Free aminoacid pool and proteolytic enzymes in Trypanosoma cruzi cultured in vitro

0020-7519/83 $3,00 + 0,00 Per&arnfln Pres.~ Ltd. © 1983 Australian Society for ParaxJtolo&V

Isffernetional Jot~rnal f o t ParoMtology I/oL 13, No. 5, pp. 43~,--4~, 1983 Printea in Great Britain.

FREE AMINOACID POOL AND PROTEOLYTIC ENZYMES IN TR YPANOSOMA CR UZI CULTURED IN VITRO JOSE A. O'DALY, LE6N E. SERRANO a n d MARIA B. R.ODRiGUEZ Cemre of Microbiology and Cell Biology, Instituto Venezolaao de lnvestigaciones Cientificas (IVIC), Apartado 1827, Caracas, Venezuela (Received 21 September 1981 ; in revised f o r m 20 December 1982)

Abslracl--O'DALv I. A,, SERRANOL. E. and RODRIGUtZM. g. 1983. Free aminoacid pool and proteolytic enzymes in Trypanosoma cruzi cultured in vitro. International Journal f o r Parasitology 13: 433.440. The intracel|ular concentrations ol"free aminoacids were measured using an aminoacid analyzer. Parasites had a characteristic pattern of free aminoacidsdifferem from that of mammalian cell lines. Glutamic acid, proline, glycine and alanine constituted 75~0 of the pool in 7". cr~zi. Protease activities (active between pH 4.5 and 8) were observed in trypanosomes while in leishmaniae, the activities were low. INDEX KEY WORDS: Trypanosoma cruzi; Protozoa; parasitic; hemoflagellates; proteases; protease inhibitors; free aminoacid pool; aminoacid analysis.

INTRODUCTION THE INTRAQELLtJLARconcentration of aminoacids has been the subject o f investigation in a number o f cell lines derived from human and animal tissues (Piez & Eagle, 1958; Mohri, 1967; Eagle & Piez, 1962; Melancon, Tayco & Nadler, 1972) for the purpose o f better understanding the animoacid and protein metabolism of cells in culture. Previous reports (Williamson & Desowitz, 1961 ) have described the aminoacid constitution of ac'id hydrolyzates of several trypanosomatides as determined by paper chromatography. These same authors also characterized the total free aminoacid content of several trypanosomes. However, the individual free aminoacids were not estimated except for alanine which accounts for about 50o/o o f the total. Several trypanosomatids have been shown to take up and digest exogenous protein molecules in their organelles (Brooker, 1971; Brown, Armstrong & Valentine, 1965; Geigy, Sleiger & Hecker, 1970; Preston, 1969; Steinert & Novikoff 1960; Langreth & Balber, 1975; Bretafia and O'Daly, 1976). Lysosomal enzymes, including acid phosphatase and other hydrolases, have been found in some trypanosomatids (Brooker & Vickerman, 1964; Bongenz & Hungerer, 1978; Avila, Casanova, Avila & Brelafia, 1979). The study o f proteinases has considerable importance in intracel]ular parasites since protein turnover is essFntial to cellular metabolism (Ballard, 1977). In this paper we analyze and compare the free aminoacid pool o f thrv¢ strains of trypanosomes and describe proteolytic enzyme activities at several pH value.

