CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
Characterization of an Antimycin A Resistant
Cell Line of
Polytomella parva
A thesis submitted in partial satisfaction of the
requirements for the degree of Master of Science in
Biology
by
Lee Alan Baresi
January, 1986
The Thesis of Lee Baresi is approved:
Charles R. Spotts, Ph.D.
Marvin H. Cantor, Ph.D.
J
eph Moore, Ph.D. (Chairman)
ii
ACKNOWLEDGEMENTS
I have been priviledged in meeting many wonderful people during
my
rather
lengthy
stay at
c.s.u.N.
Each possessing different and
special characteristics that made the long hours,
late
nights
and
boredom associated with the completion of this work tolerable.
The
first person to which I owe a great deal is Dr. Joe Moore.
His patience was streched to
procrastination.
areas
outside of
its
breaking
point
by my constant
He also served as a source of wisdom and advice in
Biology.
For his guidance throughout these many
years I am extremely grateful.
Warmest thanks go to Dr. cantor for his helpful {and relatively
painless) critique of this masterpiece.
and
succint.
Without
with nary any hope left.
agreeing
to
his
His
comments
were
honest
help I would be in for another semester
My deepest respects
to
Dr.
Spotts
for
serve on my committee (that's what you get for being
available during the semester break!)
To the stockroom personel: Pam (aka Mom), Stan the Man, captain
Jeff and Scott (why are you here so latel)
with my constant demands for antimycin.
v.
thanks for putting up
I know that your life
will
be much easier now that I am gone (but will it be as much fun?) Your
help and friendship is treasured.
Fondest
appreciation
goes
;;;
to
Don
(the lab scratching post)
Hawken for doing the piddling things I had neither the time nor
patience
for
in
the
the last year. Special thanks to Ed Koprowski for
making life at Science South room 219 anything
but
dull,
and
for
showing me how to deal with adversity with style and panache.
~
deepest
friends.
though
17-9AA.
thanks
to
Amerigo
and Kim
To Amerigo goes the credit for the
it
for being such good
isolation
(fortuitous
may have been!) of the soon to be world renown P. parva
Thanks for the good times, friendship, honesty and
concern
(not to mention the great sound effects!)
In the long list of friends that have been a source of help or
inspiration to me I now address the one and only Mark Sussman.
Well
Mark, if it had not
have
finished
this
for
you
I
would
probable never
thesis. Your seemingly constant needling was a great
incentive for me.
extend my
been
If this was
deepest
thanks.
intentional
If
it
was
on
your
part
fellow
student (a neccessary requirement for admission to heaven if
I understand correctly).
for
I
not intentional then you
deserve credit for spontaneously and unconsciously helping a
grad.
than
the
Thanks for the use of
your
computer
and
time you spent putting in all the control characters (even
if they didn't work!).
Thanks for the sagely advice and for
giving
me a different outlook on life in general.
Thanks
to
all
the exceptional people I met while at
whose names do not appear here (yes, this means you Mina,
Rhonda etc.).
Thanks
to
all
(most?)
iv
c.s.U.N.
Stanley,
of my 100L, 150L and l51L
students for memorable times.
In
conclusion,
for
all
of
you
grad.
students who have to
listen to everybody ask "are you STILL at C.S.U.N." I
fire
method
have
sure
to. insure the sucessful completion of your candidacy.
First give up _all semblance of a normal social life.
Second give up
all semblance of a normal life. Finally tell those people
their
a
to
stuff
insensitive inquisitiveness right up their ••• Best of luck to
all, it can be done!
DEDICATION
This thesis is dedicated to my
parents, Olympia and LOuis Baresi, as
well as the rest of my family for
your undying faith and love.
vi
TABLE OF CONTENTS
Acknowledgements
iii
Dedication
vi
List of Figures
viii
Abstract
ix
Introduction
1
Materials and t1ethods
11
Results
16
Discussion
32
Bibliography
39
vii
LIST OF FIGURES
Page 7 - The electron transport chain.
Page 18 - Antimycin A dose-response growth curves.
Page 20 - Growth curves for control and resistant cells.
Page 28 - The effect of antimycin A on oxygen consumption.
viii
ABSTRACT
Characterization of an Antimycin A
resistant cell line of
Polytomella parva.
by
Lee Baresi
Master of Science in Biology
A cell
line
of
Polytomella parva resistant to the metabolic
inhibitor antimycin A was isolated.
These cells are resistant at
a
concentration of 2 X lB-SM.
Studies
on
the growth of this resistant cell line (designated
P. parva 17-9AA) showed significant changes
cells.
Resistant
cells
over
that
of
control
in the presence of antimycin have a longer
lag phase and generation time than control cells. Resistant cells in
antimycin also show an extended stationary phase compared to control
cells, and lack the ability to produce cysts.
in a medium without inhibitor
show a
Resistant cells grown
decrease
in
lag
time
and
generation time.
Respiration
rates
were
significantly diminished in resistant
cells. Resistant cells in the presence of antimycin A respire
ix
at
a
rate approximately 80% that of control cells.
Resistant
of
both
cells
succinate
Cytochrome
c
show a 1.5 fold increase in specific activity
and
oxidase
NADH
dehydrogenase
activity
over
control
cells.
in resistant cells was 78% that of
control values consistant with the notion of a block in the electron
transport chain between respiratory complexes I or
II
and
complex
III.
The
specific
activity
mitochondrial fractions
resistant
cells
activity may
compensates
approximately 1.6 times
greater
in
compared to control cells. This increase in ATPase
provide
for
was
of ATPase in both crude homogenate and
the
a
mechanism whereby
inhibition of
antimycin.
