Detergents Linked to Polysaccharides : Preparation and Effects on Membranes and Cells

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1 Eur. J. Biohem. 94, (1979) Detergents Linked to Polysaharides : Preparation and Effets on Membranes and Cells Josef PTHA, Karol KOCOLEK, and Mar G. CARON Laboratory of Cellular and Moleular Biology, Gerontology Researh Center, National nstitute on Aging, National nstitutes of Health, Publi Health Servie, US. Department of Health, Eduation and Welfare, Bethesda, Maryland; Baltimore City Hospitals, Baltimore, Maryland; and Department of Mediine, Duke University Medial Center, Durham, North Carolina (Reeived June 14, 1978) Residues of Triton X-1 [C~H~-C~H~-(O-CH~-CH~)~ were bound by ether bonds to inulin, dextran, amylose, and ellulose-yielding ompounds ontaining 1-3, 5, 19 and 6 % (w/w) of Triton X-1 residue respetively. The water-soluble inulin derivative was studied in detail. This ompound was frationated on the basis of moleular weight by gel hromatography and on the basis of degree of substitution by adsorption to polystyrene resin. Even the residues of Triton X-1 whih were bound to a single inulin maromoleule were able to form a mielle; in addition to these monomoleular mielles the inulin derivative was able to form polymoleular mielles as well. The inulin derivative was effetive in solubilizing proteins and phospholipids from membranes of human erythroytes and liberated reverse transriptase ativity from the membrane-enveloped virions of murine leukemia. The Triton X-1 inulin derivative abolished binding of the 3H-labelled antagonist dihydroalprenolol to solubilized preparations of P-adrenergi reeptors from frog erythroytes in a dose-related manner similar to the inativation produed by Triton X-1, while digitonin, a detergent ontaining a bulkier hydrophobi group, did not ause inativation. On the basis of its Triton X-1 ontent, the inulin derivative was found to be less detrimental to the growth of murine erythroleukemi ells in vitro than Triton X-1 alone when short (2-4 h) exposures were used, but this differene disappeared at longer (1-3 day) exposures. Results thus suggest that the inrease in size of the hydrophili part of the detergent brings about moderation in those effets of the detergent whih are dependent on the rate of diffusion, while the solubilizing and inativating effets of the detergent were not hanged. t is probably the size of the hydrophobi part whih is important in protein-inativating properties of the detergent. Biohemial studies of membrane omponents and studies of metabolism of permeabilized ells rely on the use of detergents [l]. ndustrial detergents have found wide-spread use in the disruption and solubilization of biologial strutures. However, in appliations where very mild ation is desired suitable detergents are still not available. f the retention of biologial ativity is desired in the solubilized membrane preparations, only a few detergent moleules should be bound to a protein in order to minimize any alteration of its struture. Nevertheless even the mild eletroneutral detergent Triton X-1 replaes all the natural lipids in lipoproteins and is massively bound to lipophili proteins [2-4,7]. Even in these unphysiologial onditions some proteins ontinue to funtion [5-71, while in other proteins, biologial ativities are lost [l]. n making ells permeable, similar deter- gents not only dissolve the plasma membrane but also penetrate and diffuse into the interior of the ells [ We reasoned that ertain improvement in all of these appliations may be ahieved by using detergents whih would have bulky hydrophili omponents, e.g. polysaharide. With suh polysaharide-based detergents only few moleules would fit around a solubilized protein. The large polysaharide residue may prevent the hydrophobi omponent of the detergent from entering into the internal hydrophobi struture of proteins. Finally, in making ells permeable, the polysaharide-based detergents may be expeted to have less tendeny to penetrate into the ells. Several saharide-derived detergents have been known and were studied; these inlude saponins [l] and gluosides of fatty alohols [12]. Furthermore,