MATERIALS AND METHODS Parasite strains and their cuhivation. Three strains of Trypanosoma cruzh FI, Ma and Y, one strain o f Leishmania donovani and one strain of L. mexicana were cultivated as previously described to study the free aminoacid pools in these parasites (O'Daly, 1975a). As a consequence of the analysis of intracellular free aminoacids, a modified culture medium has been devised (O'Daly, Rodriguez, Garlin, Giar. dina & Zaidenberg, in preparation), in which 7". eruzican be cultured at 26, 30, 34 and 37°C and L. rnexicona and L. donovani at 30°C. Parasites from this recently developed cultured medium were uscci for the proteolytic enzyme assay with radiolabelled Veto cell substrate protein. Preparation o f parasite extracts. Trypanosomes from IO.-day-old cultures and leishmaniae from 5-day-old cultures were obtained as described previously (O'Daly & Aso, 1979). Parasite pellets were resuspended in $ ml of PBS (0.0l phosphate, 0.15 ~ NaCI, pH 7.3) and sonicated for 5 rain at 4°C (Ultrasonics Ins. Model W-185, cell disruptor) under sterile conditions. The parasite suspension was maint ained inside a recirculating water cooler (Savant Instruments) in order to keep the temperature at 4~C. One millilitre aliquots of each parasite extract were subsequently incubated at 37°C for 0 and 24 h. Vero ceils, prepared and cultured as described previou sly (Macpherson & St oker, 1962)were used as control (O'Daly & Aso, 1979). Aminoacid analysis. After incubation, all extracts were deproteinized by precipitation with an equal volume of 10¢/0 (w/v) TCA [trichloroacetic acid) and centrifuged at 1,00(3 I for 30 rain at 4°C. The supernatant fluids were filtered through 0.22 ~m Millipore membranes to avoid contamination with proteins and extracted three times with 5ml of diet hyl et her to remove excess TCA, The fin al aqueous phases we.relyophilized, dissolved in 3ml ofO.2 N sodium citrate pH 2.2 (Beckman buffer for dilulion of samples) and analyzed in

433

434

Josl~ A. O'DP.LY, LE6r~ E. SEr~rt^r~oand M^kl^ B. Rot)r~mu~z

a Multichrome B aminoacid analyzer (Beckman) with the standard technique for acidic and neutral aminoacids (M-8! resin) and basic aminoaeids (M-71 resin). The aminoacids were also separated with the W-3H resin using the Beckman lilhium citrate buffers methodology far aminoacids in physiological fluids. Absolute values for each aminoaeid were expressed in areal/rag of protein originally present at zero time. The Beckman calibration standard for the M-81 and M-71 resins, and this standard supplemented with the aminoacids (all from Sigma) that are resolved by W-3H resin were used as controls. Each value given in the tables is the average of three analyses ±S. D. Jbt-labelting o f substrata proteins. 80 ml of Veto cells prepared and cultured as described (Maepherson & Stoker, 1962) were labelled with 37 × 103 KBq/ml of ~H-leueine (NEN, 4.4 × 105 GBq/mmol) for 3 days. Extracts, prepared as exNained above, were precipitated with an equal voluroe of 10070(w/v) TCA, washed twice with 5070(w/v)TCA and dissolved in 5 ml of 8 ~i urea containing 0.1 ra TrisHCI (Tris(hydroxymethyl) aminomethane), pH 8,2. Subscquently, the protein solution was totally reduced and alkylated with 0.05 M dithiothreilol and iodoaeeti¢ acid as published (O'Daly & Cebra, 1971). After the reaction was completed the protein was desalted in a G-25 Sephadex column equilibrated with 0. I m Tris pH 8.2. The radioactivity of the subs(rate was 153 e.p.m./vg. Radioactive assay for pro(eases. One hundred V~ of labelled subs(rate were tested with 10 ~g of trypsin from bovine pancreas (DPCC treated, from Sigmal in 2 mt of PBS pHg.0at37~CforlS, 30,60,90and 120 rain (Fig. 3). After incubation 500 ul aliquots were mixed with equal volumes of 10070TCA, centrifuged at 20,800g for 10 rnin at 4~C and 250 ul of the supernatant counted in a Packard scintillation spectrometer using 2.5 ml of aquasol (NEN) as scintillation fluid. The optimum pbt of the proteinases from parasite extracts was determined with the following 0.2 ra buffers: phi 2.5, glycine buffer; pbI 4.5, sodium acetate buffer; pH 6, 7 and 8, sodium phosphate buffer and pH 9.0 Tris-HCl buffer: 2.5 ml of reaction mixture, containing 40og/ml o flabelled subst rate protein, 400 ~g/ml of parasite extract and different buffers were incubated for 15, 30, 60, 90 and 120 rain, at different temperatures (see Figs. 3 and 4). At the conclusion of the incubation 500 vl aliquots were preci pirated with 10070TCA centrifuged at 20,800 £ for 10 rain at a°C and 250/al o f the supernatant counted as described above. Theradioactivity found in the supernatant as a result of proteolytic activity is expressed as °7o of the total radioactivity present in the amount of labelled subs(rate used in each experiment, At each pH, the labelled Veto cell subs(rate alone was incubated as control. The pH was measured at the beginning and at the end of the reaction and no variations in theabove mentioned values z'ere observed.

