Inhibition of conjugation in Tetrahymena pyriformis by concanavalin A

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in Sweden @ 1977 by Academic Press, Inc. AN rights of reproduction in any form reserved ISSN 0014-4827

Cbpyrighr

Experimental

INHIBITION

Cell Research 106 (1977) 293-301

OF CONJUGATION IN TETRAHYMENA BY CONCANAVALIN A

Binding

of Concanavalin Starvation

A to Material and to Washed

A. FRISCH, HANNAH Depurtmenr

of Biological

Chemistry,

LEVKOVITZ

The Hebrew

University

Secreted Cells

PYRZFORMZS

during

and A. LOYTER of Jerusalem,

Jerusulem,

Israel

SUMMARY A group of glycoproteins which form an insoluble complex with concanavalin A (ConA) are secreted during starvation of the mating types strain, as well as by the non-mating types of Tetrahymena pyriformis. These glycoproteins were isolated by Sepharose-ConA, characterized, and their relevance to the conjugation process studied. The isolated ConA binding proteins (CBM) contain about 26% of their total weight, a phenol sulfuric acid-positive material, presumably carbohydrates: and exhibit about 5-g major bands by gel electrophoresis analysis. The possibility that ConA inhtbits the conjugation process by precipitating with this material was tested. Addition of isolated CBM restored conjugation previously inhibited by ConA. However, incubation of isolated CBM with either of the mating types or with both did not have any effect on the kinetics of the conjugation process. Antibodies prepared against CBM-secreted by both mating types did not prevent conjugation when added to the conjugation medium. The data suggest that CBM does not directly participate in the conjugation process. Inhibition of conjugation by ConA is probably due to its interaction with specific membrane-bound glycoproteins.

The mating process of protozoa, during which complementary mating types specifically interact and form permanent contact, is a suitable system for investigating the molecular events which are involved in cell-cell recognition. During the last few years, we have studied in our laboratory the conjugation process of Tetrahymena pyriformis in an attempt to gain a better understanding of processes such as specific cell adhesion and membrane fusion [l-3]. The events leading to conjugation in Tetrahymena pyriformis have been divided into two stages: the “initiation” period, during which each of the mating types can be separately starved and subsequently, the “co20-771817

stimulation” period, which is induced immediately after mixing of the mating types and requires the actual contact between them [4]. Elucidation of the molecular changes induced during the starvation period will undoubtedly lead to a better understanding of the whole conjugation process. Specititally, this might provide information on the chemical and structural nature of the cell surface components which are required for the pairing process. Appearance of new sites which actively participate in the pairing between the mating types of Paramecium have been noted during the starvation period [S]. In addition, mating subExp

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Rex

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Levkovitz

and Loyter

stances which were able to induce specific contact between the appropriate cells have been isolated from Paramecium [5], Chlamydomonas [6] and yeast [7]. In the case of Chlamydomonas [6] and yeast [7] these components appear to be glycoproteins, while in the case of Paramecium their chemical nature is still unknown. In these cases, the mating substances are tightly associated with the exterior surface of the membrane. Further support for the view that glycoproteins mediate mating reaction between cells, were obtained from experiments showing that ConA prevented mating between opposite mating types of Chlamydomonas [8] and inhibited the fertilization of sea urchin [9]. Recently we have found that relatively low concentrations of ConA specifically inhibit the conjugation process in Tetrahymena pyriformis when added to a mixture of the mating types. The inhibition of conjugation by ConA appeared to be reversible upon addition of a-methyl mannoside (crMM) and coincided with dissociation of bound r3H]ConA [2]. In addition, we have shown that tunicamycin, which specifically prevented incorporation of [3H]glucosamine into cells of Tetrahymena pyriformis, also inhibited conjugation [3]. These observations clearly indicate that glycoprotein molecules participate in the conjugation process, either directly, by serving as a binding substance between cells, or indirectly. From results of previous work [2] it is not clear whether ConA binds to specific sites on the surface of the ceIla or whether it reacts with a material secreted into the medium. In the present work, we tried to differentiate between these two possibilities. After finding that most of the ConA precipitates with material secreted into the meExp

