222

Biochimica et Biophysica Acta, 550 (1979) 222--232 © Elsevier/North-Holland Biomedical Press

BBA 78231 PLASMA MEMBRANE VESICLES ISOLATED FROM EPIMASTIGOTE FORMS OF T R Y P A N O S O M A CR U Z I

J. FRANCO DA SILVEIRA *, PAULO A. ABRAHAMSOHN ** and WALTER COLLI *** Instituto de Quimica, Departamento de Bioquimica, Universidade de S~o Paulo, S~o Paulo (Brazil)

(Received May 9th, 1978) Key words: Glycoprotein; Cell vesiculation; Epimastigote (Trypanosoma cruzi membrane)

Summary Membrane vesicles can be obtained from epimastigote forms of Trypanosoma cruzi by incubating cells with either cross-linking reagents or acid pH. Acetate, phtalate or citrate, at pH 4.0, but n o t at higher pH values, were able to induce plasma membrane vesiculation. Vesicles have been purified by sucrose density centrifugation and their membrane origin was demonstrated by the following criteria: (a) Vesicles are 5--10 times richer in protein-bound iodine when they are prepared from cells previously labeled with '31I by the lactoperoxidase catalysed reaction. (b) Electron microscopy of vesiculating cells shows physical continuity between cell plasma membrane and vesicle membrane. (c) Antibodies prepared against purified vesicles are able to agglutinate epimastigote forms of T. cruzi with sera dilutions up to 1 : 256 to 1 : 512. (d) Freeze-fracture studies of the purified vesicles have shown images of faces P and E compatible with known images of the intact cell plasma membrane. Typical preparations of acetate vesicles present the following characteristics: total carbohydrate : protein = 1.5--2.0; orcinol : protein = 0.07 and absence of diphenylamine reaction. Vesicles contain 0.2--0.5% and 0.3--1.0% of the total homogenate protein and carbohydrate, respectively. The presence of 10 major protein bands and a 30--50-fold enrichment of the four sugar-containing macromolecules present in epimastigote forms of T. cruzi have been demonstrated in these preparations.

Introduction The biological cycle of T r y p a n o s o m a cruzi, a digenetic parasitic protozoon and the causative agent of Chagas' disease [1,2], shows important feature as * F r o m t h e I n s t i t u t o B u t a n t a n and in p a r t i a l f u l f i l l m e n t for a D o c t o r ' s degree. ** P e r m a n e n t a d d r e s s : I n s t i t u t o d e C i ~ n c i a s B i o m ~ d i c a s , U n i v e r s i d a d e d e S~o P a u l o , S a o P~lulo, B r a z i l . *** To whom correspondence should be sent.

