EXPERIMENTAL PSRASITOLOGY 31, %0-88 (1972)

Crithidia fascicolata: Acriflavine-Induced Changes in Soluble

Enzyme Levels’
Cyrus J. Bacchi

Naskins Laboratories at Pace College, Department of Biology, Pace College,

41 Park Row, New York, New York 10038

and
George C. Hill”

University of Kentucky, Department of Biochemistry, Lexington, Kentucky 40606

(Submitted for publication, 20 July 70)

BACCHI, CYRUS J. AND HILL, GEORGE C. 1972. Crithidia fasciculata: ilcriflavine-

induced changes in soluble enzyme levels. Experimental Parasitology 31, 290-298.
Chhidia fasciculata ATCC 11745 was grown in the presence of 5 PM acriflavine.
Drug-treated organisms had -50% less respiratory activity and total growth than the
controls and accumulated larger amounts of pyruvate and ethanol in the growth me-
dium. Supernatant fractions were prepared by the alumina method. The activities of
seven enzymes in the supernatant (cytosol) fraction were compared with correspond-
ing enzyme activities in control cells. Hexokinase, glucose&phosphate dehydrogenase,
malate dehydrogenase, alcohol dehydrogenase, and a-glycerophosphate dehydrogenase
were at least 30% higher in acriflavine-treated preparations. ar-Glycerophosphate dehy-
drogenase increased most (85%) and was the most active of all enzymes measured.
Glucose-g-phosphate dehydrogenase, an enzyme of the pentose phosphate pathway,
increased 66%. NADP-linked isocitrate dehydrogenase and malic enzyme had lower
levels (50 and 20%. respectively) in the acriflavine-treated cells. The concentrations
of NAD’ and NADP’ remained relatively constant in both types of cells. The signifi-
cance of these changes with regard to the metabolism of dyskinetoplastic trypanoso-
matids is discussed.
INDEX DESCRIPTORS : Acriflavine, Trypanosomatidae ; Crithidia fasciculata; Enzyme
regulation; Metabolism; Hexokinase; (EC 2.7.1.1) ; Glucose-g-phosphate dehydro-
genase (EC 1.1.1.49); a-Glycerophosphate dehydrogenase (EC 1.1.1.8) ; Malic
dehydrogenase (EC 1.1.1.39) ; Alcohol dehydrogenase (EC 1.1.1.1) ; Malic en-
zyme (EC 1.1.1.40) ; Isocitrate dehydrogenase (EC 1.1.1.42).

A conspicuous role of the kinetoplast in ages kinetoplast DNA and mitochondrial

the life cycle of trypanosomes is amply doc- structure. Trager and Rudzinska (1964)
umented (Newton 1968 ; Vickerman 1970, and Miihlpfordt (1963a,b) indicated that
1971; Hutner et al. 1972). Acriflavine dam- acriflavine crippled replication of the kine-
toplast DNA with retention of the kineto-
‘This work was supported by Public Health plast membrane. Simpson (1968) found
Service grant AI-0789 to S. H. Hutner from the
National Institutes of Allergy and Infectious Dis- t,ional Institute of Allergy and Infectious Dis-
eases, by an American Philosophical Society grant eases.
(Penrose Fund, 5057) and by a Merck Co. Foun- * Present address: University of Cambridge,
dation Faculty Development grant to C. J. Bacchi: The Molten0 Institute of Biology and Parasi-
G. C. Hill was supported by U.S. Public Health tology, Downing Street. Cambridge CB2 3EE,
Service Fellowship No. AI-36,847 from the Na- England.

