Molecular and Biochemical }'arasitology.

1 ( 1980) 2 ~9- 289 279

© Elsevier/North-Ho!la~dBiomedical Press

CHARACTERISTIC PROTEINS OF MICRONEMES AND DENSE GIL~,NULES FROM

S A R C O C Y S T I ~ T E N E L L A ZOITES (PROTOZOA, COCCIDIA)

JEAN-FRANCOISDUBREMETZa and CLAUDE DISSOUS~

Laboratoire de Biologic Animale and ~ Laboraroire de 6~imie Biologique III, Uni~ersit~ des Sciences
et Techniques de Lille, 59655 Vffleneuve d'.4sctl Cedex, France
(Received 26 February 1980; accepted 9 April 1980)

A subcellular fractionation procedure has been developed, which allowed the recovery of purified
fractions of microne~'aes,dense granules and pellicles from Sarcoc),stis tenella zoites. As expected,
~ u m dodecyl sulpltate polyactyl~mide gels eletrophoresis of the pellicles showed a fairly hetero-
genous prot~ha content, in contrasL or~l~~ one major component (42 000 daltons) was found in the
dense g~anules, whereas two major bands (20 000 and 22 000 daltons) were observed for the micro-
heroes. The~e characteristic proteins ~ r ¢ al~3 major components of the whole zoite and might have
important functions i~ the physiology of the or?~ani~n.

Keywords: Sarcocysta~tenella, Coeeidia, Sub~llu!ar fractionation, Mieronemes,Pellicles.

INTRODUCTION

Coccidia are intra~llular parasitic Protozoa responsible for a number of diseases of

man or animals (coccidiosis~ malaria, sarcosporidiosis, toxoplasmosis), Invasion of the
host cell is accomplished by a motile, ~rmiform stage: the zoite, which has a ¢harac-
teristic organJ~tion, remarkably uniform within the group [ 1],
Among other features highly ~pecific to this invasive cell, these o ~ , l i s m s possess two
sets o f enigmatic apical organdies which have been described as rhoptfies and micro-
heroes on the basis o f morphological electron micros~pic criteria (in some organisms, a
third type occurs: the dense granules), All these~are more or less elongated membrane
born"Ideal bags ruled with electron dense material. Their origLn during zoite genesis has not
been ¢leariy ~ a t a b ! ~ , Their functions remain unknown although some results hare
suggested that r h o p ~ s might excrete ~ r ~ factor n~ediati~ the ho~.celi invasion [2].
The bioehemie~ demonstration o f such an exoey-t~sis, as well as the elucidation of
the function o f these organelles can be ~btained Pray after their isolation and the charac-
terization o f their contents. Ree~nt rest lts on a urtique histidine rich prote~a from dense

Abbr~:ci~tions:PBS, ~ l ~ t e ~ b u f f ~ r e d ~t~ne; SOS, ~d~um d~ecy~ ~iph~te; '~rA, trichlo~,acetic

a¢~l,
 280

granules of Plasmodium lophurae [3. 4] or on a 'pcneilatio~ c~thancg~ fact~.~r" fr,'~m

Toxoplasma gondii [ 5 - 7 ] sug,~est that proteins might be invtfl~ed in par::site.- ht~st~¢ell
interactions~ either by enzymatic or ionic properties. Theretbre, a [~).~ible ~ay to eluci-
date the functions of the apical organdies could be through the d~aracteri~ztion of their
prote~n co,tents,
The intraccllular nature ofCoccidia o~ten make~ the purificatioa ~f la:ge anjou.ms ~f
¢eUs difficult, The best organism t~r suetl a project appeared Io be the c,ystic zoile of
Sarcocystis teneila. The morphology of this species is well known [ 8 . I~;)], Mth~ugh its
complete life cycle and its true coccidian nature were only recently established II t].
These zoites have rhoptries, dense granules and a large number of micronemes (Fig. I);
they can be obtained in rather large amounts from parasitized sheep oesophagus and their
purification is very easy [12].
The results presented in this paper are concerned widl subcdlut~" ftactk~nation of
S. tenella cystic zoites and analysis of the protein contents of purified f?action~ of mkro-
heroes, dense granules and pellicles from these zoites.