223 y e t uncovered, namely the nature of the signals which determine transformation of a given cell type into the other, the mechanisms involved in the interaction between parasite and vertebrate host cells and, finally, the nature of the immune response [2,3]. These features depend both on receptor-ligand interactions and on cell recognition and constitute important membrane-dependent phenomena in several cell systems studied to date [4--6]. Thus, knowledge of the chemical composition and topography of T. cruzi plasma membrane is a fundamental step to the understanding of the biological problems involved in the evolutive cycle of this protozoon. Several cells and e r y t h r o c y t e ghosts shed fragments (vesicles) from their surface membranes to the outside environment, when subjected to specific treatments [7--16]. These treatments yield fairly pure membrane fragments enriched in certain components [17--19] allowing a reasonably precise analysis of the plasma membrane topography [20,21]. In the present work a method of isolation of plasma membrane vesicles from T. cruzi epimastigotes along with a general analysis of their properties are described. Materials and Methods Cell cultures. Epimastigote forms of T. cruzi, Y strain [22], were cultured at 28°C in a r o t a t o r y shaker (New Brunswick) at 120 rev./min. Cells in late exponential growth (80--100 h) were harvested by centrifugation at 1000 X g, 4°C, and washed 3 times with 150 mM NaC1. Cell numbers were determined in a Coulter counter. Vesicle isolation. 1 • 109--5 • 109 cells/ml were incubated at 37°C for up to 60 min with the vesiculating agent (see legends to the figures and tables) dissolved in a solution of 150 mM NaC1/1 mM CaC12/0.02% NAN3. After incubation the cells were centrifuged at 4000 X g for 10 min at 4°C and the supernatant was collected. The cell pellet was washed twice with cold 150 mM NaC1 (1 ml per 1 • 109--2 • 109 cells) as described above and the vesicles containing supernatants were pooled. Vesicles were then precipitated at 100 000 × g for I h at 4°C. The pellet was resuspended in 10 mM Tris-HC1, pH 7.5, containing 150 mM NaC1, and was further purified in a discontinuous sucrose gradient in order to eliminate contaminating cells and cell debris. The gradients were performed in Spinco SW27 rotor tubes by sequentially layering 5 ml each of the following sucrose solutions in 10 mM Tris-HC1, pH 7.5: 40%, 35%, 30%, 25%, 20% and 8%. After layering 4--5 ml of a vesicle suspension (0.2--1.4 mg protein) the preparation was centrifuged at 16 000 rev./min in the SW-27 Spinco rotor for 16--18 h at 4°C. Fractions of 1 ml were collected from the top with the help o f a peristaltic pump. The fractions containing the vesicles (tubes 10--20) were washed twice by centrifugation at 105 000 × g with a large excess of 1 mM Tris-HC1, pH 7.5/1 mM EDTA. The preparation was kept at --20°C in 10 mM Tris-HC1, pH 7.5, at a protein concentration of 1 mg/ml. Lactoperoxidase-catalyzed radioiodination. Washed cells were incubated in 0.1 M phosphate buffer, pH 7.4 { 0 . 7 . 1 0 s - - 1 . 2 • l 0 s cells/ml) containing

224 150 mM NaC1, 50 ttg/ml lactoperoxidase, 10--20 pM K1 and 12--45 uCi/ml of Na131I. H202 was added in four steps at 1.5-min intervals in amounts equivalent to 8 pM final concentration. H202 concentration was estimated before each experiment by the absorbanc, e at 230 nm t c = 7 2 . 4 1 . m o 1 - 1 - c m -1) [23]. Incubation was carried out at 30°C for 6 min. After incubation cells were washed with large volumes of 150 mM NaC1 and then added to non-labeled cells before vesiculation. Radioactivity incorporated into cells and vesicles was determined after two precipitations with cold 7% trichloroacetic acid. The precipitates were resuspended in 0.5 ml 0.5 M NaOH and counted in a Nuclear Chicago gamma counter at an efficiency of 40%. The results shown were not corrected for such an efficiency. The iodination procedure did n o t impair cells as judged by their normal motility under phase contrast microscope. The estimated iodination efficiency was 0.3--1.5%. Omission of lactoperoxidase and/or H20,. decreased iodine incorporation to 1.0--2.0% of the maximum. Na131I was obtained from the Instituto de Energia AtSmica, S~io Paulo. and the lactoperoxidase was prepared as previously described [24]. Analytical procedure. Protein was estimated by the m e t h o d of Lowry et al. [25] using bovine serum albumin as standard. Nucleic acids were extracted as described [26]. DNA was measured by the diphenylamine reaction modified by Giles and Myers [27] and RNA by the orcinol m e t h o d [28]. Total carbohydrate was measured by the phenol-sulfuric acid reaction [29] in trichloroacetic acid-precipitated vesicle material after extensive washing and dialysis against 10 mM Tris-HC1, pH 7.5. Sugar-containing macromolecules were analysed by electrophoresis in 15% polyacrylamide gels containing 0.1% sodium dodecyl sulfate as previously described [30]. Proteins were analysed in 10% gels in the presence of 0.1% sodium dodecyl sulfate [31]. For protein analysis cell homogenates and vesicles were previously extracted twice with chloroform/methanol (2 : 1, v/v), twice with n-butanol, dialyzed against 1 mM EDTA followed by H20, and lyophilized. Gels were stained with periodic acid-Schiff for carbohydrates [32] and Coomassie Blue for proteins [33]. Antibody preparation. Rabbits were injected with vesicles obtained from cells treated with 200 mM acetate buffer, pH 4.0, in the presence of 150 mM NaC1 and 1 mM CaClz for 30 min at 37°C. Animals were weekly injected with 0.3 mg vesicle protein in 0.1 ml 150 mM NaC1. In the first week an equal volume of Freund's complete adjuvant was injected with the antigen. In the following 4 weeks the latter was replaced by Freund's incomplete adjuvant. In order to test for antibody production 10 ~l of appropriately diluted serum, previously inactivated at 56°C for 60 min, were added to 106 cells in 10 pl 150 mM NaC1 placed in glass slide wells. Agglutination was followed under phase contrast microscope after 15 min incubation at room temperature. Controls were made with 1 : 10 dilutions of rabbit sera collected before antigen injection. Electron microscopy. Pellets from both cells and vesicles were fixed in 2% (w/v) redistilled glutaraldehyde buffered with 0.1 M sodium phosphate, pH 7.2, for 2 h at 4°C. Fixed pellets were washed overnight with phosphate buffer and post-fixed in 1% (w/v) OsO4 in phosphate buffer for 2 h at 4°C. The pellets were then dehydrated in a graded series of ethanol and embedded in Araldite.

