Comparative Biochemistry of Parasites || ADAPTATIONS IN OXIDATIVE METABOLISM DURING THE TRANSFORMATION OF TRYPANOSOMA RHODESIENSE FROM BLOODSTREAM INTO CULTURE FORM

22

A D A P T A T I O N S IN O X I D A T I V E M E T A B O L I S M D U R I N G THE T R A N S F O R M A T I O N OF TRYPANOSOMA RHODESIENSE

FROM B L O O D S T R E A M INTO C U L T U R E FORM.

I.B.R. Bowman, H.K. Srivastava* and I.W. F lynn Department of Biochemistry

University of Edinburgh Medical School

Teviot place

Edinburgh, EH8 9 A G , U.K.

Types of infect ion by Τ rhodesiense in mammalian blood may vary f rom those of an acute character w i th rapidly increasing parasitaemia and early death of the host animal to chronic types w i t h many remissions and relapses. The variat ion can be related to the degree of p leomorphism of the strain used: monomorph ic strains consisting of long slender (LS) trypomastigotes cause an acute infect ion whereas pleomorphic strains cause the more chronic type of in fect ion. In a relapsing infect ion long slender forms predominate as the parasitaemia increases and in the remission phase they are replaced by a high percentage of short s tumpy (SS) cells. A th i rd morphological cell type found in p leomorphic strains, the intermediate short s tumpy (ISS) f o rm , may represent a developmen-tal stage of the SS fo rm. Loss of the pleomorphic character as occurs in syringe passaged, rodent adapted strains is accompanied by a loss of infect iv i ty to other mammals and to the tsetse f l y and these LS forms can not be cul tured. From this it is concluded that the SS fo rm is an essential transit ional stage between the blood f o r m and epimastigote. In addit ion to considerable differences in morphology a number of bioche-mical features distinguish LS & SS trypomastigotes f rom culture epimasti-gotes. In summary the LS fo rm lacks a mi tochondr ion , a t r icarboxy l ic acid cycle and a cytochrome system

( 1-

2 ) . Oxygen uptake is mediated by

L-glycerol-3-phosphate (a GP) ox idase( 3)

located in discrete ext rami to-chondrial pa r t i c les

( 4 ). In contrast the culture epimastigote has a highly

developed m i t o c h o n d r i o n( 5 )

, possesses a funct ional T C A c y c l e( 6)

and a cytochrome mediated electron transport s y s t e m

( 7 ). It is the SS fo rm

* Present address: Department of genetics, Haryana agricultural university, Hissar, India.

329

I. Β. R. B O W M A N , H. K. S R I V A S T A V A , A N D I. W. F L Y N N

Substrate ox idat ion by the blood stream forms

Table 1 shows that the most str ik ing feature of the metabolism of the SS forms is the abi l i ty to oxidise a-oxoglutarate in addi t ion to glucose and glycerol ; on ly the latter two substrates are oxidised by the LS fo rm. This f inding of α-oxogIutarate supported ox idat ion is not surprising as i t had been shown previously

(8<

9· 1 0)

t o maintain the mo t i l i t y of the SS forms selectively. None of the other substrates tested was oxidised by whole cells. However, it can be seen that in lysed cell preparations pyruvate is oxidised almost as well as α-oxoglutarate suggesting that pyruvate is impermeable to the intact trypanosome membrane. Pyruvate and a-oxo-glutarate oxidative decarboxylases have been synthesised by the SS stage and distinguish clearly the SS or ISS f rom LS forms. It is l ikely that the activities of other enzymes of the TCA cycle are l imi t ing in the SS fo rm as the intermediates of this cycle are poor ly oxidised. The products of metabolism of glucose, α-oxoglutarate and pyruvate are shown in Table 2. The carbon balances w i th glucose and a-oxoglutarate were obtained w i th intact cells, pyruvate w i t h lysed cells. It is signif icant that only 60 % of the glucose carbon used is metabolised to pyruvate in the SS forms - the LS fo rm produces ( 90 % as pyruvate. 10 % of carbon is found in CO2, 7 % in succinate and 9 % in acetate none of which is produced t o any signif icant extent by LS s t a g e s

