or competing sugar(10 to 100 mM) at 4 to 6°C (on a cold plate) for 15

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EFFECT OF SURFACE MODIFIERS ON ECTOENZYME 477

min, unless otherwise stated. Similar incubations withperiodate solutions (10 uM to 10 mM) were done at370C. Between incubation periods, the monolayerswere washed at least six times in 0.9% NaCl. 5'-Nucle-otidase assays were then performed by incubating thewashed monolayer cells with 1 ml of assay medium at370C for 15 to 20 min.

(ii) Plasma membrane and solubilized enzymesystems. In typical experiments, duplicate enzymesamples (0.1 ml each) were suspended in 0.5 to 0.7 mlof KRP containing a particular concentration of lectin,periodate, or sugar. Unless otherwise specified, 15-minincubations were conducted at 370C for samples con-taining periodate and at 40C for all other samples.Further incubation periods, if any, were initiated byadding 0.1 ml of the specified concentrated materialto the enzyme suspension. In contrast to the situationdescribed above, washes could not be performed be-tween incubation periods. The 5'-nucleotidase assaywas begun by adding 0.2 ml of concentrated assaymedium containing 5 mM [iiP]adenosine 5'-mono-phosphate and 25 mM p-nitrophenylphosphate toeach tube (bringing the total volume to 1.0 ml) andincubating the solution for 15 to 30 min at 370C (6).The presence of 0.05% DOC in the assay mixtures

for solubilized 5'-nucleotidase did not cause activationof the enzyme. DOC concentrations up to 0.5% onlyslightly increased background levels for this assay.Nevertheless, blanks containing appropriate amountsof DOC were used routinely in experiments involvingsolubilized enzyme.

Assays. 5'-Nucleotidase, adenosine triphosphatase,and p-nitrophenylphosphatase assays were performedon cell monolayers as described by DePierre and Kar-novsky (6). p-Nitrophenylphosphate (phosphatasesubstrate 104; Sigma) at 5 mM was present in alladenosine triphosphatase and 5'-nucleotidase assaysto ensure that hydrolysis of the two nucleotides bynonspecific phosphatases was inhibited. [iiP]adeno-sine 5'-monophosphate was purchased from New Eng-land Nuclear Corp., and [yy-3P]adenosine 5'-triphos-phate was from Amersham/Searle.

Protein was determined by the method of Lowry etal. (17), using bovine serum albumin as a standard.

Radioactivity. Radioactivity was determined in atoluene-ethanol-based scintillation fluid (1), usingOmnifluor (New England Nuclear Corp.) as a scintil-lator.

All data reported are averages of experiments car-ried out in duplicate at least. Average deviations fromthe mean did not exceed 10%. Km and V,,,,. values wereobtained from inverse plots of not less than four pointsfor each condition; lines were drawn by least-squaresregression analysis. The r values of the lines werebetter than 0.9.

RESULTSResponses to various concentrations of

surface modifiers. (i) Intact cells. Incubationof guinea pig granulocyte monolayers withConA, RCA, WGA, S-ConA, or periodate re-sulted in inhibition of 5'-nucleotidase activity.Some other ectoenzyme activities (namely, ecto-adenosine triphosphatase and ecto-p-nitrophen-

ylphosphatase [6]) were only moderately af-fected (Fig. 1). None of the agents activated theenzyme at any of the concentrations tested (0.1to 1,000 g/ml). Unlike the results of Carrawayet al. (2, 3), all inhibitions were characterized byHill coefficients (13) of 1 or less. For WGA, RCA,and ConA, the coefficient was 1 at low lectinconcentrations and decreased to 0.4 at higherconcentrations; for S-ConA and periodate, theHill coefficient was 0.4 at all concentrations.

Double-reciprocal plots of inhibition versusconcentration of lectin or periodate were linear.From such plots it was possible to calculate themaximum theoretical enzyme inhibition (alwaysless than 100%) and the concentration requiredto produce 50% of maximal inhibition. As Table1 shows, ConA and RCA were powerful inhibi-tors of 5'-nucleotidase of intact cells. WGA andperiodate inhibited to a somewhat lesser degree(periodate-induced inhibition was not affectedby later treatment with excess NaBH4), whereasS-ConA was even less effective. The relativeefficiencies of the native lectins with regard toinhibition of 5'-nucleotidase are noteworthy; a 2to 3 ,M concentration of lectin monomersachieved the same degree of inhibition as 50 to100 ,M periodate.

(ii) Cell fractions. RCA and ConA appearedto inhibit plasma membrane 5'-nucleotidasemore effectively (Table 1) than intact cell en-zyme. Although theoretical inhibition was com-parable in the two cases, half-maximal inhibitionwas obtained with plasma membrane-enrichedfractions at lectin concentrations one-fifth the

1oo0

75

50s

JRCAM Con A*S-ConA

25

5'NUCLEOTIDASE ATPase p-NPPaseFIG. 1. Monolayer cells were preincubated with

300 pg ofRCA per ml, 600( g of ConA per ml, or 500pg of S-ConA per ml for 15 min at 5°C. They werethen washed and assayed for 5'-nucleotidase, aden-osine triphosphatase (ATPase), or p-nitrophenyl-phosphatase (p-NPPase) activity. Residual specificactivities (per unit of cell protein) of these enzymesare presented as percentages of the specific activitiesmeasured for control (untreated) cells. The datashown represent average values of duplicate mono-layers in a representative experiment. A large excessof lectin was employed to obtain near maximal inhi-bition of enzyme.