Cell Res 106 (1977)

dium, we have characterized the soluble binder, and studied its relevance to the conjugation process. MATERIALS

AND

METHODS

Cells Tetruhymena pyriformis mating type I (strain WH-6), mating type III (strain WH-52) and the non-mating type GL, were obtained from the American Type Culture Collection. The cells were grown in a medium containing: 0.5 % proteose peptone, 0.5 % bactotryptone, 0.01% yeast extract, 0.1% sodium acetate and 0.1% KH,PO, (final pH 7.4) under sterile conditions at 26°C.

Medium The medium used for washing of cells, starvation and conjugation experiments was 20 mM tricineNaOH, pH 7.4.

Induction and determination of conjugation Cells were starved by incubating each of the mating types (106 cells/ml) in 20 mM tricine-NaOH, pH 7.4, at 30°C for 24 h. Coniugation was induced and determined essentially as described by Elliott & Hayes [lo] and in a previous work [2]. For conjugation a suspension of two starved mating types (about 106 cells/ml) were mixed by introducing 0.5 ml of each suspension to a 20 ml glass flask, and then incubated at 30°C without shaking. The extent of conjugation was determined after 4 h of incubation [2].

Isolation of ConA binding material (CBM) by Sepharose ConA column A suspension of starved cells, 5-10 1, was centrifuged in a continuous flow Basket Centrifuge (MSE). The supematant obtained was filtered twice through a fiber alass filter oauer tvne C (Whatman) and keut in the cold. All subdqueni steps‘were performed inthe cold (OXC). ConA binding material (CBM) was isolated from the supematant b; the following way: SepharoseConA (Pharmacia), containing 8 mg of ConA/ml packed material, was introduced into glass columns (25 mm 0) and washed extensively with 20 mM tricineNaOH pH 7.4 containing 5 mM MnCl,. MnCI, was added in order to improve binding to SepharoseConA. The clear supematant to which 5 mM MnCl, was added was loaded on the SenharoseConA column using 3 ml of packed Sepharbse ConA for each liter of fluid. The flow rate used was 50 ml/h. After loading, the column was washed with the above buffer until the absorntion of the effluent at 280 nm decreased to buffer levee The effluent contained about 90-95 % of the total protein content of the supematant. This fraction was completely devoid of material precipitable by [3H]ConA. CBM was eluted by the above buffer

Inhibition

of conjugation

in Tetrahymena

by ConA

295

after the last booster. The serum was collected and the y-globulin were precipitated by 37% ammonium sulphate and further purified on DEAE-cellulose as described in [ 121. Protein was estimated by the method of Lowry et al. [ 131and sugar by the phenol-sulfuric method [14].

RESULTS 1

0

2

4

6

8

10

Kg. I. Abscissa: starvation time (hours); ordinate: [3H]ConA precipitated/system kg). O-O, Mating type I; O-O, mating type III. Increased precipitation of [3H]ConA with a cell suspension of 7etrahymena pyriformis during starvation. At different times during the starvation period samples of 1.8 ml were withdrawn and incubated with 200 pg of [“H]ConA (2x IOr’ cpm/mg) in a final volume of 2 ml.

containing 0.1 M (YMM. Protein eluted with aMM was monitored spectrophotometrically, and the 280 nm peak was collected, dialysed with five changes against 100 vol of 20 mM tricine-NaOH, pH 7.4, and lyophihzed. The powder obtained was dissolved in the above buffer to give 2-4 mglml of protein and dialysed again as described above.