X
the
respiratory
resistant,
complex
III
cell
by
INTRODUCTION
History
Polytomella agilis is a flagellate protozoan first described by
Aragao in 1910. Subsequent work by Doflein (1916), Kater (1925) and
Kater and Burroughs (1926) outlined the life cycle and general
morphology of the organism.
In a recent review of the Culture
Collection of Algae at the University of Texas at Austin by starr
(1978) stock culture L 193 originally designated as P. agilis from
Pringsheim was reclassified as P. parva.
It should be noted that
earlier work on P. agilis (Sheeler et. al., 1970) is on the
identical organism as the one used for this study.
Growth Characteristics
The life cycle of P. parva may be divided into four stages:
motile vegatative, pre-cyst, cyst and post cyst.
is a free swimming quadriflagellate.
The trophic stage
During the trophic stage
Polytomella reproduces by binary fission and by sexual reproduction
(Lewis et. al., 1974, Moore and CUshing, 1979).
The mean generation
time is approximately 4-Shr in batch culture at 25C.
The growth
curve for P. parva displays three stages:
l)Lag phase : The cell number remains relatively constant as
the cells undergo a change in chemical composition prior to an
increase in cell number The duration of the lag phase varies from
one organism to the next and upon the chemical composition of the
media. In complex medium, P. agilis has a lag time of 2-4hrs
1
2
(Sheeler et. al., 1970).
Mean cell volume increases as the rate of
cell growth exceeds the rate of cell division (Sheeler et. al.,
1968)
2) Log (exponential) phase :
This is a state of balanced
growth whereby a doubling of the biomass is accompanied by a
doubling of all other measurable properties of the populat.ion.
number increases exponentially with time.
Cell
In P. agilis mean cell
volume decreases during early logarithmic growth to a level that
remains essentially constant throughout the remainder of this
phase.
3) Stationary phase - The density of the population is limited
either by the exhaustion of available nutrients, the accumulation of
toxic waste products, or other environmental changes (e.g. pH,
oxygen tension).
The rate of growth declines and eventually stops.
Mean cell volume increases during stationary phase in P. agilis
(Sheeler et. al., 1968).
Cells in log phase are typically utilized for studies of
metabolism because all major cellular parameters change at a
constant rate.
In Polytomella , subcellular parameters such as dry
weight/ml, protein/ml and carbohydrate/ml increase at a constant
rate during log phase (Sheeler et. al., 1970).
The transistion of Polytomella into stationary phase is one of
unbalanced growth where mean cell volume and carbohydrate levels
increase with no change in levels of protein (Sheeler et. al.,
1968).
3
Encystment and excystment occur in many free-living and
parasitic protozoa.
According to Trager (1963) cyst formation is a
"reversible differentiation which enables the organism to survive
the harsh effects of an unfavorable environment." The ability to
encyst and excyst involves a considerable degree of differentiation
and reorganization of cellular structure.
concomittant changes in enzyme activity.
There are also
There are associated
ultrastructural changes in the mitochondria, proplastid network,
endoplasmic reticulum, Golgi etc.
al., 1970).
(Moore et. al., 1968, Moore et.
Encystment begins as early as the mid-logarithmic phase
of growth and is complete by the beginning of the stationary phase.
In P. parva, at most 15% of the motile cells encyst The cause of
encystment is not known.
The process of cyst formation in Polytomella seems to differ
from that seen in other protozoa in that environmental conditions
known to trigger encystment (depletion of essential nutrients,
increase in pH, changes in oxygen tension, increase in the levels of
toxic waste products etc.) in these organisms fail to do so in
Polytornella (Baresi, unpublished data).
Encystment in Polytomella
is probably not due to the exhaustion of nutrients in the medium or
the accumulation of waste products since conditioned media (i.e.
media isolated from actively growing cultures) support further
growth (Sheeler et. al., 1970, Baresi, unpublished observations).
Metabolism
4
Acetate supports the growth of many protozoa (Pringsheim, 1937)
including Euglena gracilis, Astasia longa (Cramer and Myers, 1952),
and Polytomella sp.
(Lloyd and cantor, 1979).
Alternative carbon
sources may also be utilized for growth by these organisms.
E.
gracilis can utilize short chain fatty acids, alcohols and
tricarboxylic acids, whereas odd numbered fatty acids fail to
support the growth of P. caeca (Wise, 1955, 1959).
P. parva is an
acetate flagellate that can readily utilize acetate and related
compounds as carbon sources.
The carbon and energy metabolism of
the acetate flagellates has been extensively studied (Lloyd and
Cantor, 1979).
These organisms represent some of the most primitive
protozoa and bridge the gap between photosynthetic and heterotrophic
modes of energy production.
cantor and James (1962,1965) showed that P. agilis can adapt to
the utilization of propionate and butyrate with associated changes
in respiration rate and lag time.
The different respiration rates
on various media suggest different processes for their utilization.
Cantor (1970) and Haigh (1964) have shown the presence of isocitrate
lyase and malate synthase, enzymes indicative of a functional
glyoxylate cycle in P. agilis and P. caeca , respectively.
The
glyoxylate cycle involves the bypass of the decarboxylation
reactions of the TCA cycle.
Respiratory Chain
Burton and Moore (1974) demonstrated that a log phase cell of
P. agilis contains a single highly convoluted mitochondrion.
At
5
some point during logarithmic growth, the number of mitochondria per
cell is considerably less than that implied by casual examination of
random thin sections.
A single reticular mitochondrion also occurs
at some stage of the life cycle of E. gracilis (Osafune, 1973).
osafune (1973) has suggested that the appearance of solitary "giant
mitochondria" is directly related to reduced respiration.
Cells of
Polytomella isolated at various times during the growth curve show
major changes in mitochondrial protein concentration and absorbtion
at 254nm (Sheeler et. al., 1970).
