Lectin-induced accumulation of large lysosomes in cultured fibroblasts

Copyright @ 1980 by Academic PRS, k. 4ii rights of reproductmn in an) iorm reeei-ved 0011.1827/110/0713133-10~02.OOiO

Experimental

LECTIN-INDUCED

Cell Rmearch

ACCUMULATION IN CULTURED

128 (1980) 133-442

OF

FIB

R. D. PORETZ, R. E. TRIEMER, A. C. St JOHN, M. MERION, R. .I. KUCHLER, 0. CRYAN, J. II. CARTER and .I. W. C. BIRD Bureau of Biological Research, Rutgevs University, New Brunswick, lV~‘O8903, USA

SUMMARY Cultured fibroblasts become highly vacuolated when exposed to the lectin, BGstaria floribur?da agglutinin (WFA). However, transformed cells are less susceptible toward this action of the lectin. Enzyme profiles of fractionated cellular components, cytochemical localization of acid phosphatase, and the characteristics of acridine orange (AO) uptake by the cells demonstrate that the vacuoles are lysosomes. WFA-induced accumulation of lysosomes is specific, as the response is not evident when cells are exposed to other iectins. Specific inhibition of the carbohydrate binding site of WFA with lactose inhibits the binding of the lectin to the cells and subsequent induction of lysosome accumulation. The cellular response to the lectin is dose-dependent, and related to the duration of exposure. Treatment of fibroblasts with WFA for up to 48 h has no apparent effect on the growth and viability of the cells; the cells revert to a normal morphology 5 days after removal of lectin. These experiments and results presented by St John et al. [40] indicate that WFA is internalized by the cells and either enhances fusion of primary lysosomes and pinosomes, or causes the inhibition of breakdown of secondary lysosomes, culminating in anlaccumulation of large lysosomes.

years, researchers have em- induce this response in macro lectins to study the role of binding have recently re of multivalent nds to the plasma mem- ~emaggluti~ating ctin from wi. ,~~ri~~~~~~t FA) causes the massive accumealabrane in the in on of specific biological acuoles in cultured mt;rine fibrorespsnses [9, 1 ]. In a number of cases, is paper we now describe the such as the mitogenic activity of concanavahn A (ConA) with T lymphocytes [5] h va~~~~~~at~Q~ on t QT t insulin-like activity of ConA and stasis of the cells and identify the vacuoles whe germ agglutinin (W’GA) on adipose as lysossmes. cells [l2, 2 11the effect is related to binding, and not internalization, of the lectin. In instances, as with the toxic activities inus communis toxin [33] and diph- Cells toxin [34, internalization of the %AL%/c 3T3 murine fibroblasts were grown in a medium composed of 90 % Eagle’s minimal essential mehgand subsequent to binding appears to be a dium with Hanks’ salts (EMEMN) containing 10% r~~u~~erne~tfor the effect. Edelson & Cohn calf serum (EMEMM,,CS1,) at 37°C in a 95 % aid 5 % CO, atmosphere. [I$] have described the vacuolization of acrophages by ConA, and Lotan et al. Lectins 6] have subsequently published that W. jlfloribunda agglutinin [lo], Ma&m pomiferu Iecrir some, but not all, multivalent lectins also [4j, W. jloribunda mitogen [22], and Sophovu japmricc

In

recent

134

Pore@ et al.

lectin [36] were prepared as previously described. Wheat germ agglutinin (WGA) was obtained from Boehringer Mannheim Biochemicals (Indianapolis, Ind.).

Dark-field fluorescence microscopy of cells vitally stained with A0 Cell cultures were incubated for 48 h with O-200 pg/ml WFA in EMEMH. After removal of the incubation medium the cells were washed with PBS and incubated for 15 min with 50 pg/ml of acridine orange (AO) in PBS. The cells were washed three times with PBS and viewed with a Leitz Ortholux II microscope equipped with a Ploem illuminator, and an “I” filter cube.