225

Ultrathin sections made with a Sorvall MT-2 ultramicrotome were stained with uranyl acetate [34] and lead citrate [35]. For freeze-cleavage studies the glutaraldehyde-fixed pellets were glycerinated by sequential immersion in 10 and 20% glycerol solutions containing 0.9% NaC1. After quick-freezing in 'Freon 22' (--155°C) the pellets were fractured in a Balzers (BAF 301) freeze-fracture apparatus. Ultrathin sections and replicas of freeze-cleaved material were photographed in a Zeiss EM9A and in a Phillips 301 electron microscope. Results

Vesiculating agents. Incubation of T. cruzi epimastigote forms with aldehydes, N-ethylmaleimide, p-chloromercuribenzoate and acid buffers induces cell vesiculation (Table I). This phenomenon, which can be followed under phase contrast microscope, is followed by liberation of the vesicles to the extracellular medium. Alkaline solutions do not induce vesiculation under the conditions used. Vesicle purification. The 4 0 0 0 × g supernatants from cells subjected to vesiculation are very rich in vesicles but contain some intact cells and cell debris. This material yields two main protein bands when centrifuged in a discontinuous sucrose gradient (Fig. 1) as described in Materials and Methods. The band corresponding to gradient fractions 10--20 contains pure vesicles. Vesicles concentration diminishes abruptly from fraction 21 to the top of the gradient. These fractions contain all remaining cells and debris. When fractions TABLE

I

CONDITIONS

FOR

T. C R U Z I P L A S M A

MEMBRANE

VESICULATION

1 0 9 c e l l s / m l w e r e i n c u b a t e d at 3 7 ° C in t h e p r e s e n c e o f 1 5 0 m M N a C 1 a n d 1 m M C a C 1 2 . V e s i c u l a t i o n w a s

f o l l o w e d u n d e r p h a s e c o n t r a s t m i c r o s c o p e . F o r t h e n e g a t i v e results t h e m a x i m u m o b s e r v a t i o n p e r i o d w a s 6 0 rain. Citrate, p h t a l a t e , a c e t a t e , N - e t h y l m a l e i m i d e a n d p - c h l o r o m e r c u r i b e n z o a t e at p H 7 . 0 w e r e in t h e presence of 10 mM Tris, pH 7.5. Agent

Concentration

pH

Vesiculation

(mM)

Tris-H C1

10

7.5

--

Sodium phosphate

200 10

7.5 7.0

---

Sodium citrate

200 200

7.0 7.0

---

Sodium phtalate

200 200

4.0 7.0

+ --

Sodium acetate

200 200

4.0 7.0

+ --

N-ethylmaleimide

200 5

4.0 7.0

+ +

10 1 10

7.0 7.0 3.5

+ + +

250 250 250

3.5 7.0 4.0

+ + +

250

7.0

+

p-Chloromercuribenzoate

Formaldehyde

Acetaldehyde

226

?

o x

E. i-

o

:-

w

~r

o

1

°.