Precipitation of [3H]ConA and isolated CBM

by crude

[“H]ConA (2-8.105 cpm/mg) was prepared according to Miller [ 111. MnCl* (final cont. 5 mM) was added to all the systems to improve binding. A suspension of starved cells (lofi cells/ml), cell-free medium derived from them, or isolated CBM, were incubated in a final volume of I-2.5 ml at 30°C for 30 min with the indicated concentration of [3H]ConA. At the end of the incubation, the mixtures were filtered through fiber glass filter papers, type A (Whatman) and washed three times with 10 ml of 20 mM tricine-NaOH, pH 7.4. The filters were dried and counted as described before [2]. Since (rMM is known to prevent the specific binding of ConA a control containing 0. I M aMM was performed to estimate the specific binding. The sugar was allowed to interact with F’HlConA for 30 min before addition to the various systems.

Production anti-CBM

and purification

of

Isolated, purified CBM (about 2 mg/ml) in 20 mM tricine-NaOH, pH 7.4, was emulsified with equal volume of Freund complete adiuvant (Difco) and iniected into rabbits intrac&neousiy and in the’ foot pads of the back legs. The first injection contained 2 mg and the two subsequent boosters, given at 2-week intervals, contained 1 mg each. Blood was taken 2 weeks

Precipitation of ConA with material secreted during starvation oj Tetrahymena pyrifbrmis Experiments using [3H]ConA have revealed that a certain amount of lectin added to a suspension of starved cell was retained on fiber glass filters with the cells (fig. I). As can be seen in this figure, the amount of ConA precipitated with the cell suspension increased during the starvation time. In the present work, we have preferred the use of the term precipitation instead of binding when [3H]ConA was retained on the fiber glass filter after incubation with either cell suspension or with cell-free medium. The observation that higher amounts of Con.4 precipitated with the cell suspension of mating type I than with mating type III (fig. 1) was reproducible and noted in many experiments. Precipitation of ConA was sensitive to LxMM, and the increase in the precipitation activity during starvation period up to 48 h was temperature-dependent and required active protein synthesis (not shown). In order to determine whether ConA precipitates with the cells, or forms an insoluble complex with glycoproteins, which might be secreted into the medium during starvation, the cells were collected by centrifugation, and binding of ConA was studied separately with washed cells and with the cell-free medium. A concentration of 15 pg/ml of ConA was chosen for this experiment since this is the minimai concentration found to inhibit conjugation comILrp Ceil RES 106 (1977)

296

Frisch, Levkovitz

and Loyter

Table 1. Precipitation of [3H]C~nA with starved Tetrahymena terial secreted into the starvation medium

System Suspension of mating types I+111 Suspension of mating types I+III+(rMM Cell-free supernatant mating types I+111 Cell-free supernatant of mating types I+III+oMM Washed cells (I+III) Washed cells (I+III)taMM

pyriformis

Precipitation of [3H]ConA/ml reaction mixture or to 106 cells (I%)

Net binding of [3H]ConA (% of total)

7.4

44

and with ma-

0.75 7.80

44

1.14 0.8 0.5

2

Cells were starved for 24 h (equal amount of suspension of each mating type was mixed) and then centrifuged at 500 g for 5 min. The starvation medium was collected and the cells were washed two times in 20 mM tricineNaOH, pH 7.4, and then suspended to the original concentration (106 cells/ml) in the above buffer. Volumes of 2.5 ml of either washed cells or the medium derived from them were incubated with 37.5 pg of [3H]ConA (8.4~ lo5 cpmlmg protein). [3H]ConA was precipitated and counted as described under Materials and Methods. Concentration of oMM 0.1 M.

pletely. Table 1 shows that the precipitation of ConA found with the cell suspension is due mostly to a complex formed with a material present in the medium. Washed cells were found to bind only a very small fraction of the added ConA (about 2%) (table 1). Essentially the same results, namely precipitation of [“H]ConA with soluble material, were obtained when medium derived from conjugating cells was studied. Experi-