290
Copyright 0 1972 by Academic Press, Inc.
 ACRIFLAVINE-INDUCED
#hat in acriflavine-treated Leishmania ta-
‘entolae, nuclear division seemed unimpaired
jut kinetoplast DNA was lost in successive
:ell division; respiration was inhibited as
imited growth proceeded in acriflavine
:oncentrations high enough to yield dyski-
retoplastic cells. Acriflavine treatment of
7rithidia fasciculata produced ultrastruc-
;ural effects on the mitochondrion and re-
luced O2 uptake, the latter attributable to a
lecrease in cytochromes a + u3, b, and c,
2nd to reduced activities of mitochondrial
:nzymes (Hill and Anderson 1969).
Conceivably, this impaired functioning of
;he cytochrome system might be compen-
sated by stimulation of other ATP-generat-
mg systems. Slender bloodstream (trypo-
mastigote) forms of the Trypanosomu bru-
:ei group have a rudimentary mitochon-
Orion and rely on glycolysis to provide
ATP. In contrast, culture (trypomastigote-
midgut) forms have a functional mitochon-
Orion with some evidence for cytochromes
beging present (Vickerman 1965 ; Ryley
1962).
We therefore surveyed the effect of acri-
Bavine-induced dyskinetoplasty on C. fascic-
data soluble (cytosol) enzyme levels, in-
cluding several enzymes of the glycolytic
pathway in acriflavine-treated C. fusoicu-
lutu. C. fusciculuta was convenient since
previous work (Hill and Anderson 1969)
had detailed changes in levels of cyto-
chromes and mitochondrial enzymes in this
organism at the acriflavine concentration
and with the carbon source (xylose) used in
this study.
MATERIALS AND METHODS
Mass Culture Procedures
C. fasciculatu (ATCC 11745) was grown
in a complex medium: yeast hydrolyzate
(Nutritional Biochemicals Corp., Cleve-
land, OH) 0.5%, N-Z Amine AS (Sheffield
Chemical Div., National Dairy Products,
Union, NJ) 0.3%, n-xylose 1.33% (0.05 M),
CHANGES 291
hemin (equine, type III, Sigma Chemical
Co., St. Louis, MO) was prepared as de-
scribed (Hill and Anderson 1969) and
added aseptically to a final concentration of
1.5 PM. The final pH of the medium was 7.9.
Cells grown in acriflavine received 5 PM
neutral acriflavine (gift of Eli Lilly and
Co., Indianapolis, IN) as described (Hill
and Anderson 1969). Usually, 6 liters of
control and 10 liters of acriflavine-treated
cells were grown. Cultures were inoculated
with 10 ml from a single flask (48-hr. cul-
ture) and grown at 24-27 C with aeration
(Bacchi et al. 1968) for 60-80 hr.
Isolation of Soluble Enzyme Fractions
The methods for harvesting cells, obtain-
ing a soluble enzyme fraction (“superna-
tant fraction”-alumina method), and pro-
tein determination (biuret method) have
been detailed (Bacchi et al. 1968).
Enzyme Assays
Enzymes in the 12,000g supernatant frac-
tion were assayed in a temperature con-
trolled (25 C) recording spectrophotometer.
Results were recorded as change in absorb-
ance (340 nm) due to oxidation or reduc-
tion of NAD+, NADH, NADP+, and
NADPH. Specific enzyme activities are ex-
pressed as units: 1 unit = 1 nmole pyridine
nucleotide oxidized or reduced/min/mg pro-
tein. Extinction coefficients for reduced pyr-
idine nucleotides were as given by Kornberg
(1957) and Horecker and Kornberg (1957) :
0.207 absorbance units = 100 nmoles pyri-
dine nucleotide oxidized or reduced.
Reactions were measured in l-cm light-
path cuvettes; the final volume of all reac-
tion mixtures was 3.0 ml. Reactions were
normally linear for 1 min. At least 3 deter-
minations of each enzyme were made at pH
7.4 in the appropriate buffer. Reference cu-
vettes contained all components of the en-
zyme assay mixture except the appropriate
pyridine nucleotide. In the assay, C. fuscic-
data protein was the limiting factor, as
292 BACCHI AND HILL