..

L..t~ I

Tt~i

A

I~

F i g . 1. P u r i f i c a t i o n o f T. c r u z i p l a s m a m e m b r a n e v e s i c l e s b y d i s c o n t i n u o u s s u c r o s e g r a d i e n t s . C e l l s ( 1 0 9 ) w e r e i n c u b a t e d w i t h l a c t o p e r o x i d a s e a n d N a 1 3 1 1 ( 0 . 3 6 m C i ) as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . T h e s e cells were w a s h e d a n d a d d e d t o a s u s p e n s i o n c o n t a i n i n g 8,8 • 1 0 1 0 n o n - l a b e l e d cells. The s u s p e n s i o n was then incubated with 200 mM acetate buffer, pH 4.0, for 30 min at 37°C in the presence of 150 mM NaCl a n d 1 m M CaCl 2 a n d vesicles were s u b j e c t e d t o g r a d i e n t c e n t r i f u g a t i o n as d e s c r i b e d in Materials a n d Methods. After centrifugation 1-ml f r a c t i o n s were c o l l e c t e d and the a b s o r b a n c e at 2 8 0 n m was determined. To each fraction 3,6 mg of bovine serum albumin were added followed by triehloroacetic a c i d t o a f i n a l c o n c e n t r a t i o n o f 7%. T h e p e l l e t s w e r e r e s u s p e n d e d i n 0 . 5 m l 0 . 5 M N a O H f o r r a d i o a c t i v i t y d e t e r m i n a t i o n s . T h e b o t t o m o f t h e g r a d i e n t is o n t h e l e f t o f t h e f i g u r e .

10--20 are pooled, concentrated and recentrifuged in a similar gradient only one peak is observed (not shown). If cells are labeled with radioactive iodine prior to incubation with the vesiculating agent, the specific radioactivity (relative to protein) of vesicles (0.08 • 106--2.6 • 106 cpm/mg) is 5--10 times higher than that of total cell homogenate (0.012 • 106--0.26 • 106 cpm/mg). It is interesting to note (Fig. 1) t h a t the radioactivity : protein ratio is higher in the gradient region where contaminating cells band compared to the vesiclerich region. This suggests that vesicles contain a relatively small number of exposed tyrosine and/or histidine residues as compared to intact cells. Based on the recovery of labeled iodine and total protein it is possible to show that t r e a t m e n t of vesiculated cells with Lubrol (final concentration, 0.001--0.01%), vortex mixing (30 s) or passage through a hypodermic needle does n o t increase the yield of vesicle recovery. Vesiculation induced by acid buffers. All acid buffers tested (200 mM, pH 4.0) induce vesiculation. (Table II). The anions acetate, citrate and phtalate (200 mM) were n o t effective at pH 7.0 (Table I). In a typical preparation vesicles obtained from cells treated with acetate (200 mM, pH 4.0) at 37°C for 30 min show the following characteristics: total c a r b o h y d r a t e : p r o t e i n = 1.5--2.0, orcinol : protein = 0.07 and absence of diphenylamine reaction. On the average, vesicles contain 1.2--4.7% of the initially bound iodine and 0.2--0.5% and 0.3--1.0% of the total cell protein and carbohydrate, respectively.

