JOURNAL OF BACTERIOLOGY, June 1968, p. 2151-2157Copyright © 1968 American Society for Microbiology

Vol. 95, No. 6Printed in U.S.A.

Respiratory Pigments of Crithidia fasciculataGEORGE C. HILL AND DAVID C. WHITE

Department ofBiochemistry, University ofKentucky Medical Center, Lexington, Kentucky 40506

Received for publication 1 April 1968

Mitochondria were isolated from the heme-requiring insect trypanosomatid,Crithidia fasciculata, which had respiratory activity, showed a P/O ratio with suc-cinate of 0.5 to 1.0, and contained 40 to 50% of the heme a and heme c found in theintact cells. Cytochromes b, c555 , possibly ci, cytochrome oxidase, a carbon monox-ide-binding pigment, and flavoproteins were detectable in the spectra of both intactcells and mitochondria. Cytochrome c555 is a basic protein that was extracted fromcells and mitochondria with salt solutions. The molar ratio of heme c to heme a wasapproximately 2:1 in both cells and mitochondria. This organism could possiblyserve as a model for studies of the respiratory activity of the pathogenic trypano-somes.

Crithidia fasciculata is a trypanosomatid thatcan be isolated from the intestine and rectum ofthe mosquito, Anopheles quadrimaculatus. Thisorganism requires mitochondrial activity forgrowth but is apparently unable to synthesizeprotoporphyrin (17). The organism can begrown in vitro in the presence of either proto-porphyrin or heme.

C. fasciculata has been reported to have un-usual cytochromes (1). Growth and respirationare inhibited by cyanide, although cytochromeoxidase was reported to be absent (17). Cyto-chromes with absorption maxima at 555 and 530m, were detected with a hand spectroscope (17).Toner and Weber (23) were able to detect cyto-chrome oxidase and cytochrome b but not cyto-chrome c. These authors found a P/O ratio withsuccinate as substrate of 0.7 for a cell-free ho-mogenate of C. fasciculata. In this study, evi-dence is presented which shows that a mito-chondrial fraction that contains the cytochromescharacteristic of the intact organism has beenisolated from C. fasciculata.

Since the pathogenic trypanosomes are verydifficult to obtain in amounts sufficient for bio-chemical studies, the related trypanosomatidC. fasciculata has been examined. It can begrown in vitro with reasonable yields. The mito-chondria of C. fasciculata resemble those foundin the stumpy form of the pathogenic trypano-somes, both in their morphology (12) and intheir response to inhibitors (13).

MATERIALS AND METHODSOrganism and conditions of growth. C. fasciculata

(ATCC no. 11745) was obtained from S. Hutner ofHaskins Laboratories, New York, N.Y. The protozoa

were grown at 25 C in a 22-liter bottle that was inocu-lated with 200 ml of cells in the logarithmic phase ofgrowth. The culture was stirred with a Teflon mag-netic stirrer and gassed with air at a rate of 24 liters/min through a glass sparger. The medium contained0.5% Trypticase (BBL), 0.5% yeast extract (Difco),0.01% liver extract concentrate (Nutritional Bio-chemicals Corp., Cleveland, Ohio), and 0.05 M sucrose.The pH was adjusted to 8.0 with KOH. After themedium was autoclaved for 2 hr at 121 C, it wascooled, and protoheme was added to a final concen-tration of 3.0 ,uM. The stock solution of protoheme wasdissolved in a small amount of pyridine and wasdiluted with 0.5 M NH4OH to a final concentration of30 mm. This solution was kept in the dark at -16 C.The protoheme was added to the medium after auto-claving because experiments showed that during auto-claving 75% of the protoheme was destroyed. The amaximum of the pyridine hemochrome of the hemeremaining after autoclaving was shifted from 556 to553 m,. Antifoam B (Dow Corning Corp., Midland,Mich.) was used to prevent excessive foaming. Thedoubling time was 5.0 hr, determined as the timenecessary to double the absorbance at 750 m,A (meas-ured in 13-mm test tubes) between absorbancies of0.01 and 0.5. The protozoa were grown to the late-logphase. This usually required 3 to 4 days.