Table 2. The amount of CBMpresent

ments to reveal whether material which is able to precipitate with ConA is secreted also during growth, were impossible due to the presence of sugars and glycoproteins in the regular growth medium. Characterization of CBM Since inhibition of conjugation by ConA might be due to its ability to precipitate with secreted material, it was of interest to

in the starvation

medium

and its sugar/protein

ratio

Strain

A CBM in starvation medium (protein, dml)”

B Total protein secreted into the medium Cf.&ml)

A/B

Amount of sugar in CBM C Phenol sulfuric positive material h/ml)

Mating type (I)

2.0

34

0.07

0.62

0.26

bG$g

2.5 1.4

26 22

0.07 0.054

0.52 0.54

0.26 0.27

type (III)

(1 The numbers are based on the amount of CBM isolated by Sepharose-ConA For experimental details of CBM isolation see Materials and Methods. Exp Cell Res 106 (1977)

C C+A

from cell-free starvation medium.

Inhibition

Fig. 2. Gel electrophoresis pattern of CBM. One hundred pg of protein were loaded on 10% acrylamide gels. A. CBM 1; E, CBM III; C, CBM GL. Acrylamide gels were prepared according to Laemmli [22] and stained according to Fairbanks [23].

isolate it and study its properties. The material secreted into the medium during starvation of Tetrahymena pytiformis, which had a ConA binding activity, was isolated by a Sepharose-ConA column (see Materials and Methods). After loading of the starvation medium on Sepharose-ConA column about 90-95s of its total protein content was found in the effluent. This fraction did not exhibit any [SH]ConA precipitation activity indicating that SepharoseConA effectively binds all the CBM present

of conjugation

in Tetrahymena

by ConA

2

in the starvation medium. A small fraction of the total protein was retained on the Sepharose-ConA column and could be eluted by (YMM. This fraction, which amounts to 5-7s of the total protein, was designated as CBM (table 2). The presence of protein material in the starvation medium is not surprising in the light of previous reports on secretion of several enzymes during starvation [ 151. Interestingly, CBM was secreted not only by the mating types but also by the nonmating strain GL (table 2). The CBM from all three strains contained a phenol sulfuric acid-positive material indicating the presence of sugar moieties. The sugar fraction consists of about 26% of the total weight (proteinfsugar, table 2). Gel electrophoresis analysis revealed about 7-8 major protein bands in isolated CBM obtained from the two mating types as compared with 5-g bands of CBM isolated from the non-mating strain GL (fig. 2). None of the bands were stained by the specific PAS reaction, probably indicating the absence of sialic acid in their sugar component [ 161. When increasing amounts of CBM were in-

l

I

-.-.-___

0 Fig.

-l... “l.

2s

3.

Abscissa:

5 0

75

YZ-.-‘LOO

CBM (pg); ordinctra. (gg). A precipitation

125

[RHjConA curve of

precipitated/system ConA-CBM. Increasing amounts of isolated CBMI were cubated with 38 pg of [3H]ConA (4x IQ’ cpmlmg) final volume of 1 ml. The complex obtained was lected by filtration as described in Materials Methods.

inin a coland

4. Interaction of CBM with various lectins and with its antibodies. (u) Interaction with various lectins. The central wells contained in a volume of 0.03 ml, 75 pg of A, CBMGL; B, CBM III. Lectins (0.03 ml containing 75 pg) were added to the following wells: I, PHA; 2, SBA; 3, WGA; 4. ConA; (h) interaction between CBM and its antibodies. The central wells contained in

a final volume of 0.03 ml 1 mg of purified anti-CBIM; anti CBMI; B, anti CBMIII. Isolated CBM (75 pg in 0.03 ml) was added as follows: 1, CBMI; 2, CBM III; 3, CBM GL. Double diffusion test was performed according to the method of Ouchterlony [24]. y-Globulins were purified as described under Materials and Methods.

