257
J. PROTOZOOL. 2 2 ( 2 ) , 257-259 (1975).
The Immobilization Antigen of Paramecium aurelia is a Single Polypeptide Chain HELEN G. HANSMAt
Department of Biological Sciences, University
of
California, Santa Barbara, California 93106
SYNOPSIS. The immobilization antigen (i-antigen) fraction of Paramecium aurelia syngen 4 is shown to contain a protease that is activated by mercaptoethanol. After the protease has been heat-inactivated, the molecular weight of the i-antigen (250,000 daltons) cannot be decreased by mercaptoethanol treatment. I t is demonstrated that the i-antigen is a single polypeptide chain. Reasons are also given why low molecular weight subunits were previously reported by other authors.
Index Key Words: Paramecium aurelia syngen 4; immobilization antigen; single polypeptide chain.
T HE
outer surface of Paramecium and other ciliates is covered with large amounts of a protein called the immobilization antigen (i-antigen). In Paramecium, this protein constitutes 30% of the total ciliary protein, which is sufficient to cover the cilia by a layer 20-30 nm thick (10). The function of this major protein is unknown, but it appears to be indispensable. Alterations in temperature, salt concentration, food source, and other environmental conditions cause a cell to produce a new type of chemically distinct i-antigen. The i-antigen derives its name from the fact that antibodies against this particular protein immobilize Paramecium. A cell normally produces only 1 chemically distinct type of i-antigen a t a time; this is called its “serotype.” T h e regulation of the production of this protein is of great biologic interest (10, 19). T h e i-antigens of Paramecium have molecular weights of 250,000 to 310,000 daltons. For the past decade, there has been a controversy about whether these proteins are single polypeptide chains or are composed of smaller subunits. I t has been reported that the size of the i-antigen decreases after reduction with mercaptoethanol (6, 16, 20). With this procedure, 1 group (20) obtained subunits with a molecular weight of 34,000, while another ( 6 ) found that the subunit molecular weight varied from 19,000 to 80,000 among samples. A few years later, Reisner et al. ( 1 1-13) made extensive measurements of the physical parameters of i-antigens. They reported (12,) that there was no decrease in the molecular weight upon treatment with mercaptoethanol and that the i-antigen was the largest known polypeptide chain. This controversy is resolved in the present paper. Much recent biochemical and genetic data has been interpreted on the basis of the “subunit model” (5, 10, 14-16, 19) although no one has proved that this model is correct. T h e results reported here cast serious doubt on this model.
-
MATERIALS AND METHODS Cells.-Paramecium aurelia of syngen (species) 4 were cultured in Cerophyl medium inoculated with Enterobacter aerogenes, following the methods of Sonneborn (18). Wild type stock 51s (non-kappa bearing wild type) was used. The serotypes of live cells were determined from the degree of immobilization of cells incubated for 2 hr at 28 C with various +t This investigation was supported by Research Grants GM 19406, from NIH, U. S. Public Health Service, and GB 32164X, from NSF, to C. Kung. t I thank Dr. Ching Kung for his helpful suggestions, support of this research, and careful reading of this manuscript. Thanks are also due Dr. John Preer for his advice on the immobilization antigens and Dr. T. M. Sonneborn for giving us the antisera.
