138 CAROTFNOID MX-TANTS OF Crppthccodiniuin cohnii

in carotenoid deficient mutants nf Chlaniydomonas. Nature (Lon- don) 182, 98-100.

14. Shimizu Y . Alam M, Kobayashi '4. 1976. Dinosterol, the major sterol with a unique side chain in the toxic dinoflagellate. Gonyaulax tamarensis. J. A m . Chem. SOC. 98, 1059-60.

15. Tuttle RC, Loeblich AR I l l . 1974. Genetic recombination in the dinoflagellate Crypthecodiniurn cohnii. Science 185, 1061-2.

16. -,- 1975. An optimal gro\\ th medium for the dinoflagellate Crypthecodinium cohnii. Phycologia 14, 1-8.

17. -, __ 1977. N-methyl-N'-nitro-A'-nitrosoguani- dine and UV induced mutants of the dinoflagellate Crypthecodi- nium cohnii. J . Protozool. 24, 313-6.

18. -, __ , Smith VE. 1973. Carotenoids of Cryp- thecodinium cohnii. J. Protorool. 20. 521.

19. Williams RJH, Davies BH, Goodwin TW. 1965. The presence of 6-zeacarotene in cultures of diphenylamine-inhibited Phycornyces blakesleeanus. Phytochemistry 4, 759-60.

20. Withers NW, Haxo FT. 1978. Isolation and characteriza- tion of carotenoid-rich lipid globules from Peridinium foliaceum. Plant Physiol. 62, 36-9.

21. ___ , Cox ER, Tomas RN, Haxo FT. 1977. Pigments of the dinoflagellate Peridinium balticum and its photosynthetic endosymhiont. I . Phycol. 13, 354-8.

22. ___ , Tuttle RC, Holz GG Jr, Beach DH, Goad LJ, Goodwin TW. 1978. Dehydrodinosterol, dinosterone and related sterols of a non-photosynthetic dinoflagellate, Crypthecodinium cohnii. Phytochemictry 17, 1987-9.

I . ProtozooE., 26(1) , 1979, pp. 138-142 0 1979 by the Socirty of Protozoologists

Respiratory Chain Components of Leishmania tropicu Promastigotes*

ELMER M A R T I N t and ANTONY J. MUKKADA* IIe~i ( i i -~ ir te i i t of Hiologicol Sciences, l'iriiwsiiy of Ciirci i innti ,

Ciirriiriiflti, Ohio -15221

SYNOPSIS. Crude preparations o f kinetoplast vesicles were uszd to investigate the respiratory chain components in Leishmania tropica promastigotes. In difference spectra from enzymically and chemically reduced preparations, cytochrome b was the predominant component. By utilizing special assays designed to minimize the influence of cytochrome b on difference spectra, cytochromes a. a3, and cj,; were demonstrated. Difference spectra from chemically reduced preparations indicated that pyridine nucleotides (NADH) and flavoproteins were also part of the respiratory chain. The presence of these components as well as their response to respiratory inhibitors and ascorbate proLide evidence for the presence of a typical trypanosomatid respiratory chain in L. tropica promastigotes.

Index Key Words: Leishmania tropica : crude preparations of kinetoplast vesicles; respiratory chain; cytochromes; difference spectra.

NLIKE other trypanosoinatids (see Ref. 1 1 1 , the respiratory U chain in Leishmania sp. has not been studied extensively. A cytochrome-dependent system v'as indicated by the sensitivity of promastigotes (9, 15, 25, 29) and amastigotes (9, 25) to classical respiratory inhibitors such as cyanide and azide. Krassner (15) demonstrated the presence of haem in Leishmania donovani, Leishmania enrietti, and Leishmania tarentolae pro- rnastigotes and provided whole cell difference spectra of L. taren- tolae that directly suggested the existence of cytochromes. A difference spectrum from the mitochondria of L . tarentolae pro- mastigotes was described as very similar to that of othcr trypano- somatids, cytochrome b being predominant with trace amounts of cytochromes a and c ; however, this spectrum \vas not pre- sented in the report ( 1 3 ) . These studies suggested the presence of a cytochrome-dependent system in at least the proniastigote form of Leishmania. The present investigation was undertaken using L. tropica as a possible model for the promastigote respira- tory chain. By a modification of the procedure of Braly et af. ( 3 ) , crude particulate suspensions of kinetoplast vesicles were prepared. These vesicles contain most of the kDNA (3 , 26) and represmt at least a large portion of the kinetoplast-mitochondria1 complex.

