STUDIES IN THE METABOLISM OF PLANT CELLS
XII. IONIC EFFECTS ON OXIDATION OF REDUCED DIPHOSPHOPYRIDINE NUCLEOTIDE
AND CYTOCHROME C BY PLANT MITOCHONDRIA
By S. I. HONDA,* R. N. ROBERTSON,t and JEANETTE M. GREGORyt
[Manuscript received October 17, 1957]
Summary
Some effects of ions on the mitochondrial oxidation of reduced diphospho.
pyridine nucleotide (DPNH) and cytochrome c were studied. Potassium, sodium,
magnesium, .calcium, chloride, and orthophosphate consistently increased the rate
of DPNH oxidation of beetroot mitochondria which had been isolated in a medium
of O· 4M sucrose containing ethylenediaminetetraacetic acid and tris(hydroxymethyl).
aminomethane and washed in sucrose solution; the assay of DPNH oxidation was
carried out in a O· 4M sucrose medium. The rate of DPNH oxidation was increased
by increasing concentration of chlorides but decreased at higher concentrations.
The optimum for divalent chloricles was lower than that for monovalent but the
Ji~OLbli0" W':'O not marked when considered on the basis of ionic strengths. The
optimal salt concentrations for stimulation of DPNH oxidation were much lower
than those for stimulation of cytochrome c oxidation. It is concluded that this
effect of salt on DPNH oxidation by mitochondria could explain salt respiration
in plant tissue.
I. INTRODUCTION
The active and passive roles of mitochondria in the systems responsible for
salt uptake by plant cells have been studied by Robertson et al. (1955) and Honda
and Robertson (1956). The influence of salts on the activity of particulate cyto.
chrome oxidase from plants has been investigated by Miller and Evans (1956).
These investigations have an important bearing on the problem of salt respiration
in intact tissue and are extended in this paper by examination of the influence of
salts on the rate of oxidation of both reduced diphosphopyridine nucleotide
(DPNH) and cytochrome C by plant mitochondria.
The use of organic acid substrates introduces relatively high concentrations
of ions which may mask the stimulating effects of inorganic ions on oxidation and
may also induce single.step oxidations which do not necessarily follow the normal
electron transport pathway. DPNH has several advantages for study of salt effects:
the oxidation of DPNH can be studied in a concentration which introduces negligible
quantities of ions in comparison with the ions added for study of salt accumulation,
the respiratory system is confined to the electron transport chain by the omission
of a dehydrogenase step, and rapid and sensitive methods are available requiring
only small quantities of mitochondria.
* Plant Physiology Unit, Division of Food Preservation and Transport, C.S.I.R.O.,
and Botany School, University of Sydney; present address: U.S.D.A. Plant Soils and Nutrition
Laboratory, Ithaca, N.Y.
t Plant Physiology Unit, Division of Food Preservation and Transport, C.S.I.R.O., and
Botany School, University of Sydney.
s.
2
I. HONDA, R. N. ROBERTSON, AND JEANETTE M. GREGORY
Some conditions under which inorganic ions can i.ncrease the rate of DPNH
oxidation by beetroot mitochondria are reported.
II.
MATERIALS AND METHODS
Since high yields of mitochondria were not required, modifications of previous
methods (Honda and Robertson 1956) were introduced to shorten the preparative
time and to decrease the centrifugal force required to sediment the mitochondria
isolated from commercial red beetroot (Beta vulgaris L.).
Ohilled beetroot (100 g) was disintegrated with a Waring Blendor for 20 sec
in no ml of O·4M sucrose containing either 0·02M tris(hydroxymethyl)aminomethane (TRIS) or 0·045M TRIS plus 0·005M ethylenediaminetetraacetic acid
(EDTA), except where noted. The brei was filtered through muslin and centrifuged
for 5 min at 325 g. The supernatant was decanted, filtered through muslin, and
recentrifuged for 15 min at 10,000 g. The resulting supernatant was retained in
order to measure its pH, which was taken as the brei pH. The sedimented particles
or mitochondria corresponded to those previously used for studies on respiratory
activity, active and passive behaviour in salt solutions (Robertson et al. 1955;
Honda and Robertson 1956), and fine structure of mitochondria (Farrant et al.
1956). These mitochondria if used without further manipulations were called
"unwashed" mitochondria. "Washed" mitochondria were obtained by resuspending
unwashed mitochondria in 7 ml of o· 4M sucrose and sedimenting at 9800 g for
5 min and "twice-washed" mitochondria by repeating this operation. The washed
and unwashed mitochondria were resuspended in 7 ml of o· 4M sucrose and placed
in a water-ice-bath until portions for nitrogen assay and assay for oxidation were
taken. All steps were carried out in a cold room, at less than 5°0, with chilled
apparatus or in a refrigerated centrifuge at -1 to 0°0.
The assay system contained 0·10 or 0·20 ml of the mitochondrial suspension,
the required amounts of the diluted stock solution of DPNH or reduced cytochrome
c, sucrose to adjust to the final indicated osmolarity, sufficient TRIS acetate buffer
to maintain the required pH, various specified additions, and water to adjust to
3·00 ml final volume. All concentrations were computed as the final concentrations
of added constituents. The osmolar concentrations were computed assuming the
apparent degrees of dissociation for various salt types were 0·86 for R+A-, 0·72
for R2+(A-)2 and (R+)2 A2-, and 0·45 for R2+A2-.*
A model DU Beckman spectrophotometer with photomultiplier attachment
was used to measure light absorption of the assay system. DPNH oxidationt by
the mitochondria was determined by measuring the decrease in optical density
at 340 mIL, the absorption peak of DPNH (-aO.D.340). The reference cuvette
contained the assay system without mitochondria and sometimes without DPNH
when dilute mitochondrial suspensions were used. All assays were carried out at
room temperature. Temperatures and pH values of the assay systems were measured
at the conclusion of each experiment. The cytochrome c oxidation rate was determined by measuring the decrease in optical density at 550 mIL.