The mitochondria of Polytomella contain a classical respiratory
chain {cantor and Burton 1975, Lloyd and Chance 1968) consisting of
cytochromes
~'
b, and c.
The presence of cytochromes
~'
b, and c in
Polytomella and P. caeca was reported by Webster and Hackett (1965),
and Lloyd and Chance (1968) showed the presence of several different
flavoproteins.
The effect of environment on both cytochrome content
and metabolism in Polytomella has been investigated. Lloyd et. al.
(1970) showed a reduction in cytochromes
~
and b in the presence of
chloramphenicol and an increase in the mean generation time and a
decrease in the peak population density.
demonstrated the absence of cytochromes
cytochrome
~
Cantor and Burton (1975)
~
and b, and a decrease in
in thiamine deficient cells of P. agilis.
The two main functions of the respiratory chain are to
reoxidize the NAD+ formed during glycolysis and the TCA cycle, and
to conserve the energy released from the oxidation of NADH and
succinate through oxidative phosphorylation in the form of the
6
terminal pyrophosphate bond of ATP. It is now generally accepted
that the fundamental oxidative and phosphorlative events are carried
out by five enzyme complexes (Figure I).
Four of these are
respiratory complexes whereas the fifth is ATP synthetase (ATPase
complex).
The four respiratory complexes that couple electron
transfer to oxidative phosphorylation are located in the inner
mitochondrial membrane and are composed of stationary electron
acceptors and donors, as well as mobile carriers (Tzagoloff, 1982).
The first enzyme, NADH-coenzyme Q reductase (complex I)
oxidizes NADH and reduces coenzyme Q.
The second enzyme,
succinate-coenzyme Q reductase (complex II) is responsible for the
oxidation of succinate and the reduction of coenzyme Q.
coenzyme Q is reoxidized by coenzyme QH2-cytochrome
Reduced
c reductase
(complex III.) The final respiratory complex is cytochrome oxidase
(complex IV) where electrons are passed on to the terminal acceptor,
molecular oxygen.
Coenzyme Q and cytochrome
£
act as the mobile
carriers shuttling electrons between the different complexes.
Antimycin A
The effects of respiratory inhibitors are diverse and far
reaching.
Lloyd et. al.
(197~)
studied the effects of
chloramphenicol, an inhibitor of mitochondrial protein synthesis, on
the growth and cytochrome content of P. caeca.
He reported no
change in mitochondrial structure in the presence of the inhibitor
although the concentrations of cytochromes a and b decreased.
Schwitzguebel and Palmer (1983) have shown in Neurospora crassa that
Complex I
NAOH •FMN -FeSt -FeS4 -FeS3 -FeS2
FeSm.
I
Cu Cu
I
I
Jo
coo- bT•bKJc,-c .. o -o3
b~~e
ATP /
Succinate • FAD ..,... FeS 5 • 1 - - F'eS 5 _3
ATP
ATP
Complex m
Complex Ill
I
FeS 5 • 2
Complex
Figure I.
n
The electron transport chain (modified from Tzagoloff, 1982).
2
8
the oxidation of NAD linked substrates become insensitive to
rotenone in the presence of chloramphenicol.
The antibiotic, antimycin A, was first discovered by Leban and
Keitt in 1948 as a fungicide produced by Streptomyces sp.
The
inhibitory effects of antimycin on mitochondrial respiration was
first observed by Ahmad et. al.
(1950).
Potter and Reif in 1952
showed that antimycin had no effect on succinate dehydrogenase,
cytochrome oxidase, NADH dehydrogenase or any of the known
cytochromes and concluded that antimycin blocks the "Slater factor"
(complex III).
Chance (1952) showed that in the presence of
substrate, air and antimycin, cytochrome b is reduced wheras
cytochromes
~
and
~
are oxidized thereby localizing its site of
action between cytochromes b and
~
This was later confirmed by
Keilin and Hartree (1955) and Lloyd et. al.
in P. caeca (1968.)
Antimycin A is a mixture of at least four compounds of closely
related structure known as antimycin Al, A2, A3 and A4.
The
antibiotic is a potent inhibitor of coenzyme QH2-cytochrome c
reductase (complex III), a multipolypeptide protein of molecular
weight approximately 230,000 daltons.
Complex III contains one
antimycin binding site (Slater 1973).
Both cytochrome band one of
the iron sulfur centers have been considered as possible targets of
antimycin and it is known that antimycin effects iron-sulfur centers
involved in the oxidation of succinate (Thayer et. al.
1982).
Rieske (1976) and co-workers have synthesized a derivative of
antimycin and used it as an affinity label. This radioactive
9
compound was found to bind covalently to a 12,500 dalton polypeptide
in complex III. Pretreatment of complex III with unlabelled
antimycin abolished the subsequent ability of the affinity label to
bind thereby implicating the low molecular weight polypeptide as the
point of binding of antimycin. Whether or not this polypeptide has
any function as an electron carrier is not known.
Metabolic Adaptation
The ability of an organism to circumvent sites of inhibition of
oxidative phosphorylation is an example of metabolic adaptation.
The adaptation may involve the synthesis of new components capable
of restoring normal or quasi-normal function.
Alternatively the
activity and/or selectivity of certain components may change in
response to the inhibition.
Cantor (1978) showed that in thiamine deprived cells of P.
agilis succinate dehydrogenase activity was reduced to 10% whereas
ATPase activity was twice that of control cells. Thiamine
deprivation had no effect on generation time but cytochrome a and b
were essentially absent and peak population density decreased.
Even
though there was a drastic change in the mi-tochondrial
ultrastructure (Cantor and Burton, 1975) the rate of oxygen
consumption did not change compared to control cells.