]
chondrion; m, microneme; N, nu¢|eils; R~ dtoptry). (a) Ge~nera~~i~w ~howing the distribution and
relative number of the organelles (× 8 100), (b) Enlarged are~ in the middle pa~t of the cel~showing
rhoptries, mictortemes and dense ~anules (X 37 800), (Bars: 1 tam).
 281

MA'tt(RIALSAND MF'I'ItODS

Materials

X amdta inf?~ted sMep oesopl~agi were obtahwd fi~m~ a local slaughtertumse, The
macroscopic cysts were excised ~om the ~lscl¢ tixs~le a~d kept in pho~phate-buft)Nd
s-aline (PBS~ pH 7,4 I131)snppk,,~ented with 50 U penicillin and 50/~'g streptomycin w r
ml at 4°C until u~d (within t or 2 wk).
Previous obsercatkms have demonstrated that trypsin treatment triggered the trans-
~brmation of the resting zoite m the motile, invasive tbnn [12]. Such a treatment was
found ve~, helpful in di~esting most of the cyst waUs at~.d non-~,oite material and was
therel\~re used routinely, Cysts were broken by repeated piF~t:ing and then digested with
0.1% (w/v) trypsin (I :250, DIFCO)in PBS for t0 rain at 37~C. Remaining cyst hulls were
rentoved by t'dtvatioa through a double layer of smgieal gauze.
Zoites were recovered by low speed centrifugation (500 × g for 5 rain) and washed
2,~3 times with PBS. They were finally resuspended into the homogenization medium
('250 mM s~uemse, i mM EDTA, 5 mM triethanotamine-ttCl, pH 7 5) at a concentration
of 10~ cel|s[n'd, All subsequent operat,ons weic t~rionned at 4~C,

Call dtmtptkm

Numerous attempts were necessa~' ~o find optimal conditions of cell breakage. Indeed,
a few cells were disrupted when using Dt~unce, Potter or Virtis komogenizers, whereas
grinding of frozen cells i~ a mortar ied to extensive dan~age to subcellular organe!les. In
some experinaents, soni.:ation yielded fairly good results, but the method could m,t be
staMardized,
The most satisfacto D, results were obtained using a French Pressure Cell (Aminco,
Silver Spring, MD) operated a~, 50 kg/em:. Unbroken cells (1 -5":, of the initial suspens,
ion) were sedimented by a 5 mi~ centfifugation at 500 × g, The supernatant was sub-
seq~ntly referred to as homogenate.

Subcctl¢d~rfi~vlion~m

The fraetionation procedtar¢ is dtustrated in Figure 2. AU sucrose solutions contained

t m M EDTA and 5 raM trieth,,amlamine-HCl, pH 7 5 . The centriNgations were perforat-
ed on a Beckman L 65-B ultracentrifuge,
The homogenate was centrifaged at 12 000 X gT~,a, for 10 rain in a SW27 rotor. Micro-
nemes, micro~mal vesicles, rib-:~somes and a few mnylopectm granules were left in the
supernatant, whereas electrot~ dense gramfies, n~tochondria mid pellicles were recovered
as a pellet, together with part~- emptied cells. Purification of oNanelles was then accom-
plished by sucrose densiLv gradieat centfifugations, as described below.
The 120 000 × gmax X rain pellet (the notation gin= × rain represents the integrated
282

(O.25m)
~,mqdc!
f~m)
(L.4M) " I

I-IIH tilt It ~t"