* "Handbook
t
of Cb,emistry and Physics."
33rd Ed. p. 1505, 1951-2.
That oxidation of DPNH and not decomposition was measured was demonstrated by
the fact that change in optical density was prevented by cyanide.
, . fT,' \
STUDIES IN THE METABOLISM OF PLANT CELLS. XII
3
The mitochondrial suspension was added to the rest of the assay system,
which was then stirred. Initial optical density readings were taken after about
1 . 5 min. The interval from initial cutting of the beetroot to the initial optical
density reading was usually about 1 hr; depending upon the activity, optical
densities of the systems were determined for as long as 30' min. Activities were
computed from the slopes of the curves of optical density plotted against time and
expressed as change in O.D.Junit timeJmg N (-ilO.D. 34oJhrJmg N). The initial
slopes were maintained until near the end of the complete oxidation of DPNH.
The rapid volume adjustment of the mitochondria to the new osmolar c<:mcentration
was generally completed within 2 min (cf. Cleland 1952).
The DPNH was a commercial preparation (made by Boehringer &, Soehne,
Mannheim, Germany) containing 64 per cent. nucleotide, virtually entirely reduced
(Dr. K. S. Rowan, personal co~munication). A stock solution of c. 50 mg per 2 ml
was made up containing 1 ml buffer (0.05M TRIS
O·OlM EDTA) , sucrose to
adjust the osmolarity to O' 4M, and water to 2 ml. * A dilute stock solution was
prepared by diluting 0·10 ml of the stock with 3·00 ml of O· 4M sucrose. These
stock solutions were stored at freezing temperatures between use.
The cytochrome c was prepared from ox heart by the method of Keilin and
Hartree as described by Potter (1951) and dialysed against distilled water to reduce
ion contamination to a minimum. The cytochrome c was reduced immediately
before use with a slight excess of sodium dithionite and the excess removed with
a stream of air.
Stock inorganic ion solutions were prepared either with TRIS or acetate as the
balancing ion. TRIS chloride was prepared by neutralizing HCI with TRIS to
pH 7· 2, and TRIS phosphate by neutralizing orthophosphoric acid with TRIS
to pH 7· 2. Sodium, potassium, and magnesium were used in the form of acetate
salts when differences in cation effects were required. For concentration studies,
chlorides of sodium, potassium, calcium, and magnesium were used.
In most experiments mitochondrial nitrogen was determined by difference
between the total nitrogen of the suspension and the supernatant from a 5-min
centrifugation of the suspension at 14,000 g. In some experiments where the mito.chondria had been washed twice, the total nitrogen of the suspension was taken as
mitochondrial nitrogen. Total nitrogen was assayed in duplicate by Nesslerization
after digestion ·of the samples with conc. H 2S0 4 with HgS04 as catalyst.
+
III.
RESULTS
(a) Effect of EDTA in Preparation of Mitochondria and of pH in Assay
Figure 1 shows the effects of pH on mitochondria prepared in TRIS alone
with orthophosphate and cytochrome c in the assay medium and on mitochondria
prepared in TRIS and EDTA. Both preparations showed increasing oxidation
rate with increasing pH between 5 and 7 but the rate for mitochondria prepared
in TRIS alone decreased between pH 7 and 8. At pH values below 5, an increase
* The concentrations of DPNH given are uncorrected for impurities. The concentration
of EDTA carried over from the stock solution of DPNH to the assay system was negligible
compared with the concentrations of ions added.
4
S. I. HONDA, R. N. ROBERTSON, AND JEANETTE M. GREGORY
in the optical density of mitochondrial suspensions was induced for periods as long
as 5 min after the addition of mitochondria to the assay system. Thereafter, the
optical density decreased as might be expected for a slow oxidation of DPNH. In
view of these results, all subsequent experiments were carried out between pH 7
and 8, both in isolating the mitochondria and in assay.
50
40
z
8"'Il:
30
!:
z
CJ
::;:
It-
-l:
~
cj(f)
20
ci
"q
I
/x
10
.."x,x_x__x..............x
x__x?
OLI__________-L__________
4
5
~
6
__________
~
__________
7
~
__________
8
~
9
FINAL ASSAY pH
Fig. I.-Effect of pH adjusted with TRIS acetate buffer on DPNH oxidation. .A. Mitochondria
isolated in TRIS, homogenate pH 7·7, washed in sucrose. Assay details: mitochondrial
nitrogen, 0·030 mg; DPNH, 55·6 p.g per ml; orthophosphate, 6·3 mM; cytochrome c,
13·5 p.M; temperature 22°0, sucrose concn. 0·38M, and osmolar concn. 0·4M. X Mitochondria isolated in TRIS plus EDTA, homogenate pH 7·9, washed in sucrose. Assay details:
mitochondrial nitrogen, 0·016mg; DPNH, 38·7 p.g per ml; sucrose concn. 0·4M; osmolar
concn. 0·41M.
(b) Effect of Tonicity
To test the effects of tonicity, mitochondria were isolated in O· 4M sucrose
plus additions and diluted in assay solutions of various osmolar concentrations.
The activity of washed mitochondria prepared in TRIS and EDTA remained low
at all osmolar concentrations (Fig. 2). The rate of DPNH oxidation of washed
mitochondria prepared in TRIS alone was greater than that of washed mitochondria
prepared in TRIS and EDTA but decreased as the tonicity was lowered below
0·22 osmolar; orthophosphate and cytochrome c added to washed mitochondria
prepared in TRIS increased the rate of oxidation. Unwashed mitochondria of both
5
STUDIES IN THE METABOLISM OF PLANT CELLS. XII
types of preparation with orthophosphate and cytochrome c added showed a marked
increase in the rate of DPNH oxidation as the tonicity fell below 0·22 osmolar.