( 1 2 ). Glycerol (8%) is

produced f rom aGP by a phosphatase which is extremely active in trypanosomes. It can be seen that α-oxogIutarate is simply decarboxyla-ted to succinate and CO2. The small percentage of pyruvate may result f rom the further slow metabolism of succinate. In lysed preparations pyruvate is decarboxylated to acetate and CO2. It can be concluded that the SS stages develop a chondr iome and w i t h it some of the enzymes of the TCA cycle in particular the oxidative decarboxylases, but succinoxi-dase seems to be l imi t ing due either to a lack of the f lavoprotein

330

which init iates the metabolic t ransformat ion or respiratory switch in Vickerman's t e r m i n o l o g y

( 8) between these disparate LS and culture

forms. Vickerman ( 8)

has shown that the SS congener has devloped a mitochondr ia l tubule containing cristae and NADH-te t razo l ium reductase act iv i ty which may be related to the acquired abi l i ty of these stages t o use a-oxoglutarate as an energy source

( 8> 9>

1 0 ). These results suggested

that a metabolic switch to in t ramitochondr ia l ox idat ion had occurred. Our e v i d e n c e s u g g e s t s that this switch is incomplete in the blood-stream stages and this report describes the sequence of metabolic changes in the transit ions LS to SS to epimastigote forms.

C O M P A R A T I V E BIOCHEMISTRY OF PARASITES

Metabolic Transformation in Culture

SS forms were isolated under sterile condit ions f rom rat b lood and transferred into the blood-agar biphasic system w i th Earle's saline overlay of Tobie, Mehlman and von Brand

( 1 3 ). The rates of oxygen uptake by

lysed cells at various stages of development in the bloodstream and of t ransformat ion in cul ture were fo l lowed polarographically at 26° using a-oxoglutarate, N A D H , succinate, L-proline and ^-glycerophosphate. The metabol ic changes occurring during transformat ion of SS into culture epimastigote f o rm were studied and the results presented in Figures 1 and 2. In Figure 1 i t can be seen that LS forms do not oxidise succinate or prol ine and the rates of oxygen uptake w i th these substrates is minimal in cell free preparations of trypomastigotes in which 75 % were in the SS or ISS fo rm. The rate of ox idat ion of a GP by bloodstream forms (LS and SS) was highest of all substrates tested (1.3 Mmol 02/mg prote in /h) . However, w i th in seven days of transfer in to cul ture, oxygen uptake supported by succinate and prol ine approaches the max imum rate of the established (»<=>) culture f o rm and the rate of prol ine ox idat ion is twice that of succinate and a GP. There is no signif icant increase in the oxidat ion rate of a GP in the culture forms. This could indicate the persistence of α-glycerophosphate oxidase, though this is not the on ly possibil i ty as this oxidat ive process becomes cyanide sensitive in the culture fo rm.

In Figure 2 i t can be seen that there is l i t t le increase in the ox idat ion rate of N A D H w i th increasing propor t ions of SS forms but w i th in 24 h of cul tur ing this rate increases to 0.5 Mmol 02/mg prote in/h and stays constant for 3-4 days then approaches a max imum of 1.3 Mmol 02/mg protein/h at 7 days, α-oxoglutarate is not oxidised by LS forms but as has already been noted, this substrate is oxidised by SS forms to about

331

dehydrogenase or to a lack of a cy tochrome electron transport system. Cytochromes are not detectable in the SS forms therefore N A D H generated in ox idat ion processes is reoxidised by a GP oxidase. It is possible that an autoxidisable f lavoprotein is present as an alternative oxidase as i t can be shown that in condi t ions in wh icha GP oxidase is inactive, where DHAP as electron acceptor is removed by gel f i l t ra t ion , there is sti l l a residual oxygen uptake in the presence of N A D H (5-15% of the original act iv i ty) which is rotenone and amytal sensitive. The metabolic t ransformat ion of blood forms to mi tochondr ia l mediated metabolism is in i t iated at the SS stage but is not completed unt i l transfer to culture or the insect vector.