" * ( ~,.,dit..t bI
) ...........
~. lllit I OIICIIIC~

~)J = --__ dt'lP,t' gl',lllillt=,

........ ~ j
~'~(o :- an~)h~l~Ctlingl,illlllt'~
A B
Fig. 2. Diagrammatic representation of the t,abes before (brackets) and after ~crose gradient centrifu.
gation of: (A) the r~uspended 120 000 × gmax × rain pellet, (B) the 120 000 × gmax X rain super-
natant. (*, interface where intact zoites equilibrate when not removed from the homogenate).
centrifugal force) was transferred into a Dounee homogenizer and resuspended in the
homogenization medium (1 ml for 109 cell equivalents) by five up and down strokes of
a loose pestle. The yield of clean ghosts was substantially increased by this treatment, but
care had to be taken to avoid disruption of dense granules that might occur concomitant.
ly. The suspension was adjusted to 1.6 M sucrose with 2,2 M sucrose, and layered into a
discontinuous sucrose gradient (2 2 M : 4 ml, 1.8 M : 10 ml, 1.6 M : 5 ml rumple, 1.4 M
: 5 ml, 1 M : 10 ml, 0.25 M : 2 ml) which was centrifuged for 1 hat 113 000 × gmax in the
SW27 rotor. The material present at each interface was collected and diluted with the szme
sucrose solution as the overhying cushion. The particles in each fraction were ff,en sedi.
mented by eentrifugation for 1 h at 113 000 × gmsx ( ~ 4 0 rotor). The pellets were then
either frozen or immediately processed for dectron microscopy or electrophoretie analy-
sis. Pure frdetiorm of pellides were obtained from the 0 2 5 - 1 M sueroseinterface,whereas
dense granules were removed from the 1~ - 2 2. M sucrose interface (see the Results section).
Micronemes were purified from the 120 000 X gram X rain supernatant. Eight-ml
portions of the latter were layered onto 30-ml Linear 1~1.4 M sucrose gradients and
centrifuged at 72 000 X grn~ for 30 r~dn in the SW27 rotor.The upper 7£~ ml (containing
the bulk of ribosomes) was then carefully removed and replaced by the same volume of
homogenization medium. The gradients were centrifuged for an additional 3 h at 113 000
X g n u , then fractionated with an ISCO gradient fractionator equipped with a UV cell
(Instrtmaents Specialties, Co, Litl~ln, NE). Tire mieroneme fraction was diluted w~th 1
vol. of homogenization medium and sedimenwt for 45 rain in a SW27 rotor at 72 000
×gnu"

Electron raic~scopy

Subcellular i~ractionswe:e fixed in t% (v/v)#utara~dd~yde in ho,nogeniza~}on medium

for 2 h at 4°C. Pellets were wa~cd overnight in the nwdium~ rinsed in 0~2 M ~)di~m
cacodylat¢, pH 7.2 and post-fLxed in 1% (w/v) osmium tetroxide in the same buffer for
2 h, Dehydration was performed through graded alcohols followed by p r o # e n e oxide.
Pellets were the~ embedded in Epon. Uitvathin sections across the whole thickness of the
 283

pellets were taken from central and peripheral zones. They were contrasted with uranyl
acetate and lead citrate and observed with a Hitaclfi tlU 11 E electron microscope.

SDS polyacrylamide gel electrophoresis

Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis was performed ac-
eordialg to Laenunli [14]. The separation gel (16 × 12 × i).l cm) was composed of a
continuous I0-18% (w/v) aeo, .lmaide gradient. The acrylamide concentration in the 2
cm high stacking gel was 5%. Samples were dissolved in Tris-HCl 62.5 mM, pH 6.8, 2%
SDS, 5%/~-mercaptoethanol, t0% sucrose, 9.2% bromophenol blue and heated at 100°C
for 3 rain. Electrophoresis was rhea carried out at 4 mA for 18 h. Gels were fixed for 2 h
in 50% triehloroacetic acid (TCA), stained :~or 8 min in 0.2% Coomasfie Blue R in 50%
TCA, de-stained in 7% acetic acid and subsequently dried under vacuum after a wash in
distilled water. Molecular weight markers (phosphorylase B: 94000, bovw,e serum
albumin: 67000, ovalbtunin: 47000, carbonic anhydrase: 30000, trypsin inhibitor:
20 100, lactalbumin: 14 400) were processed along with the saa~ples.

RESULTS

Aforphological analysis of the firactions

The procedure described in, the Materials and Method.~ section 'allowed simultaneous
recovery of purified pelticles, dense granules and micmnemes. Because no enzymatic
assay was available to test the purity of sporozoan organdies, electron microscopy was
the only way of evaluating the fractionation procedure.
Pellicles were found among the rapidly sedimented material and floated to the 0.25 -1
M interface when subjected to gradient purification. Light microscopic observations (data
not shown) revealed that many fragments h~d kept the shape of the organisms, especially
the pointed anterior part. Electron microscopy demonstrated the preservation of :he
three-membrane structure typical of sporozoan zo~tes (Fig. 3b). The subpellicular micro-
tubules were ~3t'te~tretaia~ed in situ (Fig. 3a) as welt as the c~moid (net illustrated). Single
membrane profdes originating from damaged pel|ictes and/or from iatracytoplasmic
membranes were also ~_~undan~ng the ghosts.
E~ctron dense granules were recovered at the 1.8--22 M suero~ interface, well
separated from the other rapid~" sedhnent~at~ components, namely pellicles, partly
emptied cells, mitoehondria, amyk~pectin granges. Figure 4 shrews that this fraction was
composed of more or ie~ intact me~brane botmded granules, up to 0.6 tam in diam,~ter.
As already outlined in the Materials and Me~hods ~ection. they seemed to be rather
sensitive to shearing forees~ Whereas intact granules kept their in situ morphology, the
broken ones had a sponge tike appearaw_~e,the eontent~ being test from the center to the
periphery. The main ~xmta~ainants of the fraction were a few amylopectin granules.
Micronemes were fou~d in t!~e 120 000 X gn~ X mill superuatant, They were sepa-
284