80
40
70
z
w
<:)
on:
60
l!)
on:
Z
<:)
<:)
.
I
'030
~
\-
20
10
o
it"
~
~
"
+.._______+
:;: 20
~ 40
ci
r::;'"
30
I-
~ 50
o
OSMOLAR
z
w
-"'-K-x.-,..""
0'1
0·2
"------<,
I
1
ci
o
__+
-=i?=
0-3
0'4
0-5
.
0-6
0-7
~
10
~g:~~
1
+-<l.
OSMOLAR CONCN. (M)
Fig. 2
"
+ 0-16
0'8
o
20
40
60
80
100
DPNH CONCN. (~G/ML)
Fig_ 3
Fig. 2.-Effect of osmolar concentration on DPNH oxidation_ ... Mitochondria isolated in
TRIS. homogenate pH 7·9, washed in sucrose. Assay details: mitochondrial nitrogen, 0·013 mg;
DPNH, 51·6 p.g per ml; orthophosphate, 22·8 p.M; cytochrome c, 0·9 p.M; TRIS, 3·1
X 10-2M; acetate, 3·5 X 10-2M; pH 7-2; temperature 22°0; non· sucrose osmolar contribution, O· 11M. 0 Mitochondria isolated in TRIS, washed in sucrose. Assay details: mitochondrial nitrogen, 0·014 mg; DPNH, 62·3 p.g per ml; TRIS, 3·1 X 10- 3 M; acetate, 3·5 X
10- 3M; pH 7·4; temperature 19°0; non-sucrose osmolar contribution, O·OIM. V Unwashed
mitochondria isolated in TRIS, homogenate pH 7 -7. Assay details: mitochondrial nitrogen,
0·010-0-013 mg; DPNH, 27·8 p.gper ml; orthophosphate, 6·3mM; cytochrome c, 6·4 p.M;
TRIS, 6-0 X lO-2M; acetate, 4·6 X lO-2M; pH 7,0-7·5; temperature 24°0; non-sucrose
osmolar contribution O·llM. 0 Unwashed mitochondria isolated in TRIS, homogenate pH
8 '1-8·3. Assay details: mitochondrial nitrogen, O' 027-0·028 mg; DPNH, 27·8 p.g per ml;
orthophosphate, 6-3mM; TRIS, 6·0 X 10- 2M; acetate, 4·6 X 10-2M; pH 7·9; temperature,
25°0; non-sucrose osmolar contribution O·llM. + Unwashed mitochondria isolated in TRIS
plus EDTA, homogenate pH 8·1. Assay details: mitochondrial nitrogen, 0·013-0·015 mg;
DPNH, 27·8 p.g per ml; orthophosphate, 6·3 mM; cytochrome c, 6·4 p.M; TRIS, 6·0 X
1O-2M; acetate, 4·6 X 10- 2M; pH 7·9-8·1; temperature 24°0; non-sucrose osmolar contribution, 0 -1M. X Mitochondria isolated in TRIS plus EDTA, homogenate pH 8·0, washed
in sucrose. Assay details: mitochondrial nitrogen, 0·014 mg; DPNH, 51·6 p.g per ml; TRIS,
3· 1 X 10-2M; acetate, 3 -5 X 10-2M; pH 7·2; temperature 20°0; non-sucrose osmolar
contribution, 0 -01M.
Fig. 3.-Effects of DPNH concn. and osmolar concn. on oxidation by unwashed mitochondria.
Mitochondria isolated in TRIS, homogenate pH 8·3. Assay details: mitochondrial nitrogen,
0·028mg; orthophosphate, 6·3mM; TRIS, 6-0 X 1O-2M; acetate, 4·6 X 1O-2M; pH 7·9;
temperature 25°0; non -sucrose osmolar contribution, 0 ·IIM.
+ Mitochondria isolated in
TRIS plus EDTA, homogenate pH 8·1. Assay details: mitochondrial nitrogen, 0 -015 mg;
orthophosphate, 6· 3 mM; cytochrome c, 6·4 p.M; TRIS, 6 -0 X 1O-2M; acetate, 4 -6 X
10- 2 M; pH 7·9; temperature 24°0; non-sucrose osmolar contribution, O·IM.
o
In high osmolar solutions, presumably hypertonic, the rate of DPNH oxidation
by mitochondria was low and largely independent of DPNH concentrations (Fig.
3). In hypotonic solutions, 0 ·18 and 0 ·16 osmolar, the mitochondria oxidized
DPNH more rapidly and the rate appeared to increase with increasing DPNH
concentration until a saturating concentration was again reached.
6
S. I. HONDA, R. N. ROBERTSON, AND JEANETTE M. GREGORY
In view of these results subsequent experiments on the effects of ions were
carried out with the osmolarity adjusted to about 0·4 or above.
(c) Effect of Cytochrome c on DPNH Oxidation Rate
Figure 4 shows that the rate of DPNH oxidation by mitochondria was
increased up to 2· 5 times by the addition of cytochrome c. The saturation con-:J
oa:
i
o
-:J
~
U
""
0250
~
W
~
a:
>-
200
z
o
~
o
~ 150
o
U
u.
180
~
160
Xcl-
H
'V K
o Na +
+
• ORTHOPHOSPHATE
o
.
