Vol.

85,

No.

November

BIOCHEMICAL

2, 1978

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages

29,1978

519-525

ULTRASTRUCTURAL AND FUNCTIONAL DIFFERENCES IN NITOCHONDRIA ISOLATED FROi3 PARAi3ECIUPI AURELIA GROWN AXENICALLY

AND MONOXENICALLY

Deborah J. Prince" and Ian Gibson School of Biological Sciences, University of East Anglia, Norwich, England eDepartment University Received

September

of Microbiology, Medical Center, of Colorado, Denver, Colorado 80262,

7,

U.S.A.

1977

S UMARY - Differences in the ultrastructure of mitochondria in P. -__ aurelia gro1.m axenlcally and monoxenically have been observed. Functional mi:ochondria have been isolated from both cultures and various metabolic narameters have been tested; respiration rates, cytochrome content, oligomycin sensitive ATPase activity, and an attempt has been made to relate the observed structure and metabolic activity of the mitochondria to the growth conditions. d

Paramecium

--aurelia

monoxenic

conditions.

teristics

have

the

study

changes

in organelle

to the

state to,

reports

concomitant also

been

with

regard

sensitivity

changes reported

to blocking

The present situations physiological

mitochondria

in the

study

differences

that

agents,

culture

under

axenic

and growth

could

or

charac-

differences conditions

observations

e.g.,

the

between (1,2).

The

be correlated

environment

variability

with

and more recently

in the cytochrome cyanide

parameters

affecting

has also

in the

metdolic

degree (6,7).

can occur environment

of The

without have

of mitochondria

activity

content

the related

mem!wane

changes

Changes

been

to the

mitochondrial

ultrastructural

functional

various

in

rate.

(8),

has isolated

with

morphology

two

inner

alterations rates

and has examined

(5)

in the

respiration

cause

to respiration

these

Structural

to indicate

to

the

of variations

(3,4).

of the

taken

general

Laboratory

and function.

or translocation

has been

in the

and biochemical

under

to see if

morphology

are numerous

on the

grmn

was initiated

energy

maintained

morphological

of mitochondria

ADP binding latter

distinct

of Paramecium

present

structure

generally

Observations

shown

same stock

There

is

(9,101

and respiratory

(10). mitochondria in an attempt

from

both

culture

to correlate

ultrastructural. 0006-291X/78/0852-0519$01.00/0 519

Copyright 0 1978 by Academic Press, Inc. All riahts of renroduction in any form reserved.

BIOCHEMICAL

Vol. 85, No. 2, 1978

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

MATEPJALSAND METHODS:Growth of Cells. The cells used in this study were Parameciumbiaurelia stock 562s. The mo=xenic cells were derived from the axa additions of Klebsiella pneumonia (2). The axenic cells by careful, sterile, cells were maintained in Soldo's 1966 axenic mediumat 26OC (11). Monoxenic cells were also maintained in grass mediumpreviously innoculated with Klebsiella pneumonia, at 26OC (12). Cells were harvested in the late stationary phase; which correspond to eight days after innoculation for axenic cells, fourteen days after innoculation for monoxenic cells (to eliminate possibilities of bacterial contamination). Large culture volumes were harvested by the method of Cullis (13) which prevents cell damage. Electron microscony. Cell pellets were fixed following the method of Herrman and Kawallik (14). The cells were stained in 1% osmiumtetroxide and then stained with uranyl acetate and lead citrate (15). The sections were viewed on a Philips 200 electron microscope. Preparation of mitochondria. The extraction of mitochondria from .._ P. ____ aurelia in a raffinose buffer has been described.(l6). The methodology here was the same excepi no EDTAwas included in the raffinose extraction buffer. Protein was estimated by the Biuret assay (17). Assays Oxygen consumption: This was done using an oxygen electrode following the method described by Stockdale (18). The reaction chamber contained 5mMpyruvate, 5mM malate, lOOti KNO3in 5mHHEPESpH 7.6, 1OmhKPO4and 100 ng mitochondrial protein. The reaction was started by addition of 0.2mM ADP. Oxygen consumption was calculated from the recorder traces (16). Gligomycin sensitive ATPase activity: This is the reaction system described by Selwyn (19). The ATPase activity is monitored by the release of PO4a/min/mg mitochondrial protein. Dual beam spectrophotometry: This procedure provides information on cytochrome content and wavelength maxima. nro, one ml mitochondrial suspensions were scanned from 400nmto 620nmafter the reduction of one sample with sodium The cytochrome content may be calculated using the molar extinction dithionite. co-efficients of the cytochrome (5). RESULTS:Electron --

micrographs.

types of mitochondria

Figures 1 and 2 show respectively

of axenic and monoxenic culture.

The axenic mitochondria

be described as in Hackenbrock's condensed configuration dria as being in the expanded, or orthodox state Oxygen consumption. The respiration tions are shown in Table 1. (>lOlO cells/ml) ATPase activity. mitochondrial ATPase activity

(20).

