Regulation of oxidase enzyme systems in trypanosomes

Comp. Biochem. Physiol., 1969, Vol. 30, pp. 61 to 72. Pergamon Press. Printed in Great Britain

R E G U L A T I O N OF OXIDASE ENZYME SYSTEMS IN TRYPANOSOMES R O N A L D A. BAYNE*, K E N N E T H E. M U S E ~ and J O H N F. R O B E R T S Zoology Department, North Carolina State University, Raleigh, North Carolina 27607, U.S.A. (Received 2 November 1968) Abstract--1. QO2, succinoxidase and glycerophosphate oxidase (GPO) levels

(0"533, 0"367 and 0"334/~moles O3 hr -1 mg protein -1) were determined for Trypanosoma conorhini.

2. Kinetoplastic and dyskinetoplastic T. equiperdum had higher QO~ (2.51 and 1.87) and GPO (2.38 and 1.15) levels but lacked measurable succinoxidase. 3. Reduced O3 availability during growth of T. conorhini gave reduced succinoxidase and increased GPO activities. 4. Acriflavin and chloramphenicol added at initiation of cultures of T. conorhini gave decreased growth and QO~ while only acriflavin decreased succinate ferricyanide reductase activity. 5. Acriflavin added to exponentially growing T. conorhini reduced only succinate ferricyanide reductase activity and increased cyanide-insensitive QO2 and GPO levels. INTRODUCTION TRYPANOSOMES have a characteristic organelle, the kinetoplast, which contains a large amount of extra-nuclear DNA. This organelle is self-duplicating and is a specialized region of an elongate mitochondrion (Clark & Wallace, 1960; Steinert, 1960; Steinert & Steinert, 1962; Vickerman, 1962; Anderson & Ellis, 1955). The D N A of the kinetoplast differs from nuclear D N A in buoyant density in CsC1, "melting" temperature and in annealing properties (duBuy e t a / . , 1955, 1966; Riou et al., 1966; Riou & Paoletti, 1967; Steinert & van Assel, 1967; Simpson, 1968). Investigators suggest that the kinetoplast D N A may play a leading role in the regulation of respiratory changes that occur during the life cycle of certain African trypanosomes. In the insect (culture) form trypanosomes, tricarboxylic acid cycle activity is coupled to a cytochrome-dependent electron transport system, but the bloodstream forms have greatly reduced tricarboxylic acid cycle and cytochromedependent electron transport activities (for reviews see: Baernstein, 1963; Trager, 1965 ; Vickerman, 1965). These latter forms use a glycerophosphate dehydrogenaseglycerophosphate oxidase electron transport system that does not contain cytochromes, but does use oxygen as the final electron acceptor (Grant & Sargent, 1960, 1961; Grant et al., 1961). * Present address: Department of Chemistry, University of South Florida, Tampa, Florida 33620, U.S.A. Supported by a USPHS Predoctoral Research Fellowship (1-F1GM-33, 043-01) from the Institute of General Medical Sciences. t Supported by USPHS Predoctoral Research Fellowship (1-F1-GM-38, 206-01) from the Institute of General Medical Sciences. 61

62

RONALD A. BAYNE, KENNETH E. MUSE AND JOHN F. ROBERTS

Loss of kinetoplast D N A (dyskinetoplasty) can arise spontaneously or be induced in bloodstream form trypanosomes. These dyskinetoplastic strains can survive in the vertebrate bloodstream, but cannot be transmitted cyclicly or grown in culture (Tobie, 1951; Hoare, 1954). T r e a t m e n t of culture-form trypanosomes with low concentrations of acriflavin results in large numbers of dyskinetoplastic culture form cells, but this condition is lethal in the culture form (Muhlpfordt, 1963, 1964; T r a g e r & Rudzinska, 1964, Trager, 1965; Steinert & van Assel, 1967; Stuart & Hanson, 1967; Simpson, 1968). Based on these observations it would appear that kinetoplast D N A is essential in the culture form, but not in the bloodstream form trypanosome. Acriflavin appears to interfere with kinetoplast D N A replication, since it inhibits 3H-thymidine incorporation into the kinetoplast, but not in the nucleus, and causes the loss of the band in CsC1 gradients attributed to kinetoplast D N A (Steinert & van Assel, 1967; Simpson, 1968). Acriflavin also appears to interfere with oxidative phosphorylation, since high concentrations have been found to inhibit respiration and succinate dehydrogenase in the trypanosomatid, Crithidia fasciculata (Hill & Hutner, 1968). T h e purpose of the research reported in this paper was to compare respiratory activities in kinetoplastic and dyskinetoplastic forms of T. equiperdum and in the normal culture form and acriflavin-induced dyskinetoplastic culture form of T. conorhini to determine if kinetoplastic D N A is involved in the regulation of mitochondrial activities. Respiratory quotients (QO2), succinoxidase and glycerophosphate oxidase (GPO) were measured in the blood stream forms. Growth, QO2, succinoxidase, GPO, succinate ferricyanide reductase (SDH), malate dehydrogenase ( M D H ) , lactate dehydrogenase ( L D H ) and kinetoplast fine structure were compared in the normal, acriflavin-treated and chloramphenicol-treated culture form.