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TABLE 1. Effect of lectinsandperiodate on 5'-nucleotidase of whole cells, plasma membrane-enrichedfractions, and solubilized preparationsMaximum inhibition (%) Concn for half-maximal inhibition

Compound Cell Plasma Plasmamono- mem- Solubilizedenzyme Cell mono- membrane Solubilized en-

layers brane layers prepn zymeprepn

ConA 88 96 95 50 10 10RCA 88 90 95 50 10 15WGA 76 95 85 100 100 400S-ConA 50 50 10 500 200 500I04- 80 15 (50-100)b 50-100 400 (1,000)

a The inhibition of the enzyme by different doses of lectin or periodate could be translated into double-reciprocal plots of inhibition versus concentration. From these data, maximum theoretical inhibition (at infinitemodifier concentration) and the concentration of modifier necessary for half-maximal inhibition were calculated.The former is given as the percentage of control activity that can be inhibited, and the latter is given asmicrograms per milliliter for lectins and as micromolar concentrations for IO4. Cell monolayers were preincu-bated with varying concentrations of modifiers for 15 min at SOC (lectins) or 370C (periodate, I04 ) and thenwashed and assayed for 5'-nucleotidase. Samples of the plasma membrane-enriched fraction (50 to 150 jyg ofprotein in 0.1 ml) were preincubated in KRP with varying concentrations of lectins for 15 min at 40C or withperiodate for 15 min at 370C. After this, 0.2 ml of a concentrated 5'-nucleotidase assay medium was added toeach tube, and incubation proceeded for 15 to 30 min at 37°C. Solubilized 5'-nucleotidase was isolated andincubated with varying concentrations of the above reagents, as described above for plasma membranepreparations. Details of all procedures are given in the text.

b Data in parentheses indicate a stimulation (see text).

level required for intact cells. However, a directcomparison between the values for the two sys-tems is probably not valid. In the whole cellsystem excess lectin was washed away beforethe 5'-nucleotidase assay; this could not be donewith membrane and solubilized preparations.Compared with the potent effects of RCA andConA, WGA was a poor inhibitor of 5'-nucleo-tidase of the plasma membrane fraction on thebasis of the amount required. Several explana-tions for this were considered, including the pos-sibility that WGA receptor sites may have beenremoved from the plasma membrane by thehypotonic medium used for cell homogenizationduring the preparative procedure or perhapscontaminating organelles in the membrane prep-aration may have competed for binding of thislectin. S-ConA and periodate were relativelyineffective against plasma membrane 5'-nucleo-tidase (Table 1). Thus, the periodate effect onwhole cells was not observed with plasma mem-brane fractions. Hill coefficients (13) of 1 wereobserved for ConA, WGA, and RCA. This valuedecreased to 0.4 for RCA at high concentrations.Both periodate and S-ConA gave values of 0.4.Table 1 also shows the effects of lectins and

periodate on solubilized 5'-nucleotidase. As withthe plasma membrane enzyme, RCA and ConAwere potent inhibitors. WGA was a significantlyweaker inhibitor than it was in the experimentsdescribed above, probably due to some of thesame reasons cited above. S-ConA, which couldinhibit the plasma membrane enzyme, did notaffect solubilized 5'-nucleotidase to any real ex-

tent; it produced only 10% inhibition at concen-trations as high as 1,700 ,ug/ml. The effects ofRCA, ConA, and WGA on the extracted enzymehad Hill coefficients (13) of 1.A surprising result was that periodate stimu-

lated the activity of solubilized 5'-nucleotidase.For these experiments, it was necessary to ex-tract the enzyme in DOC solutions buffered withsodium phosphate or HEPES (N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid), as thetris hydroxymethyll)aminomethane -hydrochlo-ride buffer normally employed consumed addedperiodate. The enzyme solubilized in this man-ner was stimulated 50 to 100% by treatment with1 to 2 mM sodium periodate; higher or lowerconcentrations of this agent produced less stim-ulation. Of all the enzyme-surface modifier com-binations examined in this work, the solubilized5'-nucleotidase-periodate system was the onlyone for which enzyme stimulation was found.Blocking and reversal of lectin effects by

hapten sugars. (i) Intact cells. Because ec-toenzyme activities can be modified by nonspe-cific means (29) (such as by protein-protein in-teractions), it was important to show that inhi-bition by lectins resulted from interactions withspecific binding sites on the cell surface. Fur-thermore, it was necessary to establish that in-hibition was not the result of internalization ofthe lectin and its receptor (22) by these phago-cytic cells, either during the preincubation pe-riod at 5°C or during the enzyme assay periodat 37°C. For this purpose, we conducted studiesin which competing hapten sugars were used to

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block or reverse the effects of the lectins.Table 2 shows the basic format of these stud-

ies. As that table shows, preincubation of mono-layer cells with RCA reduced 5'-nucleotidaseactivity by 70%. When the competing sugar ga-lactose was included in the preincubation me-dium along with RCA, inhibition was only 26%.This blocking study showed that most of theinhibition by this lectin resulted from a specificinteraction of the carbohydrate-binding site ofthe lectin with an appropriate receptor on thecell surface. An experiment to test reversibility,in which RCA was first bound to the cells andthen eluted in a subsequent incubation withgalactose, led to an inhibition of only 21%. Be-cause the inhibition could be reversed compa-rably to the blockade with galactose, it is evidentthat lectin-receptor complexes were not beinginternalized (i.e., made inaccessible both to en-zymatic assay and to elution by competing hap-ten) during incubations at 50C. However, suchinternalization could conceivably occur duringincubation at 370C (e.g., during the 5'-nucleotid-ase assay). If this were taking place, lectin-in-duced inhibition would no longer be reversibleafter a 370C incubation. As Table 2 shows, theeffect of RCA was still largely reversible undersuch conditions. Of the enzyme inhibition thatwas specific for RCA, about one-third could beattributed to internalization during the 5'-nucle-otidase assay. This conclusion is supported bythe observation that the time course of enzymeactivity was linear both before and after lectin