This suggests
an alternate or modified electron transport system. The ability of
organisms to adapt their electron transport systems to different
growth conditions is well documented.
Henry et. al.
(1983) working
with Paracoccus nitrificans showed that under aerobic conditions
10
with hydrogen as the electron donor the bacterium synthesizes a
hydrogenase as part of the electron transport system.
In anaerobic
conditions in the presence of nitrate as the sole electron acceptor,
the bacterium synthesizes nitrate reductase, nitrite reductase,
nitric oxide and nitrous oxide reductase.
cytochrome b and c are increased.
Also the levels of
Cantor and James (1965) have
suggested that metabolic adaptation can be explained in terms of
induced synthesis of specific enzymes, changes in cell membrane
permeability, or by the availability of cofactors such as ATP, NAD,
NADP etc.
This thesis involves the partial characterization of a strain
of P. parva resistant to the respiratory inhibitor antimycin A. The
purpose was to gain insight into the mechanism of resistance.
The
resistance could be attributed to a change in cell permeability,
resulting in the restriction of the antibiotics access to the
electron transport system, degradation of the inhibitor or genetic
adaptation resulting in the synthesis of alternate pathways or
agents that specifically block the inhibitory action of antimycin.
MATERIALS AND METHODS
Growth Conditions
P. parva were grown in the dark at 22C in a complex medium
consisting of 0.2% sodium acetate, 0.2% yeast extract and 0.1%
tryptone.
Cells resistant to the respiratory inhibitor antimycin A
were subcultured in complex media supplemented with antimycin A to 2
X 10-5M.
The antibiotic was always present in the media of
resistant cells.
Antimycin A was added as an ethanolic solution by
sterile filtration.
Isolation Of Antimycin Resistant Cells
Normal cells from late-log phase were placed in sterile 50 X
9mm Falcon petri plates and irradiated for 45 sec. at a distance of
29cm with a ultraviolet light source of 260nm wavelength. The plates
were then scanned under the microscope with a 20X phase objective
and individual cells were isolated using a micropipette.
These
cells were placed in fresh complex media with 5 X 10-6M antimycin
A.
Of the initial 20 cells isolated, 417 was chosen because it
showed normal growth characteristics.
The inhibitor concentration
was increased over a period of 5 months to 2 X 10-5M. After several
months of growth individual cells were reisolated and a new culture
was started from the ninth isolate.
designated P. parva 17-9AA.
11
This resistant strain is
12
Growth curves
CUltures were started from inocula from late-logarithmic or
early stationary phase cultures such that the initial density was
approximately 6000 cells/ml.
Cell counts were made of 200ul samples
using a Model B coulter electronic particle counter equipped with a
100u orifice.
Preparation Of Extracts
Cells from logarithmically growing cultures were harvested at
4C by centrifugation at 600Xg for 15min and washed three times in
0.05M Tris-HCl buffer pH 7.40.
Cells utilized for ATPase activity
were resuspended in a buffer containing 0.32M sucrose-10mM
Tris-HCl-0.5mM MgC12 (pH 7.40), whereas cells assayed for succinate
dehydrogenase, cytochrome c oxidase and NADH dehydrogenase were
resuspended in 20mM potassium phosphate buffer pH 7.40.
The crude homogenate was prepared by sonication using a Branson
sonifier (30sec, setting i6.) The mitochondrial fraction was
obtained by centrifuging the sonicate at 600Xg for 15min to remove
nuclei, membranes and starch granules followed by centrifugation of
the supernatant at 11,000Xg for 15min to isolate the mitochondrial
pellet.
The resulting pellet was resuspended in the appropriate buffer
and enzyme activities were determined at room temperature within
2hrs after sonication.
13
Enzyme Assays
Succinate Dehydrogenase
The enzyme was assayed on the mitochondrial pellet by following
the reduction of 2-6 dichlorophenolindophenol (DCPIP) at
66~nm
using
a Beckman Model 24 recording spectrophotometer with a full scale
expansion of 2A at 25C. The reaction mixture contained
~.5ml
of
~.1M
succinate, 9.lml of 1 X 19-3M KCN, and 2ml of the appropriately
diluted extract such that the reaction was linear with time.
The
reaction was started by the addition of 0.lml of 2.5 X 19-3M DCPIP.
Specific activity was calculated as change in absorbance/min/mg
protein.
Cytochrome c Oxidase
Enzyme activity was determined on the mitochondrial fraction by
following the oxidation of reduced cytochrome £ at 559nm.
reaction mixture contained
S~~ul
potassium phosphate buffer (pH
phosphate buffer and
19~ul
The
of 1% reduced cytochrome c in 20mM
7.~0),
19~ul
of 29 mM potassium
of the appropriately diluted extract such
that the reaction was linear with time.
Cytochrome c was reduced
with sodium dithionite (0.25mg/ml) and dialyzed against three
changes of
2~mM
phosphate buffer (pH
7.~~)
at 4C to remove residual
reducing agent. Enzyme activity was calculated as change in
absorbance/min/mg protein.
NADH Dehydrogenase
14
The enzyme was assayed on the mitochondrial fraction by
following the decrease in absorbance of NADH at
mixture contained
7.~~)
and
3~~ul
6~~ul 2~mM
NADH, 1ggu1
of extract diluted with
2~
34~nm.
The reaction
mM phosphate buffer (pH
2~mM
phosphate buffer so
that the decrease in absorbance was linear with time. Specific
activity is expressed as change in absorbance/min/mg protein.
ATPase
ATPase activity was determined on both the crude homogenate and
the mitochondrial fraction using the modified Fiske-Subbarow method.