dilutions of antisera (8, 17). The rabbit antisera to Paramecium of serotypes A, B, C, and D were kindly supplied by Prof. T. M. Sonneborn. Paramecia all change to serotype A when grown at 32 C and to serotype B when grown a t 17 C (10). Cells were harvested by centrifugation for 3 min at 500 g in an oil testing centrifuge ( I E C Model HN-S) and washed in Dryl’s salt solution ( 3 ) . Isolation of Immobilization Antigens.-I-antigens were purified by the method of Preer (9). Cells of known serotype were extracted in salt-ethanol solution [0.45% (w/v) NaCl, 5 mM Na phosphate, 15% (v/v) ethanol, p H 7.01. This extract was fractionated by acid precipitation at p H 2.0; the supernatant fluid was adjusted to p H 7.0 and further fractioned by (NH4)Z SO, precipitation. I-antigens A and B were precipitated in the (NH,),SO, fraction of 35% to 61% saturation (23 C ) . T h e precipitate was redissolved and dialyzed against a solution of 1 mM Tris-0.1 mM EDTA, p H 7.0. The dialyzed i-antigens were stored frozen. Protein was measured by a modified Folin phenol method (7). Electrophoresis.-Samples were incubated for 1-2 h r at room temperature with 1% (w/v) SDS and 1% (v/v) mercaptoethanol in Tris-acetate buffer, p H 7.4 ( 4 ) . Polyacrylamide gels were prepared by Fairbanks’ method ( 4 ) with 0.1% SDS. Electrophoresis was carried out for 2-2% hr a t 6 mA/tube. Gels were stained with Coomassie blue ( 4 ) . Molecular weights ( M W ) were calculated from the standard curve of peptides of known molecular weight. RNA polymerase from Escherichia coli was used as a standard peptide mixture. I t was purified by the procedure of Burgess & Travers ( 1 ) by G. Bitter and was a gift from Dr. D. Kohl. Some i-antigen samples were reacted with iodoacetic acid under the following range of conditions: 0.15-0.4 M iodoacetic acid, p H 8-9, 10 min or more at room temperature. Some samples were reduced with mercaptoethanol (- 20 mM) before treatment with iodoacetic acid. SDS (1%) and mercaptoethanol (1%) were added after iodoacetate treatment. Reagents.-Sodium dodecyl sulfate (SDS) and polyacrylamide gel reagents were Electrophoresis Purity Grade from Bio-Rad. RESULTS I-antigen isolated by the procedure described above was preincubated in buffer containing 1% SDS-l% mercaptoethanol and electrophoresed on SDS-polyacrylamide gels. The result is shown in Fig. la. There is only a broad band of less than 15,000 daltons. Since this pattern suggested proteolytic degradation, the i-antigen was boiled before preincubation. Under these conditions, a single band of 200,000-300,000 daltons was obtained (Fig. l b ) , although sufficient mercaptoethanol was present to reduce disulfide-bridged subunits.
258
IMMOBILIZATION ANTIGENSOF Paramecium cluded that the 250,000-dalton i-antigen contains smaller subunits that are joined by disulfide bridges. We have shown, however, that no subunits are produced upon reduction of the i-antigen after the protease has been inactivated; i.e. the 250,000-dalton i-antigen is a single polypeptide chain. The protease activity could be present either in a lower MW contaminant or in the i-antigen molecule itself. The former alternative seems more likely, since Macindoe & Reisner (8) purified their (NH,) $04-precipitated i-antigen on a DEAE cellulose column and found that mercaptoethanol did not cause degradation of the i-antigen. It was previously reported that the i-antigen has small subunits (6, 16, 20). Steers’ purification procedure was identical to ours and to that of Preer ( 9 ) , but Jones ( 6 ) and Sommerville (16) further purified the (NH,) 2S04-precipitated i-antigen on a SE-Sephadex column. They also used P . aurelia syngen 1 instead of syngen 4, but the i-antigens of these 2 species are quite similar in molecular weight and amino acid composition. The reproducibility of subunits in syngen 1 i-antigens was poor. One author reported a subunit MW of 16,000-80,000 from sedimentation equilibrium measurements of reduced or reduced carboxymethylated i-antigens (6). According to another, a variable pattern of 3 to 5 bands was obtained by acrylamide disc gel electrophoresis of i-antigens treated with mercaptoethanol ( 16). Thus it is possible that the i-antigen preparations from syngen 1 also had protease activity. Research on the biosynthesis of antigens and the regulation of serotypes is currently being pursued in many laboratories with at least 3 syngens of Paramecium aurelia (2, 5, 14, 15, 19). For this research, it is essential to know whether the i-antigens have subunits. It has been shown in this report that the i-antigens of 300,000 daltons, syngen 4 are single polypeptide chains of being thus among the largest polypeptide chains, as was stated by Reisner et al. (11, 12). The previously reported subunits presumably were artifacts caused by proteolytic degradation.