This investigation was supported by grants from the Research

* Present address: Department of Bacteriology, University of

* To whom reprint requests should be directed.

Coyci l of the University of Cincinnati.

California. Los Angeles, California 90024.

MATERIALS AND METHODS

Organisin and Growth Conditions.-All data reported here \\-ere obtained from the promastigote stage of L. tropica. They were maintained as previously described (21 ) . A monophasic liquid medium (24) was used for routine cultivation.

Reagents.-The following reagents were obtained from Sigma Chemical Co. (St. Louis MO) : bovine serum albumin (BSA) ( Fraction V ) , morpholinopropane sulfonic acid (MOPS), EDTA, ethyleneglycol-bis- (a-arninoethyl ether) N , N-tetra-acetic acid ( E G T A ) , cytochrome c (horse heart), sucrose (Grade I ) , pan- creatic deoxyribonuclrase ( DNase I ) , sodium ADP, antimycin A, and N , N , N' , N'-tetramethylphenylenediamine (TMPD) . All other reagents used were purchased from the Fisher Chemical Go. (Cincinnati OH).

Preparation of Kinetoplast Vesicles.-Kinetoplast-mitochon- drial vesicles were prepared by modifying the procedure of Braly et al. ( 3 ) . Promastigotes were harvested during late log or early stationary phases of growth. Cells ( - 300 ml culture) were haivested hy centrifugation in a Sorvall superspeed RC-2 at 3,300 g for 5 min. All operations were performed at 4 C. A sufficient volume of a hypotonic buffer ( 1 mM MOPS, pH 7.7; 1 mM EDTA) was added to the pooled pellets to obtain a density of - 1 x 10" cells/ml. The suspension was left undisturbed €or 15 niin for the cells to swell. The swollen promastigotes were forced through a syringe with a 26 gauge needle into a swirling mixture containing enough sucrose and BSA to yield final concentrations of 0.25 hi and 0.3% (w/v ) , respectively. The preparation was

Leishmania : RE s PIRATORY CHAIN 139

monitored microscopically for cell breakage and centrifuged at 12,000 g for 15 min. The supernatant fluid was discarded, and the particulate portions were resuspended in 10 ml DNase buffer [0.02 M MOPS, p H 7.7; 0.35 M sucrose; 0.3% (w/v) BSA; 5 mM magnesium acetate, 1 mM EGTA, 100 pg/ml DNase I, and 0.6 m u CaCI,] for 30 min. The suspension was diluted by adding 2-3 volumes of isolation buffer (0.02 M MOPS, p H 7.7; 0.3% BSA; 0.35 M sucrose; 5 mM magnesium acetate, and 1 mM EGTA) and immediately centrifuged as described above. The supernatant fluid was removed, and the particulate fraction washed and centrifuged once more, then resuspended in isolation buffer for experimental use.

Difference Spectra.-Absorption spectra were measured at 22 C in a Double Beam Spectrophotometer (Coleman 124). The slit width was 0.5 mm. Cuvettes with a 10-mm light path were used in all experiments. Cuvettes whose contents were to be reduced were sealed with 7 x 11 mm serum caps and incubated for 10 min with different electron donors (succinate, ascorbate or sodium dithionite) as indicated for individual experiments. Oxygen was bubbled for 1 min through material to be oxidized in cuvettes. The amount of protein [estimated by the Folin- phenol method (20)]/cuvette was 6-15 mg.