TABLE 2. Effect ofRCA on intact cell 5'-nucleotidase: blocking and reversal'

Component(s) present during incubation: 5'-Nucle-otidase sp

1 2 3 act (% ofcontrol)

- - RCA 30.6- - RCA + Gal 73.7RCA Gal - 79.3RCA -(370C) Gal 61.4- - RCA + GalNAc 29.9

a Each of three successive incubation periods (in-cubations 1, 2 and 3) was for 15 min, and the compo-nents present during each incubation are indicated onthe table. Between incubation periods, all monolayerswere washed extensively. The concentrations of thereagents used were as follows: RCA, 300 jg/ml; galac-tose (Gal), 100 mM; N-acetyl galactosamine (GalNAc),100 mM. -, Incubations with KRP alone. The tem-perature was 5VC, except where indicated. The 5'-nucleotidase specific activities (on the basis of cellprotein) for the experimental samples (duplicatemonolayers) are expressed as percentages of the activ-ities for control cells, which were incubated with KRPonly. See text for further details.

treatment; no transient change in enzyme activ-ity was observed.Although the RCA had been purified by a

standard technique, it was important to showthat a trace of a possible toxic contaminant, ricin(20), was not responsible for inhibition of 5'-nucleotidase. As Table 2 shows, when N-acetylgalactosamine (which blocks binding of ricin butnot RCA) was included in the incubation me-dium along with RCA, no relief of the lectin-induced inhibition was observed. Thus, RCA,not ricin, was responsible for the inhibition of 5'-nucleotidase.

Similar results were obtained for WGA (Table3). It is evident that internalization of lectin-receptor complexes at 370C could account for nomore than 50% of the total 5'-nucleotidase inhi-bition (Table 3).The situation with ConA was quite different

(Table 4). Incubation with ConA inhibited 75%of the 5'-nucleotidase, an effect that could beblocked by the presence ofthe competing haptensugar a-methyl mannoside. However, inhibitioncould be reversed only slightly; reversibility wasnot affected by intervening incubations at 5 or370C. This was the behavior that was expectedfor a lectin that binds specifically to the surfacesof these cells (and can be blocked) but which isinternalized once bound (22). It should be notedthat such internalization would have had to takeplace originally at 50C, and not during the 370Cincubation of the 5'-nucleotidase assay; other-wise, substantial reversal of the inhibition wouldhave occurred (Table 4) when the cells weremaintained at 5VC until the assay. The effect ofS-ConA was similarly difficult to reverse witha-methyl mannoside (data not shown).

It was found that inhibition of 5'-nucleotidasedue to ConA could be reversed if a-methyl man-noside was present during the enzyme assay.Thus, enzyme inhibition was in this case notactually due to internalization of lectin-receptorcomplexes. The observation was consistent withtwo hypotheses. (i) Once bound to specific cellsurface receptors, ConA might be held to thecell by some additional nonspecific hapten-re-sistant bonds. In this manner, the lectin wouldremain attached to the cell throughout normalreversal and washing procedures. (ii) The ConAeffect might actually be reversible only at 370C,a condition employed in the 5'-nucleotidase as-say, but not at 50C, the temperature normallyemployed for reversal of lectin binding by spe-cific haptens.The data in Table 5 are most simply explained

by the second hypothesis. This table shows theinhibition of 5'-nucleotidase after incubationwith ConA and indicates the extent to whichthis inhibition could be blocked with a-methyl

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mannoside. Table 5 also shows results ofreversalstudies with the competing sugar present in theenzyme assay during the customary 50C incu-bation and during an incubation at 370C. Onlyin the last case, when reversal with methyl man-noside was attempted at 370C (followed bywashing of the cell monolayers), was activitysubstantially restored to the level seen when thehapten was present during the assay.

(ii) Cell-free enzyme systems. To demon-strate that the lectin-induced inhibition of iso-lated plasma membrane 5'-nucleotidase was spe-cific, competing hapten sugars were used toblock and reverse the effects of the lectins (Fig.2). As Fig. 2 shows, the effects of ConA, RCA,and WGA could be blocked and reversed byapplication of the appropriate hapten sugars.Reversibility of ConA-induced inhibition in thecase of the plasma membrane preparation (com-pare Table 5) probably was the result of thepresence of competing sugar during the 5'-nucle-otidase assay at 370C. Similar results were ob-tained when solubilized enzyme preparationswere used (data not shown). Thus, as with intactcells, the effects of the lectins on 5'-nucleotidaseactivity appeared to be due to specific interac-

TABLE 3. Effect of WGA on intact cell 5'-nucleotidase: blocking and reversal'

Component(s) present during incubation: 5'-Nucle-otidase sp


- - WGA 52.6- - WGA + GlcNAc 82.2- WGA GlcNAc 88.5WGA - GlcNAc 97.1WGA - (370C) GlcNAc 76.5a The concentrations of the reagents used were as

follows: WGA, 100 Isg/ml; N-acetyl glucosamine(GlcNAc), 100 mM. See Table 2, footnote a and textfor further details.