MATERIALS AND METHODS Trypanosomes were grown, harvested and homogenized in isotonic sucrose solutions as previously described (Bayne & Roberts, 1969). Stock solutions of an acriflavin-riboflavin mixture (Trager & Rudzinska, 1964) were prepared containing 0"1 mg/ml acriflavin-HC1 (Sigma Chemical Co.) and 0"04 mg/ml ribofavin (Eastman-Kodak Co.) in water. The solutions were sterilized by filtering (Millipore, 0"45/~) or autoclaving and were stored in the dark at 5°C. Stock solutions of D-chloramphenicol (Sigma Chemical Co.) were prepared in 95% ethanol at a concentration of 400 mg/ml. Inhibitors were added when the cultures were inoculated or to 3-day-old cultures at a dilution of 1 : 100 to give final concentrations of 1/~g/ml acriflavin, 0'4/zg/ml riboflavin and 4 mg/ml chloramphenicol (CAP). Cultures were harvested after 5 days in the presence of the inhibitors. Protein and growth measurements Growth was determined by hemocytometer counts of cells fixed in 0"1% glutaraldehyde in Locke's solution. Protein was estimated by the method of Lowry et al. (1951) using bovine serum albumin as the standard.

R E G U L A T I O N O F T R Y P A N O S O M E OXIDASES

63

Polarographic measurements Oxygen consumption was measured on trypanosomes washed and suspended in KrebsRinger solution (Ryley, 1955) containing 0"25% glucose on a Gilson Oxygraph equipped with a vibrating platinum electrode. The reaction chamber was maintained at 25°C and contained a total volume of 2 ml (0"5/zmole 03). Succinoxidase and GPO were measured in sucrose homogenates (0"25 M sucrose, 0"1 mM EDTA, 25 mM Tris-HCl, pH 7"5) in the same manner. The succinoxidase reaction mixture (2 ml) contained 10 mM succinate, 0.1 mM ADP, 10 mM potassium phosphate buffer, pH 7"5, and 0"2 ml of homogenate (N 1-3 nag protein). The GPO reaction mixture was the same except that succinate was replaced with 20 mM DL-e~-glycerophosphate. ADP was omitted from the T. equiperdum oxidase assays. QO2 and oxidase levels are reported as/zmoles O2 consumed/hr per mg protein.

Spectrophotometric measurements MDH and L D H were assayed as previously described (Bayne & Roberts, 1969). Activities are expressed as units/min per mg protein. One unit is defined as an absorbance change of 0"001. Succinate ferricyanide reductase (SDH) was assayed at room temperature on a Beckman DU-2 Spectrophotometer equipped with a recorder by following ferricyanide reduction at a wavelength of 410 n-qz. Reactions were conducted in a total volume of 3 ml contained in cuvettes with a 1-cm light path. The reaction mixture was composed of 10 mM succinate, 0"03 mM CaCI~, 1 mM K2Fe(CN)e, 6 mM KCN and 10 mM potassium phosphate buffer, pH 7"5. Homogenates (0.2-0.4 ml) were pre-incubated in the reaction mixture for 5 rain before adding the substrate. SDH activity is reported in units in which 1 unit equals an absorbance change of 0"001. Specific activities are reported as units/hr per nag protein.