TABLE I formed/min/mg protein. The excess of pure

Sources of Biochemicals for Enzyme Assays glucose-6-phosphate-dehydrogenase ensured
that the C. fasciculata enzyme did not in-
Glucose-B-phosphate Boehringer-Mannheim
Corp., New York, NY
terfere with the reaction.
NADP Boehringer-Mannheim In assaying hexokinase, we found that
Corp., New York, NY with higher (up to 1 mM) levels of Mg2+
Glucose-6-phosphate Sigma Chemical Co., (as MgC12*6Hz0) in the reaction mixture,
dehydrogenasea St. Louis, MO the enzyme’s specific activity increased, but
Adenosine-5’-triphos- Sigma Chemical Co.,
phate St. Louis, MO then the rates increased more than linearly
Dihydroxyacetone Sigma Chemical Co., with increasing supernatant protein. At-
phosphateb St. Louis, MO tempts to elucidate this nonlinearity were
Oxaloacetic acidc Boehringer-Mannheim unsuccessful, hence the reaction mixture
Corp., New York, NY used routinely had 0.1 mM Mg2+-a con-
NADH Sigma Chemical Co.,
St. Louis, MO
centration at which the rates were linear
Acetaldehyde Eastman Organic Chem- with increasing protein but overall activity
icals, Rochester, NY lower. Because Mg2+ was to some extent
L-malic acid Sigma Chemical Co., St. limiting, the actual level of hexokinase in
Louis, MO all preparations may be twice that in Table
nL-isocitrate Sigma Chemical Co.,
St. Louis, MO
II.
Glucose-6-phosphate dehydrogenase (G-
a 325 FM units/mg of protein. 6-PDH; EC 1.1.1.49). Assay mixture:
b Supplied as the dimethylketal di-monocyclo- 0.5-1.0 mg supernatant protein, 1 mM
hexylamine salt. Prepared according to manu-
NADP, Tris-HCl buffer; 2 mu glucose-6-
facturer’s instruction.
c Prepared fresh daily as a KOH-neutralized phosphate was added to start the reaction.
solution. a-Glycerophosphate dehydrogenase (a-
GPDH; EC 1.1.1.8). Assay mixture:
indicated by control reactions in which sub- 100-200 pg supernatant protein (diluted
strate and cofactor levels were varied. 1: 20 in Tris-HCl buffer) 0.125 mM NADH,
Sources of biochemicals are in Table I. Tris-HCl buffer, 10 mM Mg2+ (as MgCl2.
Molar notations refer to final concentra- 6H2O) ; 2 mM dihydroxyacetone phosphate
tions in t’he cuvette. was added to start the reaction.
Hexokinase (HK; EC 2.7.1.1). This en- Malic dehydrogenuse (MDH; EC
zyme was measured by linking glucose-6- 1.1.1.39). Assay mixture: 50-150 pg super-
phosphate production to glucose-6-phos- natant protein (diluted 1:20 in Tris-HCl
phate dehydrogenase; the NADPH formed buffer), 0.125 mM NADH, Tris-HCl buffer;
was used as the index of hexokinase activ- 25-50 PM oxaloacetate was added to start
ity in the following assay mixture: 2.3 mg the reaction. Excess oxaloacetate inhibited
supernat’ant fraction protein, 0.1 mM Mg MDH. Therefore the optimal substrate
(as MgCla* 6HzO), 1630 units of glucose-g- level was determined for each preparation
phosphate dehydrogenase; 40 mM glucose, and the specific activity calculated from
0.1 Y Tris-HCl buffer, pH 7.4 (Tris-HCl rates obtained at the optimal concentration.
buffer). After a 2-min temperature equilibra- Alcohol dehydrogenase (ADH; EC
tion, 3 rnM ATP was added to start glucose- 1.1.1.1). Assay mixture: 24 mg superna-
phosphate formation. The reaction was al- tant protein, 0.125 mM NADH, Tris-HCl
lowed to proceed for 7 min then 1 mu NADP buffer; 1.66 mM acetaldehyde was added to
was added to start the glucose-6-phosphate start the reaction. Since acetaldehyde boils
dehydrogenase reaction. Specific activities at 21 C, all ADH reactions were measured
were calculated as the rate of NADPH at 19 C.
 ACRIFLAVINE-INDUCED CHANGES 293

TABLE II
Levels of Supernatant-Fraction Enzymes in Crithidia fasciculata Grown on o-Xylose; Results with
Untreated and Acrijlavine-Treated Cells