TABLE 4. Effect of ConA on intact cell 5'-nucleotidase: blocking and reversal'

Component(s) present during incubation: 5'-Nucleo-tidase sp


- - ConA 24.3- - ConA + MM 91.7- ConA MM 33.5

ConA - MM 36.6ConA - (37-C) MM 36.9a The concentrations of the reagents used were as

follows: ConA, 200 Ig/ml; a-methyl mannoside (MM),50 mM. See Table 2, footnote a and text for furtherdetails.

TABLE 5. Effect of ConA on intact cell 5'-nucleotidase: blocking and reversibility at 37OCa

Component(s) present during: 5'-Nucleo-tidase sp

Incubation Incubation act (% of1 2 control)

- ConA - 36.7- ConA + MM - 97.0- ConA MM 78.5

ConA MM - 47.4ConA MM (370C) - 84.0a The concentrations of the reagents used were as

follows: ConA, 80 pg/ml; a-methyl mannoside (MM),50 mM. (a-Methyl mannoside was present during theincubation for assay of enzyme at 370C.) See Table 2,footnote a and text for further details.

El Lectin Alone r Blockade *Reversal

IOOF

k

L.j

75F

50 F-

25H

O0Con A RCA WGA

FIG. 2. Blockade and reversal of inhibition: 5'-nu-cleotidase ofplasma membrane fraction. Samples ofplasma membrane were incubated with 15 pg ofConAper ml, 15 jg ofRCA per ml, or 100 pg of WGA per mlfor 15 min at 40C. For blocking studies, incubationwith lectin was performed in thepresence ofa haptensugar. For reversal studies, lectin incubation wasfollowed by exposure to a hapten sugar for 15 min at40C. In both cases, the second 15-min incubation wasinitiated by adding 0.1 ml of concentrated stock tothe suspension; washes were not performed betweenincubations (compare whole cells). The competinghaptens were 50 mM a-methyl mannoside for ConA,100 mM galactose for RCA, and 100 mM N-acetylglucosamine for WGA. See Table 2, footnote a forfurther details.

tions of their carbohydrate binding sites withcell surface receptors.Lectin combinations. It was of interest to

determine whether the three native lectinswould interact with each other with respect toinhibition of 5'-nucleotidase. Such studies in-volved incubating cells with combinations oflectins and then reversing the binding of one ormore lectins with a competing sugar. To assessthe results of these experiments, it was firstnecessary to determine the effects of the three

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sugars on inhibition by each lectin.A series of blocking tests (more comprehen-

sive than those shown in Tables 2 through 4)was performed, in which the effect of each of thethree sugars on lectin-induced inhibition of 5'-nucleotidase was tested (Fig. 3). As Fig. 3 shows,inhibition by RCA and WGA could be blockedby their specific sugars alone. However, the ef-fect of ConA could be blocked to some extent byall three sugars; a-methyl mannoside was themost effective.The combined interactions of RCA and WGA

on intact cell 5'-nucleotidase are shown in Fig.4. The enzyme was inhibited 71% by RCA (Fig.4, column a) and 54% by WGA (Fig. 4, columnb). Successive incubations with both lectins pro-duced 77% inhibition, regardless of the order ofaddition (Fig. 4, columns c and d). Removal ofRCA by galactose after exposure to WGA (Fig.4, column e) restored 5'-nucleotidase activitytoward the level expected for WGA alone (Fig.4, column b), although not completely. Removalof WGA by N-acetyl glucosamine after RCAbinding (Fig. 4, column f) restored activity tothe level expected for RCA alone (Fig. 4, columna). Thus, the prior binding of either lectin didnot appear to interfere substantially with theeffect of the other lectin on 5'-nucleotidase.The interactions of ConA and WGA were

tested in a similar manner (Fig. 5). Attempts toremove bound ConA after WGA binding did notrestore enzyme activity to the level expected forWGA alone (Fig. 5, columns b and e) since