2 12 Detergents Linked to Polysaharides dextrans substituted with fatty aids were prepared and used as a membrane probe n this work we prepared polysaharides arrying amphili substituents derived from Triton X-1 and studied their suitability 'as detergents. EXPERMENTAL PROCEDURE Materials Sepharyl S-2 was purhased from Pharmaia (Pisataway, N.J., U.S.A.); Bio-Bead SM-2 was obtained from Bio-Rad Laboratories (Rihmond, Calif., U.S.A.) and Moloney mouse leukemia virus was from Eletronuleonis Laboratories (Bethesda, Md., U.S.A.). Erythroleukemi ells were murine ells transformed by Friend leukemia virus lone Cl1 [14] and were obtained from Dr D. Tiemaier (National nstitutes of Health, Bethesda, Md., U.S.A.). Chemials and other reagents used were obtained from ommerial soures. Analyses of the Synthesized Compounds The onentration of Triton X-1 residues in the water-soluble polysaharide derivatives was determined by the omparison of absorption oeffiients at 276 nm with that of the starting Triton X-1. With insoluble ompounds the number of --CH2-CH2-- groups was measured by Zeissel method (performed by Gailbraith Labs, Knoxville, Tenn., U.S.A.) and then ompared with the orresponding value found for Triton X-1. The presene of polysaharide in various preparations was asertained by anthrone tests [15]. The presene of ontamination by Tritonlike ompounds in the polysaharide derivatives was determined by thin-layer hromatography on silia gel G, using a mixture of ethanol and hloroform (l/l) as eluent. While the polysaharide derivatives were immobile (RF = ), the ontaminating ompounds have RF values in the range Ultraviolet light or iodine vapors were used for the detetion of ompounds on hromatography plates. Preparation of Dextran Derivative 3 Sheme 1 shows the struture of the different ompounds. Triton X-1 (5 g, about 77 mmol) was dried in benzene solution (2 ml) by anhydrous MgS4; after filtering off the dessiant, pyridine (8 g, 1 mmol) and thionyl hloride (12 g, 1 mmol) were added and the solution was refluxed for 11 h. Then the mixture was extrated by 1 M HCl, dried by anhydrous MgS4 and evaporated. Compound 2 thus prepared is a yellow oil (55 g) ontaining 3.9 % of hlorine and possessing no hydroxyli groups that ould be deteted by infra-red spetrosopy in the 36-3 m-l region. The alkylation of dextran by ompound 2 was performed in the following way : NaH (1 g) was dissolved in 1 ml of dimethysulfoxide in anhydrous onditions at 6 "C. After ooling to room temperature, a solution of dextran (2 g) in dimethylsulfoxide (1 ml) was added. Compound2 (12g) was added drop-wise to this solution and the mixture was stirred overnight. Then the mixture was deomposed by water, neutralized and dialyzed against water to separate the produts from dimethylsulfoxide. Thereafter the solution was extrated with hloroform, separating the produt from the deomposed reagent 2. The aqueous solution ontaining ompound 3 was then dialyzed against water and freeze-dried; ompound 3 thus obtained (1.3 g) ontained 1.3% of Triton X-1 residues. Preparation of Compound 5 Triton X-1 (7 g, about 1 mmol) was dried by azeotropi distillation with toluene. After omplete evaporation of the solvent in vuuo, SnC4 (5 P) and epihlorohydrin (14 g, 15 mmol) were added to the rest and the mixture was kept at 9-1 "C overnight. Thereafter, the mixture was evaporated in vauo, diluted with ether and extrated by aqueous NaOH (5%) at "C. The ether layer was then dried by anhydrous MgS4. Evaporation of solvents gave ompound 5 in yields 6-9%. This ompound had a slightly different RF value (.4) from that