Controls using whole cell preparations

conditions.

might contaminate the mitochondrial

520

of II. pneumonia

ATPase activity

for the two

Controls using -K. pneumonia gave no Any possible cellular

preparations

oligomycin to the assay system.

prepara-

rates (less than 1% of monoxenic rate).

The levels of oligomycin-sensitive types are shown in Table 1.

car

and the monoxenic mitochon

rates obtained for both mitochondrial

gave minimal respiration

under similar

two characteristi

ATPases that

are excluded by the addition

of

BIOCHEMICAL

Vol. 85, No. 2, 1978

AND BlOPHYSlCAL

RESEARCH COMMUNICATIONS

Figure 1 Axenically cultured P. aurelia cells showing mitochondria x 25600. m = mitochondrion, k kinetosome, r = ribosomes, E.R. = endoplasmic reticulum. q

Figure 2 Plonoxenically cultured P. aurelia cells showing mitochondria x 25600. m = mitochondrion, nu = nucleolus, ch = chromatin body, nm = nuclear membrane, E.R. = endoplasmic reticulum.

Table 1 Oxygen consumption and oligomycin sensitive ATPase activity in mitochondria from monoxenically and axenically cultured -P. aurelia

Oxygen consumption (nmoles 02/min/mg protein) Oligomycin sensitige ATPase (umoles PO4 released/ min/mg protein)

Axenic mitochondria

Monoxenic mitochondria

Wk.6+5 -

1079.9+100 -

2.2320.2 x lo-3

2.76t0.2 -

x 10-3

The results are the average of at least six assays. The mitochondria results are the average of at least six assays. The mitochondria used were extracted from cells in the late stationary phase of growth. Kitochondria from both preparations not affected by uncouplets.

BIOCHEMICAL

Vol. 85, No. 2, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

\

Y 1 .

3

Figure 3 Abscissa: Wavelength in nm. Ordinate: ,. . . . . . ._ Extinction spectropnotometry. Scan A. 'monoxenlc' mitochondria, protein (Scale 16cm = 0.1 ext.) Scan B 'axenic' mitochondria, protein -35 mg. (Scale 4cm = 0.1 ext.) Scan C K. pneumonia, protein The reference cell must be aeramfore any spectrum can be 0.1 ext.) T'ne scans are representative and were carried out at 26V.

Cytochrome axenic the

and monoxenic

values

mitochondrial (or

content.

negligible)

obtained

Figure

3 shms

the

mitochondria, for

the

preparations. under

of at least

the

total

dual

and whole cytochrome

Bacterial conditions

three

separate

mitochondria

beam spectra

obtained

cells content

contamination used.

522

increment. Dual beam concentration: 10 mg. concentration: concentration: 12 mg. obtained (Scale 8cm =

of -K. pneumonia. of the

axenic

of the monoxenic

preparations

for

isolated

Table

2 shows

and monoxenic scan

is

nil,

Vol. 85, No. 2, 1978

Table

BIOCHEMICAL

2 Cytochrome

content

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of monoxenically

and axenically

grown

Axenic mitochondria Cytochrome

content

(mM/mg protein)

Percentage

comp-

osition

of

cytochromes

extinction

1.8 x lo-5

0.45

x lo-5

C

1.9 x lo-5

a

1.3

x 1o-5

5.5 1

NH1

0.6 x 1o-5

11

x lo-5 -5 x 10 -5 x 10

b

32.1

2.4

C

33.7

29.7

a

22.3

5.3

NH1

11.7

62.5

co-efficients

beam spectrophotometer

559nm - 575nm)

= 20 x 10W3cm-'M-'

w c Cyt a

552nm - 540nm)

q

(A ext.

445nm - 465nm)

= 9lcm-'mM-'

(A ext.

465nm - 510nm)

= 11 x 10-3cm-1M-1

above

are

the

axenic

in

Tetrahymena

is

a difference

spectrum. pyriformis

chrom.es

are

present

results

from whole

physiological

the

axenic.

This

band

which

situations broad

may correspond

which

(4) peaks

in the

spectra

the

compare

present

unusual

as an a2 band.

at 560.9nm

except

at 612nm not

to a similar

show that

and the

are similar,

shelf

designates

The preparations

band Also

monoxenic

found

there

preparations

a, b and c type

chto-

well

reported

with

other

(1,22).

The results

presented

divergence

between

and monoxenically.

two culture

Lloyd

in -P. aurelia, cells

the

scans.

show an extra

in the bl band in

19 x 10-3cm-1M-1

of several

mitochondrial

and 562.3nm

DISCUSSION: --

representative

maxima between

monoxenic

scans

(3)

(A ext.

The wavelength

tally

dual

(A ext.

The results

in the

the

Cyt b

NH1

that

Monoxenic mitochondria

b

The cytochrome content was determined from (see Fig. 3) using the following data: Molar

P. -- aurelia

here

suggest

the mitochondria

The possibilities

523

that

there

isolated

of such

is

a metabolic

from

a divergence

cells were

and

grown

axeni-

seen

in the

BIOCHEMICAL

Vol. 85, No. 2, 1978

EM study (seen ted

and are borne

in the with

tion

levels

than

exception

Kung obtains

than

is

resembles

of A-N cells staining

matrix

axenically chondria bolic

are

of environment Considering

A-El,which tions with

the

(a):

where

cell

basically

(b):

that

environment

for

the

axenic

observed

phosphorylatin

study

raises

the

question

the

growth

of -P. aurelia.