EjLectin Alone

ERLectin + ca-MMELectin + Gal

ELectin + GlcNAc

75kk

~-50

25h_

~~~~~XO_LV. _RCA WGA Con A

FIG. 3. Effects of RCA, WGA, and ConA on 5-nucleotidase of intact cells: blocking by haptensugars. Cell monolayers werepreincubated for 18 minat 50C with 80 pg of ConA per ml, 30 pg of RCA perml, or 100 pg of WGA per ml, as indicated on thefigure. Where specified, these incubation media alsocontained 50 mM a-methyl mannoside (a-MM), 100mMgalactose (Gal), or I 00mM N-acetylglucosamine(GlcNAc). The specific activity of5'-nucleotidase wasdetermined for each ofthese samples and is expressedas a percentage of the specific activity measured forcontrol cells.

100Io

75

50

25

01

RCAWGA

Gal IGIcNAcI

_

...... :..... ........GA :WMGARCGWGA.RCA....... RCA GA Gal RCA

. ..WGA RCA GIcNA

a b c d e fFIG. 4. Effects of both RCA and WGA on 5'-nucle-

otidase of intact cells. Successive incubation periodswere for 18 min at 5VC with 40 pg ofRCA per ml, 70pg of WGA per ml, 100 mM galactose (Gal), or 100mM N-acetylglucosamine (GlcNAc). The sequence inwhich incubations wereperformed is indicated by thevertical order printed in the histogram (e.g., samplefwas incubated first with WGA, then with RCA, andfinally with N-acetyl glucosamine). Reversal of theseparate lectin effects by appropriate hapten sugarsis indicated by the dashed areas in columns a and b.Washes were performed between incubation periods.

0oo0

k75k

50k

25h

0O

:WGAIGIcNAcI

(;ConA I.. *.mmmI M M I| I.........

WGA

......... .........Con

a b c d e f

FIG. 5. Effect of both ConA and WGA on 5'-nucle-otidase of intact cells. Successive incubation periodswere for 18 min at 50C with 50 pg ofConA per ml, 100pg ofWGA per ml, 50mM a-methyl mannoside (MM),or 100 mM N-acetyl glucosamine (GlcNAc). See leg-end to Fig. 4 for an explanation of the format.

inhibition by ConA was not reversible at 5VC(see above; Table 4). However, removal ofWGAwith N-acetyl glucosamine after ConA treat-ment restored more activity than that expectedin cells treated with ConA alone (Fig. 5, columnsa and f). This probably cannot be attributed tothe action of N-acetyl glucosamine on the bind-ing of ConA, since it was not as effective ahapten as a-methyl mannoside (Fig. 3) in thisregard. Thus, it appears that prior binding ofWGA inhibited the effect of ConA on 5'-nucle-otidase, perhaps by partially blocking interac-tions of the latter lectin.

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Similar experiments with ConA and RCAwere also performed (Fig. 6) and this combina-tion was the most interesting. Even though theRCA effect was reversible (Fig. 6, column b),attempts to remove bound RCA with galactoseafter ConA binding did not restore activity tothe activity level expected for ConA alone (Fig.6, columns a and f ). This was so despite the factthat galactose interfered slightly with ConA-in-duced inhibition (Fig. 3). Thus, posttreatmentwith ConA caused the inhibition of 5'-nucleotid-ase due to RCA to become apparently irrevers-ible.

It was found that ConA-RCA mixtures formedprecipitates in vitro, as originally reported byPodder et al. (23). This behavior was not ob-served with any other lectin combination. Sincea-methyl mannoside, but not galactose, com-pletely blocked precipitate formation (turbidi-metric measurement) (data not shown), it ap-peared that ConA bound determinants on RCAmolecules. In view of this, the simplest expla-nation for the data of Fig. 6 is that the multiva-lent nature of ConA allowed these lectin mole-cules to bind determinants present both on thecell surface and on RCA. RCA molecules elutedfrom their own cellular receptors by galactoseremained attached to the cell surface via linksthrough ConA. After the hapten sugar relevantto RCA was washed off, RCA reattached tonearby receptor sites on the membrane and onceagain inhibited the activity of 5'-nucleotidase.Thus, treatment with ConA could make RCAinhibition apparently irreversible.This is further demonstrated by the experi-

ment shown in Fig. 7. The irreversibility of theRCA effect after ConA treatment is shown inFig. 7, column d. However, reversal to levelsexpected for ConA alone (Fig. 7, column b) was

100-

75 ,RCA:. r Gal

Con A:50 - MM

25 ConA .......:X:::~::3 RCA

aL L::::::1...A.R RCA ConRAConA MM Gal

c~~~de f

FIG. 6. Effect of both ConA and RCA on 5'-nucle-otidase of intact cells. Successive incubation periodswere for 18 min at 5°C with 50 ug of ConA per ml, 50pg ofRCA per ml, 50mM a-methyl mannoside (MM),or 100 mM galactose (Gal). See legend to Fig. 4 foran explanation of the format.

INFECT. IMMUN.

100

k75_

50_

25[_

O0a b c

........

RCAConA

:RCA Gal.Con A MM

d e

FIG. 7. Effect of both RCA and ConA on 5'-nucle-otidase of intact cells. Successive incubation periodswere for 18 min at 50C with 50 pg of ConA per ml, 70pg ofRCA per ml, 50mM a-methyl mannoside (MM),or 100 mM galactose (Gal). The third incubation insample e contained both sugars in the medium. Seelegend to Fig. 4 for an explanation of the format.

achieved when both galactose and a-methylmannoside were employed (Fig. 7, column e).Since the effect ofConA itself on 5'-nucleotidasewas not substantially reversible at 5°C and sincea-methyl mannoside did not affect RCA binding(Fig. 3 and Table 4), it is apparent that bothRCA-cell and RCA-ConA (but not ConA-cell)bonds were dissociated by the combined sugartreatment; free RCA was washed from themonolayer plates, and the residual enzyme ac-tivity reflected only the presence of the ConAwhich was not eluted.Enzyme kinetics. The kinetic parameters of