Preparation
Enzyme
Acriflavine-treated Untreated To Change

Hexokinase 305 (254-388) 228 (191-267) f33%

Glucose-6-Phosphate dehydrogenase 458 (418-530) 282 (234-362) -t-66%
a-Glycerophosphate dehydrogenase 1285 (1186-1438) 730 (570-794) +W%
Malate dehydrogenase 1090 (1019-1144) 845 (705-1000) +30%
Alcohol dehydrogenase 29 (24-34) 20 (16-24) +e%
NADP-Isocitrate dehydrogenase 12 (7-16) 23 (1234) -48%
Malic enzyme 252 (243-263) 323 (292-358) -21%

a Values shown are average rates expressed as nmoles pyridine nucleotide oxidized or reduced/min/mg
of protein; ranges in parentheses. For details of assay procedures see “Materials and Methods”. Per
cent change is based on the following:
Units in acriflavine cells-units in control cells
x 100
units in control cells
Each value is based on the average of 3 runs.

Malic enzyme (EC 1.1.1.40). Assay mix- used with heavy cell suspensions (e.g., 5 g
ture: 0.5-2 mg supernatant protein, 1 mM wet wt of cells diluted to a total volume of
NADP, 0.1 mu Mg2+ (as MgC12*6H20), 15 ml with distilled water) of control and
Tris-HCl buffer; 10 mM L-malate was acriflavine cells. Results are reported as
added to start the reaction. rmoles pyridine nucleotide/mg cell protein.
Isocitrate dehydrogenase (IDH; EC Pyruvate and ethanol determinations.
1.1.1.42). Assay mixture: 2-4 mg superna- After growth, cells were removed from the
tant protein, 1 mM NADP, 1 mM Mn2+ (as medium by centrifugation and the pH of
MnSO* *H20), Tris-HCl buffer; 1 mM DL- the medium was adjusted to 7.4. The pyru-
isocitrate was added to start the reaction. vate assay mixture (3.0 ml final volume)
O2 polarography. This was done in a tem- contained: 2.0 ml growth medium, 0.117
perature-controlled (25 C) Yellow Springs mM NADH, 0.85 ml pH 7.4 Tris-HCl
Instrument Co. (Yellow Springs, OH) bio- buffer (0.1 M). Lactate dehydrogenase (30
logical O2 monitor (Model 53), equipped PMolar units, Type II, Sigma) was added
with a Varian G-14 recorder (Varian In- to start the reaction; the changes in absorb-
strument Co., Palo Alto, CA). Acriflavine- ance was monitored for 5 min. The ethanol
grown or control cells (approximately 8 mg assay contained: l.O-ml growth medium, 2.0
protein) were suspended in 3.0 ml of 0.1 M ml 0.1 M pyrophosphate-semicarbazide
Tris-HCl buffer, pH 7.4, with 0.25 M su- buffer (pH 8.8)) 30 PMolar units of yeast
crose and 1 mM EDTA added. Suspensions alcohol dehydrogenase (Sigma, crystalline).
were temperature-equilibrated for 3 min be- The reaction was started by the addition of
fore measurements. Rates were calculated 1 mM NAD+ and the change in absorbance
on the basis of 265 nmoles 02 dissolved per monitored for 15 min. Uninoculated me-
3 ml of distilled water. Protein was deter- dium was assayed for pyruvate and
mined on whole cells as described (Bacchi ethanol; the final concentrations given in
et al. 1968). Results are expressed as Table V have been corrected for amounts
nmoles O2 consumed/min/mg protein. present in these controls. For protein deter-
NAD+ and NADP+ determinations. mination of cells/ml of growth medium,
The methods of Klingenberg (1965a,b) were small aliquots (5 ml) of growth medium
294 BACCHI AND HILL