.o
w
>-
a:
z
o
;::
o
~ 100 r'--'-_..J3L--~5---'7~-~9--'-I"I---:"3
0-
AMg
X
x
o
o
200
Z
300
CYTOCHROME C CONCN. (I'M)
Fig. 4
I
~
o
0
2
4
6
8
10
12
14
'6
FINAL ADDED CONCN. (MM)
Fig. 5
Fig. 4.-Effect of cytochrome c concn. on DPNH oxidation by unwashed mitochondria.
Isolated in O· 02M TRIS, homogenate pH 8· O. Assay details: mitochondrial nitrogen,
0·015 mg; DPNH, 55·6 /Lgjml plus EDTA, 3·2 X 10-4M; orthophosphate, 6·2 mM; TRIS,
6·0 X 10-2M; acetate, 4·6 X 1O- 2 M; sucrose, 0·29M; osmolar,0·4M; pH 7·9; temperature
24°0; control rate, -LlO.D.jhrjmg N, 10·5. 0 Isolated in 0·07M TRIS plus O·OlM EDTA,
homogenate pH 8·5. Assay details: mitochondrial nitrogen, 0·018 mg; DPNH, 55·6/Lgjml
plus EDTA, 3·2 X 10-4M; orthophosphate, 6·2 mM; TRIS, 6·0 X 1O- 2M; acetate, 4·6 X 10-2M;
sucrose, 0·29M; osmolar, 0·4M;pH 7·9; temperature 25°0; control rate, -LlO.D.jhrjmg N,
6·6. X Isolated in 0·045M TRIS plus 0·005M EDTA, homogenate pH 7·8. Assay details:
mitochondrial nitrogen, 0·030 mg; DPNH 38·7 /Lgjml plus EDTA 8·0 X 1O- 5M; TRIS,
3·1 X 10-3M; acetate, 3·5 X 10-3M; sucrose, 0·39M; osmolar, 0·4M; pH 7·1; temperature
18°0; control rate, - LlO.D.jhrjmg N, 10·4.
o
Fig. 5.-Effect of inorganic cation and anion concentrations on DPNH oxidation by washed
mitochondria isolated in 0'045M TRIS plus 0'005M EDTA. Assay details: DPNH, 62·3 /Lgjml;
EDTA, 1·1 X 10-4M; TRIS, 3·1 X 1O- 3M; acetate, 3·5 X 10-3M; sucrose,0·4M; osmolar,
0·4lM. ~ Mg++, homogenate pH 7·7. Assay details: mitochondrial nitrogen, 0·0l5mg;
pH 7·3; temperature 20°0; control rate, -LlO.D.jhrjmgN, 6·l. 0 Na+. homogenate pH 7·6.
Assay details: mitochondrial nitrogen, 0·007 mg; pH 7·2; temperature 20°0; control rate
- LlO.D.jhrjmg N, 23·5. • Orthophosphate, homogenate pH 7· .6. Assay details: mitochondrial
nitrogen, 0·007 mg; pH 7·6; temperature 19°0; control rate, -LlO.D.jhrjmg N, 19·2.
X 01-, homogenate pH 8·0. Assay details: mitochondrial nitrogen, 0·006mg; pH 7·5; temperature 19°0; control rate -LlO.D.fhrjmgN,27·1. V K+, homogenate pH 7·6. Assay details:
mitochondrial nitrogen, 0·014 mg; pH 7·2; temp. 20°0; control rate, -LlO.D.jhrjmg N, 83·5.
centration of added cytochrome c was below 1O-6M for mitochondria prepared in
both TRIS and TRIS plus EDTA.
(d) Effect of Ions on DPNH Oxidation Rate
Under certain conditions the addition of different inorganic cations and anions
was found to increase the rate of DPNH oxidation by washed mitochondria. Un-
STUDIES IN THE METABOLISM OF PLANT CELLS.
xn
7
washed mitochondria showed an increased rate of DPNH oxidation with cytochrome c but usually not with increasing ionic concentrations of Na+, K+, Mg++,
Cl-, or orthophosphate.
Four types of washed mitochondrial preparations were examined for salt
effects:
(i) Mitochondria prepared in TRIS and assayed at constant osmolar concentration.
(ii) Mitochondria prepared in TRIS and assayed at constant sucrose concentration but increasing osmolar concentration.
(iii) Mitochondria prepared in TRIS and EDTA and assayed at constant
osmolar concentration.
(iv) Mitochondria prepared in TRIS and EDTA and assayed at constant
sucrose concentration.
Figure 5 shows representative experiments on effects of ions at low concentrations upon DPNH oxidation by washed mitochondria prepared in TRIS and
EDTA and assayed at constant sucrose concentration. Increased rates of DPNH
TABLE 1
COMBINED EFFECTS OF SOME IONS, ATP, AND CYTOCHROME C ON DPNH
OXIDATION BY WASHED MITOCHONDRIA, ISOLATED IN O' 045M TRIS + O· 005M
EDTA + 0·4M SUCROSE, ASSAYED UNDER CONSTANT OSMOLAR CONCN.
