Osmotic properties of mitochondria isolated from Acanthamoeba sp

Experimental Cell Research 19, 133-155 (1960)

133

OSMOTIC PROPERTIES OF MITOCHONDRIA FROM ACANTHAMOEBA SP. R. L. KLEINland Department

of Biology,

Vanderbilt

ISOLATED

R. J. NEFF

University,

Nashville,

Tennessee, U.S.A.

Received March 28, 1959

MANY recent

demonstrations of the existence of a functional mitochondrial membrane [ 1, 8, 10, 14, 19, 28, 401 have lead the authors to infer that some relationship may exist between the osmotic functions of mitochondrial and cellular membranes. In addition to the important role established for mitochondria in overall energy metabolism and active transport, it is proposed that small changes in cellular ion concentrations or water content, both of which have been shown to influence mitochondrial swelling and respiration [2, 5, 9, 13, 14, 311, may be devices of steady state whereby mitochondrial energy output is regulated to meet the cell membrane’s requirements of the moment. The fact that such speculation is possible constitutes ample reason for wishing to know more of the real functional interrelationships of mitochondrial and cell membranes. Difficulties arise in finding suitable cells for comparative studies between mitochondria and their whole cells. Few mammalian tissues suitable for mitochondrial preparations are also satisfactory for whole cell studies. Neither mammalian mitochondria or whole cells can be studied over a wide osmotic range. A cell ideally suited for such studies is a small soil amoeba, Acanthamoeba sp. [25]. As will be demonstrated, approximately 25 per cent of the cell volume is composed of mitochondria which are osmotically active over a wide concentration range. The whole cell shows rapid osmotic response in hypotonic solutions by secreting water via contractile vacuoles [26]. Pure clonal cultures can be grown in large numbers and in ordinary liquid media. Large volumes of mitochondria can be isolated by simple fractionation procedures. The present study has been undertaken as a first step in exploring the functional relationship between mitochondrial and cellular membranes. It deals only with Acanthamoeba sp. mitochondria and includes the following 1 Submitted to the graduate faculty in partial fulfillment of the requirements for the Ph.D. degree. Supported by a Predoctoral Fellowship, Public Health Service, National Cancer Institute, CF-5892. Present address: Dept. Pharmacology, University of Mississippi Medical Center, Jackson, Miss., U. S. A.

Experimental Cell Research 19

R. L. Klein and R. J. Neff topics: the volume of amoeba mitochondria in various suspending media; a comparison of actual mitochondrial volume with volume determined by the optical density method; mitochondrial metabolism at different volumes; and interpretation of results. METHODS Growth and maintenance of culfures.-Cultures of Acanfhamoeba sp. were grown in a liquid medium as previously described [21, 251. Ten to 15 ml of packed cells were produced from 650. ml of aerated medium in 7 days at 29” to 32°C. The maximal concentration of cells was 3 to 4 x IO8 cells/ml. Harvesting of cells and mitochondrial preparation.-Centrifugation for 5 minutes at 600 g was sufficient to pack the cells. They were washed twice in 0.25 M sucrose which was 0.01 M in Na, versene. In general, the sucrose method of Schneider and Hogeboom [32] was followed. Washed cells were homogenized in the ice cold sucrose-versene solution in an Elvehjem-Potter type homogenizer. The extent of cell disruption was checked by successive microscopic observations. Mitochondrial isolation and purification were performed at 2°C. Homogenates were centrifuged at 800 g to remove whole cells, cell debris and nuclei. The supernatant consisting primarily of mitochondria, microsomes and fat droplets was centrifuged at 10,000 g for 35 minutes to obtain a mitochondrial pellet. Several resuspensions and centrifugations of the various fractions were necessary to achieve maximal mitochondrial purity in every instance and quantitative particle recovery when required. The final supernatant was discarded and the pellet containing purified mitochondria was resuspended in the desired experimental solution and maintained at ice bath temperature until used in volume or metabolic studies. Determination of mitochondrial volumes by cenfrifugal packing.-Determinations of mass mitochondrial volumes were made using a modified hematocrit technique first employed by Hedin in 1891 [29]. Tubes were prepared from Pyrex capillary tubing (9.5 cm long, 0.4 cm diameter, 0.25 cm bore), which were flame-sealed at one end to yield a flat inner bottom. All determinations were done in quadruplicate. Small capillary pipets were used to fill these tubes. Since the capillary tubes proved fragile in the high speed angle-head centrifuge employed, quadruplicate tubes were wrapped tightly with rubber bands throughout their lengths, wrapped in gauze, and placed in small plastic test tubes filled with water. They were centrifuged at 10,000 g for 35 minutes. As the tubes were of uniform bore and their bottoms flat, the length of the mitochondrial pellet divided by the total length of mitochondria plus suspending medium (measured with calipers) gave the fraction of the suspending medium occupied by the particles. Multiplying this figure by 100 gave the percentage volume occupied by the mitochondria.