Activity was determined by measuring the inorganic phosphate
liberated following the addition of 3mM ATP at 25C to a medium
containing
1~0rnM
KCl, SmM MgC12 and 10mM Tris-HCl buffer (pH 7.40)
in a final volume of 2.5ml. Enzyme activity is expressed as umoles
Pi liberated in 10 min/mg protein.
Oxygen Consumption
Rates of oxygen consumption were determined in intact cells
using a Model 53 Yellow Springs Instruments oxygen electrode at 22C.
Measurements of respiration rates in intact cells were made on 3ml
samples taken directly from the culture.
The effect of antimycin A
on normal and resistant cells was determined by the addition of 33ul
of a 1 mg/rnl antimycin A solution to a 3rnl sample after a baseline
rate had been established.
Rates of oxygen consumption are
expressed as umoles 02/hr/1E6cells.
15
DNA Quantification
Total cellular DNA was determined by a modified diphenylamine
method of Burton (1956) on 7.5L cultures of mid-log phase cells.
The cells were collected by centrifugation at 600Xg and washed twice
in 0.05M Tris-HCl buffer pH 7.40, resuspended in 20mls of 0.01M NaCl
and sonicated. cellular protein was removed by treatment of 8ml
portions with an equal volume of 25% TCA and the resulting pellet
hydrolyzed twice with 2mls of diphenylamine reagent (100mls glacial
acetic acid :
1.5mls cone.
sulfuric acid : 1.5g diphenylamine :
30ul cold acetaldehyde) for 30 min at 70C.
The supernatants were combined and assayed using calf thymus DNA
(Sigma t D-1501) as the standard.
Protein Determination
Total cellular protein was determined by the method of Lowry
et. al.
(1951) using bovine serum albumin as the standard.
@ '
RESULTS
GROWTH CHARACTERISTICS
Figure II shows three dose-response growth curves of antimycin
A treated P. parva.
The inhibitory effects of antimycin first
appear at a concentration of 10-9M with complete inhibition of
growth at 10-7M.
Inhibition of growth was complete within one hour
after the addition of 10-7M antimycin.
cultures were inoculated from stock cultures in stationary
phase insuring the presence of a lag phase. Both control and
antimycin A resistant cells show typical growth curves (Fig. Ilia,
IIIb).
However the length of time spent in each of the phases
differed considerably.
Control cells had a much shorter lag phase
compared to the resistant population.
The lag time for control
cells was approximately 6hrs whereas resistant cells in media with
antimycin A had a lag time of approximately 24hrs (Table I).
Resistant cells when placed in fresh complex medium without
antimycin A show a 30% decrease in their lag time compared to cells
in antimycin A, suggesting that the resistant cell has an impaired
ability to adapt to fluctuations in the environment due to the
presence of the drug.
Control cells incubated with 2 x 10-5M
antimycin a fail to survive (Figure Ilia).
These data show that the
cells utilized for this study are resistant to a concentration of
antimycin A 200 times the lethal dose.
16
17
Figure II.
Antimycin A dose-response growth curves for P. parva.
Antlh·mycin A was added to mid-logarithmic celTs at the
12t hour of growth.
CJ- P. parva (control) in complex medium.
)( - P. parva
~-
~
(control~
1 X 10-
~1
in complex medium with
anti myc i n A•
P. parva (control) in complex medium with
1 X 1o-9~1 antimycin A.
-f. parva
(contro~) in complex medium with
1 X 10- M antimycin A.
18
*
0
L(')
19
Figure I II a.
Growth curves for control and resistant cells of
P. parva.
0
- P. parva (control) in complex medium.
X - p. parva (control) in complex medium with
2 X 1o-5M antimycin A.
A
- P. parva 17-9AA in complex medium.
#.-f. parva 17-9AA in complex medium with
2 X 1o-5M antimycin A.
The last data point at 1200hrs is of sonicated
material and therefore represents only the cyst
po pu 1at ion •
Figure Illb.
Growth curves for control and resistant cells of
P. parvf. The graph is an expansion of the first
~Ohrs o growth in Fig. lllb so as to illustrate
the differences in generation times.
D- f. parva (control) in complex medium.
)\- f. parva (control) in complex medium with
2 X 1o-5M antimycin A.
A- P. parva 17-9AA in complex medium.
#- P. parva 17-9AA in complex medium with
2 X 10-5M antimycin A.
20
7
8
D
5
!::!
"iii
....u
CJI
4
0
•
J
2
40
80
100
80
120
TIME (HRS X 10)
7
8
5
J
2
10
20
30
40
TIME (hrs)
50
60
70
80
Maximum
~1edium
P. parva (control)
P. parva 17-9AA
P. parva 17-9AA
Table I.
Complex
Lag Time
(hrs)
6.0 ± 1.5
Generation
Time (hrs)
4.25 + 0.5
Populat~on
Size (X10 cells/ml)
2.45 ± 0.5
Cysts
(% Peak
Population)
13.6 :!: 2.4
Complex + 24.0 ± 0.9
antimycin
6.00 ± 0.3
2.63 ± 0.3
0.16 + 0.1
16.0 :!: 1.2
5.00 + 0.7
2.82 ± 0.5
1.01 + 0.4
Complex
Growth parameters for resistant and control cells of f. parva in thg presence and
absence of antimycin A. The concentration of antimycin A = 2 X 10- M. Data represent
the mean ±the standard deviation of two experiments. Statistical analysis by the
Student's t test show the differences in generation time between control and resistant
cells (in the presence of antimycin) to be statistically significant, p<0.05. The
differences in lag time between control and resistant cells (in the presence of
antimycin), p<0.001; control and resistant cells (in the absence of antimycin),
pc::0.05; and resistant cells (in the presence and absence of antimycin), p~0.01
are all statistically significant.