Electron microscopy T. conorhini pellets were fixed for 2 hr in cold 3 % glutaraldehyde in MiUonig's phosphate buffer, pH 7"3 (Sabatini et al., 1963) and post-fixed in 2% osmic acid in phosphate buffer, pH 7"3, for 2 hr (Millonig, 1961). Segments of the fixed pellets were dehydrated in a series of graded ethanols (30-100%) in 15 rain intervals and embedded in Epon by the method described by Luft (1961). Thin sections (~ 80 m/z) were cut on a LKB Automatic Microtome with a Ge--Fe-Re knife and stained with uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963). Sections were examined in a Siemens Elmiskop 1 A microscope at an accelerating voltage of 80 kV. RESULTS

QO~ and oxidase levels Table 1 compares normal QO~ and oxidase levels in trypanosomes.

T.

conorhini QO2 was 79 per cent lower than the T. equiperdum quotient. T. conorhini homogenates demonstrated both succinoxidase and G P O at the same levels under the assay conditions used. T. equiperdum showed a high level of G P O as compared to T. conorhini, but the succinoxidase level was too low to measure accurately. T h e G P O of T. equiperdum was not inhibited by cyanide. Dyskinetoplastic T. equiperdum had a lower QO 2 (25 per cent) and lower G P O (50 per cent) levels than the kinetoplastic 71. equiperdum. QO2 and oxidase levels were dependent on the type of culture vessels used in growing the T. conorhini cells (Table 2). Cultures from vessels with a larger

RONALDA. BAYNE,KENNETHE. MUSE AND JOI-INF. ROBERTS

64

surface-to-volume ratio than the tubes routinely used showed a slight stimulation in QO2, but the succinoxidase was strongly stimulated and G P O was depressed. TABLE 1--COMPARISONSOF QO 2 AND OXIDASELEVELSIN TRYPANOSOMES /zmoles O2/hr Organism

QO 2

T. conorhini T. equiperdum

GPO

Succinoxidase

0'533 (0"407-0'670) 9 0-334 (0"244-0-490) 6 0"367 (0'266-0"536) 6

Kinetoplastic Dyskinetoplastic

2'51 (1.98-3"25) 4 1'87 (1"35-2-15) 5

2"38 (1"73-2"96) 4 1"15 (0'88-1"32) 5

< 0-01 < 0"01

Figures within the parentheses represent the range of activities. Figures following the parentheses represent the number of preparations assayed in duplicate and the figures in front of the parentheses represent the means. TABLE 2--EFFECT OF CULTURECONDITIONSON QO 2 AND OXIDASELEVELSIN

T. conorhini Enzyme (/~moles OJhr)

Flask cultures

Tube cultures

QOz GPO Succinoxidase

0"425 0-252 0"432

0'410 0"332 0"383

Surface : Volume (cm2/ml)

1"30

0'13

Flask cultures were prepared in 250-ml Erlenmyer flasks and contained 30 ml of blood-agar base overlaid with 30 ml of Locke's solution.

Effects of acriflavin and C A P on T. conorhini Acriflavin inhibited cell division when it was added just after the cultures were inoculated (Fig. 1); C A P affected cell division in a similar manner. I f the acriflavin was added to 3-day-old cultures in which cells were in exponential-phase growth, cell division was only slightly affected (Fig. 1). T h e relative QO 2 decreased in cells treated with acriflavin and CAP during lag-phase growth (Fig. 2). T h e response was immediate with acriflavin, but there was a lag with CAP before inhibition was noted. After 5 days in the presence of the inhibitors, the QO 2 was 50 per cent lower than in untreated cells. S D H was strongly inhibited in the acriflavin-treated cells, but not in the CAP-treated cells (Table 3).

REGULATION OF TRYPANOSOME OXIDASES

65

6.2"

6.0-

E (n

5.6-

._1 ..J bJ (.1 ,. 5.4O nUJ

m 5.2=E Z

O- 5.0O 4.8 I +ACK 4.6-1

4.4 I

T I M E (DAYS) Fro. 1. Effect of acriflav/n (ACF) on growth of T. conorhini when added at culture initiation (m) and 3-day-old cultures (O) as compared to normal growth (O).