-
Fig. 1. SDS-polyacrylamide gel electrophoresis of Paramecium i-antigens. a. I-antigen incubated with 1% SDS and 1% mercaptoethanol (ME). b. Boiled i-antigen incubated with SDS ME. c. Standard peptide mixture (E. coli RNA polymerase) inME. d. Standard peptide mixture cubated with SDS i-antigen (not boiled), incubated with SDS ME. e. I-antigen, preincubated 10 min with 0.4 M iodoacetamide, then incubated ME. with SDS
+
+
+
+
+
REFERENCES 1. Burgess RR, Travers AA. 1971. DNA-Dependent RNA Polymerase (EC 2.7.7.6) in Cantoni GL, Davies DR, eds., Procedures in Nucleic Acid Research, Harper & Row, New York 2, 851-63. 2. Capdeville Y. 1971. Allelic modulation in Paramecium aurelia heterozygotes. Molec. Gen. Genetics 112, 306-16. 3. Dry1 S. 1959. Antigenic transformation in Paramecium aurelia after homologous antiserum treatment during autoaamv and conjugation. J. &otozool. 6 (Suppl.), 25. 4. Fairbanks G. Steck TL, Wallach DFH. 1971. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10,2606-17. 5. Finger I, Onorato F, Heller C, Wilcox HB, 111. 1966. Biosynthesis and structure of Paramecium hybrid antigen. J . Mol. Biol. 17,86-100. 6. Jones IG. 1965. Studies on the characterization and structure of the immobilization antigens of Paramecium aurelia. Biochem. I . 96,17-23. 7. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-75. 8. Macindoe H, Reisner AH. 1967. Adsorption titration as a specific semi-quantitative assay for soluble and bound Paramecium serotypic antigen. Austral. 1. Biol. Sci. 20, 141-52. 9. Preer JR, Jr. 1959. Studies on the immobilization antigens of Paramecium. 11. Isolation. J . Immunol. 83, 378-84. . 1969. Genetics of the Protozoa, in Chen T, ed., 10. Research in Protozoology, Pergamon Press, New York 3, 129-278. 11. Reisner AH, Rowe J. 1969. Intrinsic viscosity of a randomly coiled polypeptide of 300,000 daltons and its effect on the solution of the Mark-Houwink equation. Nature 222, 558-9. 12. , Rowe J, Macindoe HM. 1969. T h e largest known monomeric globular proteins. Biochim. Biophys. Acta 188, 196206. 13. -, , Sleigh RW. 1969. Concerning the tertiary
-
The existence of protease activity in the i-antigen is further shown by its ability to degrade a standard protein mixture. The protein mixture (E. coli RNA polymerase) is shown in Fig. lc; it is degraded completely by incubation with i-antigen in SDS and mercaptoethanol (Fig. I d ) . Our results are consistent with the interpretation that the protease appears to have an essential sulfhydryl group. As shown in Fig. le, the i-antigen is not degraded if the sample is treated with iodoacetate before incubation with SDS and mercaptoethanol. Similarly, the i-antigen is not degraded if it is first reduced with mercaptoethanol, then immediately reacted with iodoacetate, and finally incubated with mercaptoethanol and SDS. Mercaptoethanol is needed to activate the protease; iantigen preparations that are incubated with SDS alone are not degraded. DISCUSSION It is evident from the results that the i-antigen of P . aurelia syngen (species) 4 contains proteolytic activity when purified by one of the standard procedures ( 9 ) . Since this protease is activated by mercaptoethanol, previous investigators have con-
I
.
IMMOBILIZATION ANTIGENSOF Paramecium structure of the soluble surface proteins of Paramecium. Biochemistry 11, 4637-44. 14. Sinden RE. 1973. The synthesis of the immobilization antigen in Paramecium aurelia: in vitro localization of antigen in ribosomal cell fractions. J . Prototool. 20, 307-15. 15. Sommerville J. 1970. Immobilization antigen synthesis in Paramecium aurelia. Synthesis in a cell-free amino acid incorporating system. Biochim. Biophys. Acta 209,240-9. 16. -1970. Serotype expression in Paramecium, in Rose AH, Wilkinson JF, eds., Advances in Microbial Physiology, Academic Press, New York, 4,131-78.
259
17. Sonneborn TM. 1950. Methods in the general biology and genetics of Paramecium aurelia. J. Exp. Zool. 113, 87-148. 18. -1970. Methods in Paramecium research, in Prescott DM, ed., Methods in Cell Physiology, Academic Press, New York, 4,241-339. 19. . Genetics of the 14 species of Paramecium aurelia, in King R, ed., Handbook of Genetics, Plenum Press, New York, in press. 20. Steers E, Jr. 1965. Amino acid composition and quaternary structure of an immobilizing antigen from Paramecium aurelia. Biochemistry 4, 1896-901.