Preparation of mitochondria. Protozoa were har-vested by centrifugation at 7,000 X g for 10 min andwashed with a buffer containing 0.25 M sucrose, 0.1mM ethylenediaminetetraacetic acid (EDTA), and 0.1M tris(hydroxymethyl)aminomethane (Tris), pH 7.6.The protozoa were transferred to a cold mortar.Alumina (neutral alumina, AG-7, 100-200 mesh,Bio Rad Laboratories, New York, N.Y.) was addedat a ratio of 2 g per g (wet weight) of protozoa. Thepaste was ground for 2 mmn, at which time examina-tion of the slurry by phase-contrast microscopy indi-cated 98 to 100% cell breakage. The mixture wascentrifuged at 1,000 X g for 10 min to remove the celldebris, whole cells, and alumina. The pellet was

2151

HILL AND WHITE

washed twice with the buffer, and all the supernatantliquids were combined. The mitochondria were col-lected from the supernatant fluids by centrifugationat 16,000 X g for 10 min. The mitochondrial pelletwas washed twice with the buffer. All the above pro-cedures were performed at 0 to 4 C. The pellet con-sisted of mitochondria and associated kinetoplastsexamined with the electron microscope (G. C. Hill,Ph.D. Thesis, New York Univ., New York, N.Y.,1968). No nuclei were present, but dense bodies whichwere observed in the intact cell were present.

Difference spectra. The difference in absorptionbetween anaerobic and aerobic suspensions of intactcells or mitochondria was measured with the tech-nique developed by Chance (3). A suspension of cellsor mitochondria with their respiratory pigments re-duced was compared to a portion of the same suspen-sion with the respiratory pigments oxidized. Spectrawere obtained with a Cary model 14 CM dual-beamrecording spectrophotometer, as described (27). Theintact protozoa had endogenous respiration whichled to reduction of the respiratory pigments in theoxidized sample. The endogenous reduction of therespiratory pigments was decreased by aerating thesuspension of cells for 15 min at 0 C before measure-ment. The mitochondria did not have significantendogenous respiration. The respiratory pigments inthe mitochondria were reduced in the presence of 10mM succinate. The reduction was complete in 60 minat 25 C. The interaction of carbon monoxide and thereduced respiratory pigments was measured by com-paring a suspension of cells with pigments reducedand saturated with carbon monoxide to a part of thesame suspension in which the pigments were reduced.Difference spectra were measured at 25 and -190 C.The low-temperature spectra were measured in anapparatus similar to that described by Estabrook (7)and constructed by T. Orr. Absolute spectra were ob-tained by comparing suspensions of protozoa to anti-foam suspensions or ground glass of proper light-scattering properties, as described (19).

Protein. Protein was determined colorimetricallywith the Folin-Ciocalteu phenol reagent (HarlecoCorp, Philadelphia, Pa.) after alkaline hydrolysis(32) with bovine albumin as standard.

Extraction of cytochrome c555. Cytochrome c555was extracted from mitochondria swollen with hypo-tonic KCI according to the method of Jacobs andSanadi (14). The cytochrome solution was dialyzedovernight against 20 mm sodium phosphate buffer,pH 8.0, and applied to an Amberlite IRC-50 columnpreviously equilibrated with the phosphate buffer.The cytochrome was eluted from the cation resinwith 1.0 M NaCl in 0.02 M phosphate buffer.

Cytochrome c555 was extracted from protozoa,essentially as described by Margoliash and Walesek(18). The cells were suspended (40 g, wet weight) in40.0 ml of 1.5 ,uM aluminum sulfate, pH 4.5, and sub-jected to ultrasonic vibration for a total of 15 min.This treatment resulted in the rupture of nearly allthe cells. The broken cell preparation was centrifugedat 16,000 X g for 5 min, and the supematant liquidwas filtered through fluted Whatman no. 588 paper.The temperature was maintained below 5 C duringthese operations. The pH was adjusted to 8.5 with

NaOH, the filtration was repeated, and the solutionwas applied to an Amberlite IRC-50 column. Thecytochrome c555 was completely reduced as isolatedfrom column chromatography.