227 T A B L E II I N F L U E N C E O F p H A N D B U F F E R S P E C I E S I N V E S I C U L A T I O N AS M E A S U R E D BY P R O T E I N A N D BOUND IODINE RECOVERY 1.7 • 109 cells w e r e i n c u b a t e d w i t h l a c t o p e r o x i d a s e a n d N a 1 3 1 I ( 0 . 6 2 m C i ) as in M a t e r i a l s a n d M e t h o d s . L a b e l e d cells w e r e w a s h e d a n d a d d e d to a s u s p e n s i o n o f 18 • 1 0 1 0 n o n d a b e l e d cells. A f t e r m i x i n g 3 • 1 0 1 0 ceils, a l i q u o t s w e r e i n c u b a t e d w i t h t h e i n d i c a t e d b u f f e r s at 3 7 ° C f o r 30 rain. V e s i c l e s w e r e i s o l a t e d as in M a t e r i a l s a n d M e t h o d s . O n e a l i q u o t ( c o n t r o l ) w a s i n c u b a t e d w i t h s o d i u m p h o s p h a t e , p H 7.0. In all e x p e r i m e n t s t h e b u f f e r c o n c e n t r a t i o n w a s 2 0 0 m M in t h e p r e s e n c e o f 1 5 0 m M N a C I a n d 1 m M CaC12. P r o t e i n a n d i o d i n e r e c o v e r y w e r e c a l c u l a t e d t a k i n g t h e i n t a c t cell v a l u e s as 100%. Treatment

Protein recovery (%)

Sodium acetate p H 4.0 p H 5.0 p H 5.7 S o d i u m citrate pH 4.0 S o d i u m phtalate pH 4.0 S o d i u m p h o s p h a t e p H 7.0

V e s i c l e - b o u n d 131 I

0.19 0.11 0.05 0.16 0.22 0

cpm

%recovery

12 6 4 11 25

1.22 0.64 0.43 1.05 2.46 0

803 740 510 103 895 0

Factors influencing vesiculation by acetate buffer. ,Vesicle production increases with time up to 30 min upon cell incubation with 200 mM acetate, pH 4.0, as measured by the recovery of vesicle-bound iodine. Examination of this phenomenon under phase contrast microscope showed that vesiculation starts as soon as acetate is added to the cell suspension. Vesiculation also occurs at 4°C but with appreciably lower yields (Fig. 2). Table II also shows the pH dependence of T. cruzi vesiculation. Better yields are always obtained at pH 4.0. Vesiculation depends also on buffer concentration. It does not occur with acetate concentrations below 20 mM. 200 mM acetate is 10 times more effi-

5,0

37°0

×

E2,5

C 1

3'°

.'o

MINUTES Fig. 2. E f f e c t o f the i n c u b a t i o n t~ne on the vesicu]ation induced b y acetate buffer. Cells (109) were incubated w i t h lactoperoxidase and 0.167 m C i o f Na 1311. A f t e r labeling, cells were washed and added to nonl a b e l e d cells. A l i q u o t s o f 2.1 • 1 0 1 0 cells w e r e i n c u b a t e d w i t h 2 0 0 m M a c e t a t e , p H 4 . 0 a t 3 7 ° C ( o ) or at 4 ° C (A) in t h e p r e s e n c e o f 1 5 0 m M NaC1 a n d 1 m M CaC12 f o r t h e i n d i c a t e d t i m e s . I n c u b a t i o n a n d vesicle i s o l a t i o n w e r e as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s .