Succinate (reduced) minus control (oxidized) difference spectra were obtained according to the method of Chance (5, 6) . The reduced sample contained: MOPS ( p H 7.5), 160 pmoles; K phosphate buffer ( p H 7.5), 45 pmoles; ADP, 0.5 pmole; K succinate, 30 pmoles; sucrose, 180 pmoles; Mg acetate, 2.5 pmoles; EGTA, 0.5 pmole; BSA, 0.05% (w/v); kinetoplast vesicles and H,O to a volume of 3 ml. The control (oxidized) material contained: MOPS ( p H 7.5), 160 pmoles; sucrose, 180 pmoles; Mg acetate, 2.5 pmoles; EGTA, 0.5 pmole; BSA, 0.05% (w/v); kinetoplast vesicles and H,O to make a volume of 3 ml.

Succinate plus cyanide (reduced) minus succinate plus anti- mycin A (oxidized) difference spectra were obtained by a modifi- cation of Hill's procedure (10) . Cyanide in the reduced sample blocked terminal oxidase ( a s ) so that all cytochromes were fully reduced. The presence of antimycin A in the oxidized sample blocked electron flow between cytochromes b and c which allowed cytochromes c and a to remain oxidized. The effect was to cancel out the influence of cytochrome b on the difference spectrum. Ten min were allowed for both samples to provide for cytochrome reduction. A 3 ml reaction mixture of the reduced sample contained in addition to those listed under succinate (reduced) minus control (oxidized) difference spec- trum : KCN, 3 pmoles, and N,N'-dimethyl formamide (DMF) , 88 pmoles. The oxidized sample also differed from the previous one in having additionally: K succinate, 30 pmoles, and anti- mycin A, 0.21 p g in 88 pmoles of DMF.

The ascorbate plus TMPD (reduced) minus controI (oxidized) difference spectra were obtained by a modification of the pro- cedure of Kusel & Storey (18) , which is based on the fact that ascorbate is an electron donor and the electrons flow through TMPD to a site near cytochrome c in mammalian systems (27) . Thus, the electrons bypass the respiratory chain up to and includ- ing cytochrome b which should minimize its influence on the difference spectrum. The reaction mixtures were essentially the same as those in the succinate (reduced) minus control (oxi- dized) difference spectrum except that in the reduced sample K succinate was replaced by Na ascorbate, 30 pmoles and TMPD, 1.8 pmoles.

The ascorbate plus TMPD plus succinate plus antimycin A (re- duced) minus succinate plus antimycin A (oxidized) difference spectrum was prepared by a modification of the procedure re- ported previously (18). Antimycin A present in both samples blocks electron flow between cytochromes b and c. While this

would cancel out cytochrome b, the addition of ascorbate and TMPD to the reduced sample would allow electrons to bypass the antirnycin A block (27) and reduce cytochromes c and a. The reaction mixture (3 ml) making up the reduced sample contained Na ascorbate, 30 pmoles; TMPD, 1.8 pmoles and anti- mycin A, 0.21 pg in 88 p o l e s DMF in addition to those listed above for the succinate (reduced) minus control (oxidized) difference spectrum. The oxidized sample contained K succinate, 30 pmoles and antimycin A, 0.21 p g in 88 pmoles D M F as well.

Na dithionite (reduced) minus control (oxidized) difference spectra were obtained by the method of Chance ( 6 ) . Both re- duced and oxidized samples contained: MOPS ( p H 7.5), 160 pmoles; sucrose, 180 pmoles; Mg acetate, 2.5 pmoles; EGTA, 0.5 pmole; BSA, 0.05% (w/v); kinetoplast vesicles and H,O to make up 3 ml volumes. The addition of a few grains of Na dithionite into one of the cuvettes served to reduce the cyto- chromes.

The concentrations of the respiratory chain components were estimated by the method of Chance (6 ) .