Extraction of the hemes. Protoheme and heme awere extracted from the cells or mitochondria asfollows. A mixture of acetone-water-concentratedHCl (50:10:2.5) was added to the protozoa or mito-chondria at 0 to 4 C. The suspension was homogenizedwith a Teflon homogenizer and centrifuged at1,000 X g for 10 min. The cold acid acetone extrac-tion was repeated four times. The pellets were sus-pended in water, and the heme c was determined bypreparing the pyridine hemochrome. The superna-tant portion of the acid acetone extraction was thenextracted with ether in the presence of HCl. The aque-ous layer was back extracted with more ether. Thecombined ether layers containing protoheme andheme a were washed with two portions of 0.27 M HCI,and the ether was evaporated to dryness in vacuo. Thepyridine hemochrome of the residue was then pre-pared as described by Falk (8). The concentration ofthe hemes was measured from the reduced minusoxidized difference spectra of the pyridine hemo-chromes by a procedure described by Falk (8). Heme awas measured as the absorbance increment betweenthe a maximum at 587 m,u and the minimum at 610m, with e = 21.7 X 103 (20); heme c was measuredas the absorbance increment between the a maximumat 553 m,u and the minimum near 535 mMA between thea and ,B maxima with E = 22.0 X 103 (15); protohemewas measured as the absorbance increment betweenthe a maximum at 556 m,u and the minimum at 540m,u between the a and , maxima with e = 20.7 X 103(30).

Oxidative phosphorylationi. Oxidative phosphoryla-tion was measured with conventional manometrictechniques (24). In the determination of oxidativephosphorylation, each vessel contained 2.0 mm adeno-sine triphosphate (ATP), 40.0 mm glucose, 20.0 mMHP04=, 6.67 mm Tris buffer, pH 7.4, 250 mm sucrose,6.67 mm MgCl2, 16.67 mm KF, 0.3 mm EDTA, and0.5 mg of yeast hexokinase (Type II; Sigma ChemicalCo., St. Louis, Mo.). The final volume was 3.0 ml.The flasks contained between 60 and 120 mg of mito-chondrial protein. Succinate (10 mM) was tipped inafter 15 min of equilibration. Phosphate disappearancein the presence of glucose and hexokinase was usedas a measure of ATP formation. Inorganic phosphatewas determined by the method of Bartlett (2), asadapted for the Technicon autoanalyzer by R. L.Lester.

Oxygen utilization. The initial oxygen uptake of-mitochondria or cells was measured poloragraphically-in the apparatus described by White (28).

Materials. The diethyl ether was washed with acidi--fied ferrous sulfate solution to remove peroxides (8).Other materials were the best grade commerciallyavailable. The chicken heart cytochrome c was pro-vided by S. K. Chan.

RESULTS

Spectra of the protozoa. The spectrum of the-protozoa with the respiratory pigments reduced

2152 J. BACTERIOL.

RESPIRATORY PIGMENTS OF C. FASCICULATA

by the activity of endogenous metabolism isgiven in Fig. 1. Cytochrome b with an a maximumat 560 m,u can be detected. The addition ofdithionite to the cell suspension produced an 11%increase in the absorbancy increment at 560 m,u.Nearly all the protoheme that could be extractedfrom the protozoa with acid acetone was ac--counted for in the cytochrome b. Approximately87% of the 3.8 mAmoles of protoheme per 10 mgof protein could be reduced by the endogenousrespiratory activity of the protozoa.A difference spectrum in which the protozoa

with pigments reduced by endogenous metabolismwere compared to cells in which the pigmentswere oxidized by bubbling with air for 15 min at'OC is illustrated in the lower part of Fig. 1.Approximately 26% of the total enzymaticallyTeducible cytochrome b can be detected in the

0.06

0.02 - /

E 500 550 600 650

zw2 0.06

zw 0.04

000 -,,

I,

400 450 500 550WAVELENGTH mjI

difference spectrum. Cytochrome b with maximaat 560, 527, and 430 m,u can be detected. The amaximum at 604 m,u and the 'y maximum at 444mu suggest the presence of cytochromes of the atype. When reduced cytochrome oxidase a3 reactswith carbon monoxide, a shift in the a maximumto 590 m,u and a shift in the y maximum to 430m,u occur (16). In the difference spectrum ofC. fasciculata between cells with pigments reducedand cells with pigments reduced but saturatedwith carbon monoxide, there was a decrease inthe absorbance at 443 m,u but no detectable in-crease in absorbance at 430 or 590 m,u.A pigment with absorbance maxima at 570,

540, and 419 m,u appeared after the reaction withcarbon monoxide. This pigment has the spectralcharacteristics of bacterial cytochrome oxidaseo (22). Complete reduction of the pigments bythe addition of dithionite resulted in a fourfoldincrease in the pigment resembling cytochromeoxidase o. This increase may be due to cyto-chrome oxidase o which is not reduced completelywith succinate. It is also possible that the CO-binding pigment is a protohemoprotein complexdue to nonspecific binding of hemin in the growthmedium.