5'-nucleotidase were examined in the hope thatsome clue might be found to the mechanisms bywhich lectins and periodate cause enzyme inhi-bition. Table 6 shows the effects of ConA, RCA,and periodate on 5'-nucleotidase. For intactcells, treatment with ConA, RCA, or periodatedecrease the Vmax of the enzyme by about 40 to60% and increased the Kin, although marginallyin the last case. The effects of WGA on thekinetic parameters of 5'-nucleotidase were dif-ferent from the above-described effects. All ofthe enzyme inhibition was attributable to a de-creased Vmax; the Km was virtually unchanged.The effects with S-ConA were the least striking,but again depression of Vmax seemed to be thecrucial factor.The effects of the lectins and periodate on the

kinetic parameters of isolated plasma membrane5'-nucleotidase were also examined (Table 6).Treatment with ConA, RCA, or WGA inhibitedthe enzyme mainly by decreasing its Vinax; theKm of the enzyme was significantly increased aswell.S-ConA inhibited plasma membrane 5'-nucle-

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TABLE 6. Effects of lectins and periodate on kineticcharacteristics of ecto-5'-nucleotidase

% Of control values'

Membrane- ExrceTreatment Intact cells enriched frac- Extractedtion enzyme

V.. K, V..x K. V..x K,ConA 59 440 37 300 39 150RCA 61 560 36 550 31 138WGA 45 92 56 300 60 100S-ConA 77 113 66 100 87 133I04- 57 154 140 1450 192 200

a The control values (mean ± standard deviation)for intact cells were Vmax = 2.49 ± 2.3 nmol/mg ofprotein per min and Km = 115 ± 33 ,uM. The controlvalues for the membrane-enriched fraction were VT,,= 10.2 ± 5.5 nmol/mg of protein per min and K. = 30± 10 jM. And the control values for extracted enzymewere V.,, = 3.0 ± 0.6 nmol/mg of protein per min andK. = 43 ± 19 AM. Four experiments were performed.Data for the lectin-treated preparations are given aspercentages of the relevant control values. Least-squares regression analyses of both Lineweaver-Burkand Eadie-Hofstee plots were used and gave valuesless than 3% apart.

otidase activity only by decreasing Vmax. Perio-date, on the other hand, greatly increased theKm and even increased V.. somewhat. Sinceordinarily 5'-nucleotidase assays were conductedat 1 mM adenosine 5'-monophosphate, this ef-fect of periodate on enzyme activity was notobserved earlier (Table 2). Thus, although per-iodate can inhibit the enzyme by only 15% (at 1mM adenosine 5'-monophosphate) its effect onthe enzyme, as revealed by kinetic data, is moreprofound.

All of the lectins affected the kinetic parame-ters of solubilized 5'-nucleotidase in a similarmanner; i.e., the Vmax was depressed, and theeffects on Km were marginal. Stimulation of sol-ubilized enzyme activity by periodate was prob-ably accomplished by the increase in Vm..; theKm was only doubled.

Solubilized 5'-nucleotidase was also obtainedby incubating a plasma membrane-enrichedpreparation (rather than whole cells) in 0.1%DOC-0.9% NaCl-10 mM tris(hydroxymethyl)-aminomethane-hydrochloride (pH 7.0) for 30min at 370C. Supernatant material was obtainedas described above for whole cell extracts. In-cubation of this solution with 500 ,Ag of RCA orWGA per ml at 370C resulted in a precipitatewhich could be isolated by centrifugation. If thispellet was resuspended in a DOC solution con-taining the appropriate competing sugar, sub-stantial 5'-nucleotidase activity could be re-covered (85% for the RCA-treated preparation

and 45% for the WGA-treated preparation). Thisobservation provides the most direct evidencethat the two lectins do indeed bind to the 5'-nucleotidase protein per se or to an oligomer ofmembrane peptides containing 5'-nucleotidase.

DISCUSSIONInhibition of 5'-nucleotidase by lectins has

been observed in a number of systems (2, 3, 14,25, 27, 28). Activation of this enzyme in somecell types at low lectin concentrations has alsobeen reported (25, 33), but was not seen in thisstudy. Activation of adenosine triphosphatasesis more common (2, 21, 31, 33), and this processhas often been cited as a model for the initialstages of mitogenesis.

Inhibition of granulocyte 5'-nucleotidase bythe native lectins ConA, RCA, and WGA can beblocked and reversed by application of appro-priate competing hapten sugars. Thus, enzymeinhibition was caused largely by specific inter-actions of the carbohydrate binding sites of thelectins with appropriate cell surface determi-nants and not by spontaneous endocytosis oflectin-receptor complexes. However, these dataper se do not imply that the determinants aredisplayed by the 5'-nucleotidase itself; enzymeinhibition might be caused by indirect means,i.e., by the interactions of lectins or periodatewith adjacent surface components other thanthose on the enzyme itself. The effects on theenzyme activity could be secondary to attach-ment of lectins or attack by periodate on neigh-bor molecules of the enzyme in the membranestructure. The ability of lectins to inhibit solu-bilized enzyme and to precipitate active 5'-nu-cleotidase suggests, however, that direct inter-actions between the lectins and the enzyme dotake place. However, it must be borne in mindthat native complexes of the enzyme proteinwith other (glyco)proteins might not be dissoci-ated by weak detergents.The effects of ConA on the kinetic parameters