RESULTS

Enzymatic Reduction of Kinetoplast Vesicle Preparations.- A typical succinate (reduced) minus control (oxidized) difference spectrum is presented in Fig. 1. The cytochromes were reduced enzymically in the presence of succinate, ADP, and inorganic phosphate. The peak at 595 nm denotes the presence of cyto- chromes a + a3. The presence of cytochrome b is indicated by the peak a t 550 nm and cytochrome c555 by a shoulder (553 nm) off the cytochrome b peak. The small, broad peak at 524 nm indicates the presence of cytochrome c jS5 . Peaks in the Soret region suggest the presence of cytochromes b (425 nm) and css5 (419 nm) . Similar spectra were obtained when succinate was substituted with proline or NADH.

Cytochrome b dominates the difference spectrum shown in Fig. 1. The preponderance of this pigment rendered determi- nation of the presence of cytochromes c and a,< difficult. To clarify this, we employed assays designed to eliminate the in- fluence of cytochrome b on the difference spectra. The spectrum of succinate plus cyanide (reduced) minus succinate plus anti- mycin A (oxidized) is presented in Fig. 2. Under these con- ditions, peaks at 554 and 446 nm for cytochromes cSs5 and a,, respectively, become distinct. The shoulder at 420 nm and the peak at 595 nm are indicative of cytochromes cJ55 and a + a,l respectively. Cytochrome b, however, is not completely elimi- nated from this spectrum, as evidenced by the peaks at 560, 526, and 425 nm. According to control experiments, the amount of DMF used as a solvent for antimycin A had no effect on these spectra.

Another method for eliminating cytochrome b interference in spectra, is by using ascorbate and T M P D in the assay. A differ- ence spectrum of ascorbate plus T M P D (reduced) minus con- trol (oxidized) is shown in Fig. 3. The presence of cytochrome c555 is evidenced by a shoulder a t 554 nm and a prominent Soret peak at 421 nm. The peak at 596 nm indicates the presence of cytochromes a+a,. Cytochrome b peaks at 558 and 530 nm have not been completely eliminated.

The ascorbate with TMPD, succinate, and antimycin A (re- duced) minus succinate with antimycin A (oxidbed) differ- ence spectrum (Fig. 4 ) shows considerable improvement in minimizing the influence of cytochrome b on the spectrum. The broad peak at 590 nm represents cytochromes a + ad , while cytochrome cSs6 has its characteristic a-peak at 555 nm. The shoulder at 445 nm indicates the presence of cytochrome a,. Cytochrome b is still indicated by the peaks at 525 and 426 nm.

Chemical Reduction of Kinetoplast Vesicle Preparations.-

140 Leishmania : RESPIRATORY CHAIN

I I I I I I I I I

410 470 530 590 6 3

WAVELENGTH (nm)

4 2 6

I

T 0.01 A

1 4

I I 1 l l l l I L

410 470 530 590 651

W AV E L E NGT H (nm)

420 \

560

446

0.0 1 A J

410 47i3 533 590 650 WAV ELENGT H (nm)

I I 1 1 1 I I I I

230 350 470 590 680

WAVELENGTH (nm)

3

I I I I I I I I I

410 470 530 590 651

WAVELENGTHhrn)

Figs. 1-5. [Difference spectra from kinetoplast preparations of Leishmania tropica promastigotes. Figs. 1-4 : the scan was monitored at from 650 to 400 nm at 22 C: scale = 0.01 absorbance U.] 1. Succinate (reduced) minus control (oxidized); 6 mg protein/cuvette. 2 . Succinate plus cyanide (reduced) minus succinate plus antimycin .4 (oxidized) ; 11.6 mg protein/cuvette. 3. Ascorbate plus TMPD (reduced) minus control (oxidized) : 11.6 mg protein,’cuvette. 4. Ascorbate plus TMPD plus succinate plus antimycin A (reduced) minus succinate plus antimycin A (oxidized) ; 6 mg proteinicuvette. 5. Na dithionite (reduced) minus control (oxidized) ; 9 mg protein/ cuvette. The scan was monitored at from 650 to 310 nm at 22 C. Scale = 0.01 or 0.1 absorbance U.