were centrifuged and the cell pellet ana- the highest percentage increase; HK,
lyzed for protein by the biuret method. Cal- MDH, and ADH activities were at least
culations were as described in Bergmeyer 30% higher in the acriflavine-treated cells.
(1965). Values are expressed as rmoles/mg Of all enzymes, the uGPDH activity in ac-
protein. riflavine cells was greatest. In contrast, spe-
cific activities of the NADP+-linked IDH
RESULTS
and malic enzyme decreased in acriflavine-
The final density of C. fasciculata acri- treated cells.
flavine-treated mass cultures was 57% To determine whether the shifts in en-
lower than control cultures (Table III). Ac- zyme levels were part of an overall physio-
riflavine-treated cells had a 52% lower O2 logical change, we also determined NAD+
uptake. These results agree essentially with and NADP+ levels in control and acrifla-
those of Hill and Anderson (1969). Super- vine-treated cells. The results (Table IV) in-
natant fractions from acriflavine-treated dicate t’hat differences in NAD levels were
cells were yellowish because some drug was minimal.
present. Acriflavine (up to 100 ,UM) added Although the mean NAD+ level of acri-
to supernatant fractions of unt’reated cells flavine-treated cells was 13% lower than
did not inhibit the reactions. that of control cells, analysis of the mean
In general, the activity of soluble en- difference indicated the decrease might not
zymes associated with glucose catabolism be as great. Levels of NADP+ were essen-
was higher in acriflavine-grown cells (Table tially the same for both sets of cells.
II). aGPDH and G6PDH activities had To obtain a “physiological profile” of
both groups of cells, culture conditions were
TABLE III
scaled down and 2 sets of cells (3 flasks/
E$ects of Acrijlavine on Cell Growth set) were grown in 500 ml Erlenmeyer
and Respirationa
flasks (100 ml of medium/flask). Cells cul-
Cell type Absorbance 02 Consumption tured under these conditions took 5 days to
achieve full growth. Acriflavine-treated
control 0.60 29.5
acriflavine 0.26 14.1 cells had -30% less growth than controls
(Table V) but caused greater decrease in
(1 Absorbance (750 nm) was taken of pooled the final pH of the medium. Oxygen con-
mass cultures at time of harvesting. 01 consump-
sumption was -50% less in acriflavine-
tion is expressed as nmoles O?/min/mg of protein.
Polarography procedures in “Materials and treated cells. The glycolytic end products
Methods” section. pyruvate and ethanol (remaining in the me-

TABLE IV
Pyridine Nucleotide Levels in Control and Acrijavine-Treated Crithidia fasciculataa

NAD+* Mean Difference” NADP+d Mean Differencec

Control 2.25 f 0.064” 1.84 f 0.23

+0.25 f 0.195 +0.026 f 0.095
Acriflavine-treated 1.95 f 0.145 1.81 f 0.17

5 Usually, 4 liters of control cells and 6 of acriflnvine-treated cells were grown for each set of deter-
minations. For assay procedures, see “Materials and Methods”.
* 4 determinations.
c Control values as compared to acriflavine values.
d 5 determinations.
6 Mean difference i SD.
 ACRIFLAVINE-INDUCED CHANGES 295

TABLE V
Comparison of Control and Acriflauine-Treated Crithidia fasciculata. Cells Were Cultured in 100 ml of
Standard Medium in 600 ml Erlenmeyer Flasks”
(nmoles, (1 X 16-)-Wn;les/mg)
m;;/t;if
Flask Absorbance Final pH
02 Consumption Pyruvate Ethanol

Acriflavine
No. 1 0.30 5.30 6.2 4.25 56.3
2 0.32 5.35 6.6 4.53 48.1
3 0.31 5.45 7.0 3.83 44.2
Control
No. 1 0.47 6.15 12.6 3.34 12.9
2 0.44 6.20 11.2 3.21 11.4
3 0.45 6.46 16.5 3.32 8.1

0 Growth was stopped after 5 days; contents were treated separately for each determination. “Pyru-
vate” and “Ethanol” refer to end products of respiration in the growth medium after cells were cen-
trifuged out and the pH of the medium adjusted to 7.4 with KOH. Other details in “Materials and
Methods” and text.