22
The generation time of the resistant cell is approximately 48%
greater in the presence of the inhibitor than in control cells (6hrs
vs. 4.25hrs respectively) and 28% greater than control cells when in
the absence of the inhibitor (g.t. = 5.88hrs, Table I).
The
generation time is the time it takes for the population density to
double and represents the time cells take to pass through the
different phases of the cell cycle before division occurs.
Abnormal
generation times can be attributed to delays in any phase or
combination of phases.
The length of time spent in log phase varies
from approximately 24hrs in control cells to 32hrs in resistant
cells in the absence of the inhibitor and 48hrs for resistant cells
in the presence of antimycin A (Table I).
The transistion from log to stationary phase occurs at about
35hrs in control cells and approximately 78hrs in resistant cells in
the presence of the inhibitor.
The stationary phase lasted 98hrs in
control cells before the rate of cell death began to exceed that of
cell division.
In contrast, the resistant cells in the presence of
the inhibitor remain in stationary phase for approximately 688hrs.
This 6 1/2 fold increase in the time spent in stationary phase is
one of the most significant differences between resistant and normal
cells and indicates a drastic change in the fundamental physiology
of the organism.
Control cells are characterized by the ability to produce
cysts.
Only a small percentage of the total number of cells in the
population ever encyst.
In control cells approximately 13.6% of the
23
peak population density formed cysts by the 7th week of growth.
Resistant cells have lost the ability to produce cysts in
appreciable numbers regardless of whether or not the inhibitor is
present in the culture medium (Table I).
Resistant cells grown in
fresh media without antimycin A show a six fold improvement in the
ability to produce cysts demonstrating that the impairment may be
reversible.
The number of cysts produced in the absence of the drug
is still less than 10% of that produced by control cultures.
Microscopic observation of resistant cultures after seven weeks of
growth with inhibitor present show a preponderance of motile cells
with exceedingly few cysts.
The cysts present are devoid of the
large starch granules prevalent in cysts of normal populations.
The
cysts of resistant cells are also more opaque than control cysts and
are slightly larger (average size app. 12 microns as compared to 10
microns for normal cysts: Table II).
There is also no difference in
total cellular DNA between resistant and control cells (0.071
pg/cell for control cells compared to 0.072 pg/cell for resistant
cells in the presence of antimycin).
The differences in lag and generation time of resistant cells
over that of control cells coupled with the prolonged time spent in
stationary phase and the inability to produce significant numbers of
cysts suggest possible changes in metabolic pathways.
Therefore the
respiration rates of both control and resistant cells were studied
to determine if the changes seen in the growth curves could be
associated with changes in respiration.
Also, oxygen consumption is
I'
,
24
urn
Complex
t~edi
P. parva {control)
P. parva 17-9AA
Complex +
antimycin A
20
Size (urn)
9.95 + 0.22
20
12.1 + 0.25
n
p <0.001
Table II.
Cyst size in control and resistant cells of P. parva.
Data represent the mean + the standard deviation of
20 observations (n). The P value was calculated by
the Student•s t test.
25
an indication of the activity of the terminal portion of the
respiratory chain.
OXYGEN CONSUMPTION
Oxygen consumption in control cells (Table III) is relatively
constant throughout the log phase and into stationary phase.
At
very high cell densities (>2.75 X 1E6cells/ml) oxygen consumption
decreases by approximately 70% (x = 0.201 +/- 0.03
umoles/hr/1E6cells, n=4).
Resistant cells respire at a rate equal
to 80% that of control cells throughout log phase up to the 7th day
of growth.
oxygen consumption of resistant cells past the 7th day
is 80% that of log phase resistant cells and approximately two fold
greater than control cells at the same stage of the growth phase.
This difference reflects the difference in growth characteristics
between the two cell lines.
On the 7th, day normal cells begin to
undergo cell death as the population density declines and the
proportion of cysts increases.
Resistant cells are still in
stationary phase on the 7th day with little or no cell death taking
place.
The effect of antimycin A on the respiration of control cells
is seen in Fig. IV.
After establishing a baseline rate for
respiration 33ul of a lmg/ml solution of antimycin A was added so
that the final concentration in the sample chamber would equal that
found in the resistant cell culture (ie 2 X 10-SM).
immediate cessation of.respiration.
There was an
To determine if this effect was
due to the antimycin A or the ethanol used as the solvent, 33ul of
26
n
P. parva (control) in
complex medium
81
P. parva (17-9AA) in
108
complex medium
with 2 x 1o-5
antimycin A
Table III.
Oxygen
Consumption
umoles 02/hr/10 6 cells
0.669
:t
0.21
0.479 + 0.14
Oxygen consumption in intact logartthmic phase cells
of P. parva. Data represents the mean + the standard
deviation and are statistically significant at
p <0. 001. The P va 1 ue was ca 1cul a ted by the
Student's t test. n represents the total number of
readings.
27
Figure IV.
The effect of antimycin A on oxygen consumption of
control and resistant cells of f. parva. The arrows
represent the addition of 33ul of either 95% EtOH or
a 1 mg/ml solution of antimycin A.
D - P. parva (control) in complex medium.
d - P. parva 17-9AA in complex medium.
~
- P. parva 17-9AA ~n complex medium with
2 X 10- Mantimycin A.
28
0
t")
L()
N
0
N
w
~
I-
L()
0
.......
0
co
0
L()
3~N'v'H8
0...,..
%
0
t")
0
N
29
95% ethanol was added to the sample chamber after a baseline rate
was established.
Except for the brief increase in total oxygen, due
to dissolved oxygen present in the ethanol, the rate of respiration
did not change significantly.
Therefore the inhibitory effect of
antimycin A is immediate and irreversible.
No effect on the
respiration rate by either ethanol or antimycin was seen in
resistant cells.