Cyanide Sensitivity of Developing Culture Forms

Table 3 shows the effect of cyanide (3 mM) on the oxygen uptake supported by the test substrates. There is a gradual increase in inh ib i t ion by cyanide in the bloodstream stages and early culture stages and after 3 days in cul ture the ox idat ion of N A D H , succinate, a-oxoglutarate and proline is who l l y inhibi ted by cyanide. The concentrat ion of cyanide at 3 mM is excessively high compared to those concentrations (10~5 - 10~6 M) required t o cause complete inh ib i t ion of cy tochrome aa3 in mammalian mitochondr ia. Lower concentrat ions (10~5 M) inhibi ted oxygen uptake by only 50 % (Figure 3) . It should be noted that a GP ox idat ion has become cyanide sensitive suggesting that the cytoplasmic a GP oxidase has been superseded by a cytochrome dependent a GP oxidase system. It is concluded that the cytochrome oxidase of the culture fo rm is not of the aa3 type but some other cytochrome oxidase less sensitive to cyanide inhib i t ion and that this is synthesised w i th in 3 days in culture.

Cytochromes of Established Culture Forms

In the light of the poor inh ib i t ion by cyanide spectral analysis of the epimastigotes was carried out to ident i fy the terminal oxidase. Interpreta-t ion of the spectra was complicated by contaminat ion of the trypanoso-me preparation by haemoglobin and haemiglobin f rom the blood agar biphasic cul ture m e d i u m

( 1 3 ). T. rhodesiense E A T R O 173 is easily

established in culture and has been sub-cultured for over one year. The culture organisms were harvested and washed nine t imes in an a t tempt to

332

50 % of the max imum oxidat ion rate. The difference in the rates of ox idat ion of a-oxoglutarate quoted in Table 1 and Figure 2 are due to the di f ferent assay temparatures, 37° and 26° respectively. Max imum rates of ox idat ion of α-oxogIutarate and N A D H are obtained w i th in 7 days of transfer to culture. The progress curve of prol ine ox idat ion is redrawn in Figure 2 for comparative purposes and shows again that its rate of ox idat ion is twice that of other substrates tested. With the exception of a GP it can be stated that there are marked increases in the rates of ox idat ion of the marker substrates w i th in 3-4 days of transfer of trypomastigotes into culture and these rates reach the values found in established culture epimastigotes w i th in 7 days at which t ime all trypanosomes have transformed morphological ly in to culture forms.


333

remove the last trace of haemiglobin and the cells were then sonicated. The difference spectrum (Figure 4 , lower curve) of d i th ion i te reduced versus oxidised (aerated) preparations shows an a band at 555-556nm, β band 524-526 nm and Soret band at 430 nm showing the presence of cytochromes b and c, but al though there is a small peak at 600 nm the absence of a peak at 444 nm shows that cytochrome aaß is absent. The upper curve (Figure 4) is the CO-difference spectrum giving peaks at 570, 540 and 418 nm which is consistent w i th the presence of cytochrome o. The absence of peaks at 590, 550 and 430 nm is a fur ther indicat ion of the lack of cytochrome aa3. It should be emphasized that a contaminant CO complex w i t h haemoglobin wou ld give the same spectrum. The CO difference spectrum also showed a small peak at 625-628 nm. Since the methaemoglobin of the culture medium may be present intracel lularly and therefore wou ld not be removed by washing of the cells, a mitochondr ia l preparation was made f rom cells sonicated in 0.3M sucro-se, 24 mM tris, 1 mM EGTA, pH 7.4 for 3 min at 3 A m p . The mixture was centr i fuged at 1250 g fo r 10 min and the supernatant f lu id centrifuged at 15,000 g for 10 min. The resulting pellet was washed 3 times by repeated centr i fugat ion and f inal ly suspended in 100 mM phosphate buffer, pH 7.4. The difference spectra of this mi tochondr ia l f ract ion are given in Figure 5. The lower curve is the reduced (di th ioni te) versus oxidised (aeration) spectrum in which the α, β and y peaks are rather sharper than w i th unfract ionated lysates. The middle spectrum is obtained when cyanide is added t o the reduced sample. No change is observed in the posit ions of the maxima but the peak heights are considerably potent iated. These spectra indicate the presence of cyto-chromes b and c and again the absence of cytochrome aaß. The small peak at about 600 nm shown in Figure 4 is also to be found in the mitochondr ia l preparations. The CO difference spectrum w i th peaks at 570 nm, 540 nm and 418 nm identif ies cytochrome o. In those experiments w i t h broken cells or mi tochondr ia l f ract ions any contami-nating methaemoglobin wou ld be reduced by d i th ion i te to haemoglobin and so give an identical spectrum w i t h CO as cytochrome o. This possibil i ty can be avoided by metabolic reduction of the cytochrome system in washed intact cells. Glucose (10 mM) was used as reducing substrate. The reduced minus oxidised spectrum is shown in Figure 6 (lower curve) along w i th the CO difference spectrum obtained by saturating a metabol ical ly reduced cell suspension w i t h CO and setting this against a metabol ical ly reduced reference suspension. The results are essentially the same as before showing the presence of cytochromes b, c and ο and the absence of aa3.