To compare the swelling

or shrinking

in different

test solu-

tions an empirical formula was used. An example of its use is given below: percentage volume of percentage volume of - mitochondria in mitochondria in 0.25 M sucrose distilled water percentage volume of mitochondria in distilled water Experimental Cell Research 19

X 100 = per cent increase or decrease in mitochondrial volume

Osmotic properties of Amoeba mitochondria

135

In the above case distilled water was used as the basis of comparison (standard), and the per cent change in volume of mitochondria in 0.25 M sucrose was calculated. If a decrease in volume occurred it was referred to as a shrinking compared to the standard; if an increase, it was referred to as a swelling compared to the standard. Determination

of mitochondrial

volumes by optical density measurement.-Measure-

ment of the optical densities of mitochondrial suspensions were performed at 520 millimicra according to the procedure used by Raaflaub [30] and Cleland [S]. Readings were ta!ten in a Beckman Spectrophotometer using 1 cm square cuvettes or in a Coleman Junior Spectrophotometer using 1 cm diameter cylindrical cuvettes. Dilution of the mitochondrial test suspension varied with the amount of mitochondria present and was adjusted to give a reading in an optical density range between 0.1 and 0.7. Where correlations of optical density and packed volume were desired, the same mitochondrial suspension was used. For purposes of comparing mitochondrial volumes determined by centrifugal packing with those determined by optical density measurements, the assumption was made that an inverse relationship existed (see Discussion) between optical density and the actual mitochondrial volume of a given suspension. Thus, we constructed a formula to yield percentage volume change by the optical density method, which corresponded exactly to the percentage volume change determined by csntrifugal packing. Below, this formula has been applied to the example given in the previous section on volume by centrifugal packing. 1 1 optical density in - optical density in distilled water 0.25 M sucrose X 100 = per cent increase or decreasein mitochondrial 1 volume optical density in distilled water

Sucrose determination.-The concentrations of sucrose in suspending media and alkaline-disrupted mitochondria were determined by the Somogyi-Shaffer-Hartmann method [15] for reducing sugars. The sucrose in the various samples was hydrolysed for 5 minutes in dilute H,SO, in a boiling water bath. The acid was neutralized before the reducing test was made. Metabolic studies.-Standard Warburg techniques [41] were employed to determine oxygen consumption. A purified mitochondrial pellet was resuspended by a brief homogenization at ice bath temperature to give approximately a 5-10 per cent particle suspension by volume with an optical density of 0.05-0.10 at 520 millimicra. One milliliter of this suspension was pipetted volumetrically into each Warburg flask. The final particle suspension volume in all flasks was 2.0 ml when the other constituents were added (plus 0.2 ml 20 per cent KOH in center well). Unless otherwise stated the conditions for the experiments were as follows: temperature, 32°C; gas phase, air; phosphate buffer, 0.02 M at pH 7.0; and MgCl, 0.005 M. The magnesium and phosphate concentrations given above were found to be optimal for this system. The sucrose concentration was variable and is indicated in each experiment.

Experimental

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R. L. Klein and R. J. Neff

136

RESULTS

,\ficroscopic oDse~urrtions.~~litc,chontlria of living Actrnfhrrnloebrr sp., obappeared to be of t\\-o types. The served with phase contrast microscopy, majority \vere spherical, approximately 0.3 to 1.0 micron in diameter, and sho\ved high contrast. ;\ small percentage \\-ere rod-shaped, showed less

Fig. 1 A. Thin section of whole Acanthamoeba, L 15,000. U, Thin section of a crude sucrose homogenate, x 40,000. The preparations were fixed in 2 % osmium tetroxide for 15 minutes and embedded in methacrylate (Rhom-Hass). CJI, cell membrane, ER, endoplasmir reticulum, L, lipid droplet, nf, mitochondria.