Incubation of perasite extracts withlaroteolytic enzyme inhibitors for aminoacid analysis. PMSF (phenyt methyl sulfonyl fluoride), TKLCK (N-o-P-Tosyl-L-lysine ehloromethyl ketone HCI and TPCK (L-To~ylamide-2phenylethyl chlorometh~l ketone)were dissolvedin PBS containing 2070 (v/v) 2-propanol at final concentrations of 0.02 ~. The inhibitors were dissolved first in propanol, PBS was added slowly, and the pH adjusted to 10 with 50% (w/v) Na0H to dissolve the precipitate which formed with PMSF and TPCK. Subsequently the pH was adjusted slowly to 7.3 with HCI. The solutions were sterilized by filtration with 0.22 vm Millipore membrane. ZPCK (N-CBZ-L-phenyl-alanine chloromethyl ketone) was used at the same concentrations, but in PBS containing 5% (v/v) methanol. Parasite strains were resuspended in the respective inhibitor solutions and in

I,LP. VOL, 13. 1983

PBS as control, sonicatcd, incubated at 37°C for 24 h and subsequently processed for aminoac~d analysis as described above. Aerytamide gels. Parasite extracts, incubated in solutions of the different inhibitors were eleetrophoresed in 10070(w/v) SDS (sodium dodeeyl sulfate, Sigma) aerylamide gels (Laemmli, 1970) with a Biorad vertical slab electrophorcr, is apparatus at 20mA/slab gel. Protein determination. The protein content of each exl raet was determined by the Fotin-phenol method (Lowry, Rosebrough, Farr & Randall, 195IL

RESULTS The concentration o f free aminoacidsin trypanosome extracts as determined with the W - 3 H resin are presented in Table I. The internal aminoaeid pool of each trypanosome strain showed a high (above 30 n m o l / ~ g ) concentration o f glutamic acid, praline, glyeine and alanine, which together composed 75% of the total free aminoacid pool at zero time, other aminoacids being at very low concentration. Precursors like phosphoethanolamine and asparagiJte; intermediates such as phosphosedne, sarcosine, a-aminoadipie acid, citruline and ornithine; oxidation products, such as taurine; aminoaeids found in fibrous tissue such as hydroxylysine and peptide: !:fund in vertebrate muscle tissue such as earnosine, were also detected in T. cruziextracts, The concentration o f all free aminoacids in Vero cells was under 15 n m o l / m g (Table 2). This was contrary to parasite extracts where a group of aminoaeids increased in concentration well over the rest at zero time. When the extracts were incubated at 37°C the free aminoaeids increased markedly in trypanosomes by 24 h. The increase in free aminoacid concentrations with time in T. cruzi extracts (Fig. 1) was ir~hibited by proteolyric enzyme inhibitors for serine-proteinases to the point that free aminoacids values remained close to those obtained at zero time (Fig, 2). These results were corroborated by the acrylamide gel pattern shown in Fig. 3, No protein bands were detected in T. cruzi extracts incubated in PBS for 24h, with the exception o f small peptides running at the bottom of the gel, By contrast, the complete spectrum o f parasite proteins was observed in those extracts containing the inhibitors. Proteolytic activity was also evident with radiolabelled proteins as substrate (Fig. 4). For T'. cruzi, proteolysis was observed at all pH values tested, with optimum liberation o f acid soluble material between pH 4.5 and 8,0. At the pH optimum values, trypanosomal extracts from parasites cultured at 30°Cand incubated at t h e s a m e temperature showed the maximum degree o f proteolysis. Trypanosomai extracts from parasites cultured at 26, 3,t and 37°C incubated at the same temperature showed a lower degree of proteolytie activity, At 37°C trypanosomal extracts exhibited a marked decrease in proteolytic activity (40% or less of the activity at 30 ° C) at all p H values. Similarly, trypanosomal extracts incubated at 37°C from parasites cultured at 30°C showed lower proteolysis than the same extracts incubated at