Pyridine Haemocromes

Pyridine haemochrome of the acid acetone extract of T. rhodesiense Pittam gave absorption bands at 556, 526 and 418 nm consistent w i t h presence of haem b. The reduced pyr idine haemochrome of the acid acetone insoluble residue had absorption peaks at 553, 520 and 415 nm similar to the values quoted by H i l l

( 1 5'

1 6) for haem c derived f rom

cytochrome C555 isolated f rom C. fasciculata or 7". rhodesiense. A similar cytochrome C555 has recently been isolated f rom C. oncopelti

(17). It is

concluded that the strains of T. rhodesiense examined here contain this atypical cytochrome C555 .

General conclusions

The epimastigote fo rm of T. rhodesiense has an atypical cytochrome system, consisting of cytochromes b, C555 and o, and this system is synthesised w i th in 3-4 days after in t roduct ion of the organisms to culture. Depending perhaps on strain differences or on culture condit ions, a second CO-binding pigment w i th some of the properties of cytochrome d may be present. Mul t ip le cytochrome oxidases have been reported in C. fasciculata^

b) and in C. oncopelti

(18).

Concomitant w i th the development of this cytochrome system in the early stages of culture T. rhodesiense EATRO 173, there is a marked development of enzyme systems for the oxidat ion of N A D H , succinate and proline, and a potent iat ion of the oxidat ive metabolism of a-oxoglu-

334

Difference Spectra o f T. rhodesiense Pittam Strain.

This established culture fo rm was grown in bulk in the blood broth medium of P i t t a m

( 1 4) and an acetone dried powder prepared. The

d i th ion i te reduced minus ferr icyanide oxidised spectrum of this material is shown in Figure 7. Peaks at 555-556, 526 and 430 n m indicate cytochromes b and c. The small peak at 608-610 nm is not indicative of aaß since there is no peak at 444 nm. Furthermore the CO-difference spectrum (upper curve) shows no band at 590 nm of aaß; instead there is a marked peak at 630 nm of some other CO binding pigment, perhaps cytochrome d. The other bands in the CO spectrum are, as in the case of freshly prepared T. rhodesiense EATRO 173, most l ikely due to cyto-chrome o. There appears to be l i t t le CO binding pigment tentat ively ident i f ied as cytochrome d in the E A T R O 173 strain, as the ext inct ion at 630 nm is very small and not present in all preparations.


tarate. The high rate of prol ine oxidat ion in the established culture

(insect mid-gut) f o rm parallels the dependence of tsetse f l y tissue on

proline as an energy s o u r c e( 1 9 )

. Whereas the bloodstream trypomastigotes

of T. rhodesiense show a str ict requirement for carbohydrate, i t is

possible that the epimastigotes rely upon the ox idat ion of prol ine and

other amino-acids as an energy source, as does the insect host.

This work was supported by grants f rom the Trypanosomiasis Panel o f

the Overseas Development Admin is t ra t ion , U.K.

References

1. R Y L E Y , J.F. (1956). Biochem. J . f 62, 215. 2. F U L T O N , J.D., and SPOONER, D.F. (1959). Exp. Parasit, 8, 137. 3. G R A N T , P.T. and SARGENT, J.S. (1960). Biochem. J . , 76, 229. 4. BAYNE, R.A., MUSE, K.E. and ROBERTS, J.F. (1969). Comp. Biochem.

Physiol., 30 , 1049. 5. V I C K E R M A N , K. (1970). The Afr ican Trypanosomiases, edited by Mull igan, H.W.

(George Al len and Unwin L td . , London) p. 60. 6. BRAND, T. von, TOBIE, E.J. and M E H L M A N , B. (1950). J. Cell Comp. Physiol.,

35, 273. 7. R Y L E Y , J.F. (1962). Biochem. J . , 85, 211 . 8. V I C K E R M A N , K. (1965). Nature, 208, 762. 9. BALIS, J. (1964). Rev. Elev. Med. Vet. Pays. Trop. , 17, 3 6 1 .