of Triton X-1 (.5), on thin-layer hromatograms using benzene-hloroform (l/l) mixture as the solvent. The nulear magneti resonane spetrum in the region of aromati protons ( ppm) is the same for ompound 5 as that of Triton X-1; thus no C-alkylation by epihlorohydrin ourred in the etherifiation proedure. Condensation of Compound 5 with Polysaharides Polysaharide (1 5 g) was dissolved (or dispersed) in 1 M NaOH (75 ml); then the ompound 5 (1 g) was added and the mixture was stirred overnight. The the mixture was dialyzed against water (4 x 4 1) and freeze-dried. The resulting semi-solid produt was extrated with benzene to separate the ondensation produts from deomposed ompound 5. The benzene extrats gave a brown olor with the anthrone reagents indiating that the extration may have removed highly substituted polysaharides from the produt. The solid material remaining after the extration was suspended in water and dialyzed against water. n the ases where the produts were watersoluble, i.e. ompounds 6 and 9, the solutions were larified by entrifugation (1 rev./min, 1 min) and freeze-dried to give olorless produts. The entrif-

3 J. Pitha, K. Koiolek, and M. G. Caron 13 CH3 CH3-C-CHz-C CH3 Clig CH3 ( - CH2 - CH2)9.1 - R R = OH R = C / R = --CH (dextran derived) \ R = -O-CH~-CHOH-Ci~C R = --CH2-CH-CH2 1 (Triton X-1) R = -O-CH2-CiOH-CH2-O-Cl$ (dextran derived) 6 \ R = -O-CH~-CiOH-CH~-O-CH~-(amylose derived) 7 R = --CH2-CHOH-CH2-O-CH2- (ellulose derived) 8 R = --CH2-CHOH-CH2-O-CH2- (inulin derived) 9 Sheme 1. Formulus of the Studied Compounds. The polysaharides are substituted by the Triton X- residues in a polydisperse manner; the average ontent of Triton X-1 residues is denoted by a number in parentheses, i.e., 9(9 %) denotes ompound 9 ontaining 9 % (WjW) of Triton X-1 residue ugation step was omitted for partially soluble ompounds 7 and 8. n several instanes, the produts were still ontaminated by Triton-like ompounds, but repetition of the extration proedure gave pure produts. The desribed proedure yielded, per one gram of starting polysaharide; 1 g of ompound 6(5%), 1.1 g of ompound 7(17%),.9 g of ompound 8(4%), and.5 g of ompound 9(21%). The above proedure and onditions were superior to a large number of others tested. Derivatization of both Sepharose and ellophane ould also be performed in the way desribed. Methods The amount of miellar phase was measured using the iodine method [16]. Membranes of human erythroytes were isolated as desribed [17], and the protein solubilized from them was measured by the Lowry method whih was modified to avoid interferenes due to the presene of Triton X-1 [18]. The solubilized phospholipids were measured as desribed previously [19]. The assay for the reverse transriptase ativity was performed as previously desribed [2]. Solubilized preparations of P-adrenergi reeptors were obtained by treatment of purified frog erythroyte membranes with 1 % digitonin and [3H]dihydroalprenolol binding to these preparations was measured as desribed before [21]. The erythroleukemi ells were grown and toxiity studies were performed as previously desribed [22]. RESULTS Preparation of Polysaharide Derivatives Triton X-1 is a mixture in whih the ompounds of formula 1 (Sheme 1) predominate; as minor om- ponents there are some ortho isomers of ompound 1 and some ompounds in whih the benzene nuleus arries an additional otyl group; nevertheless all of these ompounds possess a primary hydroxyli group [23]. By reating Triton X-1 with thionylhloride in pyridine the primary hydroxyli group was onverted into the hloro derivative 2, whih was reated with sodium salt of dextran in dimethylsulfoxide, giving the detergent 3(1.3 %). The disadvantage of this approah is a slight disoloration of Triton X-1 upon reation with thionylhloride and diffiulty in obtaining ompounds of type 3 ontaining higher perentage of detergent. Alternatively