cubated with a constant amount of [“HIConA, a typical antigen-antibody precipitation curve was obtained (fig. 3). The equivalence point was reached at a ratio of 1 : 0.3 ConA to ConA binding material, obtained from the starvation medium of mating type I (CBM I). Essentially the same precipitation curves were obtained with ConA binding material obtained from the starvation medium of mating type III (CBMIII) and CBM GL, although the equivalence points were slightly different from those obtained for CBM I. Previously, it was reported that among the various lectins tested only ConA was

found to inhibit conjugation [2]. It was, therefore, of interest to check whether the isolated CBM will interact only with ConA or whether it contains receptors for other lectins. The double diffusion technique of Ouchterlony widely used for detecting precipitation of specific antigen-antibody complexes, was used to study the interaction between CBM and the various lectins. It can be seen in fig. 4u that CBM interacts specifically with ConA giving a heavy and sharp precipitation line. Wheatgerm agglutinin @VGA), soybean agglutinin (SBA) and phytohemagglutinin (PHA) gave no reaction with CBM (fig. 40).

Fig.

Table 3. Restorution

conjugation

Time of addition of either (YMM or CBM (min after mixing of the mating types)

Addition None CB.M CB,M ConA ConA ConA ConA ConA

of ConA-inhibited

A,

by isolated CBM

Conjugation (%I

Restoration of conjugation (%I

65 70 70

I III and and and and

aMM CBM I CBM I CBM I

0 ,200 240

60” 65 ii:

90” 100 90 loo

When CBM was added at zero time it was incubated with ConA for 15 min at 30°C before addition. In experiments where CBM was added after mixing of the mating types, conjugation was determined 4 h after its addition. Concentrations: ConA 25 pg/ml, aMM 50 mM; 9.7 mg/ml; CBM, 50 &ml. Lip

Cell

RPI 106 (1977)

Inhibition

Table 4. Occurrence presence

of Sepharose

of conjugution

of conjugation

in the ConA and anti-CBM

Addition

Conjugation

None ConA Sepharose-ConA Anti-CBM 1 Anti-CBM III Anti-CBM I+111

70 5: 68 68 72

ConA 25 pg/ml, Sepharose-ConA (containing 80 pg of ConA/ml) and anti-CBM (120 /*g/ml) were added at zero time of conjugation. When a mixture of the antibodies was checked, 120 &ml of each were added.

The relevance of CBM to the conjugation process

The ConA-CBM complex, due to its granular nature, might stimulate digestive vacuole formation [17] and thus, serving as a nutrient, break the starvation and inhibit conjugation. Indeed, experiments using fluorescein-ConA, which will be published shortly, showed that a CBM-ConA complex is taken in by digestive vacuoles [18]. To determine whether the ConA-CBM complex affects conjugation, a preformed complex was added to the conjugatio’i system. Table 3 shows that such a complex did not affect conjugation when added to a mixture of the mating types. The effect of the exogenous CBM on the conjugation system, inhibited by ConA, was studied. The exogenous CBM might substitute for the endogenous CBM eliminated by precipitation with ConA, and whose presence might be essential for conjugation. Exogenous CBM effectively restored conjugation previously inhibited by ConA for 2-4 h. As can be seen in table 3, isolated CBM was 200 times more effective on weight basis than aMM in restoration of conjugation (50 pg of CBM were equivalent to 9.7 mg of aMM).