of 5'-nucleotidase have been reported in somestudies. Riordan and Slavik (25) found thatConA produced both a depression of Vma. andan increase ofKm for rat liver plasma membrane5'-nucleotidase. Similar results were observedwith fetal calf cell 5'-nucleotidase (4). V.,x de-pressions without corresponding Km changeswere found for ConA inhibition of intact cell,plasma membrane, and solubilized 5'-nucleotid-ase preparations from rat mammary ascites car-cinoma (3) and of pig lymphocyte plasma mem-brane 5'-nucleotidase (8). Both Vmax and Kmwere decreased for the enzyme of intact C6glioma cells (28).The effects of the surface modifiers on the

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kinetic parameters of the three granulocyte 5'-nucleotidase preparations may be summarizedas follows. All four lectins decreased the Vma. ofthe enzymes. For intact cell 5'-nucleotidase, Kmwas increased by RCA and ConA. RCA in-creased the Km of the enzyme most significantlyin plasma membrane-enriched fractions (25),whereas none of the lectins greatly affected theKm of detergent-solubilized enzyme (3). Thus, asthe disruption of the system increases (in goingfrom intact cell to plasma membrane to deter-gent extract) and the potential for indirect inter-actions that might affect the enzyme decreases,the ability of the lectins to produce Km changesseems to be progressively decreased. These re-sults are consistent with the hypothesis thatVMax decreases are produced by direct interac-tions with the enzyme, whereas increases in Kmcould be due to indirect interactions.

Periodate-induced inhibition has strikinglydifferent properties from the lectin-induced ef-fects. At the intact cell level, periodate affectsVmax and, marginally, Km similarly to ConA andRCA. The enzyme of isolated plasma membranehas a very high Km and a slightly increased Viaxafter periodate treatment. This result is uniquein this series of experiments; the Vmax of plasmamembrane 5'-nucleotidase was strongly de-pressed by all of the other agents. At the nextstage of disruption (namely, detergent solubili-zation), the modest increase in Vmax observedwith plasma membrane 5'-nucleotidase predom-inates; the Km is, however, now only doubled.The result is that periodate produces a substan-tial stimulation of 5'-nucleotidase activity. Thus,the periodate results are also consistent with thehypothesis that direct reactions with the enzymeprobably affect Vma., whereas indirect reactions(i.e., with carbohydrate entities in the environ-ment) affect the Km of the enzyme.That five agents with different carbohydrate

specificities could interact with a single glyco-protein is not inconceivable; a glycoprotein iso-lated from human blood group 0 erythrocytescontains receptors for a large number of agglu-tinins (11). However, the presence or absence ofspecific sugars in the glycoprotein structure doesnot necessarily determine whether it is boundby a lectin; the above-mentioned glycoproteincontains some galactose but little N-acetyl glu-cosamine, yet it interacts with WGA but notRCA. A single short carbohydrate sequence maycontain the determinants for several lectins (30).It is not difficult to find glycoproteins whichcontain all three specific sugar residues forConA, RCA, and WGA; a human gamma Gmyeloma protein (15) and porcine thyroglobulin(10) are examples. On the other hand, as indi-cated above, various lectins might attach to dif-

ferent neighbors of the nucleotidase in a com-plex, each of which could manifest functionalinteraction with the enzyme. Furthermore, thedata indicate that none of the surface modifierscan completely inhibit 5'-nucleotidase. Thismight reflect microheterogeneity of the glyco-protein structure; however, among other expla-nations, it is equally possible that binding of thelarge lectin molecules to the carbohydrate de-terminants might never completely block accessof a small substrate.Presumably, inhibition of access of substrate

to enzyme by bound lectins would be expectedto increase Km without necessarily affectingVmax. Although distortion of the enzyme mole-cule, which could be responsible for changes inVmax, might be readily accomplished by the bind-ing of a large lectin molecule to certain carbo-hydrate moieties, the effect of periodate on 5'-nucleotidase is more difficult to explain. Underneutral conditions periodate might not causevery extensive oxidative attack on oligosaccha-ride chains or terminal sialates, for example. Itis not obvious how the subtle alterations ofpolysaccharide moieties produced by periodatemodify enzyme activity. The production of al-dehyde groups per se is probably not responsiblefor enzyme inhibition since postincubation withNaBH4 does not reverse the effect of periodate.Because the physiological role of granulocyte

5'-nucleotidase is not clear, the exact relevanceof the effects of cell surface modifiers on thisectoenzyme cannot yet be determined. However,this system could serve as a model for processessuch as mitogenesis, phagocytosis, chemotaxis,and others in which events at the cell surfacepromote dramatic changes.

ACKNOWLEDGMENTThis work was supported by Public Health Service grant

AI-03260 from the National Institute of Allergy and InfectiousDiseases.

LITERATURE CITED1. Butler, D. R. 1962. A simple scintillation counting tech-

nique for assaying C"402 in a Warburg flask. Anal.Biochem. 4:413-417.

2. Carraway, C. A. C., G. Jett, and K. L. Carraway.1975. Cooperative effects in the perturbation of mem-brane enzymes by concanavalin A. Biochem. Biophys.Res. Commun. 67:1301-1306.

3. Carraway, K. L, D. D. Fogle, R. W. Chestnut, J. W.Huggins, and C. A. C. Carraway. 1976. Ecto-enzymesof mammary gland and its tumors. Lectin inhibition of5'-nucleotidase of the 13762 rat mammary ascites car-cinoma. J. Biol. Chem. 251:6173-6178.