TABLE 3--EFFECTS OF ACRIFLAVIN AND CAP, ADDED WHEN CULTURES STARTED, ON THE RELATIVE LEVELS OF ~ O 2 AND SDH IN ~-DAY-OLD CULTURES OF T. c 0 n o r h i m

Enzyme

Control (%)

Acriflavin (%)

CAP (%)

QO, SDH

100 100

46 31

46 94

66

RONALD A. BAYNE, KENNETHE. MUSE AND JOHN V. ROBERTS

Exponentially growing cells treated with acriflavin on the third day of culture also had lowered S D H , but near normal levels of M D H and L D H after 5 days in the presence of the inhibitor (Table 4). CAP did not significantly affect the levels of S D H , M D H and L D H in cells treated in a similar manner (Table 4). These cells showed near normal QO~ and GPO, but these activities became increasingly

90

80

Z

0 In--

¢n UJ

7O

n~

60

50

40 0

t

t

I

2

3 DAYS

4 IN

5

6

7

CULTURE

FIG. 2. Relative QO a of T. conorhini grown in the presence of acriflavin (0) and CAP (©). Inhibitors added when cultures inoculated.

REGULATION O F T R Y P A N O S O M E

67

OXIDASES

cyanide-insensitive (Table 5). This response was variable as demonstrated in the various experiments reported in Table 5. The variability is probably due to the complex culture conditions and media. TABLE 4---EmmcTs o F ACRIFLAVIN A N D CAP, ADDED T O 3 - D A Y - O L D CULTURES, O N T H E RELATIVE LEVELS O F SEVERAL O X I D O R E D U C T A S E S IN 8 - D A Y - O L D C U L T U R E S O F

T. conorhini

Enzyme

Control (%)

Acriflavin (%)

CAP (%)

SDH MDH LDH

100 100 100

27 79 83

95 83 86

T A B L E 5 - - E F F E C T S O F ACRIFLAVIN~ A D D E D T O 3 - D A Y - O L D CULTURES, O N LEVELS AND CYANIDE-SENSITIVITY OF Q O 2 AND GPO IN 8 - D A Y - O L D CULTURES OF

T. conorhini

Control

Acriflavin

Activity (%)

Plus CN(%)

Activity (%)

Plus CN(%)

002 GPO

100 100

37 30

86 120

85 89

2 QOa GPO

100 100

~

105

--

15

107

89

13 30

46 83

31 80

Experiment 1

3

QO2 GPO

100 100

The plus CN- percentages represent the remaining activity after the addition of 6 mM KCN relative to the control activity. The ultrastructure of a kinetoplast from an untreated T. conorhini cell is shown in Fig. 3. CAP did not change the fine structure characteristics to any noticeable extent. In cells treated with acriflavin during lag-phase growth, the electron-dense fibrillar material was in the collapsed, condensed state that has been described by others (Trager & Rudzinska, 1964; Kusel et al., 1967; Steinert & van Assel, 1967). In cells treated with acriflavin during exponential-phase growth, the tightly packed, highly ordered kinetoplast fibrillar genophore became dispersed and disorganized and not condensed (Fig. 4). Thin strands can be seen extending to the kinetoplast membrane. Similar connections have been seen in mitochondrial genophores (Nass & Nass, 1964). Other homologies have been seen in kinetoplast genophores and will be discussed elsewhere.

68

RONALD A. BAYNE, KENNETH E. MUSE AND JOHN F. ROBERTS

DISCUSSION Kinetoplast DNA appears to be involved in the regulation of oxidase activities in both bloodstream and culture (insect) form trypanosomes. This conclusion is based on the comparisons reported here for kinetoplastic and dyskinetoplastic bloodstream form trypanosomes and the effects of culture conditions and acriflavin on the culture form trypanosome. Isolated mitochondria appear only to synthesize a hydrophobic group of structural proteins that may serve as attachment sites for mitochondrial enzymes synthesized in the cytoplasm, such as M D H and cytochrome c (for review see: Roodyn & Wilkie, 1968). We have found that only membrane proteins are synthesized in mitochondrial fractions isolated from T. conorhini (Bayne et al., 1969b). The culture form T. conorhini contains low numbers of spherical bodies in the cytoplasm (unpublished data) that resemble the GPO bodies isolated from the bloodstream T. equiperdum (Bayne et al., 1969a). T. conorhini may therefore contain low levels of the cyanide-insensitive GPO that is found in the bloodstream form trypanosomes. T. equiperdum contains, in addition to GPO bodies, a significant number of mitochondrial structures (unpublished data). These mitochondria must be in a repressed state because of the low levels of succinoxidase and mitochondrial M D H (Bayne & Roberts, 1969). The presence of the mitochondrial M D H in T. equiperdum does seem to indicate a low level of typical mitochondrial activity in these organisms. In an attempt to correlate the above data and the data reported in this paper with kinetoplast-mitochondrial complex function in trypanosomes, we postulate six systems of nucleo-cytoplasmic regulation (Table 6). System 1 describes the condition found in the normal bloodstream form trypanosome. Since there are GPO bodies and cyanide-insensitive GPO activity in the dyskinetoplastic form trypanosome, we conclude that the synthesis of this enzyme system contained in the GPO bodies is directed by the nucleus. This is the predominant oxidase system in the bloodstream form trypanosome and must be rapidly being synthesized. Since there is a low level of succinoxidase and what appears to be a mitochondrial M D H in these organisms, we conclude that there is a low level of mitochondrial structural protein and mitochondrial catalytic protein synthesis being conducted. System 2 is a hypothetical model of the condition found in the dyskinetoplastic strain of the bloodstream T. equiperdum. If the assumption that kinetoplast DNA is responsible for the synthesis of structural proteins which offer binding sites for mitochondrial membrane-bound enzymes is valid, then the absence of kinetoplast DNA would result in totally inactive mitochondria or a complete absence of mitochondrial structures. Since the GPO level in the dyskinetoplastic form is 50 per cent lower than in the kinetoplastic form, it is possible that some sort of interaction of nucleus and kinetoplast is necessary to maintain maximal cyanideinsensitive GPO levels. System 3 depicts the regulation of oxidases in the culture form trypanosome. Environmental conditions in the insect or in culture induces the synthesis of