Chemical reduction by dithionite is an alternate method to identify respiratory chain components in assays involving dif- ference spectra ( 6 ) . A sodium dithionite (reduced) minus con- trol (oxidized) spectrum of kinetoplast vesicle preparations is shown in Fig. 5. As in enzymatically reduced preparations, cyto- chrome b is the dominant component of the spectrum (562, 533. 431 n m ) . A small pcak at 610 nni and a slight shoulder at 446 nm indicate the presence of cytochronies a and a,>, respectively. Other components of the respiratory chain were also revealed by chemical reduction. A trough at 465 nm is typical of flavo- proteins, while the peak at 345 nni wggests the presence of pyridine nucleotides (e.g. NADH j ( 6 ) . Chemical reduction, hmvever, failed t o give any indication of the presence of cyto- chrome c j j 5 .

Concentration of Respiratory Chain Components.-From the data obtained by the spectral assays. estirnatcs of the concentra-

tlons of the variou.; reqpiratory chain components were made according to the method of Chance ( 6 ) . As shown in Table 1, c ) tochrome b has the highest concentration; it is 3 times more concentrated than a,{, 9 times more than cS5; , and 22 times more than a. Flavoprotein( s ) and pyridine nucleotide( s ) characteris- tic all! ha\e concentrations higher than those of the cytochromes.

DISCUSSION

Enzymic and chemical reduction of crude kinetoplast vesicle preparations from L. tropica promastigotes led to the identifica- tion of a respiratory chain similar to that found in other trypano- Foniatids ( 11 ) . As in most trypanosomatidq, cytochrome b is the predominant component of difference spectra; its concentra- tion i3 at least 3 times that of Q , and 9 times that of cytochrome c . The predominance of cytochrome b in simple difference spectra

Leishmania: RESPIRATORY CHAIN 141

TABLE 1. Concentration of respiratory chain components in Leish- mania tropica promastigotes.*

Wavelength pairst Concentration (nmole/mg

Component Maximum Minimum AE* protein)

C ytochromes b 562 575 2 2 0.18 csss 555 540 19.1 0.02 a 610 615 16 0.008 as 445 465 91 0.05

Flavoprotein (s) 465 510 11 0.25

Pyridine Nucleotide(s\ 340 3 74 6.2 19.1

* All data except that for csrs are from chemically reduced prepa- rations; they are the averages of a t least 4 separate assays. The value for csss was calculated from enzymatically reduced prepara- tions.

t The wavelength pairs were from Chance (6) . * Millimolar extinction coefficients (cm-' mM-l) from Chance ( 6 ) .

from Crithidia fasciculata (17) and Trypanosoma sp. (23) was thought to indicate the absence of cytochrome c in these organ- isms. However, cytochrome c555 from Crithidia fasciculata has subsequently been isolated and partially characterized (4, 12, 19) . I t is evident from the data included in the present report that cytochromes c and a could be demonstrated in difference spectra of kinetoplast vesicles if the assay procedures were manipulated in such a way as to eliminate or a t least minimize the expression of cytochrome b. In such assays, the expression of cytochrome b, however, could not be entirely eliminated possibly due to electron leakage past the respiratory inhibitors. Such leakage has been previously demonstrated in heart muscle mitochondria ( 2 7 ) .

In our preparations of kinetoplast vesicles, cytochromes a + a, had an a-peak at 590-596 nm, in contrast to the 600-605 nm peak characteristic of their mammalian counterparts. Leishmania tropica is not unique in this respect; a + a, peaks in the 590-596 nm range have been reported in Trypanosoma rhodesiense ( 2 ) , C. fasciculata (18) , Moniexia sp. (8) and Mycobacterium phlei ( 1 ). The demonstration of cytochromes a + a, indicates the presence of a terminal oxidase route for the promastigote respiratory system. Additional oxidases, especially cytochrome 0, have been reported in culture forms of several trypanosomatids (10, 11). These include Blastocrithidia culicis (13, 16), C. fasciculata ( 13, 14, 16) , Crithidia oncopelti (28), Herpetomonas muscarum (13) , Leishmania tarentolae (13, 16) , Leptomonas sp. (13) , Trypanosoma conorrhini, T. cruzi, T. lewisi (13, 16) and T. mega (16, 2 2 ) .