Assay details for control: mitochondrial nitrogen, 0·0095 mg; DPNH,
38·7 p.g/ml; EDTA, 8·0 X 10-5M; sucrose, 0·39M; osmolarity, 0·4M;
TRIS, 3·1 X 10-3M; acetate, 3·5 X 1O-3M; pH 7·0; temperature 20°C;
rate, -110.D./hr/mg N, 18·8. Assay details for complete medium: as in
the control with Mg++, 1·3 roM; orthophosphate, 2·5 mM; ATP (sodium
salt), 1· 1 mM; TRIS chloride, 8·4 mM; cytochrome c, 0 . 4 p.M
Treatment
Control
Control + TRIS chloride, 8·4 mM
Control + TRIS chloride + cytochrome c, 0·4 p.M
Complete medium
Complete medium, less Mg++
Complete medium, less orthophosphate
Complete medium, less ATP (sodium salt)
Complete medium, less TRIS chloride
Complete medium, less cytochrome c
DPNH Oxidation
Rate as Per Cent.
of Control
100
162
240
259
257
264
259
264
206
oxidation with the additions of Mg++ and Na+ as acetates and with TRIS orthophosphate were found for all types of washed mitochondrial preparations. K +
added as acetate had little or no effect on the rate of DPNH oxidation with the
exception of mitochondria prepared in TRIS and EDTA. Demonstration of
8
S. 1. HONDA, R. N. ROBERTSON, AND JEANETTE M. GREGORY
chloride effects was obtained consistently only with washed mitochondria prepared
in TRIS and EDTA under conditions of constant sucrose concentration and for
brief transient times for washed mitochondria prepared in TRIS. The sodium
salt of adenosine triphosphate (ATP) neither increased nor decreased the oxidation
rate of DPNH by any type of mitochondrial preparation.
Table 1 shows that withdrawal of one ion from a mixture of several ions with
cytochrome c did not decrease the maximal DPNH oxidation rate by washed mitochondria prepared in TRIS and EDTA. Only the withdrawal of cytochrome c
TABLE 2
COMBINED E]-FECTS OF SOME IONS AND CYTOCHROME C ON DPNH OXIDATION
BY WASHED MITOCHONDRIA ISOLATED IN 0·02M TRIS AND ASSAYED UNDER
CONSTANT OSMOLAR CONCENTRATION
Assay details for control: mitochondrial nitrogen, 0·0098 mg; DPNH,
62· 3 fLg/ml; EDTA, 1·1 X IO-4M; sucrose, O· 4M; osmolar, O· 4M;
TRIS, 3·1 X IO-3M; acetate, 3·5 X IO-3M; pH 7·3; temperature
20°C; rate, - 6.0.D./hr/mg N, 32·3. Assay details for complete medium:
as in the control with Mg++, 5 mM; orthophosphate, 7·6 mM; ATP
(sodium salt), 1 ·1 mM; TRIS chloride, 8·4 mM; cytochrome c, 0·4 fLM
Treatment
Control
Control
Control
+ TRIS
+ TRIS
0·2 fLM
chloride, 7·5 mM
chloride, 7·5 mM
I
+ cytochrome
Control + cytochrome c, O' 2 ~
Control + Na+, 7'OmM
Control + Na+ (7·0mM) +Cl- (7·5mM)
Complete medium
Complete medium, less Mg++
Complete medium, less Mg++, less orthophosphate
DPNH Oxidation
Rate.as Per Cent.
of Control
100
116
c,
236
193
120
125
215
211
252
decreased the rate from the maximal and only chloride plus cytochrome c was
required to establish the maximal rate. Table 2 also shows that, even for washed
mitochondria prepared in TRIS, cytochrome c plus chloride alone was sufficient
to establish the maximal rate of DPNH oxidation although chloride itself only
slightly increased the rate.
Of the other ions, when added with cytochrome c, only Na+ was as effective
as chloride in establishing the maximal rate of DPNH oxidation by washed mitochondria. When chloride alone had little effect and chloride plus cytochrome c
did not establish maximal oxidation rates, the addition of Na+, Mg++, or orthophosphate, but not K +, gave maximal oxidation rates. When three or more ions were
added with cytochrome c to washed mitochondria, the omission of anyone did
not depress the rate of DPNH oxidation.
STUDIES IN THE METABOLISM OF PLANT CELLS.
xn
9
These results suggest that endogenous salts were sufficient to maintain maximal
rate of DPNH oxidation by unwashed mitochondria, so added salts had no additional
effect. The endogenousIsalt could be removed by washing, but ion effects were
found consistently in washed mitochondria only when EDTA was added to the
isolation medium prior to washing. This might suggest that EDTAremoved, from
the mitochondria, cations normally required to stimulate the oxidation; this would
be consistent with our observation that effects of cations added to such mitochondria
were easy to demonstrate. Since. the withdrawal of anyone anion or cation from a
mixture of three or more ions did not depress the induced rapid rate of DPNH
oxidation, specificity of the ions was not established.
15
.....J
~'i
o w
... Cl
10
w 0
:J 1r
o !:
w z
"
... Cl
0(
:f
'"
",""
",,"
,fI' "
~'
o '"
'" ""
d'0 _""."..
'" It
~~
W 0
~ ci
W "l
'"
5
I
~-
o
0'01
0·02
CONCN
0'03
0'04
OF SALT (M)
Fig. 6.-Effect of increasing concentration of KCl and NaCl on DPNH oxidation by twicewashed mitochondria isolated in o· 045M TRIS plus O· 005M EDTA plus O' 4M sucrose. Assay
details: DPNH 64·5 p.g/ml plus EDTA 1·7 X 10-4M; cytochrome c, 2~; TRIS, 3·1 X
1O-3M; acetate 3·5 X 10-3M; sucrose, 0·4M. X KCl effect;. homogenate pH 7·9. Assay
details: mitochondrial nitrogen, 0·031 mg; pH 7·3-7·2. + KCI effect; homogenate pH 8·2.
Assay details: mitochondrial nitrogen, 0·022mg; pH 7·3-7·2. 0 NaCI effect; homogenate
pH 7·9. Assay details: mitochondrial nitrogen, 0·016mg; pH 7·2-7·1. • NaCl effect;
homogenate pH 8·1. Assay details: mitochondrial nitrogen, 0·014 mg; pH 7· 5-7' 2.