Electron microscopy Samples were fixed in a solution containing 2% glutaraldehyde and 0.1 M phosphate buffer, pH 6.8, for 2 h at 4°C. The cells were then washed 3~ (15 min each) in cold buffer and post-fixed for 2 h in 2% buffered 0~0, in the. dark at 4°C. Samnles were rinsed in distilled water and rapidly dehydrated through a graded ethanol series followed by two changes in 100% acetone. Cells were infiltrated and embedded in Epon, and polymerized at 60°C for 48 h. Silver sections were cut on a Sorvall MTZB ultramicrotome using a DuPont diamond knife. Some sections were post-stained with lead citrate and/or uranyl acetate before examining on a Siemens Elmiskop 1A transmission electron microscope operating at 80 kV. Acid phosphatase localization was achieved utilizing /3-glycerophosphate as a substrate and lead as a capture reagent according to the method of Dauwalder et al. [13]. P-Glycerophosphate was omitted from the reaction mixture of the control samples. All samples were incubated at 37°C for 20 min.

Distribution of enzyme activities and A0 following differential centrifugation of cell homogenates Cells were maintained in a serum-free EMEMH with or without 200 pg/mI WFA for 48 h. Before harvest, the cells were incubated with acridine orange (AO) (10 pg/ml) for 15 min at 37°C followed by a 10 min chase period in fresh EMEMH. Cells were harvested and homogenized by a modification of the method of Wibo & Poole [43] using 0.25 M sucrose, 2 mM EDTA and 20 mM KCl, pH 6.8, as homogenizing medium. The homogenates were fractionated bv differential centrifuga&n as developed by Canon&o & Bird [8]. The five fractions collected were the nuclear (N, 600 g for 10 min. washed once), the heavy mitochondrial (H, 11400 g for S min), the light mitochondrial (L, 22.500 g for 15 min), the microsomal (P, 105000 g for 45 min) and the soluble. The substrates for the five lysosomal enzymes assayed were p-nitrocatechol sulfate for acyl sulfatase 1381, Nbenzoyl-DL-arginine-2-naphthylamine for cathepsin B [3], denatured hemoglobin for cathepsin D [6], and the Exp CdRes

IZN(1980)

p-nitrophenyl derivatives for acid phosphatase and N-acetvl-/3-D-nlucosaminidase 171.The suantitation of enzymatic activities satisfied- the linearity requirements of valid assay conditions. The distribution of the A0 dimer was determined by measurement of the relative intensity of fluorescence at 660 nm, with excitation at 330 nm, in acidified, alcoholic extracts of the fractions [8]. The mean relative specific activities which were normalized to 10 were calculated as described by deDuve et al. [14]. The specific activities of the above enzymes were determined on the total cell homogenates and the average recovery of enzymatic activities and protein were: acid phosphatase, 83 %; N-acetyl-/3-n-glucosaminidase, 78 %; arylsulfatase, 102%; cathepsin B, 98 %; cathepsin D, 86 %; AO, 107%; and protein, 100%.

Growth offibroblasts in the presence and absence of WFA A replicate series of BALB/c 3T3 cell cultures was set up in 35 mm diameter plastic Petri dishes in 3 ml of a medium composed of 90% minimum essential medium with Hanks’ salts containing 10% calf serum (EMEMHXS,,,). After incubation at 37°C for 24 h, three cult&es*“were harvested and counted as described below, and the remaining cultures were divided into four sets. The media on the cultures in the four sets were replaced respectively with 3 ml of fresh media of the following composition: EMEMH; EMEMH+200 Kg/ml WFA; EMEMH&&; and EMEMH&S,,+200 tie/ml WFA. At various time intervals, triplicate cult&& in each set were harvested by decanting the medium and replacing it with 2 ml of 0.1 M citric acid containing 0.1 g/l crystal violet. The lysed cells were scraped off the surface, diluted appropriately and the stained nuclei were counted in a hemocytometer.