Na dithionite reduced minus oxidized difference spectra re- vealed the presence of flavoprotein(s) (trough at 465 nm) and pyridine nucleotide(s), such as NADH (peak at 345 nm). Al- though pyridine nucleotides typically have a peak at 340 nm, it is not unusual to find small deviations from the normal. In yeast and rat liver mitochondria, Chance ( 6 ) obtained a pyridine nucleotide peak at 335 nm. Such a peak would be absent from nonphosphorylating preparations of rat liver mitochondria ( 7 ) . Since phosphorylation requires intact mitochondria, our data suggest that kinetoplast vesicles employed in the present study retain typical mitochondria1 characteristics. Thus, the respira- tory chain in L. tropica promastigotes includes flavoproteins, pyridine nucleotides, as well as cytochromes b, c, and a.

In view of the evidence obtained from difference spectra as well as the effect of respiratory inhibitors and electron donor by- pass systems on these spectra, we propose the following tentative

scheme for the respiratory chain in L. tropica promastigotes: NADH-Flavoprotein-Cyt b--+Cyt c+Cyt a + a,+OL'. Further support for this scheme is provided by our unpublished data from studies using purified kinetoplast-mitochondria1 com- plexes of this organism.

ACKNOWLEDGEMENT

We are greatly indebted to Dr. Douglas G. Winget for stimu- lating discussion and advice.

REFERENCES

1. Asano A, Brodie AF. 1964. Oxidative phosphorylation in fractionated bacterial systems. XIV. Respiratory chains of Myco- bacterium phlei. J . Biol. Chem. 239, 4280-91.

2. Bowman IBR, Srivastava HK, Flynn IW. 1972. Adapta- tions in oxidative metabolism during the transformation of Trypano- soma rhodesiense from bloodstream into culture form, in Van den Bossche H, ed., Comparative Biochemistry of Parasites, Academic Press, New York, pp. 329-42.

3. Braly P, Simpson L, Kretzer F. 1974. Isolation of kineto- plast-mitochondria1 complexes from Leishmania tarentolae. J .

4. Chan SK, Hill SC. 1969. Purification of cytochrome csss from Crithidia fasciculata. Bacteriol. Proc. 69, 142.

5. Chance B. 1952. Spectra and reaction kinetics of respira- tory pigments of homogenized and intact cells. Nature 169, 215-21.

6. - 1957. Techniques for the assay of the respiratory enzymes. Methods Entymol. 4, 273-329.

7. - , Williams GR. 1955. Respiratory enzymes in oxi- dative phosphorylation. 11. Difference spectra. J. Biol. Chem. 2 17,

Prototool. 21, 782-90.

395-407. 8. Cheah KS. 1972. Cytochromes in Ascaris and Moniezia,

in Van den Bossche H, ed., Comparative Biochemistry of Parasites, Academic Press, New York, pp. 417-32.

9. Fulton JD, Joyner LP. 1949. Studies on protozoans, Part I. The metabolism of Leishman-Donovan bodies and flagellates of Leishmania donouani. Tr.ans. R. SOC. Trop. Med. Hyg. 43, 273-86.

10. Hill GC. 1972. Recent studies on the characterization of the cytochrome system in Kinetoplastidae, in Van den Bossche H. ed., Comparative Biochemistry of Parasites, Academic Press, New York, pp. 395-415.

11. ~ 1976. Electron transport systems in Kineto- plastidae. Biochim. Biophys. Acta 456, 149-93.

12. ~ , Chan SK. 1969. Properties of purified cyto- chrome cjs6 from Crithidia fasciculata. J. Prototool. (Suppl.), 16, 13.

13. - , Cross GAM. 1973. Cyanide-resistant respiration and a branched cytochrome system in Kinetoplastidae. Biochim. Biophys. Acta 305, 590-6.

14. ___ , White DC. 1968. Respiratory pigments of Crithidia fasciculata. J . Bacteriol. 95, 2151-7.