(e) Effect of Concentration
The effects of concentration of chlorides on DPNH oxidation were examined
with twice-washed mitochondria in O'4:M sucrose solution (Figs. 6 and 7). In such
. mitochondria, a basal rate of oxidation in the absence of salt was always obtained
and varied from preparation to preparation. Increase in concentration of chlorides
increased the rate of oxidation at lower concentrations but decreased the rate at
higher concentrations. No marked differences were observed between sodium and
potassium chlorides but calcium chloride showed a peak in the stimulated respiration
at lower normality than the monovalent ions. Results of experiments over a narrow
concentration range are shown in Figure 6.
10
S. I. HONDA, R. N. ROBERTSON, AND JEANETTE M. GREGORY
(.f) Comparison of DPNH Oxidation and Cytochrome c Oxidation
The possibility that the effect of salt was entirely due to its action on the
cytochrome oxidase system was examined by comparing DPNH oxidation with
cytochrome c oxidation on replicate preparations (Fig. 7). Three different preparations
were used and, since some variation was to be expected, the effect of potassium
chloride on both DPNH and cytochrome c oxidation is given in each preparation.
The differences between the curves for potassium chloride effects are only small and
sodium chloride and potassium chloride resemble each other (Fig. 7(a)). Thedivalent
salts, calcium and magnesium chlorides, are different from monovalent salts in
their effects on both DPNH oxidation and cytochrome c oxidation (Figs. 7(b) and
7(c)). The effects of these chlorides on oxidation rate of cytochrome c are very
o
KC!, CYTOCHROME c • KCI, DPNH
x NaCI, CYTOCHROME c + NaCl. DPNH
(a)
Z 200r
w
o
KCt, CYTOCHROME c
o CaCI 2 .CYTOCHROME c
• KCI, DPNH
• CaC1z.oPNH
(b)
200r-
o KCl.
CYTOCHROME C
•
KCl. DPNH
t::. MgC1z. CYTOCHROME c ... MgC1z. DPNH
(c)
200r
l!)
~
1501-
~
50
1/
~---"-
"'Z
"irl!)100~
:
ci..,
I
0
0
100~ F\
50
-. ----.--__ .. _
0'05 0·1
0,15
150
0·2 0'25
p'
,
/'
-
",,_.
"
o
0
0·05
0'1
0'15 0'2
0'25 0·3
Fig. 7.-Effects of sodium, potassium, magnesium, and calcium chlorides on DPNH and cytochrome e oxidation by twice-washed mitochondria isolated in o· 045M TRIS plus o· 005M
EDTA plus 0'4M sucrose. Assay details: DPNH oxidation: DPNH, 64·5 p,gjml; EDTA.
1· 7 X 10-4M; cytochrome c, 2 p,M; TRIS, 3·1 X 10- 3M; acetate, 3·5 X 1O- 3M; sucrose
0·4M. Cytochrome c oxidation: reduced cytochrome c, 41· 33 p,M; TRIS, 3 - 1 X 1O- 3M;
acetate, 3·5 X 1O- 3 M; sucrose O· 4M. (a) KCI and N aCI effect: mitochondrial nitrogen, 0·049 mg.
(b) KCI and CaCh effect: mitochondrial nitrogen, 0·087 mg. (e) KCI and MgCh effect: mitochondrial nitrogen, 0·053 mg.
similar to those obtained by Miller and Evans (1956) with plant mitochondria from
four other sources. The cytochrome c results differ from the DPNH results, however. Generally speaking, increase in salt concentration stimulates DPNH oxidation
to its maximum at a lower concentration than the maximum for cytochrome c
oxidation. Subsequently, there is a slow fall with increasing concentrations. The
divalent chlorides produce a peak in both oxidations at lower normalities than the
monovalent salts but this difference is much less when the results are plotted against
ionic strength (Fig. 8). At higher ionic strength, the divalent salts cause a decrease
in the rate of oxidation which is more marked than that caused by the monovalent
salts.
These results show that, while both cytochrome oxidation and DPNH oxidation
are stimulated by the presence of ions in vitro, the DPNH oxidation is more sensitive
to depressant effects at higher concentrations.
IV.
DISCUSSION
Some characteristics of DPNH oxidation by lupine mitochondria have been
reported (Humphreys and Conn 1956). The isolation and assay methods which
STUDIES IN THE METABOLISM OF PLANT CELLS.
xn
11
differed from those used for our beetroot mitochondria may account for some
difference in DPNH oxidation by lupine mitochondria. For beetroot mitochondria,
saturation levels of cytochrome c enhancing the rate of DPNH oxidation were
about 10-20 times lower than for lupine mitochondria. Without added cytochrome
c the rates of DPNH oxidation were generally two to three times greater for lupine
mitochgndria than beetroot mitochondria. These greater activities were obtained
in solutions of low tonicity, probably hypotonic, where greater activities may be
120
(a)
eo
40
z
0
0·3
0'2
0"
o'!!
0'4
"'
0
I!)
~ 200
(b)
Z
I!)
:f
tt
-!
,,
,,
,
'60
ci
ci
"'l
\
120
\
\
\
\
"
eo
\
MgCl z ' CYTOCHROME C
\
"~,
40
........
o
0·1
0'2
' ......
...... ........
....
MgCI 2 , OPNH
~
...
--.---""!------0'3
0'4
o'!!
IONIC STRENGTH
Fig. S.-Effects of sodium, potassium, magnesium, and calcium
chlorides on DPNH and cytochrome c oxidation plotted against
ionic strength; same data as in Figure 7. (a) KCl and CaCh
effect. (b) KCl and MgCh effect.
expected compared with those of beetroot mitochondria in hypertonic solutions.