Ls.p. VOL, 13, 1983

435

Free aminoacids and proteinases in Trypanosoma cruzi

TAaLe 1--FRnE ^MIr~OACIOPOOLIr~ nmol/mg Paor~;t~ ~v STRAINSY, MA AND FL OF Trypanosoma cruzi Y

MA

0 Phosphoserine Taurine Phosphoethanolamine Asparticacid Threonine Serine Asparagine GIutamicaeid Glutamine Sarcosine a-Amino adipicacid Proline Glyeine Alanine Citrullin¢ o-Amino butyric acid Valine Cystine Methionine lsoleucine Leucine T?crosine Phenylatanine Hydroxylysine Ornithin¢ Lysine blistidlne 3- Methil-histidia ; Anserine Carnosine Arginine

5,7 ± 0 27.5± 9.3± 6.7± 12.2-,0 79.8 3= 10.9 ± 0 0 115,1 -*101.1 ± 266.6 ± 2.7± 0 12,6 ± 1,1 ± 4,9 ± 6,1± 16,9± 6.1± 7.9 ± 1.1 ± 0,6± 23.4 • 1.7 ± 0 0 0 27.8 +-

0.9 3.7 1.3 0.9 0.6 1.1 0.l 3.0 1.5 1,1 0.I 0.2 0.1 0.1 0.5 0.2 0.8 0.9 0.7 0.2 1.1 0.1

0.4

FL

24

0

24

0

10.9 :t: 1.5 4.3 _ 1.4 26.5± 3,4 92.7± 4.0 168.0 ±21,1 217.8±19.8 48.5 ± 7.7 287.4_ 7.6 102.5 m 8.6 40.8± 7.1 0 206.1 ± 15.2 292.4.- 5.7 529,0± 24.8 6 . 0 ± 0.4 7.5 +- 0.4 203.2 ~ 5.7 22.1 :t: 1,1 92.0 ± 2.4 142.5± 5,6 214.7±11.1 i10.4± 1.5 139.8 ± 3.1 ~ . 8 ~ 0.6 1 . 6 - 0.5 203.4 ± 2.2 3 . 1 - 0.2 0.8 ± 0.3 0 14.4 ± 2.7 22|.4 +- 1,9

5.2 ± 0.2 0 12.0.- 3,3 5 . 0 ± 0.1 3.7*- 0.3 6 . 1 ± 0.1 0 56.5 ± !.7 6.1 ± 0.2 0 0 55.7 *- 0.6 108.7 ± 2.8 364.6:z 15.8 0.7-* 0.1 0 8.4 ± 0.5 0,~ ± 0,3 2.8 *- 0.2 4,7__. 0,2 9 . 0 ± 0.3 3 . 4 ± 0.2 4.5 ± 0.7 0.5_+ 0.1 0 . I ± 0.4 19.9 -* 2.7 1.8± 0.2 0 0 0 18.7 +_ 0.8

14.4 ± 1,2 4.0 *- 1.3 25.0*_ 4.3 58.1± 1.9 121.5 *- 8.9 190.9_14.7 86.6 ± 5.2 211.! ± 8.7 95.4 ~ 3.6 17.6± 0.4 1.8 ± 0.7 111.8 __. 4.3 285.0.- 20,1 6Q1,8 +_-29.3 3.4-* 1.7 3.1 ± 0.7 196.1 ± t9.7 14.5 ± 4,5 80.8 *- 3..t 118.2--12,9 271.6__. 0.9 96.2-,- 7.5 120.7 ± 9.6 6.3 ± 0.9 0 . 8 ± 0.1 176.4 ___ 13.6 3.1-¢- 0.6 1.4 *- 0.4 0 16.9 ± 2.4 203.7 +_ 21.7

6,5 ± 0 25.2--7.0_+ 5.8*t1.1.0 73.5 ± 9.3 ± 0 0 95,8 ± 86.3 ± 235,5 ~ 3.0± 0 13.3 1,2 5.1 *6.1± 15.5 + 5.7* 8.3 ± 0.7± 0.3± 16.9 _* 2.0± 0 0 0 29,8 •