in Tetrahymena

bv ConA

299

A more direct approach to the elimination of the CBM from the conjugation system was the use of Sepharose-ConA. which has been proved to bind CBM with high efficiency (see Materials and Methods). Because of the large dimension of the Sepharose beads (40- 190 pm) and the high motility of Tetrahymena cells, SepharoseConA is unable to reach the cell membranes. It does not, therefore, form permanent linkage to the cells (microscopic observations), and binds only glycoproteins present in the medium. Addition of Sepharose-ConA (containing 80 pg of ConA) had no effect on the conjugation process (table 4), although at the concentration used, i! binds all the CBM present in the conjugation medium (see Materials and Methods). In contrast, addition of 15-25 kg of soluble ConA was sufficient to completely prevent conjugation (table 2). A closer analogue to the soluble ConA might be an antibody to CBM which will act effectively and specifically. Antibodies against CBM were prepared by injecting isolated CBM into rabbits (see Materials and Methods). A double diffusion test shows that an anti-CBMI interacts with its own antigen, giving both a specific sharp precipitation line and a second diffuse line. The diffuse line was also obtained with CBMIII, while CBM from the strain GL gave almost no reaction (fig. 4b). An antiCBM III interacts with CBM from both mating types almost to the same extent. giving multiple bands, while CBM-GL gave only one precipitation line (fig. 4b). A precipitation curve of CBM- and anti-CBM was performed using CBM obtained from all mating types. The equivalence point was reached at a ratio of 1 : 0.3 (antibodylisolated CBM). As can be seen in table 4) conjugation was not affected even when amounts of antibodies far beyond the

300

Frisch,

Levkovitz.

and Loyter

equivalence point (120 pg) were added. Addition of anti-CBM at the equivalence point also did not prevent conjugation. Further indications that CBM does not directly participate in the conjugation were obtained by the following observations: (a) Addition of isolated CBM at high concentration to the conjugation medium neither had any effect on the kinetics of conjugation (not shown) nor on its final percentage (table 3); (h) incubation of isolated CBM from one mating type with cell suspension of the complementary mating type did not produce any change in the behaviour of the cells. DISCUSSION Two kinds of mechanisms have been suggested for the recognition and agglutination stages of conjugation processes in protozoa [19]. One involves the participation of the membrane-bound mating substances, as is the case, e.g., in the conjugation processes of Paramecium [S] and Chlamydomonas [6}. The second mechanism, deduced from studies in Blepharisma intermedium, involves soluble macromolecules, among them glycoproteins. These substances induce specific chain reactions which eventually lead to agglutination and conjugation of complementary mating types [ 193. Participation of membrane-bound proteins in the conjugation process of Tetrahymena pyrvormis have been suggested by Bruns [4] and by Allewell et al. [20]. In contrast to this view, Phillips has suggested involvement of soluble substances, based on experiments showing that conjugation can be stimulated by cell-free conjugation medium [21]. Participation of either soluble or membrane-bound glycoproteins in the conjugation process was inferred from recent experiments in our laboratory show-

ing that the lectin ConA [2] and the antibiotic tunicamycin [3] inhibit pairing between the mating types. In the present work we have studied the inhibition of conjugation in Tetrahymenu pyriformis by ConA, in order to gain insight of the participation of glycoproteins in the mating process. The data in the present work show that, under minimal inhibitory conditions, only 2% of the added ConA precipitated with washed cells, whereas 45 % precipitated with a soluble material secreted from either of the mating types during starvation. It was found that CBM was secreted by the two mating types, as well as by the non-mating type GL and consisted of several polypeptides of different molecular weights. Isolated CBM was shown to interact with soluble ConA, resulting in a complex which precipitated and was retained on a fiber glass filter, probably due to its high molecular weight. These results explain the observation that most of the ConA added to starved mating types or to conjugating cells precipitated with an extra-cellular soluble material. Inhibition of conjugation by ConA was not due to the presence of such a complex, since a preformed CBM-ConA-insoluble complex did not affect conjugation when added either before or during conjugation. Interestingly, addition of exogenous CBM effectively restored conjugation prevented by ConA. Restoration of the conjugation process by exogenous CBM might be explained in two ways: (a) The presence of free CBM is absolutely required for induction of conjugation, and when the endogenous CBM is eliminated by addition of ConA, conjugation is inhibited. Under these conditions exogenous CBM would restore conjugation by substituting for the bound CBM. (6) It is possible that a small