FIG. 3. Ultrastructure

of the kinetoplast in untreated T. conorhini. Basal bcBdies The horizontal line represents 1 CL.

03 ; kinetoplast (K); kinetoplast genophore (G); magnification, x 36,000.

Effect of acriflavin on the kinetoplast of an exponentially growin g 7’. ~hini cell. Acriflavin added after 3 days of growth and the cells were bar\-fested of acriflavin. Kinetoplast genophore ((;I; and fixed 26 hr after the addition magnification x 39,000. The horizontal line represents 1 ,I*.

FIG. 4.

REGULATION OF TRYPANOSOME OXIDASES

69

mitochondrial proteins and enzymes and represses maximal synthesis of cyanideinsensitive GPO. Due to the presence of what appear to be GPO bodies, and the inability to obtain complete inhibition with cyanide, we feel repression is not complete. TABLE 6 - - P o s S I B L E MECHANISMS OF NUCLEO-CYTOPLASMIC REGULATION IN TRYPANOSOMES

Bloodstream form ( T. equiperdum) System 1 : Kinetoplast . . . .

Nucleus

~- Structural proteins 1

F - - ~ " Catalytic Proteins-f . . . . "

Mitochondrion

" GPO bodies

System 2: Kinetoplast

(no DNA) Nucleus

~ - -:

Catalytic proteins GPO bodies Culture form ( T. conorhini) Structural proteins 7

System 3 : Kinetoplast

Mitochondrion Catalytic proteins - ] Nucleus L - - ~ ' G P 0 bodies System 4: Kinetoplast . . . . .

Nucleus

Structural proteins-l_ 1 [ --=- Catalytic proteins- -J | GPO bodies

Structural proteins-] (dispersed DNA) -. ~-] Catalytic proteins - - ~ Nucleus I "~ GPO bodies

Mitochondrion ~w~Os

System 5 : Kinetoplast . . . . .

-'~- Mitochondrion

System 6: Kinetoplast

(condensed DNA) Nucleus [?-~- Catalytic proteins /

L?--~. GP0 bodies The solid lines represent a high rate of synthesis and the dashed lines represent a low rate of synthesis.