Triton X-1 was onverted to an epoxyderivative. This was ahieved optimally by an unusual type of etherifiation [24] ; Triton X-1 was reated with epihlorohydrin under atalysis of tin tetrahloride to give the orresponding 3-hloro-2-hydroxypropyl ether, ompound 4 in Sheme 1. That ompound was onverted by aqueous sodium hydroxide into ompound5, a olorless liquid. Compound 5, when allowed to reat with dextran, yielded the derivative 6. The onditions known to lead to a high substitution of Sepharose by epoxides [25] gave in the present ase produts whih ontained only 5 % of ovalently bound Triton X-1 residues. Dextran is a polysaharide whih ontains only seondary hydroxyls; sine the reation of epoxides with primary hydroxyls is known to proeed faster than with the seondary one [26], the same proedure was applied to polysaharides ontaining primary hydroxyls. Cellulose and amylose gave produts 8(5 %) and 7(19%). The degrees of substitution of these ompounds were apparently not high enough to solubilize these polysaharides properly and produts thus had only limited solubility. Possibly these ompounds may find their use as adsorbents in hydrophobi hromatography [27]. nulin, a polysaharide

4 14 Detergents Linked to Polysaharides ; 1. 4 n.5 Fig. 1. Gel hromatography of ompounds and 9(9%j on a olumn of Sepharyl S-2 (3 x 75 m), water was usedas the eluant. (----) The elution profile of Triton X-1 (5 mg sample). (-) The elution profile of detergent 9(9 %) (4 mg sample). Frations olleted are noted by Roman numerals. Rehromatography of part of fration (...). The insert shows the onentration dependene of absorbane at 4 nm, whih represents the sattered light; (-) detergent 9(9%), (O----o) fration of detergent 9, (a- -. -@) fration 1 of detergent 9 whih also ontains primary hydroxyls, gave derivative 9 with 9-3 % of ovalently bound Triton X-1 and all these produts were soluble. Physial Properties From the ompounds prepared, the inulin derivative 9 was studied in detail. Aqueous solutions of ompound 9 (9 %) sattered light slightly; the dependene of sattered light on the onentration of ompound 9 (9 %) indiated that some self-assoiation ourred at onentrations higher than 1 mg/ml (insert of Fig. 1). The dispersity of moleular weight and the self-assoiation of ompound 9 was studied by gel hromatography. The miellar form of Triton X-1 (M, 9, aording to [l]) was eluted lose to the void volume while the monomeri form was eluted lose to the total bed volume of the olumn (Fig. 1). The inulin derivative 9(9 %) was eluted in three frations, all of maromoleular harater (Fig. 1). Fration was a solid after freeze drying and ontained 8 % of Triton residues by weight. This fration sattered light. However, the sattering was not due to an aggregation, as the amount of sattered light dereased linearly with the onentration (insert of Fig. 1) and the rehromatography of that ompound did not lead to an appearane of frations with a lower moleular weight (Fig. 1). Fration 1 was also a solid and ontained 21 % of Triton by weight and did not satter light. Fration 11 was a relatively minor one - 1.".- 3 a ul.- 'x, 6 - Lo a U 3 'z 4 A 2 z,, >'.- + b%....,...,., l,, Amount of polystyrene beads (mg / rng of Triton X-1 residue) Fig.2. Adsorption of Triton X-1 und detergent 9(9%) to polystyrene resin (Bio-Beads SM-2j. Water was used as a solvent and the adsorption was performed by overnight inubation at room temperature and was liquid after evaporation and ontained 13 % of Triton X-1 by weight. Triton X-1 is adsorbed to polystyrene beads [28]. nulin derivative 9 (9 %) was adsorbed less avidly than Triton X-1 (Fig. 2). Adsorption ofompound 9(9 %) by three times its weight of polystyrene beads left unadsorbed a fration of 9 whih ontained only 4 of Triton X-1. Apparently the highly substituted inulin moleules were adsorbed preferentially and the frationation of produts by their