228 cient than 20 mM acetate. Similar results, however, are obtained with 200 and 500 mM acetate. Electron microscopy. Figs. 3--5 show cells incubated with 200 mM acetate, 150 mM NaC1 and 1 mM CaC12, pH 4.0, for 5 min at 37°C. Vesicles are formed from a plasma membrane evagination where the unit membrane can be easily recognized. In general, the forming vesicle appears as a head-like structure and, occasionally, as a narrow protrusion. Vesiculation is observed in the cell body plasma membrane as well as in the flagellar membrane (Fig. 6). Cell organelles are excluded from the vesicles. Purified vesicles have a homogeneous appearance under the electron microscope with a diameter of approximately 0.5 ttm (Figs. 7 and 8). As can be seen, some of the vesicles are made of several overlapped sheets resulting in a denser appearance. This might reflect a tendency to aggregate which is more pronounced if vesicles are frozen and thawed. After 30 min incubation with acetate a certain degree of cytoplasm vacuolization may be observed. However, internal organelles are easily identifiable as reasonably intact structures. Vacuolization is intense after 60 min incubation. At this time cells loose approximately 30% of their protein. Freeze-fractured vesicles show P and E faces from cleaved membranes; a few smooth membranes (with no particles or with very few particles) were also observed (Fig. 9). Density and diameter of the intramembranous particles are comparable to those found in intact cell membranes [36]. Polyacrylamide gel electrophoresis of the isolated vesicles. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the vesicle preparation reveals the presence of about 10 major protein bands as seen by the Coomassie Blue staining (Fig. 10). When gels are stained with periodic acid-Schiff reagent

m

5

t

!

Figs. 3 - - 5 . P l a s m a m e m b r a n e e v a g i n a t i o n (*) a n d i s o l a t e d vesicle ($). Fig. 3, 28 0 0 0 X ; Figs. 4 a n d 5, 45 000X. Fig. 6. P l a s m a m e m b r a n e e v a g i n a t i o n in a f l a g e l l u m . 4 5 0 0 X .

Figs. ‘I and 8. Vesicle Fig. 8. 50 000X.

pellet

depicting

single membrane

Fig. 9. P, E and smooth (S) faces of freeze-cleaved shadowing direction. 50 000X.

and multilamellated

isolated

vesicle membranes.

vesicles.

Fig. 7, 28 500X;

The arrow

indicates

the

(Fig. 10) the typical pattern for T. crzui glycoproteins appear [37]. The faster component is the lipopeptidophosphoglycan previously described [ 38,391. The component which migrates with and ahead of the marker dye can be stained (not shown) with the Schiff reagent without previous periodate oxidation, suggesting the presence of reactive carbonyl functions in this component. Densitometric analyses revealed that vesicles are 30-50 times enriched in these sugar-containing substances when compared to the cell homogenate. A precise

Fig. 10.

Polyacrylamide gel electrophoresis of isolated acid-Scbiff. The letters refer to sugar-containing was stained with Coomassie Blue. Periodic

vesicles. The gel on the left was stained with substances A, B, C and D. The righthand gel

230 quantitative analysis, however, of the composition of the vesicles with respect to these substances will be published elsewhere. Antibodies. Sera obtained from rabbits previously injected with a vesicle preparation can readily agglutinate suspensions of epimastigote cells up to a dilution of 1 : 512. This result ftLrther shows the identity, at least in part, between plasma membrane and vesicles antigens. Discussion The vesicles obtained from epimastigote forms of T. cruzi upon incubation of the cells with cross-linking reagents or acid buffers are, in all probability, derived from the plasma membrane. This conclusion is drawn from the following experimental results: (1) the specific activity of vesicle-bound radioactive iodine is 5--10 times higher than that found in homogenates prepared from cells previously labeled with ~311 by the lactoperoxidase-catalysed reaction; (2) pictures obtained at the electron microscope from vesiculating cells reveal continuity b e t w e e n cell b o d y (or flagellum) plasma membrane and vesicle membrane; (3) vesicles analyzed by the freeze-fracture technique have a general appearance consistent with the plasma membrane topography; (4) antisera prepared against purified vesicles are able to agglutinate intact living trypanosomes. As in other cellular systems [14,40], vesiculation induced by cross-linking membrane reagents (e.g. aldehydes, N ~ t h y l m a l e i m i d e and p-chloromercuribenzoate) may be due to the binding of these c o m p o u n d s to free sulfhydril or amino groups from plasma membrane proteins. Acid buffers also induce'vesiculation in T. eruzi. The pH, rather than the chemical species, seem to be responsible for cell vesiculation since all anions tested are increasingly uneffective as the pH rises. Vesiculation induced by acetate buffer is also dependent on buffer concentration, time and temperature. As it has been found for other cells [14] the formation of surface blebs and vesicles in T. cruzi is much m o r e e f f e c t i v e at 37°C than at 4°C. E r y t h r o c y t e ghosts are also able to vesiculate at acid pH through an intramembranous aggregation of protein particles, giving as a result protein-depleted vesicles [10,41,42]. That acid-induced T. cruzi vesicles are not protein depleted can be inferred from the sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, taking into consideration that in T. cruzi vesicles the carbohydrate : protein ratio is 1.5--2.0 and glycoproteins are 30--50 times more represented than in the total cell homogenate it is possible to conclude that vesicles are relatively richer in sugar-containing substances [ 37,38] as compared to proteins. This conclusion is apparent if one considers that, in general terms, biological membranes are composed of approximately 10% carbohydrates and 60% proteins [43]. The suggestion can thus be made that either vesiculation takes place at specific membrane regions or that vesiculating compounds increase lateral diffusion of intrinsic membrane macromolecules through the lipid bilayer. Alkaline treatment of sheep erythrocyte ghosts has also been reported to yield glycoprotein-enriched vesicles [ 19]. If one speculates that glycoproteins A, B and C from T. cruzi (Fig. 10 and