J. PROTOZOOL. 22(2), 259-261 (1975).
Antigenic Differences Among Epimastigotes, Amastigotes and Trypomastigotes of Trypanosoma cruzi* JUDITH KLOETZEL,t* MARIO E. CAMARGO and VERA LUCIA GIOVANNINI§ Instituto de Medicina Tropical de Sio Paulo, Caixa Postal 2921, Sio Paulo, B r a d
SYNOPSIS. Antigenic differences were demonstrated among trypomastigotes, amastigotes, and epimastigotes of Trypanosoma cruzi by the indirect fluorescent antibody method. Tests using cross-absorbed sera were included in the study. Index Key Words: Trypanosoma cruzi; amastigotes; trypomastigotes; epimastigotes; antigenic differences.
C
ULTURE forms of Trypanosoma cruzi have generally been employed as antigens in diagnostic serologic tests and in attempts at immunization ( 7 ) . T h e serologic tests included complement fixation, agglutination, precipitation and immunofluorescent antibody. There are some indications that the several developmental stages of T . cruzi may differ immunologically. Thus, Muniz &, Borriello (8) demonstrated that normal guinea pig serum lysed epimastigotes while sparing metacyclic and bloodstream trypomastigotes. Adler ( 1) incorporated specific antisera into culture media and, with certain concentrations of the sera, observed the appearance of bizarre forms, the absence of epimastigotes, and a large number of amastigotes, which divided directly. Afchain & Capron (2) obtained cross-reaction in immunoelectrophoresis between culture and bloodstream forms, but not all precipitating bands appeared in both systems. These observations, however, were not conclusive, because a different trypanosome strain was employed for obtaining bloodstream and culture antigens. I n contradistinction to the foregoing, no significant differences in antibodies against epimastigotes and bloodstream trypomastigotes could be demonstrated by the direct fluorescence antibody method in sera of mice ( 11) and of a horse ( 16) immunized with live culture forms of T . cruzi. The present work was undertaken to demonstrate possible antigenic differences between some of the forms T . cruzi assumes during its life-cycle (trypomastigotes, amastigotes, and epimastigotes) , by the indirect fluorescent antibody method.
* This investigation was supported by Research Grants from FundaGIo de Amparo A Pesquisa do Estado de SIo Paulo and Conselho Nacional de Pesquisas. t Fellow of the FundaGIo de Amparo B Pesquisa do Estado de SIo Paulo. *The senior author thanks Dr. Maria P. Deane for suggestion of the subject of this investigation and for the many fruitful discussions. §We wish to thank Mesdames Cacilda Rebonato Carletti and Herta B. Wiillert Telles de Souza for excellent technical help.
MATERIALS AND METHODS
Organisms Strain Y of T . cruzi (14) was used throughout this study. This strain has been maintained in our laboratory for several years, both in cultures kept in Yaeger’s LIT liquid medium (6) by biweekly transfers and in 30-day-old outbred laboratory mice. Passages in mice were done weekly. Culture forms.-Cells cultured in LIT medium were harvested by centrifugation at 500 g on the 7th day of growth, when no more than 5% of metacyclic trypomastigotes were observed, and washed 3 X in 0.15 M NaCl solution. T h e trypanosomes were resuspended in this solution and adjusted to the desired concentration on the basis of hemocytometric counts. This suspension was inoculated into mice to obtain antisera. For absorption of antisera, washed trypanosomes were spun down, and small samples of sediment were kept in individual tubes at -20 C to be used when needed. As antigen for immunofluorescence, washed trypanosomes were suspended in 2% (v/v) formalin in phosphate buffered saline (PBS) (0.15 M NaCl, 0.01 M phosphates, pH 7.2), kept at room temperature for 4 hr, washed again 3 X, and suspended in PBS. This suspension was placed with a pipette on glass slides in small areas drawn with nail varnish, 1 drop/area, dried a t 37 C for a few min and stored a t -20 C until use. Bloodstream forms.-Trypomastigotes for absorption of antisera were collected into 3.8% (w/v) sodium citrate solution by heart puncture of 30-day-old mice on the 7th day of infection. T h e sample was centrifuged for 1 min a t 2,000 g, left a t 37 C for 15 min, the plasma and buffy layers collected, and organisms contained in this layer were washed as above. For each batch of bloodstream trypomastigotes 40 animals were used, and several batches were necessary for the absorption of 0.1 ml of antiserum diluted to 1 :5. For fluorescent antibody tests, the bloodstream form antigen was obtained from infected hamsters by heart puncture and prepared by the method described for the culture antigen.