Respiration rates of resistant cells in media
without inhibitor is not significantly different from that of the
control cells.
The respiration of resistant cells continues in the
presence of antimycin A, albeit at a reduced rate.
These results
tend to support the belief that the resistance is not due to a
change in cellular permeability but to an intracellular change that
confers resistance to the effects of antimycin. Otherwise resistant
cells in media without inhibitor would be expected to show the same
respiration rate as those in the presence of the inhibitor.
ENZYME ACTIVITIES
All of these data prompted an investigation of the activity of
several respiratory enzymes.
Table IV shows the specific activity
of several mitochondrial enzymes.
In the mitochondrial fraction of
resistant cells there is a 1.5 fold increase in specific activity of
both succinate and NADH dehydrogenase over that of control cells.
Cytochrome oxidase activity in the resistant population was
approximately 78% of that of the control value.
The data are
consistent with the slight reduction in oxygen consumption of the
resistant cells.
Enzyme
n
Specific Activity
Crude Homogenate
Control
Succinate
Dehydrogenase
5
NADH
Dehydrogenase
5
Cytochrome c
Oxidase
5
ATPase
5
Table IV.
Resistant
Specific Activity
Mitochondrial Fraction
Control
6.6 :!: 0.53
X 1o-3
Resistant
10.5 + 1.4
X 1o-3
p<O. 001
6.94 + 1.11
X 1o- 3
10.3 + 1.2
X 10- 3
p<O. 005
2.54 + 0.14
1.92 + 0.2
X 10-2
X 16-2
p<0.001
0.760 ± 0.10
1.22 ± 0.13
P< 0.001
0.706 :!: 0.1
1.18 ± 0.14
pc::::O. 001
Specific activities of enzymes involved in electron transport and oxidative
phosphorylation. Specific activity is calculated as change in absorbance/min/mg
protein. n represents the number of experiments. The P values were calculated
by using the Student's t test.
w
0
31
It is known that three molecules of ATP are generated during
the passage of a pair of electrons down the respiratory chain from
NADH to oxygen by the process of oxidative phosphorylation.
There
are three sites along the electron transport chain in which
decreases in free energy occur sufficient for the formation of ATP
from ADP.
One of these is between cytochrome
partially impaired cytochrome
£
~
and oxygen.
A
oxidase could indicate interference
with ATP production. Therefore the activity of ATPase (the enzyme
responsible for converting ADP to ATP) was examined in the two cell
types.
The ATPase activity of both crude homogenates and mitochondrial
fractions is greater in resistant cells than control cells.
In
control cells, 93% of the total ATPase activity was localized in the
mitochondrial fraction wheras resistant cells retained 97% of the
total ATPase activity in the mitochondrial fraction.
The specific
activity of the ATPase of resistant cells in both the crude
homogenate and mitochondrial fractions was approximately 1.6 times
that of the corresponding fractions from control cells.
DISCUSSION
The growth characteristics and lack of encystment in the
antimycin a resistant cells strongly suggests an impaired ability of
these cells to adapt to fluctuations in the environment due to the
presence of this drug.
These changes are further reflected in
altered metabolism as indicated by the decrease in rates of oxygen
consumption and in the altered activities of respiratory enzymes.
This might be explained by a mutation in these cells.
Mutations conferring resistance to antibiotics which inhibit
mitochondrial function may be encoded in either the nucleus or the
organelle itself.
Indeed, many proteins functioning within the
mitochondria are transcribed on cytoplasmic ribosomes and are coded
for by nuclear genes.
The synthesis of several mitochondrial
enzymes appear to be the combined effort of the nucleus and
mitochondria. For example, of the seven polypeptides that make up
cytochrome oxidase, four are synthesized on cytoplasmic ribosomes
and three on mitochondrial ribosomes (Sheeler and Bianchi, 1980).
Mutations resulting in antibiotic resistance can take many forms.
Resistance to an antibiotic could be due to a change in permeability
of either the plasma or inner mitochondrial membranes thereby
restricting access of the inhibitor to its proposed site of action.
Resistance could also be due to a change in a single biochemical
pathway thereby circumventing the site of inhibition, or the
mutation could result in a lower binding capacity of the target site
32
33
to the inhibitor.
There is evidence from other sources that prove that antibiotic
resistance can be induced by mutagenic substances.
Munro (1983)
succeeded in isolating an antimycin A resistant human cell line
capable of continous growth in lSuM antimycin due to a
cytoplasmically localized determinant.
Cytoplasmically
transmissible mutations that confer resistance to other antibiotics
have also been shown (Kearsey, 1981). Early work with yeast resulted
in resistant cells where the mutation affected the respiratory chain
and not the permeability to antimycin (Burtow et. al., 1968).
The
results of the present work provide evidence for a cytoplasmically
located mutation conferring resistance to antimycin A.
Early work by Slater (1973) and Rieske and Zaugg (1962)
localized the point of action of antimycin in the cytochrome b c
region of the electron transport chain.
Studies with isolated
coenzyme QH2-cytochrome c reductase (complex III) confirmed their
results and showed that antimycin inhibits not only enzymatic
activity but also cleavage of the complex by bile salts.
In P.
parva there were significant increases in succinate dehydrogenase
(SDH) and NADH dehydrogenase (NADH DH) activity in the resistant
cells compared to control cells.
If this were a permeability mutant
the activity of SDH and NADH DH should not differ between resistant
and control populations.
In fact, it could be argued that a cell
with an altered permeability would have either no change in SDH or
NADH DH activity or, more likely, a reduction in activity due to the
34
absence of needed cofactors or substrates.
The increase in activity
is consistant with the idea that there is a block in electron
transport further down the electron tronsport chain. The increases
in SDH and NADH DH activities is supportive of the notion that the
block of electron transport is past respiratory complexs I and II.