231

ref. 37) are transmembrane macromolecules, as was demonstrated for other membrane glycoproteins from several cell systems [44,45], and that they are s o m e h o w associated to the conspicuous net of subpellicular microtubules present in this p r o t o z o o n [46,47], a suggestion can be made that vesiculating agents induce breakage of these bonds, increasing glycoprotein mobility. This in turn, would result in vesicle formation through membrane destabilization. Nicolson [48] has shown that clustering of sialic acid-containing macromolecules is associated with projections or distortions on the surface of the ghost membrane. This hypothesis is reinforced by the well known rigidity of the kinetoplastidae plasma membranes [49], attributed to the existence of underlying microtubules [50], and by recent results on the relatively small mobility of concanavalin A receptors in T. cruzi epimastigotes (De Souza, W., personal communication). Therefore, T. cruzi acetate vesicles might be a convenient material to study particle mobility without possible microtubule interference. Freeze-fracture studies with the acetate vesicles show the great majority of vesicles rich in intramembranous particles, whereas only a minor proportion is represented by smooth vesicles. The latter are most probably derived from the flagellar membrane since it has been reported that these membranes from T. cruzi are almost devoid of intramembranous particles [36]. More work has to be done in order to assess the sidedness of the isolated vesicles. However, the great majority of P faces seen in the freeze-fracture micrographs are convex, suggesting a predominance of right-side-out membranes. T. cruzi membranes prepared by classical methods of cell fractionation contain an enriched activity of adenylyl cyclase [51]. However, it has n o t been possible to demonstrate such activity in vesicles. This can be explained by an inactivation of the enzyme during acetate incubation which also occurs under standard assay conditions. A systematic study of vesicles enzyme and protein composition will be published elsewhere. Finally, it must be said that as the isolated vesicles are antigenic they are a convenient material for immune protection studies during experimental infection. Acknowledgements This research was supported by grants awarded to W. Colli by the Conselho Nacional de Desenvolvimento Cientffico e Tecnol6gico (CNPq-FINEP) and Funda~ao de Amparo fL Pesquisa do Estado de Sao Paulo (Projeto BIOQ/ FAPESP). We are indebted to Dr. M.J.M. Alves for the electrophoresis, to Dr. A. Sesso from the Faculdade de Medicina, USP, w h o kindly allowed us to use the freeze-fracture equipment and the Philips electron microscope, and to Dr. M.C.S. Armelin for helpful suggestions during the preparation of this manuscript. The invaluable technical assistance of Carmen Martins Pinto is greatly acknowledged.

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