J. PROTOZOOL. 2 2 ( 2 ) , 257-259 (1975).
The Immobilization Antigen of Paramecium aurelia is a Single Polypeptide Chain HELEN G. HANSMAt
Department of Biological Sciences, University
of
California, Santa Barbara, California 93106
SYNOPSIS. The immobilization antigen (i-antigen) fraction of Paramecium aurelia syngen 4 is shown to contain a protease that is activated by mercaptoethanol. After the protease has been heat-inactivated, the molecular weight of the i-antigen (250,000 daltons) cannot be decreased by mercaptoethanol treatment. I t is demonstrated that the i-antigen is a single polypeptide chain. Reasons are also given why low molecular weight subunits were previously reported by other authors.
Index Key Words: Paramecium aurelia syngen 4; immobilization antigen; single polypeptide chain.
T HE
outer surface of Paramecium and other ciliates is covered with large amounts of a protein called the immobilization antigen (i-antigen). In Paramecium, this protein constitutes 30% of the total ciliary protein, which is sufficient to cover the cilia by a layer 20-30 nm thick (10). The function of this major protein is unknown, but it appears to be indispensable. Alterations in temperature, salt concentration, food source, and other environmental conditions cause a cell to produce a new type of chemically distinct i-antigen. The i-antigen derives its name from the fact that antibodies against this particular protein immobilize Paramecium. A cell normally produces only 1 chemically distinct type of i-antigen a t a time; this is called its “serotype.” T h e regulation of the production of this protein is of great biologic interest (10, 19). T h e i-antigens of Paramecium have molecular weights of 250,000 to 310,000 daltons. For the past decade, there has been a controversy about whether these proteins are single polypeptide chains or are composed of smaller subunits. I t has been reported that the size of the i-antigen decreases after reduction with mercaptoethanol (6, 16, 20). With this procedure, 1 group (20) obtained subunits with a molecular weight of 34,000, while another ( 6 ) found that the subunit molecular weight varied from 19,000 to 80,000 among samples. A few years later, Reisner et al. ( 1 1-13) made extensive measurements of the physical parameters of i-antigens. They reported (12,) that there was no decrease in the molecular weight upon treatment with mercaptoethanol and that the i-antigen was the largest known polypeptide chain. This controversy is resolved in the present paper. Much recent biochemical and genetic data has been interpreted on the basis of the “subunit model” (5, 10, 14-16, 19) although no one has proved that this model is correct. T h e results reported here cast serious doubt on this model.
-
MATERIALS AND METHODS Cells.-Paramecium aurelia of syngen (species) 4 were cultured in Cerophyl medium inoculated with Enterobacter aerogenes, following the methods of Sonneborn (18). Wild type stock 51s (non-kappa bearing wild type) was used. The serotypes of live cells were determined from the degree of immobilization of cells incubated for 2 hr at 28 C with various +t This investigation was supported by Research Grants GM 19406, from NIH, U. S. Public Health Service, and GB 32164X, from NSF, to C. Kung. t I thank Dr. Ching Kung for his helpful suggestions, support of this research, and careful reading of this manuscript. Thanks are also due Dr. John Preer for his advice on the immobilization antigens and Dr. T. M. Sonneborn for giving us the antisera.