10. R Y L E Y , J.F. (1966). Proc. Intern. Congr. Parasitol., Rome, (Pergamon Press, Oxford) p. 4 1 .

11. BOWMAN, I.B.R., F L Y N N , I.W. and F A I R L A M B , A . H . (1970). J. Parasit., 56, 402. 12. GRANT, P.T. and F U L T O N , J.D. (1957). Biochem. J. , 66, 242. 13. TOBIE, E.J., von B R A N D , T. and M E H L M A N , B. (1950). J. Parasit, 36, 48. 14. P ITTAM, M.D. in D I X O N , H. and W I L L I A M S O N , J. (1970). Comp. Biochem.

Physiol., 33, 127. 15. H I L L , G.C. and WHITE , D.C. (1968). J. Bacteriol., 95, 2151 . 16. H I L L , G .C , GUTTERIDGE, W.E. and MATHEWSON, N.W. (1971). Biochem.

Biophys. Acta (in press). 17. PETTIGREW, G. and MEYER, T. (1971). Biochem. J. (in press). 18. S R I V A S T A V A , H.K. (1971). FEBS Letters (in press). 19. BURSELL, E. (1966). Comp. Biochem. Physiol., 19, 809.

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Mmol 0 2 / m g Protein/h

Substrate Whole Cells Lysate

Glucose 8.3 (12 ) * 2.00 (6)

o:-oxoglutarate 3.95 (4) 1.00 (11)

Glycerol 7.95 (3) -

L-a-glycerophosphate - 2.40 (3)

Pyruvate 0.00 (5) 0.78 (9)

Succinate 0.00 (4) 0.23 (5)

Citrate 0.00 (2) 0.23 (3)

Isocitrate 0.00 (2) 0.28 (2)

Fumarate 0.00 (2) 0.08 (4)

Malate 0.00 (2) 0.11 (3)

* Figures in parenthesis denote numbers of determinations.

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Table 1. Substrate ut i l isat ion by 7". rhodesiense E A T R O 173 SS Substrates (25 μητιοΙ) were incubated w i t h whole cells (1.1 mg protein) in Krebs saline (3 ml) in conventional Warburg respirometers w i t h KOH in centre wells. Cell lysates (5.7 mg protein) were suspended in a reaction mixture (3 ml) containing 3 mM EDTA, 25 mM nicot inamide, 5 mM KCl, 5 mM MgCl2, 66 mM potassium phosphate buffer, pH 7.4, 30 mg bovine plasma albumin, substrates (25 Mmol) and cofactors ADP and NAD (5 μητιοΙ). Rates of oxygen uptake were measured manometr ical ly at 37° over the f i rst 30 minutes.


Substrate

Product Glucose a -oxoglutarate Pyruvate

Pyruvate 6 0 3 —

C 0 2 1 0 2 0 3 1

Succinate 7 7 5 -Glycerol

00 - -Acetate 9 - 6 2

Citrate < 1 - -Hexose phosphate < 1 - -Phosphoenolpyruvate < 1 - -

94% 9 8 % 9 5 %

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Table 2. Metabolic products of T. rhodesiense E A T R O 173 SS Whole cells ( 1 . 1 mg protein) were incubated w i t h glucose ( 2 5 Mmol) or

a-oxoglutarate ( 2 5 Mmol) in saline ( 3 ml ) . Samples were taken at zero

t ime and after 3 5 minutes, deprotenised w i t h HCIO4 ( 0 . 5 ml , 0 . 3 3 M )

and neutralised w i t h K2HPO4 ( 0 . 6 M ) prior to analysis. Lysates ( 5 . 7 mg

protein) were incubated w i t h pyruvate ( 2 5 μ π γ ι ο Ι ) in the fo r t i f ied reaction

mixture given in Table 1 . Results are expressed as a percentage of

substrate uit l ised.


Table 3.

The effect of cyanide on the oxidative metabolism of lysates of T. rhodesiense

Condit ions were the same as those described in Figure 1. A f te r a steady rate of oxygen uptake was recorded w i t h each of the substrates, KCN (0.1 ml , 30 mM) was injected in the chamber.