Increase in specific activity might indicate that the system is
attempting to compensate for the blockage to electron flow.
Similiar increases in SDH activity have been noted in glucose
repressed yeast (Poole and Lloyd, 1973).
The decrease in oxygen consumption and cytochrome oxidase
activity is consistant with all the data showing antimycin
inhibition occurring at complex III.
The 1.7 fold increase in mitochondrial ATPase activity suggests
modified energy metabolism. The cells must produce enough ATP to
grow and divide, albeit at slower rates.
Normally the ratio of
"high energy" adenylate concentrations to total adenylate
concentration is approximately 0.9 (Lehninger, 1982). If this ratio
were decreased, this could account in part for the slower growth
rate of the resistant cells. Such a decrease could be achieved by
the increased hydrolytic activity of ATPase.
On the other hand, increased synthesis of ATP could arise by
either a modified ATPase or by the blockage of an ATPase inhibitory
protein.
It has been shown in antimycin resistant yeast that
increases in ATPase activity may be due to blockage of an ATPase
35
inhibitor.
The inhibitor is coded for by yeast mitochondrial DNA,
synthesized on cytoplasmic ribosomes and transported back into the
mitochondria (Yoshida et. al., 1983).
Similiar increases in ATPase
activity were shown in thiamine-deprived cultures of P. agilis
(Cantor, 1978).
Further experimentation especially on the respiratory chain
would help elucidate the mechanism of adaptation in the resistant
strain.
Alexandre and Lehninger (1984) have shown that
antimycin-inhibited electron flow from succinate to oxygen in rat
liver mitochondria can be reactivated by the addition of an
artificial electron donor like DCPIP.
Many protozoans manifest
different forms of cytochrome oxidase with slightly different
characteristics.
In Acanthamoeba castellanii three different
terminal oxidases exist, each inhibited by a different drug (Lloyd
et. al., 1982) It seems as though the terminal oxidase changes from
one form to another during the cell cycle and more than one oxidase
may be present at the same time.
In A. castellanii antimycin A at a
concentration of SUM antimycin results in a large decrease in the
reduction of cytochromes
~
and
~·
Therefore it might be useful to
analyze cytochrome content in the present strain and determine what
changes, if any, have taken place.
Also, a study of "respiratory
control" (i.e. the control of electron transport by the
concentration of ADP) would be neccessary to further elucidate the
biochemical basis of this mutation.
When ADP is absent from a cell
free system the rate of respiration is very low and no
36
phosphorylation occurs.
This condition is known as state 4
respiration and represents the resting state.
When a known amount
of ADP is added, the oxygen uptake increases dramatically and the
added ADP becomes phosphorylated.
This is called state 3 or active
respiration and lasts only as long as there is ADP present,
returning to state 4 upon the complete phosphorylation of all the
added ADP.
The ratio of state 3 to state 4 respiration is a measure
of the integrity of isolated mitochondria. Studies on the ratio of
ATP synthesis to oxygen consumption would also be useful in
assessing the ability of resistant cells to couple oxidative
phosphorylation to electron transport.
The main purpose of this thesis was not to determine the site
of action of antimycin A; that has been shown conclusively elsewhere
(Slater, 1973).
The purpose, rather, was to determine if the mode
of resistance was due to a change in cell permeability or by a
cytoplasmically localized determinant.
The evidence presented here
(i.e. a 1.59 fold increase in SDH,a 1.48 fold increase in NADH DH, a
reduced cytochrome c oxidase and a 1.7 fold increase in ATPase
activity) suggests against a change in either cell or mitochondrial
permeability.
This resistant cell line should be extremely useful in the
elucidation of several facets of protozoan physiology, including
reproduction and encystment.
Conjugation in P. parva has been shown
to occur in newly excysted cultures when cell density is increased
by gentle centrifugation (Baresi, unplublished observations).
The
37
fate of the zygote has escaped observation due to the inability to
keep the cells alive long enough under the microscope for visual
observation.
Frequency of conjugation as well as the fate of the
zygote could be more easily studied in cell lines with
distinguishing genetic or cytoplasmic markers.
Conjugation in P.
parva unlike that in bacteria involves the complete fusion of the
two parental cells and not just an exchange of genetic information
(Moore and cushing, 1979).
TWo separate cell lines each resistant
to a different metabolic inhibitor could be utilized to study the
frequency of conjugation since only the zygotes would survive in
media with both inhibitors present.
The advantage of using cell
lines with characteristics like P. parva 17-9AA is that it would
allow for the demonstration of survival on a population scale and
not by examination of a few isolated fusions.
It also, in
conjunction with another resistant cell line, could potentially be a
source of large numbers of zygotes which would make genetic analysis
much easier to study.
Perhaps the greatest potential for P. parva 17-9AA is the
elucidation of the cause(s) of encystment. Encystment has been
extensively studied in Acanthamoeba by Neff et. al.
Roti (1974) and Matsusaka (1979).
(1964), Roti
In this soil amoeba, synchronous
encystment can be induced by placement of the cells into a nutrient
free salt solution at high pH. Neff (1972) found that inhibitors of
DNA synthesis were the most effective inducers of differentiation
and speculated that certain deoxyribonucleosides were repressor
3B
molecules which normally inhibit differentiation.
When these
molecules become depleted the cells accumulate in S phase and
differentiation may be induced. There is also evidence that RNA and
protein synthesis accompany encystment.
to encyyst in this manner.
P. parva cannot be induced
However P. parva 17-9AA has a defective
mechanism leading to cyst formation. By characterizing the
differences between P. parva 17-9AA and control P. parva the
mechanism of encystment in particular and cellular differentiation
in general may be better understood.
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