degree of substitution was easily ahieved. Triton X-1 at onentrations above about 2 pg/ml forms mielles [l, 23,291 (Fig. 3). The mielle formation in the inulin derivative 9 was investigated using the differene in olor whih elementary iodine assumes in non-polar mielles and in the surrounding aqueous medium. n the experimental arrangement used the differene of light absorption as plotted in Fig. 3 is proportional to the amount of non-polar, i.e. miellar, phase [3]. The result in Fig. 3A indiates that all preparations of detergent 9 studied form ertain amounts of miellar, or non-polar phase, but in ontrast to Triton X-1 its formation is a gradual proess and some small amount of this phase seems to be present even at the lowest studied onentrations. t is instrutive to evaluate this phenomenon on the basis of the onentration of Triton X-1 residues (Fig. 3 B). The amount of hydrophobi spae then depends linearly on the onentrations of inulin derivative 9 and the lines extrapolate through the origin. Prepara-

5 J. Pitha, K. Koiolek, and M. G. Caron 15 tions 9(4%) and 9(9 %) formed as muh hydrophobi spae per weight of Triton X-1 residue as Triton X-1 itself, whereas preparation 9(21%) formed only about half of that amount. Effets on Membranes The ability of inulin derivative 9 (9 %) to solubilize membrane omponents was tested on human erythro- iri. % _-- a, my,. _---/-.a-- " -... a 1 t l / a L Conentration of Triton X-DO residue (pg/rnl) Fig. 3. The hange in adsorhane of aqueous iodine solutions observed upon additions of Triton X- or of various polysaharide detergents of type 9. The inrease in the absorbane at 38 nm is proportional to the amount of non-polar spae formed by detergents ytes and on murine leukemia virions. The proteins and the phospholipids were solubilized effiiently from the isolated erythroytes membrane (Fig. 4). The reverse transriptase ativity of murine leukemia virus is not manifested unless the envelope of the virus is perturbed by a detergent; the results in Fig. 5 indiate that this was ahieved by the inulin derivative 9 (9 %) about as effiiently as by Triton X-1. /3-Adrenergi reeptors present on frog erythroyte plasma membranes an be effetively and funtionally solubilized by treatment with the plant glyoside digitonin [21]. As found previously [21], numerous other detergents and reagents seem to be ineffetive in solubilizing funtional binding sites. Moreover, it appears that several of these detergents, even nonioni, will ompletely abolish the ability of the antagonist [3H]- dihydroalprenolol to bind speifially to digitoninsolubilized preparations when present during the inubations. Fig. 6 shows that Triton X-1 produes suh an inativation and the inulin derivative 9 (9 %) also perturbs the binding ativity at about the same extent as equivalent onentrations of Triton X-1 residues. Effets on Cells in vitro Eletroneutral detergents are known to affet growth of ells in vitro espeially if the ells are treated in the absene of serum. When TritonX-1 and inulin derivative 9 (9 %) were ompared at equivalent onentrations of Triton X-1 residues during relatively short exposures (2-4 h), the inulin derivative aused less ell damage (Fig.7A). When ells were exposed ontinuously to detergent the toxi effets on ell growth were idential for both Triton X-1 and 9 (9 %), (Fig. 7 B). DSCUSSON The intention of this work was to prepare a polysaharide-ontaining detergent whih would be ele-.s 1,Triton X Conentration of detergent (mglrnl) Fig. 4. Solubilization of proteins and phospholipids from the membranes of human erythroytes by Triton X-1 and by 9(9 %). Membranes were equilibrated with detergent for 3 h at room temperature and the protein and organi phosphate were measured in the supernatant after entrifugation of the mixture. n the absene of detergent no protein was released. Nevertheless, some phospholipids were regularly released; in this experiment the release of 42 pg of phosphorous per 1 ml of membrane preparation was deteted