The decreased rate of DPNH oxidation by lupine mitochondria in solutions of low
sucrose concentration compared with that in high sucrose concentration, in the
absence of added cytochrome c, recalls the behaviour of washed beetroot mitochondria prepared in TRIS and assayed without added cytochrome c. The addition
of cytochrome c to both lupine and beetroot mitochondria permitted the demonstration of high rates of DPNH oxidation in hypotonic solutions compared with
hypertonic solutions. This could be due to replacing endogenous cytochrome c
12
S. I. HONDA, R. N. ROBERTSON, AND JEANETTE M. GREGORY
which had been removed during the preparation, an effect which in beetroot mitochondria can be duplicated by omission of washing the mitochondria, and, in part,
pretreatment with EDTA during the isolation procedure. It could also be due to a
non-specific effect of the cytochrome c protein, since protein among other things
can stabilize the activity of succinoxidase (cf. Keilin and Hartree 1947).
.
The use of DPNH as a respiratory substrate for study of salt respiration of
mitochondria raises the question of associating physiological behaviour of intact
mitochondria with the oxidation of exogenous DPNH. Liver mitochondria effectively
carried out phosphorylation coupled with the oxidation of exogenous DPNH
(Lehninger 1951) only if subjected to extreme hypotonicity. It was suggested that
mitochondrial permeability to DPNH was increased, or the mitochondrial structure
altered by the incubation in distilled water. Our results with beet mitochondria at
different tonicities (Figs. 2 and 3) could also have been due to more sites for DPNH
oxidation becoming available in hypotonic solutions. If, for instance, the particle
in hypertonic or isotonic solutions was highly impermeable to exogenous DPNH,
then oxidation would take place only at sites on the surface and increasing concentration of DPNH would have little effect (Fig. 3). When the particle swelled in
hypotonic solutions, the permeability might increase, more internal sites for DPNH
oxidation become available, and the rate of oxidation be limited by diffusion of
exogenous DPNH to these sites. If little change in membrane permeability occurred
in the hypertonic to isotonic range, the same activity would be expected at all
concentrations as is suggested by Figure 2. At hypotonic concentration, however,
the increased swelling may result in increased accessibility of the internal sites;
unless too much disorganization occurs which may have happened with the TRISwashed mitochondria which showed decreased rate of DPNH oxidation at lower
concentrations. It is, of course, possible that EDTA inhibits the activity of the
internal sites at low osmolar concentrations and that this explains the difference
between the mitochondria prepared in TRIS and those prepared in TRIS plus
EDTA. Cytochrome c and orthophosphate and the omission of the washing appeared
to stabilize the internal sites.
The ion effects which increased the rate of DPNH oxidation occurred consistently only with mitochondria which were apparently intact. For example, the
addition of chloride increased the rate of DPNH oxidation most consistently for
mitochondria isolated in EDTA solutions. EDTA was found to prevent the increase
in permeability of rat-heart sarcosomes (Cleland 1952), part of which effect may be
attributed to protection by chelation of calcium (Slater and Cleland 1952). Calcium
may activate ATPase (Potter, Siekevitz, and Simonson 1953), and lowered endogenous ATP content was correlated with the swelling of mitochondria (BrennerHolzach and Raaflaub 1954). Both ATP and EDTA may complex calcium within
the mitochondria (Raaflaub 1955). Isolation of beetroot mitochondria in EDTA
solutions was found to decrease the content of mitochondrial calcium (Honda and
Robertson 1956) and to increase both the rate of oxidations of succinate, malate,
and a-ketoglutarate and the accompanying phosphorylations (Robertson and Tobin,
unpublished data). Although previous experiments did not show beneficial effects
of EDTA in the demonstration of salt accumulation by mitochondria (Honda and
STUDIES IN THE METABOLISM OF PLANT CELLS. XII
13
Robertson 1956) or improvement in the resolution of their fine structure (Farrant
et al. 1956), the modifications in preparative methods have now shown effects on
mitochondrial oxidation of DPNH.
The ion effects which increased the rate of DPNH oxidation cannot be attributed
to the DPNH oxidation step alone but may act on other steps in the mitochondrial
respiratory chain. The increased rate of oxidation could, for instance, be due to the
ions stimulating the cytochrome c-cytochrome oxidase steps, if these steps had
been rate limiting. The fact that the further increase in concentration reduces the
rate of DPNH oxidation while still stimulating cytochrome c oxidase suggests,
however, that cytochrome c oxidation is not the rate-determining step in DPNH
oxidation at these ionic concentrations. It is possible for salt to affect, directly or
indirectly, the electron transport chain in several different ways and at the present
stage of our knowledge, speculation on how it is affected would seem premature.
When no salt was added to the twice-washed mitochondria, the rate of DPNH
oxidation was about eight times that of added cytochrome c oxidation (in terms
of electron transport), assuming E340m,u DPNH = 6·22 X 106 cm 2 jmole and
AE550m,u cytochrome c = 1·96 X 10 7 cm 2 jmole. In the presence of added salt the
maximum rate of DPNH oxidation was about twice the maximum rate of added
cytochrome c oxidation. This observation, taking into consideration tlie low concentration of cytochrome c necessary to give maximal rate of DPNH oxidation,
suggests that the endogenous cytochrome c, together with the small amount added
to replace what had been removed from the particles during preparation, is efficient
in the electron transport from DPNH to oxygen. This system is' more efficient
than that in which reduced cytochrome c is supplied, probably because DPNH
molecules can reach the sites of their oxidation more readily than reduced cytochrome c molecules. Alternatively, centres of oxidation of cytochrome c may be
blocked by oxidized cytochrome c already occupying these centres. The ratio of
the DPNH oxidation to cytochrome c oxidation changes at higher concentrations
of salts, when the DPNH oxidation becomes depressed relatively more than the
cytochrome oxidation.