dilutions of antisera (8, 17). The rabbit antisera to Paramecium of serotypes A, B, C, and D were kindly supplied by Prof. T. M. Sonneborn. Paramecia all change to serotype A when grown at 32 C and to serotype B when grown a t 17 C (10). Cells were harvested by centrifugation for 3 min at 500 g in an oil testing centrifuge ( I E C Model HN-S) and washed in Dryl’s salt solution ( 3 ) . Isolation of Immobilization Antigens.-I-antigens were purified by the method of Preer (9). Cells of known serotype were extracted in salt-ethanol solution [0.45% (w/v) NaCl, 5 mM Na phosphate, 15% (v/v) ethanol, p H 7.01. This extract was fractionated by acid precipitation at p H 2.0; the supernatant fluid was adjusted to p H 7.0 and further fractioned by (NH4)Z SO, precipitation. I-antigens A and B were precipitated in the (NH,),SO, fraction of 35% to 61% saturation (23 C ) . T h e precipitate was redissolved and dialyzed against a solution of 1 mM Tris-0.1 mM EDTA, p H 7.0. The dialyzed i-antigens were stored frozen. Protein was measured by a modified Folin phenol method (7). Electrophoresis.-Samples were incubated for 1-2 h r at room temperature with 1% (w/v) SDS and 1% (v/v) mercaptoethanol in Tris-acetate buffer, p H 7.4 ( 4 ) . Polyacrylamide gels were prepared by Fairbanks’ method ( 4 ) with 0.1% SDS. Electrophoresis was carried out for 2-2% hr a t 6 mA/tube. Gels were stained with Coomassie blue ( 4 ) . Molecular weights ( M W ) were calculated from the standard curve of peptides of known molecular weight. RNA polymerase from Escherichia coli was used as a standard peptide mixture. I t was purified by the procedure of Burgess & Travers ( 1 ) by G. Bitter and was a gift from Dr. D. Kohl. Some i-antigen samples were reacted with iodoacetic acid under the following range of conditions: 0.15-0.4 M iodoacetic acid, p H 8-9, 10 min or more at room temperature. Some samples were reduced with mercaptoethanol (- 20 mM) before treatment with iodoacetic acid. SDS (1%) and mercaptoethanol (1%) were added after iodoacetate treatment. Reagents.-Sodium dodecyl sulfate (SDS) and polyacrylamide gel reagents were Electrophoresis Purity Grade from Bio-Rad. RESULTS I-antigen isolated by the procedure described above was preincubated in buffer containing 1% SDS-l% mercaptoethanol and electrophoresed on SDS-polyacrylamide gels. The result is shown in Fig. la. There is only a broad band of less than 15,000 daltons. Since this pattern suggested proteolytic degradation, the i-antigen was boiled before preincubation. Under these conditions, a single band of 200,000-300,000 daltons was obtained (Fig. l b ) , although sufficient mercaptoethanol was present to reduce disulfide-bridged subunits.
258
IMMOBILIZATION ANTIGENSOF Paramecium cluded that the 250,000-dalton i-antigen contains smaller subunits that are joined by disulfide bridges. We have shown, however, that no subunits are produced upon reduction of the i-antigen after the protease has been inactivated; i.e. the 250,000-dalton i-antigen is a single polypeptide chain. The protease activity could be present either in a lower MW contaminant or in the i-antigen molecule itself. The former alternative seems more likely, since Macindoe & Reisner (8) purified their (NH,) $04-precipitated i-antigen on a DEAE cellulose column and found that mercaptoethanol did not cause degradation of the i-antigen. It was previously reported that the i-antigen has small subunits (6, 16, 20). Steers’ purification procedure was identical to ours and to that of Preer ( 9 ) , but Jones ( 6 ) and Sommerville (16) further purified the (NH,) 2S04-precipitated i-antigen on a SE-Sephadex column. They also used P . aurelia syngen 1 instead of syngen 4, but the i-antigens of these 2 species are quite similar in molecular weight and amino acid composition. The reproducibility of subunits in syngen 1 i-antigens was poor. One author reported a subunit MW of 16,000-80,000 from sedimentation equilibrium measurements of reduced or reduced carboxymethylated i-antigens (6). According to another, a variable pattern of 3 to 5 bands was obtained by acrylamide disc gel electrophoresis of i-antigens treated with mercaptoethanol ( 16). Thus it is possible that the i-antigen preparations from syngen 1 also had protease activity. Research on the biosynthesis of antigens and the regulation of serotypes is currently being pursued in many laboratories with at least 3 syngens of Paramecium aurelia (2, 5, 14, 15, 19). For this research, it is essential to know whether the i-antigens have subunits. It has been shown in this report that the i-antigens of 300,000 daltons, syngen 4 are single polypeptide chains of being thus among the largest polypeptide chains, as was stated by Reisner et al. (11, 12). The previously reported subunits presumably were artifacts caused by proteolytic degradation.