Difference spectra of the mitochondria. Differ-ence spectra of the mitochondria isolated fromC. fasciculata are illustrated in Fig. 2. In thesespectra, the respiratory pigments were allowedto reduce in the presence of 10 mm succinate for

0.04

600 650 2z 0.03z

FIG. 1. Spectra of Crithidia fasciculata. In theupper curve, the straight line represents the absorptionof intact protozoa in which respiratory pigments werereduced by the endogenous metabolism ofthe cells com-pared to an aqueous suspension of antifoam B of suit-able light-scattering properties (29); dashed line indi-cates the spectrum in which the respiratory pigmentswere reduced in the presence ofdithionite. In the lowerpart ofthe figure, straight line represents the differencespectrum between a cell suspension in which respiratorypigments were reduced by endogenous metabolism com-pared to a portion of the same suspension in which therespiratory pigments were partially oxidized by vigor-ous aeration for 15 min at 0 C; dashed line repre-sents the difference spectrum ofthe cell suspension withpigments reduced by endogenous metabolism and satu-rated with carbon monoxide compared to part of thesame suspension in which the respiratory pigments werereduced. The cells were suspended in 0.25 M sucrose,0.1 mM EDTA, and 0.1 m Tris buffer at pH 7.6 at a

density of 10 mg of protein per ml. The temperaturewas 25 C, and the optical path length of the cuvettes.was 10 mm.

2: 0.02z

4 0.01

4D OD

-0.01

I -

I, -- - - /-

360 400 440 460 520 560 600 640WAVELENGTH mFL

FIG. 2. Difference spectra of mitochondria ofCrithidia fasciculata. Straight line represents the differ-etice spectra of mitochondria with pigments reduced inthe presence of 10 mM succinate compared with mito-chondria with the pigments oxidized by aeration. Noadditional reduction ofpigments occurred after 60 minin the presence of succinate. Dashed line representsthe difference spectrum ofmitochondria reduced in thepresence of succinate and saturated with carbon mon-oxide compared with mitochondria reduced in the pres-ence of succinate. The mitochondria were suspendedin buffer at a concentration of1.5 mg ofmitochondria-prote in per ml as in Fig. 1.

2153.VOL. 95, 1968

HILL AND WHITE

60 min at 25 C. Incubation for longer than 60min did not result in further reduction of thecytochromes. Other substrates such as reducednicotinamide adenine dinucleotide (NADH),DL-a-glycerol phosphate, and pyruvate + malateat 10 mm did not decrease the time required forreduction of the cytochromes or the amounts ofthe cytochromes that were reduced. Addition ofdithionite to the mitochondria resulted in thereduction of 50% more cytochrome, indicatingthat in mitochondria prepared with aluminafrom cells grown in 3.0 ,uM hemin only half thecytochrome is enzymatically reducible.There was a marked sharpening of the a

absorbance maxima if the spectra were measuredat -190 C. Figure 3 illustrates the differencespectrum of mitochondria reduced in the presenceof succinate minus the absorbance of mitochon-dri:i whose pigments were oxidized. In additionto the a maximum of cytochrome b at 556 m,u,there was a distinct shoulder near 554 m,u. Theshoulder was more prominent in a differencespectrum of mitochondria reduced in the presenceof succinate minus mitochondria reduced in thepresence of succinate and 10-4 M antimycin.

Properties of extracted cytochrome c555. Acytochrome c with an a maximum at 555 m,ucould be extracted from both mitochondria andruptured cells with salt solutions. The cytochromewas adsorbed on Amberlite IRC-50 at pH 8.5and eluted from this resin with 1.0 M NaCl in0.02 M sodium phosphate buffer, pH 8.2. Theseare properties very similar to those of mammaliancytochrome c. The absolute spectrum of theisolated cytochrome C555 is illustrated in Fig. 4.There were maxima at 555, 525, and 420 m,u at25 C. When measured at -190 C, there weremaxima at 555 and 551.5 m,u, 531 and 523.5 m,u,and 419 mu. The pigment showed maxima at563, 526, and 413 m,u when oxidized in the

iABSORBANCE INCREMENT (crn)

I 001

420 440

500 520 540 560 580 600 620WAVELENGTH mM

FIG. 3. Difference spectra of the mitochondria ofCrithidia fasciculata measured at -190 C. Differencespectra of the mitochondria reduced in the presence ofsuccinate compared with mitochondria oxidized byaeration, whose absorbance is illustrated with the solidline in Fig. 2, were measured in leucite cuvettes with1-mm optical path at -190 C. In Fig. 2 the opticalpathwas 10 mm.