15. Krassner SM. 1966. Cytochromes, lactic dehydrogenase, and transformation in Leishmania. J . Protorool. 13, 286-90.

16. Kronick P, Hill GC. 1974. Evidence for the functioning of cytochrome o in Kinetoplastidae. Biochim. Biophys. Acta 368,

17. Kusel JP, Weber MM. 1968. Enzymatic activities and cytochrome components of the terminal transport pathways of Crithidia fasciculata. Bacteriol. Proc. 68, 139.

18. ___ , Storey BT. 1973. Low temperature spectral prop- erties of the respiratory chain cytochromes of mitochondria from Crithidia fasciculata. Biochim. Biophys. Acta 305, 570-80.

19. - , Suriano JR, Weber MM. 1969. Isolation, purifi- cation, and characterization of Crithidia fasciculata cytochrome clss, Arch. Biochem. Biophys. 133, 293-304.

20. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. 1951. Protein measurement with the Fohn-phenol reagent. J . Biol. Chem.

21. Mukkada AJ, Schaefer FW 111, Simon MW, Neu C. 1974. Delayed in vitro utilization of glucose by Leishmania tropica promastigotes. /. Prototool. 21, 393-7.

22. Ray SK, Cross GAM. 1972. Branched electron transport chain in Trypanosoma mega. Nature (NB) 237, 174-5.

23. Ryley JF. 1956. Studies on the metabolism of the pro- tozoa. VII. Comparative carbohydrate metabolism of eleven species of trypanosomes. Biochem. J . 62, 215-22.

1 73-80.

193, 265-75.

142 Leishmania : RESPIRATORY CHAIN

24. Schaefer FW 111, Bell EJ. Etges FJ. 1970. Leishmania for a study of oxidative phosphorylation. Methods Enzymol . 10,

25. Simpson L. 1968. The leishmania-leptomonad transfor- 28. Srivastava HK. 1971. Carbon monoxide-reactive haemo- proteins in parasitic flagellate Crithidia oncopelti. FEBS Lett. 16, 189-91.

26. __ 1972. The kinetoplast of the hernoflagellates. Znt. 29. von Brand T, Johnson EM. 1947. A comparative study of the effect of cyanide on the respiration of some Trypanosomat-

27. Slater EC. 1967. Application of inhibitors and uncouplers idae. J. Cell. Comp. Physiol. 29, 33-49.

tropica: chemostatic cultivation. E x p . Parasitol. 28. 465-72. 48-57.

mation of Leishmania donovani: nutritional requirements. respira- tory changes and antigenic changes. J. Protozool. 15, 201-7.

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I . P?otozooZ., 2 6 ( 1 ) , 1979, pp. 142-146 0 1979 by the Society of Protozoologists

Partial Purification and Characterization of a Bacteriolytic Enzyme Secreted by Tetrahymena*

G. WESLEY VICK, III,t HARRY A. GALLIS,* ROBERT WHEAT,* and J. J. BLUMt t I l r p n i t i i t m / \ of Plt\ctology and *AftrtolJiology, Duke Z'nrueisrty Medical Center,

Diith(iiti, .Yoith C ( i i o l i ~ i ~ 27710

SYNOPSIS. Tetrahymena pyriformis strain HSM secretes large quantities of acid hydrolases into the culture medium. An enzyme secreted by the ciliate and capable of degrading walls of streptococci was identified and purified to a considerable degree. The p H optimum of this enzyme was 3-4, and it was eluted after cytochrome c from Sephadex G-75 columns. Unlike lysozyme, the enzyme was thermolabile at p H 2.9. but relatively thermostable at pH 8.1. It degraded 14C-labeled cell walls of streptococci releasing reducing groups. Cell walls prepared from different strains of streptococci differed in susceptibility to this enzyme, the most sensitive strain tested being of group A. type T12. It was shown in immunologic studies that this hydrolase released the group-specific carbohydrate from the walls. Secretions of Tetrahymena from early stationary-phase cultures had more bacterio- lytic activity than those from cells from late stationary-phas- cultures. Further, cells from cultures grown in glucose- supplemented medium secreted less of the enzyme than ciliates of coniparable age grown in unsupplemented proteose-peptone. The newly isolated bacteriolytic enzyrnc. presumably of lysosomal origin. may be helpful in characterizing streptococcal cell walls.