-
Fig. 1. SDS-polyacrylamide gel electrophoresis of Paramecium i-antigens. a. I-antigen incubated with 1% SDS and 1% mercaptoethanol (ME). b. Boiled i-antigen incubated with SDS ME. c. Standard peptide mixture (E. coli RNA polymerase) inME. d. Standard peptide mixture cubated with SDS i-antigen (not boiled), incubated with SDS ME. e. I-antigen, preincubated 10 min with 0.4 M iodoacetamide, then incubated ME. with SDS
+
+
+
+
+
REFERENCES 1. Burgess RR, Travers AA. 1971. DNA-Dependent RNA Polymerase (EC 2.7.7.6) in Cantoni GL, Davies DR, eds., Procedures in Nucleic Acid Research, Harper & Row, New York 2, 851-63. 2. Capdeville Y. 1971. Allelic modulation in Paramecium aurelia heterozygotes. Molec. Gen. Genetics 112, 306-16. 3. Dry1 S. 1959. Antigenic transformation in Paramecium aurelia after homologous antiserum treatment during autoaamv and conjugation. J. &otozool. 6 (Suppl.), 25. 4. Fairbanks G. Steck TL, Wallach DFH. 1971. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10,2606-17. 5. Finger I, Onorato F, Heller C, Wilcox HB, 111. 1966. Biosynthesis and structure of Paramecium hybrid antigen. J . Mol. Biol. 17,86-100. 6. Jones IG. 1965. Studies on the characterization and structure of the immobilization antigens of Paramecium aurelia. Biochem. I . 96,17-23. 7. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-75. 8. Macindoe H, Reisner AH. 1967. Adsorption titration as a specific semi-quantitative assay for soluble and bound Paramecium serotypic antigen. Austral. 1. Biol. Sci. 20, 141-52. 9. Preer JR, Jr. 1959. Studies on the immobilization antigens of Paramecium. 11. Isolation. J . Immunol. 83, 378-84. . 1969. Genetics of the Protozoa, in Chen T, ed., 10. Research in Protozoology, Pergamon Press, New York 3, 129-278. 11. Reisner AH, Rowe J. 1969. Intrinsic viscosity of a randomly coiled polypeptide of 300,000 daltons and its effect on the solution of the Mark-Houwink equation. Nature 222, 558-9. 12. , Rowe J, Macindoe HM. 1969. T h e largest known monomeric globular proteins. Biochim. Biophys. Acta 188, 196206. 13. -, , Sleigh RW. 1969. Concerning the tertiary
-
The existence of protease activity in the i-antigen is further shown by its ability to degrade a standard protein mixture. The protein mixture (E. coli RNA polymerase) is shown in Fig. lc; it is degraded completely by incubation with i-antigen in SDS and mercaptoethanol (Fig. I d ) . Our results are consistent with the interpretation that the protease appears to have an essential sulfhydryl group. As shown in Fig. le, the i-antigen is not degraded if the sample is treated with iodoacetate before incubation with SDS and mercaptoethanol. Similarly, the i-antigen is not degraded if it is first reduced with mercaptoethanol, then immediately reacted with iodoacetate, and finally incubated with mercaptoethanol and SDS. Mercaptoethanol is needed to activate the protease; iantigen preparations that are incubated with SDS alone are not degraded. DISCUSSION It is evident from the results that the i-antigen of P . aurelia syngen (species) 4 contains proteolytic activity when purified by one of the standard procedures ( 9 ) . Since this protease is activated by mercaptoethanol, previous investigators have con-
I
.
IMMOBILIZATION ANTIGENSOF Paramecium structure of the soluble surface proteins of Paramecium. Biochemistry 11, 4637-44. 14. Sinden RE. 1973. The synthesis of the immobilization antigen in Paramecium aurelia: in vitro localization of antigen in ribosomal cell fractions. J . Prototool. 20, 307-15. 15. Sommerville J. 1970. Immobilization antigen synthesis in Paramecium aurelia. Synthesis in a cell-free amino acid incorporating system. Biochim. Biophys. Acta 209,240-9. 16. -1970. Serotype expression in Paramecium, in Rose AH, Wilkinson JF, eds., Advances in Microbial Physiology, Academic Press, New York, 4,131-78.
259
17. Sonneborn TM. 1950. Methods in the general biology and genetics of Paramecium aurelia. J. Exp. Zool. 113, 87-148. 18. -1970. Methods in Paramecium research, in Prescott DM, ed., Methods in Cell Physiology, Academic Press, New York, 4,241-339. 19. . Genetics of the 14 species of Paramecium aurelia, in King R, ed., Handbook of Genetics, Plenum Press, New York, in press. 20. Steers E, Jr. 1965. Amino acid composition and quaternary structure of an immobilizing antigen from Paramecium aurelia. Biochemistry 4, 1896-901.