Fig. 3. Electron micrographs of the pelticte ftacliom (a) Ge~erat view f~rtows point to s~bpelt~;u',a~
microtubules; x 10 800) (Bar: 1 /am~. (b) I!nlarg~darea sh~wing tl~e ?~c~e~va~,~nel" the l}~:eom,~'~v.
brane structure (P. plasmalemma: C. ironermembra;w ct~rnplcx:x | 12 500} ~l?~a,~:O I ~m!.

rated from the microsomal membraae and ribosomes by ce,urifl, gation lhrough li~ear su-
crose gradient. As shown in Figure 5 they were small (50 × 250 rim) membrane bounded
bags filled with homogenous dense materiM. No difference was fotmd betweea ti~e micm~
nemes i:r~the zoite and in the purified fraction (compare Figs. t a~d 5), No ~Nniiqcant
contm~zinants were found in that fraction.

SDS poO~a'crytami&,gel decm~phoresis of ehe purifi~d fr~¢t,ions

Eleetropboresis of file pel!ide fraction revealed about 20 p~'¢dominam pr~-~tea~c~nder

90 fKk3dattons (Fig. 6c)~
Co~ttrasting with tltis complex p a t t e r ~ o~3y two m~or prot,~'h~s(20 ~ 0 and 22 (}00
daltons, respectively) of equal aaini~g int~:Y~i~ywere detected upo~l electrop~,oresk~ of
a purified fraction of micronemes ( F ~ 6b). A few minor bands were also ~prt~ducibly
detected in the fraction (30 000, 32 000, 96 000. ~arger ~i~;m 100 ~30 da|to~s). The two
major polypeptide chains were not linked together by dis~tfide boads si~ce :::heomissio~
of the reducing agent from the sample buffer did not mod~f) t}~c electr~.~phe~-eti.cpattern,
Eleetrophon~sis of the dense gat~ule fraction demo~strated o~c mNc, r prote~'~ of
approx.. 42 000 daltons (Fig, 6e). Another COl~lp~nellt t~f slightly lowe~ l ~ t : c t k~r weigkt
was also presem, often kidden by the lane band of the mNor comlo~onem (Fig. :tit3.
Minor proteins of 32 000,33 000, 34 000.40 0OO and 78 000 dailens were ag~ detecled~
 2S5

Fig 4~ Fl~:ct,or, m~et~}grap~s,~f the der~aegranule t:?ra~fi~n, gt)Gerte~al view (X 10 800). (b) Detail (a.
;~rr'~y|owctien gram~le;D, ~en~ graaule: x .';4 0001(Ba~: 1 .urn)-

DIS('t SSION

The presem paper is the l}rst report o,~ the is.eta{ion m~d electwpho.vetic characteriza-
lion o f the speci*'ic s~b~eth, lar o ~ m e i l e s o f st.x~rozoar~, zoites. The main ,:esult of this
work was to give a distinct ide~tiD ~(morpholo~'. sedimer, tation behavior, protein con-
te~ts) to organdies which w e ~ oniy kt}ow~;: from electroa microscopica~ observatious
of fixed cctls.
'l_'he aatme ~-,f'micr~w~e~ of &~-c~<vsl~ s~'cies had long been c, mtroversial since
286

Fig, 5. t~tectron micmgraplls of lhe microneme fraction. (a) General view (× 10 ~;00) (B~r: t ;~m), (b)
Detail (X 90 000) (Bat: 0, I ~m)
 287