RESULTS The nature of the lectin-induced vacuoles offibroblasts Cultures of murine BALB/c 3T3 fibroblasts incubated in the presence of 200 pug/ ml WFA for up to 48 h were found to accumulate massive cytoplasmic vacuoles when compared with untreated cells. The vacuolization is quite apparent when an electron micrograph of a thin section of a cell treated with 200 pg/ml WFA for 24 h is compared to an untreated cell (fig. I a, b). The vacuoles in the affected cells are as large as 2-3 km in diameter and have a morphology reminiscent of lysosomes. Consistent with these results, fig. 1c, d

FIR. I. Electron microscopy of BALB/c 3T3 fibroblasts. (a) Untreated cells. A few single membranebound vesicles are present in the cytoplasm (arrows). (i?) Large vacuoles accumulate in the cytoplasm foilowing treatment of the cells with 200 pg/ml WFA fos 20 h. (c. d) Cytochemistry. (c) The large vacuoles

in cells treated with 200 pg/ml WFA for 8 h stair? positively for acid phosphatase activity indicative flf lysosomes. (d) Higher magnification view of a single lysosome e&biting a positive reaction for acid phosphatase following 8 h incubation with 2OC&mk WFA N, nucleus: M, mitochondrion: I,, :ysosome

Table 1. Effect of WFA treatment of 3T3 cells on the specific activity of acid hydvolases Percentage activity of untreated cells % t SE. (n) Cathepsin D Cathepsin B Aryl sulfatase N-Acetyl-fi-D-glucosaminidase Acid.phosphatase

129422 (4) 1095~20(4) 107513 (4) 140+30 (4) 102 (2)

Specific enzymatic activities,on;control cells and cells treated with 200 pg/ml WFA for 48 h were determined as described in Materials and Methods. Numbers in parentheses represent number of experiments, each experiment represents a pool of 2-8 cultures of lectintreated and control cells. The results are reported as + S.E.

homogenates were similar to the specific activities of these enzymes in homogenates of untreated cells (table 1). Rate-sedimentation cell fractionation exPERCENT OF TOTa PROTElN RECOVEREO periments clearly demonstrate that the lecFig. 2. Distribution of enzyme activities and acridine orange following differential centrifugation of homo- tin causes a redistribution of the popula: genates of BALB/c 3T3 fibroblasts. Cells were maintained in a serum-free EMEMH with or without 200 tions of lysosomes. Fig. 2 shows that lectin pg/ml WFA for 48 h. Cells were incubated with AO, treatment r.esults in cells containing a harvested, homogenized and enzymes were assayed as greater proportion of lysosomal hydrolases described in the text. in the heavy mitochondrial fraction than in the light mitochondrial fraction. This is in demonstrates that acid phosphatase is lo- contrast to the untreated cells which exhibit calized in the large vacuoles of cells treated a greater. amount of hydrolase activities in with 200 pg/ml WFA for 8 h prior to cyto- the light mitochondrial fraction. The inchemical staining for the enzyme. crease of the lysosomal enzyme activities In order to determine the biochemical associated with the soluble fraction oPthe nature of the large vacuoles induced by lectin-treated,‘cells may reflect the fragile WFA, total cell homogenates were pre- nature of the large lysosomes formed in the pared, treated with 0.2% Triton-X 100 to WFA-treated cells. Furthermore, when up lyse the lysosomes and analysed for specific to 600 pg/ml WFA was added to the lysolysosomal hydrolases. Although celk ex- some-enriched cell fraction, no change was posed to 200 pg/ml of WFA for 48 h were observed in the specific? .activities of the vacuolated, the specific activities of the above enzymes. This suggests that the lysosomal enzymes acid phosphatase, N- intralysosomal-lectin probably does not inacetyl-P-D-glucosaminidase, arylsulfatase, hibit the enzymes studied nor does it afcathepsin D and cathepsin B, in total cell fect the assay of these enzymes.

Lectin-induced accumulatioa of large lysosomes i~~~bi,~~~~~~~ts137

F$. 3. Dark-field fluorescence microscopy of cells (b, e) and 200 pg/mi (c, ,f) of WFA in EMEMH. vitally stained with AO. Cultures of BALB/c 3T3 After removal of the inccbation medium the ceils were fibroblasts (a, b, c) and their MSV transformants S end incubated for 15 min with 50 (d, e, f) were iricubated for 48 h with 0 (n, d), SO &ml of acridine orange in PBS.