While there is evidence for some differences in the effects of different ions
supplied, there is no evidence in these experiments for the suggestion that the
stimulation is an anion effect as originally proposed by Lundegardh (1940). It appears
that the stimulation is due to the presence of free ions and that cations are important.
These results, like those of Miller and Evans (1956), may have some bearing on the
explanation of the effects of salts on respiration in intact tissue. J\filler and Evans
rightly point out that the concentration of monovalent ions (0 ·IM) required for
maximum oxidation of cytochrome c is considerably higher than the concentration
of salts (approx. 0·005-0·01M) necessary for maximum salt respiration in tissue.
Our results show that the optimum stimulation of DPNH oxidation occurs at about
o·OIM and that of cytochrome oxidation at about 0 ·15M. Our results, taken with
those of Miller and Evans, suggest that the DPNH oxidation is more sensitive to
salt than the cytochrome oxidation and reaches its maximum at an external concentration similar to that giving maximal stimulation of respiration in carrot tissue
(Robertson and Wilkins 1948). It is possible that the mitochondria of intact tissue
14
S. 1. HONDA, R. N. ROBERTSON, AND JEANETTE M. GREGORY
may be even more sensitive in their response to free ions than mitochondria tn
vitro.
The salt respiration is observed only in tissue which has been treated (e.g.
washing for approved periods in distilled water) in such a way as to reduce the
concentration of ions in the external medium and in the free space, including the
cytoplasm of the tissue. The fact that leakage of free ions from such aerated tissue
is negligible suggests that the free ion concentration of the free space in the cytoplasm is very low and that the ions in contact with the surfaces of the mitochondria
are present in negligible amounts. Under such conditions, the salt respiration would
be negligible. When salt is applied to the external solution, some ions diffuse into
the free space and mobile ions come into contact with the mitochondria. If they
exert a stimulatory affect similar to that observed in extracted mitochondria, salt
respiration would result.
V. ACKNOWLEDGMENTS
The authors wish to thank Mr. K. T. Glasziou, Mr. M. D. Hatch, and Mr.
N. F. Tobin for interest and assistance, Dr. F. E. Huelin and Professor R. K. Morton
for helpful criticism of the manuscript, and Dr. J. R. Vickery, Chief, Division of
Food Preservation and Transport, C.S.I.R.O., and Professor R. L. Crocker, Botany
School, University of Sydney, in whose laboratories the work was carried out.
The work described in this paper was carried out as part of the joint research
programme of the Division of Food Preservation and Transport and of the Botany
School, University of Sydney.
VI. REFERENCES
BRENNER-HoLZACH, 0., and RAAFLAUB, J. (1954).-Die Korrelation zwischen der Schwellung
isolierter Mitochondrien und dem Abbau der intramitochondrialen Adenosinnucleotide
(ATP, ADP, AMP, Co. A). Helv. Physiol. Acta 11: 242-52.
CLELAND, K. W. (1952).-Permeability of isolated rat heart sarcosomes. Nature 170: 497.
FARRANT, J. L., POTTER, C., ROBERTSON, R. N., and WILKINS, M. J. (1956).-The morphology
of red beet mitochondria. Aust. J. Bot. 4: 117-24.
HONDA, S. 1., and ROBERTSON, R. N. (1956).-Studies in the metabolism of plant cells. XI.
The Donnan equilibration and the ionic relations of plant mitochondria. Aust. J. BioI.
Sci. 9: 305-20.
HUMPHREYS, T. E., and CONN, E. E. (1956).-The oxidation of reduced diphosphopyridine
nucleotide by lupin mitochondria. Arch. Biochem. Biophys. 60: 226-43.
KElLIN, D., and HARTREE, E. F. (1947).-Activity of the·cytochrome system in heart muscle
preparations. Biochem. J. 41: 500-2.
LEHNINGER, A. L. (1951).-Oxidative phosphorylation in diphosphopyridine nucleotide-linked
systems. In "Phosphorus Metabolism". Vol. 1. (Ed. W. D. McElroy and B. Glass.)
(Johns Hopkins Press: Baltimore.)
LUNDEGARDH, II. (1940).-Investigations as to the absorption and accumulation of inorganic
ions. Ann. Agr. Coll. Sweden 8: 234-404.
MILLER, G. W., and EVANS, H. J. (1956).-The influence of salts on the activity of particulate
cytochrome oxidase from roots of higher plants. Plant Physiol. 31: 357-64.
POTTER, V. R. (1951).-"Manometric Techniques and Tissue Metabolism." (Ed. W. W. Umbreit,
R. H. Burris, and J. F. Stauffer.) (Burgess Publ. Co.: Minneapolis.)
POTTER, V. R., SIEKEVITZ, P., and SIMPSON, H. (1953).-Latent adenosine-triphosphatase activity
in resting rat liver mitochondria. J. BioI. Chem. 205: 893-908.
STUDIES IN THE METABOLISM OF PLANT CELLS. XII
15
RAAFLAUB, J. (1955).-Komplexbildner als Cofaktoren isolierter Zellgranula. Helv. Ohim.
Acta 38: 2.7-3.7.
ROBERTSON, R. N., and WILKINS, M. J. (1948).-Studies in the metabolism of plant cells. VII.
The quantitative relation between salt accumulation and salt respiration. Aust. J. Sci.
Res. B 1: 17-37.
ROBERTSON, R. N., WILKINS, M. J., HOPE, A. B., and NESTEL, L. (1955).-Studies in the metabolism of plant cells. X. Respiratory activity and ionic relations of plant mitochondria.
Aust. J. Biol. Sci_ 8: 164-85_
SLATER, E- C_, and CLELAND, K_ W. (1952),-Stabilization of oxidative phosphorylation in
heart-muscle sarcosomes. Nature 170: 118-19_