contrast than the spherical particles, but were of similar diameter and approximately three diameters in length. ,4t times the rods appeared to possess independent flexibility. Mitochondria isolated and purified in 0.25 M sucrose were of the spherical variety and the same size as those in the living cell. When suspended in plain sucrose, they often formed small clumps. In the presence of 0.001 M versene, there were few or no clumps. The addition of 0.0001 M CaCl, caused clumping in a short time and eventually resulted in large masses, each composed of hundreds of particles. Isotonic SaCl caused more clumping than isotonic sucrose. mitochondria.1 Notice Fig. 1 is an electron micrograph of Acanthamoeba the membrane surrounding the particles and the dense concentration of irregularly folded membrane-like structures within. 1 We gratefully acknowledge the permission of Dr. Rose E. Cerroni, Ph.D., Biology Dept., Vanderbilt University, and Dr. John I,. Norris, M.D., Howard Hughes Investigator, Anatomy Dept., Vanderbilt, University Medical School, to use their electron micrograph of Acanthomoeba sp. mitochondria. Experimental

Cell Research 19

Osmotic properties

of Amoeba mitochondria

137

Other observations of homogenized cell suspensions indicated an excellent ability for membrane repair by ruptured cells. This resulted in all sizes of new cells. Some had much diluted cytoplasm such that the major portion appeared optically empty under phase contrast. Enucleated cells were prevalent. Often membranes of isolated nuclei were swollen away from their nuclear contents. Nucleoli appeared to swell in various suspending media, particularly when salts were present. Weights, volume and water content of mitochondria compared to whole cells Complete recovery of mitochondria was attempted by differential centrifugation of amoeba homogenates. Complete recovery was obviously difficult and the results must be considered as minimal. Mitochondria occupied approximately 25 per cent of the whole cell volume. They comprised approximately 30 per cent of the wet weight and dry weight of the cell. Mitochondrial water content has been found to be approximately 70 per cent [21]. Assuming that the mitochondrial volume changes in various sucrose concentrations were osmotic phenomena, and by applying a modification of Boyle’s Law [IS], P(V -b) = K, the osmotically inactive volume (b) of the mitochondria was calculated. By substituting the figures found for the mitochondrial volume changes in a series of plain sucrose concentrations (Fig. 2, Curve 1) into simultaneous equations, the inactive volume was determined to be approximately 70 per cent of the total mitochondrial volume. This suggested that approximately + of the particle water content was not directly involved in osmotic volume changes. Sucrose en trance A number of mitochondrial volume changes (presented in following sections) suggested that sucrose entered the particles with time. The results varied considerably from one experiment to the next. It was shown in Table I that sucrose entered the mitochondria during the normal isolation and puritlcation procedure and was released at different rates from versene, CaCl, and KC1 treated preparations. More sucrose was released from particles treated with KC1 or versene than with CaCl, in a given period of time. The CaCl, treated particles retained more sucrose despite the fact that they were approximately 25 per cent smaller than the versene treated ones and approximately 20 per cent smaller than the KC1 treated ones. On the basis of 70 per cent water content, it was calculated that the molarity of the sucrose inside was approxiExperimental

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R. L. Klein and R. J. Neff

138

mately 10 per cent of that in the environment, after being suspended for 1 to 1.5 hours in 0.50 M sucrose-O.01 M versene during mitochondrial isolation. Mitochondrial

volume studies

Mitochondrial volume studies in sucrose, versene, succinate, calcium and a salt mixture.--The initial volume experiments were exploratory in nature and were designed to determine the effect of various experimental solutions on TABLE

I. Entry

of sucrose

mg Sucrose in supernatant intact mitochondria Suspending Medium

into mitochondria. mg Sucrose in disrupted mitochondria

First wash

Second wash

Acid soluble fraction

Acid ppt. fraction

Total mg sucrose

A. 0.01 CaCl, 0.01 Versene

1.15 2.30

0.55 0.73

1.05 0.48

0.65 0.55

3.40 4.06

B. 0.01 CaCI, 0.01 KC1

2.16 3.42

0.68 0.58

0.43 0.25

0.74 0.61

4.01 4.86

M

A and J3 are results from two typical experiments. A single large preparation of mitochondria was centrifuged from 0.50 M sucrose-O.01 M versene isolation medium. The pellet was resuspended in distilled water by brief homogenization, divided into equal aliquots and brought up to 40 ml in each of the suspending media indicated in A and B. Each solution was centrifuged and the first wash (supernatant) saved. Each remaining pellet was resuspended in 4 ml of the same medium and centrifuged. The second wash (2nd supernatant) was saved. A wash required approximately 40 minutes. The twice-washed mitochondrial pellets were then disintegrated in 0.1 M NaOH by vigorous shaking, and treated as described in the Methods.

TABLE

II. Effect of time on mitochondrial volumes in distilled versene and sucrose. Actual percentage volumes in centrifugal packing tubes

Suspending medium M

DW, 0.001 Versene DW DW, 0.0001 CaCl, 0.88 Sucrose plus 0.0001 CaCl,

water, calcium,

Percentage change

145 min.