Characteristics of WFA-treated cells at, under these To further investigate the nature of these conditions, lysosomes are t vacuoles, fibs&lasts treated with WFA able of ~Q~~~~tr~~~~~ were vitally stained with the metachromatic acridine orange to a level t dye, acridine orange (AO). Dark-field fluorescence microscopy demonstrates that IX-treated cells concentrate AQ within e large vacuoles, resulting in their red rescence (fig. 3). The control cells that e not exposed to lectin contain only a vacuoles. Allison & Young

138

Poretz et al.

Table 2. Relative vacuolating response offibroblasts to lectins

BALB/c BALBlc BALB/c BALBlc BALB/c BALBlc BALB/c BALB/c HeLa BALB/c BALBlc BALBlc BALBk

Relative vacuolization”

Additions

Cells 3T3 3T3 3T3 3T3 3T3 3T3 (S+L-) 3T3 (S+L-) 3T3 (S+L+) 3T3 3T3 3T3 3T3

None 50 pg/ml 100pg/ml 200 pg/ml 200 pg/ml No lectin 200 pglml 200 pg/ml 200 pg/ml 200 pg/ml 200 pg/ml 200 pg/ml 200 pglml

WFA WFA WFA WFA+O.l M lactose

agglutinin wheat germ agglutinin S. japonica agglutinin W. floribundu mitogen M. pomifern

2 if i-++ ++++ 2 + ++ ++ ++ IL XL + rt

a Measured by intensity of vacuolar fluorescence of A0 in the red region of the spectrum. Cell cultures were stained with A0 and viewed microscopically as described in the text and the legend of fig. 3.

It is evident from fig. 3 that the vacuolating response of the cells is dependent on the dose of the lectin. Thus, cultures treated with 50 pg/ml WFA have fewer vacuolated cells and the affected cells have accumulated fewer large lysosomes than those cells treated with 200 ,ug/ml WFA (fig. 3). Transformed mammalian cells have been shown to have altered responses to lectin treatment [ 311. Therefore, we examined several transformed cultures including BALB/c 3T3 cells transformed by murine sarcoma virus (S/L+ and S/L-) and HeLa cells for their response to treatment with WFA. The transformed cells are less responsive than normal cells (table 2, fig. 3). Fig. 3 shows that when S/L+ MSV transformants of BALB/c 3T3 cells are exposed to 200 pg/ml WFA for 48 h, the cells accumulate lysosomes to a degree similar to normal cells exposed to only 50 pg/ml WFA. The response of the cells to WFA is specific since several other lectins tested do not cause lysosome accumulation in BALB/ c 3T3 cells. Treatment of the cells for 24Ex.0 Cell Res I28 fIY80)

48 h with WGA, M. pomifera lectin, S. japonica agglutinin or the mitogenic lectin from W. Jloribunda seeds does not result in the accumulation of large lysosomes (table 2). However, unlike the effect of lectins on macrophages [26] the unique characteristic of WFA is not solely related to its valency. The 132000 mol. wt species employed here is tetravalent [lo, 251, as are the non-vacuolating lectins, S. japonica agglutinin [42] and WGA [30, 371. The specific vacuolization of fibroblasts by WFA treatment is dependent on the interaction of WFA with the cell surface. Lactose, a simple sugar inhibitor of the hemagglutinating ability of the lectin [lo], completely inhibits the lysosome inducing property of WFA when cells are treated simultaneously with 200 pg/ml WFA and 0.1 M lactose (table 2). Immunofluorescence studies and experiments employing lz51-labeled WFA verified that lactose virtually eliminated the ability of the lectin to bind to the surface of the cells [29]. Experiments were designed to explore the effect of exposure time of fibroblasts to WFA on the ability of the lectin to cause