J. PROTOZOOL. 22(2), 259-261 (1975).
Antigenic Differences Among Epimastigotes, Amastigotes and Trypomastigotes of Trypanosoma cruzi* JUDITH KLOETZEL,t* MARIO E. CAMARGO and VERA LUCIA GIOVANNINI§ Instituto de Medicina Tropical de Sio Paulo, Caixa Postal 2921, Sio Paulo, B r a d
SYNOPSIS. Antigenic differences were demonstrated among trypomastigotes, amastigotes, and epimastigotes of Trypanosoma cruzi by the indirect fluorescent antibody method. Tests using cross-absorbed sera were included in the study. Index Key Words: Trypanosoma cruzi; amastigotes; trypomastigotes; epimastigotes; antigenic differences.
C
ULTURE forms of Trypanosoma cruzi have generally been employed as antigens in diagnostic serologic tests and in attempts at immunization ( 7 ) . T h e serologic tests included complement fixation, agglutination, precipitation and immunofluorescent antibody. There are some indications that the several developmental stages of T . cruzi may differ immunologically. Thus, Muniz &, Borriello (8) demonstrated that normal guinea pig serum lysed epimastigotes while sparing metacyclic and bloodstream trypomastigotes. Adler ( 1) incorporated specific antisera into culture media and, with certain concentrations of the sera, observed the appearance of bizarre forms, the absence of epimastigotes, and a large number of amastigotes, which divided directly. Afchain & Capron (2) obtained cross-reaction in immunoelectrophoresis between culture and bloodstream forms, but not all precipitating bands appeared in both systems. These observations, however, were not conclusive, because a different trypanosome strain was employed for obtaining bloodstream and culture antigens. I n contradistinction to the foregoing, no significant differences in antibodies against epimastigotes and bloodstream trypomastigotes could be demonstrated by the direct fluorescence antibody method in sera of mice ( 11) and of a horse ( 16) immunized with live culture forms of T . cruzi. The present work was undertaken to demonstrate possible antigenic differences between some of the forms T . cruzi assumes during its life-cycle (trypomastigotes, amastigotes, and epimastigotes) , by the indirect fluorescent antibody method.
* This investigation was supported by Research Grants from FundaGIo de Amparo A Pesquisa do Estado de SIo Paulo and Conselho Nacional de Pesquisas. t Fellow of the FundaGIo de Amparo B Pesquisa do Estado de SIo Paulo. *The senior author thanks Dr. Maria P. Deane for suggestion of the subject of this investigation and for the many fruitful discussions. §We wish to thank Mesdames Cacilda Rebonato Carletti and Herta B. Wiillert Telles de Souza for excellent technical help.
MATERIALS AND METHODS
Organisms Strain Y of T . cruzi (14) was used throughout this study. This strain has been maintained in our laboratory for several years, both in cultures kept in Yaeger’s LIT liquid medium (6) by biweekly transfers and in 30-day-old outbred laboratory mice. Passages in mice were done weekly. Culture forms.-Cells cultured in LIT medium were harvested by centrifugation at 500 g on the 7th day of growth, when no more than 5% of metacyclic trypomastigotes were observed, and washed 3 X in 0.15 M NaCl solution. T h e trypanosomes were resuspended in this solution and adjusted to the desired concentration on the basis of hemocytometric counts. This suspension was inoculated into mice to obtain antisera. For absorption of antisera, washed trypanosomes were spun down, and small samples of sediment were kept in individual tubes at -20 C to be used when needed. As antigen for immunofluorescence, washed trypanosomes were suspended in 2% (v/v) formalin in phosphate buffered saline (PBS) (0.15 M NaCl, 0.01 M phosphates, pH 7.2), kept at room temperature for 4 hr, washed again 3 X, and suspended in PBS. This suspension was placed with a pipette on glass slides in small areas drawn with nail varnish, 1 drop/area, dried a t 37 C for a few min and stored a t -20 C until use. Bloodstream forms.-Trypomastigotes for absorption of antisera were collected into 3.8% (w/v) sodium citrate solution by heart puncture of 30-day-old mice on the 7th day of infection. T h e sample was centrifuged for 1 min a t 2,000 g, left a t 37 C for 15 min, the plasma and buffy layers collected, and organisms contained in this layer were washed as above. For each batch of bloodstream trypomastigotes 40 animals were used, and several batches were necessary for the absorption of 0.1 ml of antiserum diluted to 1 :5. For fluorescent antibody tests, the bloodstream form antigen was obtained from infected hamsters by heart puncture and prepared by the method described for the culture antigen.