70

RONALD A. BAYNE, KENNETH E. MUSE AND JOHN F. ROBERTS

System 4 is based on data presented here and elsewhere (Bayne & Roberts, 1969) on the effects of the length of the 0 2 diffusion path on oxidoreductase levels. In this case, low 0 2 concentrations or compounds interfering with oxidative phosphorylation could call forth feed-back mechanisms that cause lowered rates of mitochondrial protein and enzyme synthesis and increased rates of cyanideinsensitive GPO synthesis. System 5 is based on the effects of acriflavin on exponentially growing cells. Acriflavin appears to cause a change in kinetoplast DNA template characteristics (Fig. 4) resulting in a lowered synthesis of structural protein with concomitant decreased mitochondrial activity, but not affecting the nuclear directed synthesis of mitochondrial catalytic proteins (normal M D H levels). There appeared to be increased synthesis of cyanide-insensitive GPO that might have resulted from a kinetoplast-nuclear interaction or as simply a compensatory mechanism to counterbalance the impaired energy production. System 6 is based on the effects of acriflavin on cells in lag-phase growth. In the acriflavin-induced condensed state, the kinetoplast D N A may not be available for transcription and the synthesis of mitochondrial structural protein is stopped, resulting in inactive mitochondria. We did not determine if there were low levels of cyanide-insensitive GPO or if mitochondrially associated tricarboxylic acid cycle enzymes were affected. If the induction of cyanide-insensitive GPO was "automatic" as suggested in System 6, then this system should also have increased levels of cyanide-insensitive GPO. It is possible though that the physiological state of cells in lag-phase growth affects this response. In conclusion, the hypothesis that acriflavin induces dyskinetoplasty by binding to kinetoplast DNA and inhibiting replication is strongly supported. An alternative hypothesis is that acriflavin induces dyskinetoplasty by inhibiting oxidative phosphorylation which more directly affects kinetoplast D N A replication than nuclear D N A replication. The subsequent stimulation of anaerobic glycolysis or cyanideinsensitive GPO-driven glycolysis furnishes energy for a reduced rate of growth and cytokinesis. These two hypotheses need not be mutually exclusive since an involvement of both in tandem may account for mitochondrial susceptibility to acriflavin. REFERENCES ANDERSONW. A. & ELLIS R. A. (1965) Ultrastructure of Trypanosoma lewisi: flagellum, microtubules and the kinetoplast. J. Protozool. 12, 483-489. BAERNSTEINH. D. (1963) A review of electron transport mechanisms in parasitic protozoa. J. Parasit. 49, 12-21. BAYNER. A. & ROBERTSJ. F. (1969) Activities and isozymes of malate and lactate dehydrogenases in culture and bloodstream form trypanosomes. Comp. Biochem. Physiol. 29, 731-741. BAYNE R. A., MUSE K. E. & ROBERTSJ. F. (1969a) Isolation of bodies containing the cyanide-insensitive glycerophosphate oxidase of Trypanosoma equiperdum. Comp. Biochem. Physiol. (In press.) BAYNE R. A., MUSE K. E. • ROBERTSJ. F. (1969b) Mitochondrial protein synthesis in Trypanosoma conorhini. J. Protozool. (Submitted.)