va~no~izatio~ caused by nificant effect on As expected, ce lacking serum stopped dividing before confluency was reached at about 7.4~IO5cells/ dish. T residual growth rn se Re medium as the same in lectin-tre an control cells. These studies also demonstrate that the Bectin is not mitogenic. Furthermore, we have found that t TIME (hrl ration of [3~~tbym~di~e into T~A-~r~~~~i~aFig. 4. Growth of fibroblasts in the presence and absence of WFA. Experimental cultures were grown ble material is the same in cells exposed under the following conditions as described in the FA as in untreated Materials and Method? section: EMEMH, Cl; EMEMH+ZOO pg/ml WFA, 69; EMEMH,,CS,,, 0; avoid the possibde EMEMH&S,,+200 fig/ml WFA, 1. lectin on the transport of extracellular thymidine through the plasa~c~rn~~at~on of lysosomes. Cell cultures were treated with 200 pg/ml WFA for varying periods of time, whereupon the medium was decanted a the cells were washed with medium c aining 0.1 M lactose to remove cell-bound lectin. Fresh medium was added to the cultures and the cells croscopic ~~a~~~at~o~ were allowed to incubate for a total of 48 h reversible. Light after initial addition of lectin. The re- of cells treated wi 200 pg/ml of lectin for SUltS monstrate that large lysosomes apate within 48 h, following from cell cultures by washing with 0. I M cells to the lectin for as lactose ~o~ta~n~n~media and the addirion cf little as 2 h. owever, microscopic ex- fresh media with or ‘thout caif serum 5 days after restained cells indicates that de~~~st~a~ed that wit both the intensity and extent of the vacuolar moval of the lectin, th mor~~Q~ogy of the entical to untreaTed e cells to the lectin increases ined in media lackg time of exposure (up to id not show appreciable The massive accumulation of lysosomes growth. the loss of the large lysosomes cacin cultutred roblasts treated with WFA is not be attributed to a ‘diluting’ of the existnot an indi ion that the lectin is toxic to ing number of 4ysosomes by contmued the cells. The growth of cells was moni- growth of the cells over the 5day period, by enumerating the cellular nuclei in but probably represents the degradation cultures in the presence or absence of 200 and turnover of the organeIles.

140

Poretz et al.

DISCUSSION Murine fibroblasts cultured in vitro in the presence of WFA accumulate large vacuoles. These vacuoles are lysosomes as evident from their concentration of A0 [ 11, the ultrastructural characteristics of these single-membrane bound vesicles [ 141, and their positive cytochemical staining for acid phosphatase [32]. It is interesting that cells treated with WFA contain no more acid hydrolase activities than control cells (table l), though they do have a greater proportion of particles containing acid hydrolases that sediment as large lysosomes than do the control cells (fig. 2). deDuve et al. [14] have previously demonstrated that the heavy and light mitochondrial fractions are enriched in the lysosomal components of fractionated cells. These results suggest that WFA is causing an increase in the fusion of small lysosomes or a decrease in the breakdown of the fused organelles thus resulting in the accumulation of large lysosomes. Alternatively, we may be witnessing the swelling of pre-existing lysosomes or the capture of fluid by lysosomes due to their enhanced fusion with endocytic vesicles. We have recently demonstrated that WFA is capable of entering BALB/c 3T3 cells in a manner consistent with pinocytosis within 15 min after exposure to the lectin [29, 401. However, the enlarged lysosomes are probably not the result of WFA acting as an intralysosomal inhibitor of acid hydrolase(s) leading to an accumulation of substrate(s), since up to 600 pglml WFA does not inhibit a number of these enzymes. Lotan et al. [26] suggest that the vacuoles in macrophages treated with ConA and certain other lectins result from lectin-enhanced fusion of primary endocytic vesicles with other vesicles. Goldman & Raz [19] have previously indicated that such fusion may in-