Q b c

V
cl e
q

2-.- 1--

2--

_..
= 3~ ,~ID
f

,..,
4--,,

_,
6--

.,

Fig. 6. SDS~PAGEof the fractions rml in par:dlci with intact v~ites. Arrows point to tx- the location of
molecular weight markers in the same experiment ( h 94 000: 2, 67 000; 3, 43 000; 4, 30 000; 5.
20 100~ 6, 14 400). a, intact zoites (60 ,ag proteins de.pos,.'ted);b, micconemes (7 vg); c, pelhcles (50
ug): d, intact zoites (150 ~g); c. dense ~anules (19 ~g): t', enlargement of the major band of det:se
granules with a .~naUersample deposited {3 ~g): two pro~e!nsare detected.

their first description as 'tubules contourn~s' (coavohited tubule 0 [8]. Their isolation ~,s
short closed bags definitiveIy establhhed thcir individuality and ailed out the hypothesis
o f a system o f canaliculi joining together rhoptries :rod micronemes [ l 5 ].
The dense granules of $,~rcoo, stis zoites did not receive much attention from cytolo-
gists and were describtxt as "probabb~ litx~p~oteinic~ [8]. We have once observed one such
granule being exocytosed i~ tile host-cell v:icuote after the invasion [16]. Their isolation
as spherical granules clearly distinguish~ tllem from the rhoptries wi::h which they were
sometimes confiJ,~ed, They bear ~m~e morphologi&d resemblaltce to and have a sedimcn.
288

tation behavior comparable with P. tophuree dense granules [3]; however, t'~e iatter do
not belong to an invasive stage.
Rho ~ries were not recovered in tlds work possibly because of tlleir very small number
(squash preparations have shown ealy two of them per zoite [1 I])~ because they anchor
in the conoid area and persist within partly emptied cells, or because of tile meci~anical
fragility of their extremely long peduncle which might be responsible for tile leakage of
their contents. We favour the first possibility because prelimina~• experimet~ts with
Eimeria nieschutzi spomzoites which contain a large number of these organelles have
allowed their recov¢~ in significant amounts [17].
The procedure described here allowed the purification of zoite peliicles. Similar to
the results obtained with trophozoites of Gregarines [18], tile thr<'e~a~embrane structure
was prese~ed, which demonstrated the cohesion between the ptasmalenuna and the inner
complex. An important difference has been found between the buoyant densities of the
ghosts: S. tenella pelli¢les are much lighter in sucrose than those of Gregarina blah, rae
(less than 1.13 g/cm 3 vs. more than 1.20 g/cm3). A possible explanation for this might
be the ceU coat which is much more de.loped on the latter. Tttese preliminary observa~
tions reed to be further developed towards the elucidation of the functions of this very
peculiar cell wall. Having detected more than 20 predominant proteins in our fraction is
not surprising (G. blaberae has about 30 [18D. Future investigations will have to locate
these components more precisely and to fred their respective roles.
Most interesting is the very small number of major protein species present in each of
the analyzed organetles. Simi!arly, P. lophurae dense granules have t~w components
[3]. The duality of the major polypeptide componem of micronemes is rather pu:~zling.
They are not linked together by disulfide bond~ and might thus come from different
organeUes or even from different cells.
S. tenella micronemes and dense granules thus seem to be bags containing a l~:fited
number of components. They are then likely to assume a limited nmnber of functiom,
The basic question concerning these organelles is to know whet~er they act witlfin or
outside the zoite, i.e. to determine whether they a~ or are not exocytosed. Whereas
Jensen and Edgar had 'strong evidence' for secretion of pro~aucts from rhoptdes during
Eimeria sporozoite invasion of the host-cell [2], the matter is far from clear with respect
to the other organellcs. In fact, the only way to demonstrate such an exocytosis would be
to detect the products outside of the zoite at a given moment of the interaction with the
hos~-ceU. The present characterization of ~he contents of micronemes and dense granules
will make such an investigation possible. It wiU also allow the biochemical analysis of
these contents, which, as illustrated for the P. lophurae prote:in [3, 4]. can yield infon'na.
tion on a pos.sibie physiological role.
Last, the ~aracterization of these orgaae!les in one species of zoites should be extend-
¢d to other species to determine if morphologically similar structures are functionally
identical
 289

ACKNOWLEDGEMEN TS

This work was supported by Centre National de la Recherche Scientifique (E.R,A,

184), The authors t h a n k Elisabeth Ferreira for expert technical assistance.

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