dium after growth) were -27% and faxiculata have diminished

C. mito-
-370% greater, respectively, in the acri- chondrial-mediated oxidation (Hill and An-
flavine-treated cells. Presumably, differ- derson 1969)) glycolytically produced
ences in production of organic acids besides NADH would have to increase due to faster
pyruvate (e.g., succinate: Hunter, 1960) but incomplete breakdown of carbohydrate.
aided in the greater pH decrease observed In addition, reducing equivalents (as
in the medium of acriflavine-treated cells. reduced products of soluble NAD-linked
dehydrogenases, e.g., a-glycerophosphate,
DISCUSSION
malate) should be oxidized more slowly as
As noted earlier, C. fasciculata grown a result of decreased mitochondrial func-
with xylose in 5.0 PM acriflavine was 85’% tion. Higher levels of NAD-linked dehydro-
dyskinetoplastic, and had decreased levels genases would then be required to recycle
of cytochromes and other mitochondrial en- NADH produced in glycolysis by 3-phos-
zymes (Hill and Anderson 1969). In the phoglyceraldehyde dehydrogenase; activity
present study, using similar conditions, five of this enzyme is controlled by the level of
of seven soluble enzymes assayed had in- NAD+ available in the cytosol as a cofac-
creased specific activities. Although only tor (Grant 1966). The possibility of faster,
HK is part of the Embden-Meyerhof path- more incomplete carbohydrate utilization in
way, the remaining 4 enzymes are function- acriflavine-treated cells seems likely, since
ally associated with glucose catabolism: the there are higher concentrations of pyruvate
NAD-linked dehydrogenases recycle and alcohol accumulated in the growth me-
NADH produced in glycolysis and GGPDH dium of these cells.
is the entrance to the pentose phosphate The increased activity of uGPDH in ac-
pathway of glucose catabolism. riflavine-induced dyskinetoplastic cells
Increases in activities of NADH-linked seems to approximate the situation in the
dehydrogenases (aGPDH, MDH, ADH) in slender bloodstream form of T. brucei-sub-
acriflavine-treated cells may denote higher group trypanosomes in so far as high activi-
glycolytic activity. Since dyskinetoplastic ties of NAD-linked crGPDH are needed to
296 BACCHI
oxidize glycolytically produced NADH.
The level of soluble UGPDH in T. rhode-
siense slender blood forms was ~50%
higher than in culture forms (Ryley, 1962).
The parallel is limited, however, since slen-
der blood forms have a cyanide-resistant
,GP oxidase (Grant and Sargent 1960;
Grant et al. 1961), while in acriflavine-
treated C. jasciculata this enzyme remains
cyanide sensitive (Hill, unpublished).
Bayne et al. (1969) found that growth in
acriflavine slightly depressed MDH (21%)
and lactate dehydrogenase LDH (17%) ac-
tivities in Trypanosoma conorhini; succinic
dehydrogenase decreased 73%. The aGP ox-
idase activity of such T. conorhini cells
proved less sensitive to cyanide. In these
studies, acriflavine was added to S-day
log-phase cultures. The above dehydrogen-
ase assays were apparently carried out on
whole-cell homogenates (see also Bayne
and Roberts 1969) and so introduce the un-
certainties in interpretation of combined
mitochondrial and cytosol protein (~44%
of total homogenate protein was attributed
to combined mitochondrial fractions : Table
III, Bayne and Roberts 1969). In the
present study, supernat’ant fractions were
cleared by centrifuging at 12,000g for 10
min, ,thus removing kinetoplasts and mi-
tochondria. Succinic dehydrogenase was not
a contaminant of the supernatant fractions
(Bacchi et al. 1968)-thus excluding mem-
brane-bound mitochondrial enzymes as con-
taminants. These considerations limit direct
comparison of the 2 systems of inducing dys-
kinetoplasty.
Further similarities between dyskineto-
plastic C. jasciculata and slender blood
forms are indicated by the lower activities
of malic enzyme and NADP-linked LDH.
In T. .rhodesiense, these enzyme levels were
60-70% lower in slender blood forms as
compared to culture forms (Ryley 1962).
Acriflavine-treated C. jasciculata cells had
20-50% lower enzyme activity as compared
with levels in control cells.
The large (66%) increase in the activity
AND HILL
of GGPDH ostensibly reflects increased ac-
tivity of the pentose phosphate pathway in
acriflavine-treated cells. It is tempting to
interpret this as a compensatory attempt by
the dyskinetoplastic organisms to maintain
a supply of NADPH. NADPH is necessary
for steroid and lipid syntheses (Simpson
and Estabrook 1969). Impairment of
NADPH-generation capacity is inferred
from the lower activity of malic enzyme
and NADP-IDH in acriflavine-treated or-
ganisms. The latter soluble enzyme may
also have regulatory functions in both the
carbohydrate metabolism and oxidative
metabolism of C. jasciculata (Marr and
Weber 1969a,b).
Alterations in the level of NAD+ - and
NADP+-linked enzymes do not seem to af-
fect concentrations of the oxidized pyridine
nucleotides in acriflavine-treated cells. The
explanation for this constancy may be that
pyridine nucleotides no longer needed for
operation of the Krebs cycle are in greater
demand for cytosol reactions.
Three of the enzymes assayed in the
study are sited in both cytosol and mito-
chondrion in mammalian cells: NADP-
IDH, MDH, and malic enzyme. The relative
amounts of mitochondrial forms of these en-
zymes contaminating the C. jasciculata
supernatant fractions are not known, how-
ever the degree of contamination may be of
minor importance in view of the following
considerations. Marr and Weber (1969a)
found high activity of NADP-IDH in the
cytosol fraction of C. jasciculata; Bayne and
Roberts (1969) found only 21% of the total
MDH activit’y of untreated T. conorhini to
be associated with mitochondrial fractions;
most of the mitochondrial malic enzyme ac-
tivity of bovine adrenal cortex was demon-
strable only after sonication of the mito-
chondrial fraction (Simpson and Estabrook,
1969). Determination of these enzyme levels
in mitochondrial preparations of dyskineto-
plastic organisms should help clarify the
source of synthetic control.
Comparison of mitochondrial and soluble
 ACRIFLAVINE-INDUCED CHANGES 297