6 16 Detergents Linked to Polysaharides.- F 1 E " 1 u 2 6 e a._ E 5 r 1 $ 2 1 Detergent onentration (rng/mi) Fig. 5. Release of reverse transr$tase ativityfrom virions ofmurine leukemia by the addition of Triton X- () or oj detergent 9 (9%). Exoyeneous template system, poly(ra) and oligo(dt), was used. nubation time was 3 rnin at 37 "C Fig. 6. The effets of addition of Triton X-1 or of detergent 9 (9%) on the speifi binding of [3H]dihydroalprenolol to P-adrenergi reeptors, in solubilized prepurations obtained from frog erythroyte membranes by digitonin treatment. Binding was performed by the inubation of the mixtures for 2 h at 4 "C troneutral and resistant to hydrolysis. These properties were obtained by forming ether linkages between polysaharides and the detergent residues. Another aim was to obtain produts whih would be water-soluble even with highly substituted polysaharides. This partiular ondition prevented the use of fatty alohols or aids as hydrophobi residues and lead to the use of Triton X-1 residues. From the ompounds prepared, the inulin derivatives were the most aessible ones. nulin itself is known not to bind to ells in vitro [31] thus providing a suitably inert arrier for the detergent residues ; the only disadvantage may be that its resistane to hemial hydrolysis is lower than that of other polysaharides [32]. The methods desribed for preparation of the inulin derived detergents lead to produts whih are heterogenous in both the amount and position of substitution. Highly substituted produts were adsorbed by polystyrene resins nearly as well as the starting Triton X-1. Considerable amounts of polysaharide detergent appear to exist in miellar form even at the lowest studied onentrations ; suh mielles are probably formed by Triton X-1 residues loated on a single moleule of substituted polysaharide. The preparations 9 (4 %) and 9 (9 %) form nearly as muh of non-polar spae per Triton X-1 residue as Triton X-1 forms by itself. The highly substituted ompound 9 (21 %) is less effiient in that respet; possibly steri interation ould prevent a omplete intramoleular assoiation of hydrophobi residues in this ase. The formation of mielles omposed from several moleules of inulin derivative 9 (9 %) was deteted only above the onentrations of about 1mg/ml, i.e. a muh higher onentration than those used for dissolution of membranes. The dissolution of a membrane in detergent solutions proeeds through the inorporation of detergent into the bilayer, followed by the formation of mixed mielles between detergent and membrane omponents [l]. The inulin derivative 9(9%) is apparently effetive in both of these proesses, sine both protein and phospholipid omponents of erythroyte membranes were solubilized as well as the enzymati ativity ontained in an enveloped virus was easily released. Eletroneutral detergents are known to inativate only a few enzymati systems [l]. Triton X-1 inativates the binding ativity of j3-adrenergi reeptor ; this protein an be solubilized in an ative form by digitonin, a detergent ontaining a steroid moiety as a non-polar part and pentasaharide as the polar one. t is interesting that the inulin derivative 9 (9 %) inativates the reeptor-binding ativity as extensively as Triton X-1 when ompared on the basis of equivalent Triton X-1 residues, indiating that the presene of polysaharide residues on Triton X-1 does not diminish the deleterious effet of Triton X-1 on this ativity. n this partiular ase probably the hydrophobi part of the Triton X-1 residue was able to penetrate into the protein struture leading to inativation, while the bulkier nonpolar part of digitonin did not penetrate into the hydrophobi portion of the protein. The effets of eletroneutral detergents on ells in vitro an be greatly modified by the onditions of exposure. Low onentrations of detergents in isotoni salt solutions ause leakiness in the ell membranes whih an be repaired by the addition of serum [ Detergents at high onentrations release the majority of ell omponents and only the ytoskeletons and nulei are left in the ells [8-11. nulin derivative 9(9%) proved to be as potent as Triton X-1 in dissolving membrane omponents. Nevertheless, inulin derivative 9(9%), on the basis of its larger moleular weight should diffuse muh slower