REGULATION OF TRYPANOSOME OXIDASES

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DEBuY H. G., MATTERN C. F. T. & RILEY F. L. (1965) Isolation and characterization of D N A from kinetoplasts of Leishmania enrietti. Science 147, 754-756. DuBLrY H. G., M A ~ C. F. T. & RILEY F. L. (1966) Comparison of D N A ' s obtained from brain nuclei and mitochondria of mice and from nuclei and kinetoplasts of Leishmania enriettii. Biochim. biophys. 11cta 123, 298-305. CLARK T. B. & WALLACEF. B. (1960) A comparative study of kinetoplast ultrastructure in the trypanosomatids..7. Protozool. 7, 115-124. GRANT P. T. & SARGENTJ. R. (1960) Properties of L-~-glycerophosphate oxidase and its role in the respiration of Trypanosoma rhodesiense. Biochem..7. 76, 229-237. GRANT P. T. & SARGENTJ. R. (1961) L-~-Glycerophosphate dehydrogenase, a component of an oxidase system in Trypanosoma rhodesieme. Biochem.oT. 81, 206. GRANT P. T., SARGENTJ. R. & RYLEY J. F. (1961) Respiratory systems in the Trypanosomidae. Biochem.o7. 81, 200-208. HILL G. C. & H u r N ~ S. H. (1968) Effect of trypanocidal drugs on terminal respiration of Crithidia fasciculata. Expl Parasit. 22, 207-212. HOAm~ C. A. (1954) The loss of the kinetoplast in trypanosomes with special reference to Trypanosoma evansi. O7.Protozool. 1, 28-33. KUSEL J. P., MOORS K. E. & WEBERM. M. (1967) The ultrastrueture of Crithidiafasciculata and morphological changes induced by growth in acriflavin. ~. Protozool. 14, 283-296. LOWRy O. H., ROSmmOUGHN., FArm A. & RANDALLR. (1951) Protein measurement with the Folin phenol reagent. O7. biol. Chem. 193, 286-290. LUFT J. (1961) Improvements in epoxy resin embedding methods. O7.biophys, biochem. Cytol. 9, 409--414. MILLONIG G. (1961) Advantages of a phosphate buffer for OsO4 solutions in fixation. o7. appl. Phys. 32, 1637. MUHLPFORDT H. (1963) ~ b e r die Bedeutung und Feinstruktur des Blepharoplasten bei parasitischen Flagellaten II Tiel. Z. Tropenmed. Parasit. 14, 475-501. MUHLPFOaDT H. (1964) l~ber den Kinetoplasten der Flagellaten. Z. Tropenmed. Parasit. 15, 289-323. NASS S. & NASS M. M. K. (1964) Intramitochondrial fibers with deoxyribonucleic acid characteristics: observations of Ehrlich ascites tumor cells. O7. natn. Cancer Inst. 133, 777-794. I~YNOLDS E. S. (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. O7. Cell Biol. 17, 208-212. RIou G. & PAOL~rTI C. (1967) Preparation and properties of nuclear and satellite deoxyribonucleic acid of Trypanosoma eruzi. O7. Mol. Biol. 28, 377-382. RIou G., PAUTaIZELLR. & PAOLETTIC. (1966) Fractiormement et caraeterisation de l'acide desoxyribonucleique de trypanosome (Trypanosoma equiperdum). C.r. hebd. Sdanc. Acad. Sci. 262, 2376-2379. ROODYN D. B. & WILKm D. (1968) The Biogenesis of Mitochondria. Methuen, London. RYLEY J. F. (1955) Studies on the metabolism of the protozoa 4. Metabolism of the parasitic flagellate, Strigomonas oncopelti. Biochem. O7. 59, 353-361. SABATINI D. D., BENSCH K. G. & BARNETTR. J. (1963) Preservation of ultrastructure and enzyme activity by aldehyde fixation. O7. Cell Biol. 17, 19. SIMPSON L. (1968) Effect of acriflavin on the kinetoplast of Leishmanla tarentolae. O7. Cell Biol. 37, 660-682. STEINERT M. (1960) Mitochondria associated with the kinetonucleus of Trypanosoma mega. O7. biophys, bioehem. Cytol. 8, 542-546. STEINERT M. & VAN ASSEL S. (1967) The loss of kinetoplastic D N A in two species of Trypanosomatidae treated with acriflavine. O7. Cell Biol. 34, 489-503. STEINERT M. & STEINERTG. (1962) La synthese de l'acide desoxyribonueleique au tours du cycle de division de Trypanosoma mega. O7.Protozool. 9, 203-211.

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RONALD A. I{AYNE, KENNETH E. MUSE AND JOHN F. ROBERTS

STUART K. D. & HANSON E. D. (1967) Acriflavin induction of dyskinetoplasty in Leptomonas karyophilus. J. Protozool, 14, 39-43. TOBIE E. J. (1951) Loss of the kinetoplast in a strain of Trypanosoma equiperdum. Trans. Am. Mic. Soc. L X X , 251-254. TRAGER W. (1965) T h e kinetoplast and differentiation in certain parasitic protozoa. Am. Nat. X C I X , 255-266. TRAGER W. & RUDZINSKA ~'I. A. (1964) T h e riboflavin requirement and the effects of acriflavin on the fine structure of the kinetoplast of Leishmania tarentolae. J. Protozool. 11,133-145. VICKERMAN K. (1962) T h e mechanism of cyclical development in trypanosomes of the Trypanosoma brucei sub-group: an hypothesis based on ultrastructural observations. Trans. Roy. Soc. Trop. Med. Hyg. 56, 487-495. VICKERMAN K. (1965) Polymorphism and mitochondrial activity in sleeping sickness trypanosomes. Nature, Lond. 208, 762-766. WATSON M. L. (1958) Staining of tissue sections for electron microscopy with heavy metals. ~. biophys, biochem. Cytol. 4, 475-478.

Key Word Index--Trypanosomes ; Trypanosoma conorhini ; Trypanosoma equiperdum ; QO~; succinoxidase; succinate ferricyanide reductase ; glycerophosphate oxidase ; acriflavin ; chloramphenicol; kinetoplast; dyskinetoplastie; growth; malate dehydrogenase ; lactate dehydrogenase; regulation ; oxidases; hemoflagellates ; protozoans ; parasites.