enzyme activities from trypanosomatids of the Society of General Microbiology 16,

281-293.
treated with other kinetoplast-specific drugs GRANT, P. T., AND SARGENT, J. R. 1960. Properties of
(e.g., ethidium bromide, Steinert 1969; p- L-cr-glycerophosphate oxidase and its role in
rosaniline, Inoki et al. 1969) may clarify the respiration of Trypanosoma rhodesiense.
whether these drugs act exactly like acri- Biochemical Journal 76,229237.
flavine in respect to the kinetoplast, i.e., GRANT, P. T., SARGENT, J. R., AND RYLEY, J. F.
1961. Respiratory systems in the Trypano-
whether the sensitivity of the kinetoplast to somatidae. Biochemical Journal 81,269296.
DNA-binders elicits a stereotyped pattern HILL, G. C. AND ANDERSON, W. A. 1969. Effects of
of dyskinetoplasty and attendant change in acriflavine on the mitochondria and kineto-
levels of oxidative vs glycolytic enzymes. plast of Crithidiu jasciculata. Journal of Cell
The shift in activities of soluble enzymes Biology 41,547~561.
HORECHER, B. L., AND KORNBERG, A. 1957. Isola-
(and quite likely of overall glycolysis at- tion of diphosphopyridine nucleotide and tri-
tending dyskinetoplasty) is only one aspect phosphopyridine nucleotide. II. Triphospho-
of the cellular adaptation resulting from pyridine nucleotide. In “Methods in Enzy-
impairment of mitochondrial function. mology” (S. P. Colowick and N. 0. Kaplan,
Other adaptational changes should be pres- eds.), Vol. 3, pp. 879-882, Academic Press,
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ent within the mitochondrion since it has HUNTER, F. R. 1960. Aerobic metabolism of
many biosynthetic and catabolic functions Crithidia jasciculata. Experimental Parasi-
besides electron transport and oxidative tology 9,271-281X
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Organisms” Vol. 18, “International Encyclo-
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