24 0,1 3.6 0.5 0.,t 0.6 2.0 0.9 1.5 5.0 ~.8

0.2 1.3 0.6 0.4 0,2 0.3 0.2 0.4 0.1 0.t 4.3 0.1

0.8

13.7 -,- 2,1 5.5 ± 0.8 17.0± 4.4 87.9_-. 9.4 178.t ±-15.8 217.1_+22.2 127.5 ± 12.8 290.1 ± 4.9 122.0 ± I2.4 49.1 + 6.7 2.0 ± 0.3 225.4 ± 8.5 334.8 *- 28.6 620.6 ± 47,4

6.t ± 1.38 O 251.5 ± 8.9 16.7 ± 1,9 95.9 - 11,3 149.8__.14 ~ 357.1 ±10.1 121.4± 9.4 151.9 ± 13.4 7 . 0 ± 0.9 1.5± 0.4 258.1 _ 22.1 5.0 + - 0.9 1.3 ± 0.3 0 16.6 ± 2.4 253.0 -,- 13.3

• 0 and 24 at the heed-; , ' i i.~, columns refer to concentrations at zero time and 24 h after incubation at 37°C. At 48 h the concentration values for ,: ,:- aminoacids were slightly higher than at 24 h for all T. cruzistrains, 30°C. The r a d i o l a b d l e d V e t o cell substrate proteins inc u b a t e d alone shoxved a negligible s p o n t a n e o u s hydrolysis at all p H values. Trypsin f r o m bovine p a n creas used as c o n t r o l had a similar profile Io 7". cruzi extracts with 6 0 % release o f radioactivity after 120 rain TABLE 2~FREE AMINOACID POOl. IN N m o l / m g PROTEIi'~ IN VERO CELLS

Lysine Histidine Arginine Aspartlc Threoniue Serine Glutamie Prolin¢ Glyeine Alanine Valirte Methionine Isoleucine Leucine Tyroslne Phenylalanine

12.8 1.8 0.6 5.0 8.0 8.5 11.6 9.3 11.4 12.2 7.9 0.3 3.5 13.1 2.9 4.2

+_ 0.4 + 0.2

± 0.1 _± 0.2 _ 0.6 + 0.1 ± 0.7 ± 0,5 ± 0.2 _+ 0,4 :t 0.3 ± 0.1 ± O. I ± 0.1 ± 0.3 ± 0.1

incubation time, Le/shman/a extracts incubated at 30°C f r o m parasites cultured at the same t e m p e r a t u r e s h o w e d values o f u p to 40°70 release o f radioactivity for L. raexicana a n d up to 2 5 % for L. d o n o v a n i w i t h p H o p t i m a o f 4 . 5 . 6 . 0 a n d 7.0 respectively (Fig. 5). DISCUSSION The present st udy indicates that t r y p a n o s o m e s exhibit a characteristic pattern o f free a m i n o a c i d s which differs f r o m the aminoacid pool previously described for cultivated m a m m a l i a n cells (Eagle, 1959; Pie.z & Eagle, 1958; M o h r i , 1967). In all these cell lines, including V e t o ceils the intracellular c o n c e n t r a t i o n s o f aminoacids at zero time arc generally low, (under 15 n m o l / m g ) a n d a similar pattern is evident in all the tissues. H u m a n skin fibroblasts have a different aminoacid p o o l t o t h o s e d e s c ~ ¢ l for other mammalian ceils (Melancon, Tay¢o & Nadler, 1972). Serine, glutamic acid, proline, glycine, alanine, lcucinc, lysine a n d arginine are the m a j o r c o m p o n e n t s a n d constitute a b o u t 6270 o f the total aminoacid p o o l o f cultivated h u m a n skin fibroblasts. This is in c o n trast with Hela a n d L cells where taurine, glutamine, glutamic acid a n d glycine are the m a j o r constituents o f