volve lysosomes. In contrast, Edelson & Cohn, who have demonstrated that ConA is a stimulator of pinocytosis in macrophages [16], believe that these ConA-induced pinosomes persist due to their markedly impaired ability to fuse with lysosomes [ 171.WFA induces the accumulation of lysosomes in fibroblasts; however, it is not clear whether it affects the fusion or inhibits the breakdown process of the organelles. To cause accumulation of lysosomes in fibroblasts, WFA must bind to the cells via its carbohydrate-binding site. The specific saccharide inhibitor of the lectin, lactose, completely abolishes the vacuolizing effect of the protein as well as its ability to bind to cells. This observation is noteworthy since WFA is a glycoprotein containing glucosamine, mannose, arabinose, xylose and fucose but lacking galactose and galactosamine. The inhibition by lactose of both the binding of the lectin to surface of the cells and the accumulation of lysosomes in fibroblasts indicates that the carbohydrates on the lectin are not interacting with the cells via a lectin-like protein on the surface of the cells as has been described by others [2, 231. Rather the carbohydrate binding site of WFA interacts with glycoconjugates on the surface of the cells. The requirement for WFA to bind to the cell surface prior to the development of large lysosomes may also be related to our observation that the action of the lectin is quite heterogeneous and time-dependent. Not all the cells become vacuolated to the same degree at any one time and the extent of cellular vacuolation is related to the length of exposure to the lectin. Such an action is consistent with a cell-cycle dependent phenomenon. Lotan et al. [26] observed a similar heterogeneity of response with lectin-treated macrophages. Others

[I I] have noted a similar behavior with the ConA induction of lymphocyte blastogenesuggested that it may be due to the eneity of the cell population studied. The variation in the response to lectin may be caused by differences either in the types of cehs or position of individual cells in the c.eU~cycIe. However, since our studies employed an established line of fibroblasts ~os~~bi~ity is not relevant. Manurger [27] have related the cellcycle dependence of the inhibition of growth of untransformed fibroblasts by succinylated ConA with the specific agglutinability of the cells by the native lectin and the cellular binding of fluorescein-labeled ese phenomena may be associhe cychcal changes in the presence and availability of a variety of cell surface glycoconjugates during the normal growth of cultured cells as reported by a number of laboratories [18, 20, 24, 411. Similarly, the re tory response of transformed cells to FA may be associated with differences e nature of cell surface ~iycoco~j~~ates associated with the transformed ~he~~ty~e [3 I 7441. Surprisingly, the WFA related accumulation of large lysosomes does not have a sig~~f~~anteffect on t e growth of the cells. Exposure of the cells to up to 200 pg/ml FA for 48 h does not alter the doubling rate of the cells. These results are in contrast to the effect of ConA on BALB/c 3T3 ~~br~~~astswhich has been reported [27] to inhibit the cell cycle in the mitotic and e of growth, as well as the early 61 arty et al. [2S] who observed report by BhaeConA inhibite DNA synthesis in Chicells. It is therefore rentially causes a of large lysosomes m transformed cells rowth of the cells.

This research has been supported in par: by a grant from the Charles and Johanna Busch Fund of the Bureau of Biological Research and by grar~ts from the NCI, nos. CA 17193md CA 20889.