Index Key Words: Tetrahymena pyr i formis strain HSM : enzyme degrading streptococcal cell walls.

U M E R O U S organisms produce enzymes partially degrading N bacterial cell walls. Such enzymes have been described in 2 species of ameba (14, 19) hut not in other protozoa. Since bacteria are a major food of Tetrahymena pyriformis ( l o ) , we tested the medium in which this ciliate had grotzn for hacterio- lytic activity. The finding that label was soluhilized from 14C- labeled streptococcal cell walls suggested that a harteriolytic enzyme was present in the growth medium, and led us to attempt to separate this activity from the other glycosidases and proteases known to be released into the medium. In this paper ive report partial purification of this enzyme and some of its properties. I n particular, we have studied the effect of this enzyme on streptococcal cell walls, and have found that i t could partially degrade certain cell-tzall preparations that resist attack hy hen egg white lysozynie (6, 8 ) . Unlike lysozyme, hacteriolytic action of the enzyme includes release of the group-specific carbohydrate rendering it potentially useful for studies on streptococcal cell wall structure. An ahstract of this \zork has heen presrnted previously (20 ) .

MATERIALS .4KD M E T H O D S

Bacterza.--Gioup A streptococci of Types 1 (T l -A\ ' ) , 3 (C203S), 4, 6, 12, and 19 ueie piokided hy R. M Cole, Lab- oratory of Streptococcal Diseases, National Institutes of Health, Rethesda MD. T h e paient stiain of TI-A\ ' (T1/155) \+as piokided by Dr. Leo Pine, Centei foi Diseaw Contiol, Atlanta

* This investigation was supported by Research Grant 5 R O I HD01269-16, from the National Institute of Child Health and Human Development. U.S. Public Health Service.

GA. Streptococci of groups B and C are from clinical isolates at Duke Hospital.

Preparation of "-Labeled Bacteria.-Bacteria were grown to stationary phase (usually 18 h ) in Todd-Hewitt broth (Difco, Detroit M I ) containing 0.5 pCi of [U-14C]glucose/ml ( 2 to 4 mCi/mmol; AniershamjSearle, Arlington Heights IL) , heat- killed, and collected as described by Gallis et al. (8) .

Preparation of Cell Walls.-Bacterial cell walls were prepared hy 2 methods. Preparations by the TCA-SDS extraction mode were made according to the following procedure. Bacteria from 2 liters of medium were washed in distilled H,O, resuspended in 100 nil of 10% (w/v) trichloroacetic acid ( T C A ) , and heated at 80 C with stirring for 30 min. T h e cell walls were washed in distilled H,O, resuspended in 100 ml of 2% (w/v) sodium dodecyl sulfate (SDS), heated a t 100 C with stirring for 30 min, kzashed again in distilled H,O, and treated with trypsin, chymo- trypsin, and ribonuclease as described by McCarty (13 ) . Only cell walls from the C203S strain of group A streptococci were prepared in this manner.

Additional preparations of cell walls were made by the proce- dure of Gallis et al. (8). Bacteria from 1-liter cultures were washed, resuspended in 25 nil of distilled H,O, shaken for 20 min bvith No. 13 glass heads ( 3 M Co., St. Paul MN) in a Mickle apparatus at 4 C. Whole cell walls were then isolated as de- scribed by Salton & Horne (16) and treated with trypsin, rhymotrypsin, and rihonuclease as indicated above.

Growth uf Ciliates.-Tetrahymena pyriformis strain HSM was gro\\.n in proteose-peptone medium (see Ref. 15). Five hundred- nil Erlenmeyer flasks, each with 110 ml of cells, a t an initial density of - 75,000 rells/ml, were grown in a gyratory shaker hnth for 24 h at 25 C (carly stationary phase). Some cultures