Jos~ A. O'DALY. LEONE. SERR^NOand MAat^ B. Rooatou~z

436

i.j.r, voL. 13. 1983

5(3O

4oo

[] ZPCK J TPCK P~SF [] TLCI~

3oo \

E E 2oo

~iEll-I II'E I LEUI TYRI~HE[LYSill/ST[~,RG F~o. I. Free aminoacid concentrations in Y strain of T. crur,i extracts at zero b and 24 h incubation at 37 oC. the aminoacid pool. Trypanosomes differ significantly from the above mentioned cell lines and tissues. Glutamic acid, proline, glycine and alanine constitute about 75% o f the total aminoacid pool in the T. cruzi strains. To interpret the data on the aminoacid distribution in parasite extracts, it must be remembered that the aminoacid concentration appears to represent a steady state and not a temporary imbalance and that metabolic reactions other than membrane transport may affect the pool size in cultures. These could include synthesis, incorporation into protein, degradation of proteins by proleolytic enzymes and several interconversions. Also as a particular feature in these parasites, degradation of foreign protein taken up by trypanosomes (Langreth & Balber. 1975; Bretafla & O'Daly, 1976) should be considered as a possible source of aminoacid for the free aminoacid pool, The nutrient mixture (EAGLE'S MEM) contains threonine, tryptophane, glutamine, valine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, arginine, cysteine and methionine but in concentrations different to that of trypanosome-free intracellular pools. The increase in free arainoacids in parasite extracts and the proteolytic activity, as measured with the labelled substrate, may be due to a sequential action of

several enzymes, endopeptidases and exopeptidases, with different pH optima, present in the extracts and inhibited with the specific enzyme inhibitors used in our experiments. Bongertz and Hungerer (1978) purified an enzyme from T. cruzi~,ith protease (at pH 8.5), esterase and transamidase activity that was inhibited by sulphydryl inhibitors. Also previous studies (Avila etol.. 1979) have reported the existence of cathepsin A (lys~somal carboxypeptidase A) with a pH optimum of 5.2 and cathepsin D (carboxylendopeptidase), pH optimum 3.8 and peptidases I, II and llI with pH optima of 4.6, 5.Sand 5.6respectively. In our st udies wecon firm and extend the existence of a wide range ofproteinasesin both Trypanosoma and Leishmania. Proteinases have been involved in degradation of polypeptides that have no current value in the cell because they have been synthesized with errors in sequence or because they ate inactive fragments or precursors of low tfiol, wt peptides. The breakdown o f such polypeptides provides a pool of aminoacids for the resynthesis of proteins and especially for the rapid adaptive increase in concentration of proteins serving functions newly required in the cell with little net change in the protein content of the cell (Bohley, Kirschke, Langner, Miehe, Rieman, Salama, Schon, Wiederanders & Ansorge, 1979). Ithas been reported that in vitrogrowth of norm al and

Free a m i n o a c i d s a n d p r o t e i n a s e s in

I.;.p. VOL, 13. 1983

Trypanosomacruzi

437

400 O-h

E]y ~FL

'

i

E o

2(3(:

E

. ~ . . ~ . . 3?

LYS!ARG Fro, 2. Free aminoacid concentrations in Y strain o f T. cruzi incubated 24 h at 37°C in the presence of proteolytic enzyme inhibitors at 0.02 ~ concentration,

malignant cells can be stimulated by proteases (Reich, Riftzia & Shaw, 1957). Previous work has demonstrated the need for serum proteins in di~sion and transformation of ttypanosoraes (O'Daly, 1975a, 19"/5b, 1976, 1979). Furthermore, these proteins enter the trypanosom¢ cytoplasm and are concentrated in dense granules (Bretafla & O'Daly, 1976). The proteolyti¢

effect described in this paper may play a fundamental role in the processing of these proteins t o the pep tide level (O'Daly, 19"/9) in order to liberate nutrients and aminoacids needed for division and growth of parasites in vitro. On the other hand, an extract factor that lyses mammalian ceils has been dc~crii:)ed in t h e e p a r ~ t e s (O'Daly & Aso, ! 979) and with L. mexicana part of the

438

Jos¢~A. O'DALY,LIZ6NE. SeRs.~ro and MARIAB. RODItlQUEZ

I.J.P. VOL. 13. 1983

Fro. 3. SDS-acrylamide gels of 7". cruzi extract* incubated 2,*hat 37°C in: (A) PBS and in PBS containing: (B and C) 0.01 M and 0.02 M PMSF; (D and E) 0.01 M and 0.02 u TLCK; (F and G) 0.01 M end 0.02 u TPCK; and (H) 0.01 M ZPCK. All

samples were loaded in tO0 ~1conteamng 400 t~gof protein. lyric activity was found in the aqueous phase of a Folch extraction. This probably indicated that proteolytic enzyme~ present in the extract are responsible for such activity. Also, the proteolyti¢ activity may have an important role in the host-parasite rdationship, sinoe all these organisms are intracellular in the vertebrate host. This proteolysis should be remembered when one deals with antigen purification from parasite extracts for the production of vaccines. Injection of parasites or their fractions will have to take into account the eventual destruction of antigenic proteins as demonstrated with acrylamide gels (Fig. 3) in a short period of time and hence impairment of the ability to elicit a protective immune response against virulent parasites.