Inhibition

of conjugation

fraction of the ConA masks specific membrane glycoproteins which mediate the conjugation process. Indeed, when the minimal amount of ConA, which still inhibits conjugation, was added to the medium, about 2% of it was found bound to the cells. Removal of the bound ConA from its membrane site by a large excess of soluble isolated CBM would result in restoration of conjugation. Under the regular conjugation conditions, the concentration of the continuously secreted CBM is never high enough to neutralize the ConA, which is added in excess. Most of the data in the present work indicate that soluble CMB does not play any active role in the conjugation process. (1) Isolated CBM did not affect conjugation when added to a mixture of mating types. (2) An antibody prepared against isolated CBM was without effect, even when added at concentrations which supposedly precipitate all the CBM present in the conjugation medium. (3) Sepharose ConA, capable of binding the CBM (Sepharose ConA was used for isolating the CBM), failed to inhibit conjugation. The discrepancy between the effect of soluble ConA and SepharoseConA can be explained by assuming that the membrane receptors are accessible to the soluble ConA but cannot be reached by the bulky Sepharose-ConA. The soluble glycoproteins (CBM) may consist of macromolecules which were shed from the membrane during starvation or, alternatively, they may be a degradative product of the membrane-linked macromolecules. The present work supports the view that conjugation in Tetrahymena pyriformis is mediated by specific membrane-bound glycoproteins. Inhibition of conjugation by ConA is due probably to its interaction with these sites. The localization of these receptors might be limited to a specific membrane

in Tetrahymena

by ConA

301

area, namely the conjugation plate, and thus their number would be small. Accurate measurements of the number of ConA molecules bound to these receptors are difficult, due to the high background of ConA which precipitates with soluble CBM. Experiments to demonstrate the localization of ConA receptors were performed by use of fluorescein-ConA and ferritin-ConA. Results showing specific binding of ConA to the membrane at the conjugation plate will be published shortly [ 181. REFERENCES

6. I. 8. 9. 10. !I. 12.

13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

Levkovitz. H, Ofer, L & Loyter, A. Israel j med sci9(1973)24. Ofer, L, Levkovitz. H & Loyter. A. J cell biol 70 (1976) 287. Frisch, A, Levkovitz, H & Loyter. A, Biochem biophys res commun 72 (1976) 138. Bruns, P J & Palestine, R F, Dev biol42 (1975) 75. Kitamura, A & Hiwatashi, K, J cell biol 69 (1976) 736. Snell, W J, J cell biol68 (1976) 48. Yen, P H & BaUou. C E, Biochemistry 13 (1974) 2428. Wiese, L & Shoemaker, D W, Biol bull 138 (1970) 88. Lallier, R, Exp cell res 72 (1972) 157. Elliott, A M & Hayes, R E, Biol bull 105 (1953) 269. Miller, I R & Great, H, Biopolymers 11 (1972) 2533. Fahey, J L, Methods in immunology and immunochemistry (ed C A Williams & M W Chase) vol. I, pp. 323-324. Academic Press, New York and London (1967). Lowry. 0 H. Rosebrough. N J. Farr, A i & Randall, R J, 3 biol them 193 (1951) 265. Du Bois,. M. Gilles, K A, Hamilton, J K, Rebers, P A & Smtth. F, Anal them 28 (1956) 350. Miiller, M, J cell biol52 (1972) 478. Spiro, R G, J biol them 239 (1964) 567. Seaman, G R, J protozool8 (l%l) 204. Frisch, A & Loyter, A. In preparation. Miyake, A, Current topics in microbiology and immunology 68 (1974) 49. Allewell. N M. Oles. J &Wolfe. J. EXD , cell res 97 (1976) 394. Phillips, R B, J protozool 18 (1971) 163. Laemmli. U K, Nature 227 (1970) 680. Fairbanks. G. Steck. T L & Wallach. D F H. Biochemistry JO (197i) 2606. Munoz. J. Methods in immunoloev and immunochemis&(ed C A Williams & M q Chase) vol. 3. DD. 147-149. Academic Press, New York and L&don (1971).

Received September 24, 1976 Accepted December 6, 1976

ExpCellHesIO6(1977)