0.

E 0.4 8 0.08 ,

005[ 0.0Ix

0~~~~~~0

cn *. 0.062 'co~ ~ ~ ~ ~ ~ I

380 420 460 500 500 540_ 580 620WAVELENGTH mIL

FIG. 4. Spectra Of reduced cytochrome C555 fromCrithidia fasciculata. Upper curve represents the abso-lute spectrum of the reduced cytochrome C555 extractedfrom cells and concentrated by column chromatographyas described in Materials and Methods and measuredat 25 C. Lower curve represents the absolute spectrumof the same preparation measured at -190 C in leucitecuvettes of I mm-path length.

presence of 10-3 M ferricyanide. The reducedpyridine hemochrome of the isolated cytochromec550 had an a maximum at 553m,0, whereas thatof chicken heart cytochrome c was 550 m2.Onthe basis of the heme content calculated from thereduced pyridine hemochrome of the isolatedcytochrome C555, the extinction coefficient at 555mlu for the reduced cytochrome was e = 29.3 X10f. Assuming a molecular weight of 12,500 andone heme per protein molecule, the cytochromeC55Crepresents 40% of the protein at this stage ofpurification.The salt extraction removed approximately 1.2

cvmoles of cytochrome C555 (measured as heme c)per kg (wet weight) of protozoa. About 19r ofthe total heme c of the cells was removed by thesalt extraction. The residual cytochrome c couldrepresent tightly bound cytochrome C555, cyto-chrome cl, or both.

Estimatiof of heme a, heme c, and protoheme.Hemes that are not covalently bound to theapoproteins can be removed by extraction withacid acetone (8). The protoheme and heme a canbe extracted with acid acetone from intact cells ormitochondria. Treatment of the protozoa ormitochondria with four portions of acid acetoneremoved 99s% of the total heme a and protoheme.The residue after acid acetone extraction con-tained heme c but not detectable heme a or proto-heme as measured by the pyridine hemochrome.The heme of cytochrome c was bound covalentlyto the apocytochrome by thioester linkages andwas not extractable with acid acetone.The relative proportions of the hemes from

three mitochondria preparations and the intact

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RESPIRATORY PIGMENTS OF C. FASCICULATA

protozoa are given in Table 1. The ratios of thehemes in both the intact protozoa and the mito-chondria were essentially identical.

These protozoa were grown with a protohemeconcentration of 3.0 m,umoles of protoheme perml of medium. With this protoheme concentra-tion there was no limitation of the growthrate of the organism. After a growth cycle during-which approximately 0.5 mg of protozoal pro-tein per ml was produced, 38% of the totalprotoheme in the medium at the start of theexperiment was present at the end of the growthcycle. The protozoa incorporated 5.7% of theprotoheme present at the start of the experimentof which 4.1% was found in protoheme, 1.0%was found as heme c, and 0.6% was found asheme a. Approximately 56% of the protohemepresent at the start of the experiment could notbe detected in the media or the protozoa as hemeat the end of the experiment. The fate of theprotoheme which has not been accounted for isunknown.Comparison of the heme c and heme a con-

centrations of the intact protozoa and themitochondria derived from these cells indicatesthat, after isolation, the mitochondria that wererecovered contained 41 % of the total heme a and46% of the total heme c that was found in theintact cells. The isolated mitochondria contained23% of the total protein of cells from which themitochondria were isolated.

Metabolic activities of the isolated mitochondria.The intact protozoa have an initial endogenousrespiratory rate of 29.4 m,umoles of oxygen per

TABLE 1. Heme a, heme c, and protohemeconcentrations in mitochondria andintact cells of Crithidia fasciculata

Alitochondria Ratio'

Component 1 ____1 __E IntactExpt Expt ExptAv cells Mito- itc

1 2 3 dvhna el

Heme ab. 0.88 0.92 0.68 0.83 0.17 1.0 1.0Heme c.. 1.99 2.37 1.45 1.94 0.38 2.3 1.6Proto-heme. 5.83 8.17 3.93 5.98 1.14 6.9 6.9

Ratio indicates the molar proportions of thehemes with heme a taken as 1.0.

b The concentration of heme a and protohemewas determined in the acid acetone extracts ofmitochondria as the reduced pyridine hemo-chromes and expressed as ms4moles of heme/10mg of protein. The reduced pyridine hemochromeof the residue after the extraction was used tomeasure heme c. The reduced pyridine hemo-chromes were prepared and measured as describedin Materials and Methods.

min per mg of protein. The isolated mitochondriahave an initial respiratory rate of 10.3 m,molesof oxygen per min per mg of protein with suc-cinate as the electron doncr. Succinate was usedas substrate for the measurement of the oxidativephosphorylation activity of the isolated mito-chondria. Five experiments were carried out withdifferent mitochondrial preparations. The P/Oratio with succinate as substrate was found to varyfrom 0.5 to 1.0.