Acknowledgement--We thank Dr. Francisco J. Castillo for his help in writing the manuscript and Mrs. Teresa AvendatIo for eacellent secretarial assistance. REFERENCES AVILAJ. L., CASANOVAM. A., AYILAA. & BRETAI~AA. 1979. Acid and neutral hydrolases in Trypanosorna cruzi. Characterization and Assay. Journal o f Protozoology 26: 304-31t. B ~ F. $. 1977. IntraceHular protein degradation. Es~ays in Biochemistry 13: 1-37. Bom.EY P., KmSCHKE H., L~dcONEP, J., MIEttE M., ~ S., SALAtt~Z., SCttONE., Wt~olERmOtm.~B. & ANSORGES. 1979. IntraceIlular protein turnover. In: BlologlcalFunctions of Proteinase$(Edited by Ho ZERH. & Tscmssc~mH.). Springer, Berlin.

BONGERTZ V. & HUNGERERK. D. 1978. Trypanosoma cruzi: Isolation and characterization of a proteas¢. Experimental Parasitology 45:8-18. BRETANAA. & O'DALYJ. A. 1976. Uptake of fetal proteins by Trypanosoma eruzi. Immunofluoreseence and ultrastructural studies. International Journal for Parasitology 6: 379-386. BgOOKER B. E. 1971. The fine structure of Critttidia fasciculata with special reference to theorganelleslnvolved in the irigestion and digestion of protein. Zeitschriftfllr Zellforschung und Mikroscopische Anatomic !16: 532-563. BItOOKERB. E. & VICKERMANK. 1964. Acid phosphatase in trypartosotrtes. Transactions of the Royal Society of Tropical Medicine and Hygiene ~ig:193-194. BI~OWNK. N., ARMSTRONGJ. A. & VALENTINER. C. 1965. f965. The ingestion of protein molecules by blood forms of

Trypano$oma rhodesiense. Experimental Cell Researck 39: 129.135. EAULEH. 1959. Aminoacid metabolism in mammalian cell cultures. Science 130:432-437. EA~tE H. & Pl~z K. H, 1962. The population-dependent requirement by cultured mammalian cells for metabolites which they can synthesize. Journal of F.xlmrimental Medicine 116: 29-43, GEIGY" R., STEIGERR. & -["[ECKERH. 1970. Beitr~tge zur Pinocytose yon Trypanoaoma (Trypano¢oon) brucei, PIimmer & Bradford, 1899.Acta Tropie¢ 27: 271-227. Low'mYO., Rostl~l~otIoltV., FAttl~L. & RANDALLR, 1951. ProtOn measurement with the Folin phenol reagent. Journal of Biological Chemistry 193:265-275. LAEMMLI U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage 1"4. Nafure (London/227: 680-685.

Free aminoaeids and proteinases in Trypanosomacruzi

LJt.P. vox.. 13. 1983 80

T~-

439 -

~3

pH 2.5 6C

40

20 >-

8 ~-

pH 6.0

(..:,

60,

1-),_J 0

40

~

pH 7.0

I--0 p-;7 LU (D "' n" I,I

0

n

pH 8.0

pH 9.0

Z"

6O

40

¢ll~fl"¢

'

L~

20

0 t5 :~1

60

90

120

MINUTES FIG. 4. Protcolytic activity o f T. cruzi extracts with 'H-icucine-labclled Vcro cell protein as substratc. Parasite extracts, obtainedfrom~rypanosomescuiturcdat26~C(A--A ); 30°C( O--O );34~C(D--O)and 37°C (A--A)wercincubatedatthe tern perature o f the origina! culture. P arasite extracts incubated at 37 °C fro m trypanosomes c u !| areal at 30 °C ( 41 ~ • ) Veto cell radiolabcllcd substratc only ( x ~ x} incubated at 30°C, Trypsin control ( l l ~ i ) . The acid soluble radioactivity as result of proteolysis was expressed as percent o f the total radioactivity present in the radiolabelled substrate.