4. Daveze, J.-L., A. Di Pietro, J. Frappa, R. Deloince,Y. Beaudry, and R. Fontanges. 1978. etude cinetiquede l'activite 5'-nucleotidase membranaire de cellulesdiploides en culture. C. R. Acad. Sci. Ser. D 286:1621-1624.

5. DePierre, J. W., and M. L. Karnovsky. 1973. Isolationof a nuclear fraction from guinea pig polymorphonu-

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clear leukocytes after controlled hypotonic homogeni-zation. Biochim. Biophys. Acta 320:205-209.

6. DePierre, J. W., and M. L. Karnovsky. 1974. Ecto-enzymes of the guinea pig polymorphonuclear leuko-cyte. I. Evidence for an ecto-adenosine monophospha-tase, -adenosine triphosphatase, and -p-nitrophenylphosphatase. J. Biol. Chem. 249:7111-7120.

7. DePierre, J. W., and M. L Karnovsky. 1974. Ecto-enzymes of the guinea pig polymorphonuclear leuko-cyte. II. Properties and suitability as markers for theplasma membrane. J. Biol. Chem. 249:7121-7129.

8. Dornand, J., J.-C. Bonnafous, and J.-C. Mani. 1978.Effects of Con A and other lectins on pure 5' nucleotid-ase isolated from lymphocyte plasma membranes. Bio-chem. Biophys. Res. Commun. 82:685-692.

9. Frazier, F., and L. Glaser. 1979. Surface componentsand cell recognition. Annu. Rev. Biochem. 48:491-523.

10. Fukuda, M., and F. Egami. 1971. The structure of aglycopeptide purified from porcine thyroglobulin. Bio-chem. J. 123:415-420.

11. Fukuda, M., and T. Osawa. 1973. Isolation and charac-terization of a glycoprotein from human group 0 eryth-rocyte membrane. J. Biol. Chem. 248:5100-5105.

12. Gunther, G. R., J. L Wang, I. Yahara, B. A. Cun-ningham, and G. M. Edelman. 1973. Concanavalin Aderivatives with altered biological activities. Proc. Natl.Acad. Sci. U.S.A. 70:1012-1016.

13. Hill, A. V. 1913. The combinations of haemoglobin withoxygen and with carbon monoxide. I. Biochem. J. 7:471-480.

14. Ikehara, Y., K. Takahashi, K. Mansho, S. Eto, and K.Kato. 1977. Contrast manifestation of alkaline phos-phatase and 5'-nucleotidase in plasma membranes iso-lated from rat liver and ascites hepatoma. Biochim.Biophys. Acta 470:202-211.

15. Kornfeld, R., J. Keller, J. Baenziger, and S. Kornfeld.1971. The structure of the glycopeptide of human yGmyeloma proteins. J. Biol. Chem. 246:3259-3268.

16. Little, J. S., and C. C. Widnell. 1975. Evidence for thetranslocation of 5'-nucleotidase across hepatic mem-branes in vivo. Proc. Natl. Acad. Sci. U.S.A. 72:4013-4017.

17. Lowry, 0. H., N. J. Rosebrough, A. L Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

18. Michell, R. H., M. J. Karnovsky, and M. L. Karnov-sky. 1970. The distributions of some granule-associatedenzymes in guinea-pig polymorphonuclear leukocytes.Biochem. J. 116:207-216.

19. Michell, R. H., S. J. Pancake, J. Noseworthy, and M.L Karnovsky. 1969. Measurement of rates of phago-cytosis. The use of cellular monolayers. J. Cell Biol. 40:216-224.

20. Nicolson, G. L., and J. Blaustein. 1972. The interaction

of Ricinus communes agglutinin with normal and tumorcell surfaces. Biochim. Biophys. Acta 266:543-547.

21. Novogrodsky, A. 1972. Concanavalin A stimulation ofrat lymphocyte ATPase. Biochim. Biophys. Acta 266:343-349.

22. Oliver, J. M., T. E. Ukena, and R. D. Berlin. 1974.Effects of phagocytosis and colchicine on the distribu-tion of lectin-binding sites on cell surfaces. Proc. Natl.Acad. Sci. U.S.A. 71: 394-398.

23. Podder, S. K., A. Surolia, and B. K. Bachhawat. 1974.On the specificity of carbohydrate-lectin recognition.The interaction of a lectin from Ricinus communesbeans with simple saccharides and concanavalin A. Eur.J. Biochem. 44:151-160.

24. Pommier, G., G. Ripert, E. Azoulay, and R. DePieds.1975. Effect of concanavalin A on membrane-boundenzymes from mouse lymphocytes. Biochim. Biophys.Acta 389:483-494.

25. Riordan, J. R., and M. Slavick. 1974. Interactions oflectins with membrane glycoproteins. Effects of concan-avalin A on 5'-nucleotidase. Biochim. Biophys. Acta373:356-360.

26. Romeo, D., G. Zabucchi, and F. Rossi. 1973. Reversiblemetabolic stimulation ofpolymorphonuclear leukocytesand macrophages by concanavalin A. Nature (London)New Biol. 243:111-112.

27. Slavik, M., N. Kartner, and J. R. Riordan. 1977. Lec-tin-induced inhibition of plasma membrane 5'-nucleo-tidase: sensitivity of purified enzyme. Biochem. Bio-phys. Res. Commun. 75:342-349.

28. Stefanovic, V., P. Mandel, and A. Rosenberg. 1975.Concanavalin A inhibition of ecto-5'-nucleotidase ofintact cultured C6 glioma cells. J. Biol. Chem. 250:7081-7083.

29. Swann, A. C., A. Daniel, R. W. Albers, and G. J.Koval. 1975. Interactions of lectins with (Na+ + K+)-ATPase of eel electric organ. Biochim. Biophys. Acta401:299-306.

30. Toyoshima, S., M. Fukuda, and T. Osawa. 1972. Chem-ical nature of the receptor site for various phytomito-gens. Biochemistry 11:4000-4005.

31. Traficante, L J., L. Shenkman, J. Rotrosen, and S.Gershon. 1978. Stimulation of the membrane-bound,magnesium-dependent adenosine triphosphatase ofmouse neuroblastoma by concanavalin A and wheatgerm agglutinin. Life Sci. 22:1059-1066.

32. Young, M. E. M., M. A. Moscarello, and J. R. Riordan.1976. Concanavalin A binding to membranes of theGolgi apparatus and resultant modification of galacto-syltransferase activity. J. Biol. Chem. 251:5860-5865.

33. Zachowski, A., D. Migliore-Samour, A. Paraf, and P.Jolles. 1975. Nonspecific effector-induced enzyme mod-ulation in isolated plasma membranes. FEBS Lett. 52:57-61.

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Documents

Effect Surface Modifiers on an Ectoenzyme: Granulocyte 5