% sensitivity t o KCN (3 χ 10"3M)

a -oxoglu- Proline Succinate N A D H ta rate

Bloodstream forms

LS 0.0 0.0 0.0 2.5

S S ( 7 5 $ ) 25.0 10.0 3.8 15.0

Culture forms

1-day 31.0 19.3 19.4 26.0

3-day 100.0 100.0 100.0 100.0

Established 100.0 100.0 100.0 100.0

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* ISS 0 25 50 75

BLOOD S T R E A M FORMS

D A Y S IN C U L T U R E

CULTURE F O R M S

Fig. 1 .

Water lysed cells were incubated in the fo r t i f i ed reaction mix ture (3 ml) given in Table 1, at 26° in a polarograph chamber f i t t ed w i th a Clark oxygen electrode. A f te r a steady endogenous rate of oxygen uptake was obtained substrates (5 Mmol) were injected by microsyringe, ® , L-glyce-rol-3-phosphate; o, L-prol ine; ^ , succinate.

3 0

2 5 -

1 2 0 -

\ 15

2 1 0

I

0-5

0 0

^ —°-\~ιί—°

L-PROLINE /

/ ? «Î-OXOGLITARATE

/ , - - - / / · — i

χ / y

L S 100 75 50 25 ' I S S 0 25 50 75 D A Y S IN C U L T U R E

η — ι — ι — ι ι ι— 1 2 3 U 5 6 7

BLOOD S T R E A M FORMS CULTURE FORMS

Fig. 2.

Condit ions as in Figure 1. X , a-oxoglutarate; • , N A D H ; o, L-proline.

3 3 9

I I «-oxoglutarate <x-glycerophosphate

100-

1

SEHS

ITIVIT

Y PE

RCEN

T a

ε

1 I 10" 5M 10 •

4M 10 3M KCN

Fig. 3. Condit ions as in Figure 1. ^-glycerophosphate or a-oxoglutone (5 μιτιοΙ) was injected in to the chamber and after a steady rate of oxygen uptake was recorded KCN freshly prepared and neutralised to pH 7.4 was added to give the concentrations shown.

tOO 500 600 700 Wavelength nm

Fig. 4. Difference spectra of sonicated preparations (5.0 mg prote in /ml) of T. rhodesiense EATRO 173, in 0.1 M phosphate buffer, pH 7.4. The lower solid line represents the difference spectrum of respiratory pigments reduced w i th d i th ion i te minus pigments oxidised by vigorous aeration at 2 ° . The upper dashed line is the difference spectrum of a preparation reduced w i th d i th ion i te and saturated w i t h CO compared w i t h a prepara-t ion reduced by d i th ioni te .


Λ I 05| Δ Extinction I 01 II * Ι * I I

1.00 500 600 ' 700 Wavelength nm

Fig. 5.

Difference spectra of a mi tochondr ia l preparation of T. rhodesiense EATRO 173 epimastigotes suspended in 0.1 M phosphate buffer, pH 7.4 at a concentrat ion of 2.5 mg protein per m l . Lower sol id curve: d i th ion i te reduced minus oxidised aerated spectrum. Middle broken curve: the reduced sample treated w i t h cyanide minus oxidised spectrum. Upper dashed l ine: the difference spectrum of mi tochondr ia reduced w i t h d i th ion i te and saturated w i t h CO minus a d i th ion i te reduced sample.

341

ÜB

1,00 500 600 700 Wavelength n m

Fig. 6. Difference spectra of suspensions of intact cells of T. rhodesiense EATRO 173 epimastigotes. The lower solid line represents the spectrum of cells metabolical ly reduced w i t h glucose (10 mM) minus cells aerated w i thou t substrate. The upper dashed line is the spectrum of metabolically reduced and CO treated cells minus cells in which the pigments were reduced by glucose metabol ism.

ι MB j 51.0 570

~m ' 600 ' 700 Wavelength nm

Fig. 7. Difference spectra of acetone dried T. rhodesiense Pittam strain sus-pended in 0.1 M phosphate buffer, pH 7.4, at a concentrat ion of 5 mg protein per ml. Lower solid line represents the spectrum of a suspension w i th the pigments reduced by d i th ioni te versus a sample oxidised by aeration. The upper dashed line indicates the spectrum of respiratory pigments after reduction w i t h d i th ion i te and saturation w i th CO minus pigments reduced by d i th ioni te.

Documents

Comparative Biochemistry of Parasites || ADAPTATIONS IN OXIDATIVE METABOLISM DURING THE TRANSFORMATION OF TRYPANOSOMA RHODESIENSE FROM BLOODSTREAM INTO CULTURE FORM