7 J. Pitha, K. Koiolek, and M. G. Caron r l l i i lo6 1 A 1-h exposure 3-h exposure h exposure 17 '" Time (days) Fig.7. Effets of Triton X- and detergent 9(9%) on the growth ojerythroleukemi ells in vitro. (A) Cells were treated with Dulbeo's balaned solution whih was without (o----o) or ontained detergent: Triton X-1, 5 mg/ml (M----M); detergent 9(9%), 5 mg/ml, (*n), for 1, 3 or 4 h at room temperature. Thereafter the ells were diluted 1 times with the growth medium and their growth reorded daily. (B) The detergents were present ontinuously in the growth media. (-) Control; ( b U ) detergent 9(9 %), 5 pg/ml; (t-m) detergent 9(9;,), 5 pg/ml; (A-A) Triton X-1, 5 pg/ml; (A-A) Triton X-1, 5 pg/ml than Triton X-1 and thus damage of intraellular omponents after the dissolution of ell membrane should proeed slower than with the lower moleular weight detergents. This expetation was onfirmed by omparing effets of inulin derivative 9(9 %) and Triton X-1, at the same onentration of Triton X-1 residue, on growth of erythroleukemi ells in vitro. n short treatments, the inulin derivative 9 (9 %) was less toxi than Triton X-1, while both Triton X-1 and derivative 9 (9 %) did not differ signifiantly in their effets when the ells were ontinuously grown in their presene. The surfae of ells is known to be overed by glyoalyx onsisting mainly of polysaharides and this polysaharide oating may represent a onsiderable barrier for maromoleules to penetrate to ells [l, 36, and referenes therein]. Nevertheless the ompound 9, whih might not be expeted to interat with polysaharides [l], is onsiderably toxi to ells. This finding indiates that some regions of the bilayer part of the ell membrane must be exposed to the surrounding medium and these regions must be large enough that a diret interation with the maromoleular speies, suh as inulin derivative, an take plae. A similar onlusion an be drawn from the data on the interation of erythroytes with alkyl agaroses [37]. While these ompounds arry some ationi harges [38] the detergent 9 is eletroneutral and is thus a less perturbing probe for the eletronegative surfae of ells. REFERENCES 1. Helenius, A. C Simons, K. (1975) Biohim. Biophys. Ata, 415, Simons, K., Helenius, A. SC Garoff, H. (1973) J. Mol. Bid. $, Makino, S., Reynolds, J. A. SC Tanford, C. (1973) J. Bid. Chem. 248, Clarke, S. (1975) J. Biol. Chem. 25, Cuatreasas, P. (1972) Pro. Nail Aad. Si. U.S.A. 69, Reynolds, J. A. & Stokenius, W. (1977) Pro. Nut1 Aad. Si. U.S.A. 74, Helenius, A. SC Simons, K. (1972) J. Bid. Chem. 247, Tsai, R. L. & Green, H. (1973) Nut. New Bid. 243, Tseng, B. Y. C Goulian, M. (1975) J. Mol. Biol. 99, Osborn, M. & Weber, K. (1977) Exp. Cell Res. 16, Miller, R. M., Castellot, J. J. C Pardee, A. B. (1978) Biohemistry, 17, Lasser, H. R. SC Ellias, H. G. (2972) Kolloid Z. u. 2. Polymers, 25, Wolf, D. E., Shlessinger, J., Elson, E. L., Webb, W. W., Blumenthal, R. C Henkart, P. (1977) Biohemistry, 16, Leder, A. C Leder, P. (1975) Cell, 5, Hassid, W. Z. & Abraham, S. (1957) Methods Enzymol. 3, Ross, S. C Olivier, J. P. (1959) J. Phys. Chem. 63, Abbott, R. E. SC Shahter, D. (1976) J. Biol. Chem. 251, Duley, J. R. C Grieve, P. A. (1975) Anal. Biohem. 64, Bartlett, G. R. (1959) J. Bid. Chem. 234, Pitha, J. (1976) Caner Res. 36, Caron, M. G. & Lefkowitz, R. J. (1976) J. Biol. Chem. 251,

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