LASGRETHS. G. & BALI~EI~A. E. 1975. Protein uptake and digestion

in

bloodstream

and

culture

forms o f 7"rypanosomabrucei. Journal of Protozoology 22: 40-$3. ~,|ACP~ERSO~ !. & STOKER M. 1962. Polyoma transformation of hamster cell clones - an investigalion of genetic factors affcctln8 cell competence. Virology 16:147-15 I, MEt.ANCON S. B., TAYCO I, & NADLER H. L. 1972. The frec am[noacid pool of cultured human skin fibroblaslS.

Proceedings of the Society of Experimental Biology and Medicine 141: 391-395.

Mourn T. 1967. Effects o f cortisol and t9-nortestosterone on free aminoacid pool and aminoacid uptake o f cultured cells. Endocrinology 81: 454-460. O'D^Lv J. A. 1975a. A new liquid medium for

Trypanosoma (Schizotrypanum) eruzL Journal of Protozoolog2 22: 265-270. O'D^Lx J. A. (1975b. Serum proleins promoting 'H-thymidine uptake by Trypanosoma (Schizotrypan,m) cruzi (Chagas) in vitro. Journal of Protozoology 22: 550-555.

1.1.~,. rot, 13. 1983

osi~ A. O'DAI.¥, LEt"INE. SERR^NO and M^klA B. ROIOI~iOUEZ

440

50~

pH 2.5

2~

>-

t--

0

I

lZ Ld cO rY uJ 13..

I

~

1

pH 7,0

pH 6.0

50 p>-. .J 0 1.4.J t0 Pi

I

25

0 5C

L

I

I

I

I

pH 9,0

pH 8.0

~5 3O

60

9O

20

MINUTES

Fro 5. Proteolytic activity of L. mexicana { A - - A ) and L.donovani (0 - - 0 ) with ~H-leucine labelled Veto ¢¢11protein as substrate. Parasites extracts obtained from cultures at 30°C were incubated at the same temperature of culture. Percent proteolysis expressed as in Fig, 4.

O'DAtv J. A. 1976. Effect of fetal calf serum fractions and proteins on division and transformation of T. cruziin vitro. Journal of Protozoology 23: 577-583. O'DALY J. A. 1979. Molecular biology of 7". cruzi, L. mexicana and L. donovanL In: The in vitro Cultivation of the Pathogens o f Tropical Diseases, chaplet 29, pp, 237-243. Tropical Diseases Research Series 3. Schwabe, Basel. O'DAI.Y J. A. & Aso P. M. 1979. Trypanosorna cruzi, Leishmania donovaniand L. mexicana: Extract factor that lyses mammalian cells, Experimental Parasitology 4"/: 222-231. O'DAtv J. A. & Czura J. J. 1971. Rabbit secretory lgA, Chemical and physicochemical studies of its component polypeptide chains. Biochemistry 10: 4154-4164. PIEz K. & EAGLE H. 1958. The free aminoacid pool of cultured human cells. JournalofBiological Chemistry 231: 533-545.

PKESTON T. M. 1969. The form and function of the cytostomccytopharynx of the culture forms of the elasmobranch haemoflagellat¢ Trypanosoma raiae Laveran & Mesnil. Journal of Protozoology 16: 320-333. R~ICH E,, RIFrzla D. B. & SHaw E. 1975. Proteases and biological control, In: Cold Spring Harbor Conferences on Cell Proliferation, Vol. 2. Cold Spring Harbor Laboratory, New York. STEIblERT M. • NOVIKO/V A. B. ].960. The existence of a cytostom¢ and the occurrence of pinocylosis in Ihe trypanosome Trypanosoma mega. Journal of Biophysical and Biochemical Cytology 8:563 -569. WfLLIAMSO.~ J. & DESOWlTZ R. S. 196]. The chemical composition of trypanosomes I. Protein, aminoa¢id and sugar analysis, Experimental Parasilology I I: 161-175.