DISCUSSION

A significant portion of the electron transportsystem responsible for the respiratory activity ofthe heme-requiring trypanosomatid C. fasciculatais localized in a fraction which contains mitochon-dria when examined in the electron microscope(G. C. Hill, Ph.D. Thesis, New York Univ., NewYork, N. Y., 1968). Evidence for the mitochon-drial localization of the respiratory system is asfollows: (i) the intact cells and the isolatedmitochondria contain the same cytochromes;(ii) these cytochromes are reversibly oxidizedand reduced; and (iii) both the intact cells and theisolated mitochondria contain protoheme, hemec, and heme a in the same molar proportions.Evidence indicates that the present isolationprocedures for the mitochondria may damage theelectron transport system. The proportion ofcytochrome b that is not enzymatically reducibleincreases from 11% in the intact cells to 50% inthe mitochondria. When the cells are grown inlower amounts of hemin (e.g. 0.76 to 1.5 ,M), theamount of the nonenzymatically reducible pig-ment at 559 m,u decreases. Although this may bedue to a nonsubstrate reducible hemoprotein,it is possible that a portion of the electrontransport system is not reduced by succinate orthe other electron donors examined in the study.The respiratory rate per mg of protein decreasesmarkedly after the isolation of the mitochondria,and the P/O ratio of the isolated mitochondria is0.5 to 1.0 and not the expected 2.0 with succinateas the electron donor. About 40 to 50% of theheme a and heme c in the intact cells are recoveredin the mitochondria isolated as described in thisstudy.

Despite previous statements that the respiratorysystem of these organisms lacks cytochromes a +a3 or cytochrome c (1, 23), the mitochondriaof C. fasciculata have many features in commonwith the mitochondria of other systems. Thelevel of cytochromes, the respiratory activity, theextractability of cytochrome C555 with solutions ofhigh ionic strength, and the ratio of heme a andheme c are all typical of other mitochondria (4-6,9, 14, 18, 19, 22, 25). More extractable protoheme

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HILL AND WHITE

is present in this mitochondrial fraction. Acytochrome c with spectra similar to cytochromec555 has been isolated from heterotrophicallygrown Euglena gracilis (31).Changes in the respiratory apparatus are

characteristic of the metamorphosis of thepathogenic trypanosomes (26). The vertebrateforms of the trypanosomes are very difficult toobtain in sufficient quantities for biochemicalstudy. C. fasciculata can be grown in vitro inlarge quantities and forms mitochondria that are

similar to the mitochondria of the stumpy formof the pathogenic trypanosomes. Similaritiesexist in the mitochondrial morphology by electronmicroscopy (12), in the behavior to inhibitors(13), and in the mitochondrial enzyme activities(26). C. fasciculata also forms low levels of the aglycerol phosphate dehydrogenase (C. J. Bacchi,Ph.D. Thesis, Fordham Univ., New York, N.Y.,1968) that is part of the cyanide-insensitiveterminal oxidase in the slender form of thepathogenic trypanosomes (10, 11). C. fasciculatamay serve as an excellent tool for the developmentof more effective chemotherapy that involves thetransition from a cyanide-insensitive to a cyanide-sensitive respiratory system. This transition isessential for the development of infectivity for thevector in the infectious cycle of trypanosomes.

ACKNOWLEDGMENTS

This investigation was supported by contractDADA-68-C-8043 from the Preventive MedicineResearch Branch of the U.S. Army Medical Re-search and Development Command. George C. Hillwas supported by Public Health Service fellowshipAI-36,847 from the National Institute of Allergy andInfectious Diseases.We gratefully acknowledge S. Hutner of the Haskins

Laboratories, New York, N.Y., who provided theorganism, and our colleagues P. Sinclair, R. L. Lester,and S. K. Chan, who provided helpful discussions ofthis investigation.

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