1. Allison, A C & Young, M R, Lysosomes in biology and pathology (ed .I T Dingle & H B Feil) vol. 2, p. 600. American Elsevier. New York (1969). 2. Ashwell, G B Morell. A, Adv enzymol 41 (1974) 99. 3. Barrett, A J, Lysosomes; a laboratory handbook (ed J T Dingie) p. 46. American Elsevier, New York (1972). 4. Bausch, 3 N & Poretz, R is, Biochemistry I6 (1977) 5790. 5. Betel, I lk van den Berg, K 3, Eur j biochem 30 (1972) 517. 6. Bird, J W C, Berg, T & Leathem. J, Proc sot exp biol med 127 (1968) 182. 7. Bosmann, H B, Exp cell res 54 (1968) 217. 8. Canonice. B G & Bird, 9 W C, .Tcell biol 43 (1969) 361. 9. Cuatrecasas, P, Biochemistry 12 (1973) 1312. IO. Cheung, 6, Maratz, A, Katar, M, Skrokov, R 8 Poretz, R D, Biochemistry 18 (1979) 1646. II. Cunningham, B A; SeEa.B-A. Yahara. 1 & Edelman, G M, Mitogens in immunobiology (cd J 3 Oppenheimer & D L Rosenstreich) p. 13. Academic Press, New York (1974). i2. Czech, M P, Lawrence, J C Jr & Lynn. W S. .i bioi them 249 (1974) 7499. 13. Dauwalder, M. Whalev, W G & KeDhart. J E. J cell sci 4 (1969) 455. ’ 14. deDuve, C, Pressman, y3C, Bianetto. ii, WaZ~aux. R & Appelmans, F, Biochem j 60 (1955) 604. 15. Edelman. GM. Science 192 11976)218 16. Edeison, P J & Cohn, Z A, j exp med 140 i ‘974) 1364. 17. - Ibid (1974) 1387. 18 Gahmberg, C G 8-cMakomori, S, res commun 59 (1974) 283. 19 GoBdman, R & Raz, A, Exp cell res96(i975: 393. 20. Hynes, R 0 & McBye, J, Cell 3 (1974) I!?. 21. .Tacobs, S, Schechter, Y. B&sell. K & Cuatrecasas, P, Biochem biophys res ccnxmup 77 (1977) 981. 22. Kaladas. P & Poretz, R D; Biochemistry 18 (1979) 4806. 23. Kaplan, A, Achord, D T A & Sly. W S. Psoc nail acad sci US 74 (1977) 2026. 24. Kraemer, P M Pr Fobey, R A, J ceil biol55 (1971) 713. 25. Karokawa, T. Tsuda, M & Sugino. Y, 5 biol them 251 (1974) 5686. 26. Lotan, R, Sharon, N & Goldman, R. P clinica! and biological research (ed .i P Henning & C F Fox) p. 531. Academic Bres\, New York (1977). 27. Mannino, R .?& Burger, M M. Nature 256 11975) 19. 28. McCarty, K S Jr, Olive, K E> Kaufman, B & McCarty; D S Sr, Fed proc 37 (1978) 1550. 29 Merion, M. St John, A, Poretz, Is. Kxhler. R,

142

30. 31. 32. 33. 34. 35. 36. 37. 38.

Poretz et al.

Bird, .I, Triemer, R&Carter, J, J cell bio179 (1978) LP1307. Nagata, Y & Burger, M M, J biol them 249 (1974) 3116. Nicolson, G L, Biochim biophys acta 457 (1976) 57. Novikoff:’ A B, Beaufay, H & deDuve, C, J biophys biochem cytol2 (1956) 179. Olsnes, S & Pihl, A, FEBS lett 20 (1972) 327. Pappenheimer, A M Jr, Ann rev biochem 46 (1977) r_ OY. Poretz, R D, Carbohydrate-protein interaction (ed I J Goldstein) p. 56. American Chemical Society, Washington, DC (1979). Poretz, R D, Riss, H, Timberlake, J W & Chien, S M, Biochemistry 13 (1974) 250. Privat, J-P, Delmotte, F & Monsigny, M, FEBS lett 46 (1974) 224. Roy, AB, Biochem j 77 (1960) 380.

Exp Ceil Rrs 128 11980)

39. Sharon, N, Mitogens in immunobiology (ed J J Oppenheimer & D L Rosenstreich) p. 31. Academic Press, New York (1976). 40. St John, A C, Merion, M, Triemer, R E, Kuchler, R J, Bird, J W C, Carter, J H & Poretz, R D, Exp cell res 128 (1980) 143. 41. Shoham, J & Sachs, L, Exp cell res 85 (1974) 8. 42. Timberlake, J W, Physical and chemical properties _ ^ of the hemagglutinin from the seeds of Sophova iaponica Dissertation, University of Kansas (1974). 43. Wibo, M & Poole, B, J cell bio163 (1974) 530. 44. Yamada, K M & Pouyssegur, J, Biochimie 60 (1978) 1221. Received October 5, 1979 Revised version received January 15, 1980 Accepted January 18, 1980

Prmted

in Sweden