524

(16).

Hicrographs

are not (a)

the

in

than

i.e.,

the

two

meta-

the its

axenic

mito-

same

structure. activity

growth

e

condi-

days compared

days.

be experiencing

of an ageing reflects a "hostile"

suitability does not

refute

is It

is

cytoplasmic

of cellular

g enzyme or phosphate

cell this.

and low metabolic

due to a build-up

It

axenic

same metabolic

under

less

seen in

experiencing

observed

spectra.

densely

may be determining

structure

of the

the

with

is

environment

ultrastructure

rather

mitochondria

either

fifteen

mitochondrial

part

cells

axenic

have the

phase

can last

may too

may be in of the

mitochondria

study.

of the

mitochondria which

and

obtaining

mitochondria

that

(1,21)

at 612nm,

as A-M mitochondria,

cytoplasmic

and the

reports

for

characteristics

disappears

which

mitochondria

environment

The present medium

phase

significantly

or in Williams'

(A-I.1 cells)

suggest

death

population

in the

due to a lack

the

peculiar

Possibly

resulting

cells

state

axenic

all

use of whole

employed

A-M mitochondria,

to itself

axenic

'hostile'

axenic

death

"toxic"

possible

possibly

the

monoxenic

might

(b)

Possibly the

Considering

The

as the

may explain

(2,l)

or

The respira-

a peak

here,

the

ageing

same physiological

similar,

case.

other

report

condensed

Other This

associa-

are

with

ultrastructure

show the

Paramecium.

are

content

observed

monoxenic

fissions)

in the

demands

type

the

and few tubules.

grown

not

techniques

that

generally

to be the

agrees

differences,

of ageing

(175-180

obtained

is

and the

to note

mitochondria

appear

Kung or William's

at 588nm which

interesting

condition

mitochondria

mitochondria.

spectra

mitochondria,

would

derived

neither

The monoxenic

and cytochrome

may be due to stock

isolated It

that

a peak

The variations

activity

absorption

assays.

in an expanded

and this

in axenically

The cytochrome the

are

state

of ATPase

seen

biochemical

microscope)

a more energized

greater

with

out by the

electron

rates,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

activity. ADP (6,7),

cations.

of the

available

the

necessity

of

Vol. 85, No. 2, 1978

a sterile

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

axenic medium, but perhaps underlines the need for an improved axenic

medium. We would suggest that research on axenic cells to date, although valid in context,

may not fully

stand up to extrapolation

to the "wild"

state.

This work was carried out whilst D. J. Prince was in receipt of an S.R.C. studentship. Wewould like to thank Drs. Selwyn and Dawsonand B. Barrett for their advice and assistance. Thanks also to G. Bedingfield and H. Baker for their help with the electron micrographs. REFEARENCES: 1. 2. 3. 4. 5. 6. 7. 0. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

Williams J. (1971). Studies on killer particles of Paramecium. Ph.D. thesis, tiniversity of East Anglia, Norwich, England. Prince D.J. (1976). The biochemistry of Parameciumaurelia. Ph.D. thesis, University of East Anglia, Norwich, England. Ilunn E A.- (1974). The Structure oi hitochondria. Academic Press. London. Lloyd D. (1974). The mitochondria of microorganisms. Academic Press. London. C'nance B, Williamsmlr)55). J. Biol. Chem. 217. 409-29. Sherer B, Klingenberg 14. (1974). Biochemistry. lcl61-70. innis M A, Beers T R, Craig S P. (1976). Exn. Cell Res. 98. 47-56. Light P A, Garland P B. (1971). Biochem. J. 124. 123-34.-Cartledge T G, Lloyd D, Ereci&k~an~e c(1972). Biochem. J. 130. 739-47. Bohringer S, Hecker H. (1975). J. Protozool. 22. 463-67. Soldo A T, Godoy G A, van Wagtendonk WT. (1965. J. Protozool.-- 13. 492-97. Sonneborn T M. (1950). J. Exp. Zool. 113. 87-147. culxs c. (1971). Cell Transformation= Paramecium. Ph.D. thesis, University of East -a, Norwich, England. Herrman R G, Kowallik K V. (1970). Cell Biol. 45. 198-202. Gibbons I H, Grimstone A V. (1960). J. Biophy. Eochem. Cytol. -7. 697-715. SuyamaY, Preer J R. (1965). Genetics. 52. 1051-58. Gornal A E. Bardawill C S. David II 11. (lG9). J. Biol. Chem. 177. 751-66. Stockdale M, DawsonA P, Selwyn M J. (1970); Eu. J. Biochem. -15. 342-51. Selwyn I4 J. (1967). Biochem. J. 105. 279-88. Hac!!enbrock C R. (1966). J.l--%ol. 30. 269-97. Kung C. (1970). J. Gen. llicro. 61 37lya. --

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