45-90 min.

45-145 min.

10.65 6.80 6.00

10.60 6.30 5.40

0 13 % dec. 18 % dec.

1 % dec. 19% dec. 26 % dec.

4.93

4.92

2 % inc.

2 % inc.

45 min.

90 min.

10.70 7.80 7.30 4.84

The mitochondria were washed once in distilled water (DW) before adding versene and CaCl,. The results are from two experiments with quadruplicate determinations in each. Experimental

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Osmotic properties of Amoeba mitochondria

139

mitochondrial volume. The solutions selected were similar to those used in isolation procedures or to those used in metabolic studies. The basic solutions were glass distilled water, 0.125 h4, 0.25 M, 0.50 M and 0.88 M sucrose. Volumes were determined in distilled water and the untreated sucrose as well as in these solutions supplemented with (1) a salt solution mixture (SSM),

(4)

,o.ae

(6) (2)

.o.so

.o.ez

10.

(71 0.1 0.1 0.4 0.a SUCROSE MOLARITY

so

0

01

0. 0-e (
0s

.o.eo 10

Fig. 2. Fig. 3. Fig. 2.-Volumes of mitochondria in various suspending media. Curves: (I) sucrose only, (2) sucrose plus 0.01 M SSM (see text for composition), (3) sucrose plus 0.01 M versene, (4 sucrose plus SSM and versene, (5) sucrose plus 0.01 M (Na), succinate, (6) sucrose plus SSM and succinate, (7) sucrose plus 0.0001 M CaCl,. Fig. 3.-Volumes and optical densities of a mitochondrial preparation suspended in a series of KC1 concentrations. A , optical density of suspended mitochondria, 0, percentage volume of an equal number of mitochondria in the same suspending medium. For comparison, an equal number of mitochondria suspended in 0.25 M sucrose gave an optical density of 0.32.

which was a modified Zamecnik and Keller [43] medium and contained 0.001 M MgC12, 0.00003 M CaCl,, 0.0025 M KCl, 0.0035 M KHCO,, 0.0014 M KH,PO, adjusted to pH 7.2; (2) 0.01 M versene; (3) 0.01 M sodium succinate; (4) SSM containing 0.01 M versene; (5) .SSM containing 0.01 M sodium succinate; and (6) 0.0001 M CaCl,. Mitochondria were allowed to incubate in these solutions for approximately 1 hour. Part of this time was involved in packing the mitochondria by centrifugation in the capillary tubes. Temperature did not seem to be critical in these experiments since identical results were obtained in experiments in which the mitochondria were incubated and Experimental

Cell Research 19

R. L. Klein nnd R. J. Neff 18-25"C, or in the cold room at 2-5°C. centrifuged at room temperature, This was true only for short term experiments where mitochondria were in the isolated state for less than two hours. Longer periods at room temperature resulted in reduction of enzymatic activity and probably slow destruction of the particles. Results are presented in Fig. 2. Each curve, exclusive of Curve 1, represents the volume of mitochondria in increasing concentrations of sucrose supplemented with various other substances (mentioned above). The percentage volumes recorded are relative to the volume of the same number of mitochondria in 0.25 M sucrose. Curve 1 represents the volume of mitochondria in increasing concentrations of sucrose alone. Each point is an average of at least five and in some cases twenty experiments with quadruplicate determinations in each. Where sucrose alone was used as the suspending medium (Curve l), all points excepting distilled water fell on a straight line. In the range of 0.125 M to 0.88 M sucrose, mitochondrial volume was inversely proportional to sucrose concentration. In contrast to this osmotic-like behavior, the addition of any of the other compounds studied in this series of experiments showed deviations from expected osmotic behavior. When SSM was added to sucrose (Curve 2), mitochondrial volumes changed little as the concentration of sucrose was increased from 0.25 M to 0.88 M. Also, SSM caused swelling (compare with Curve 1) which became more marked at higher sucrose concentrations. By contrast SSM caused shrinking when compared to distilled water (see Table III). The addition of versene to plain sucrose (Curve 3) always caused an increase in volume of mitochondria, which became more pronounced as the sucrose concentration was increased. A small decrease in volume occurred with increasing sucrose concentrations between 0.25 M and 0.88 M when versene was present. In sucrose plus SSM, the percentage increase in volume caused by adding versene (Curve 4) was approximately the same at all sucrose concentrations (compare with Curve 2). Versene increased the volume in distilled water slightly. The addition of succinate to plain sucrose (Curve 5) or to sucrose plus SSM (Curve 6) always caused swelling compared to the respective solutions without succinate. The increase in volume with added succinate in plain sucrose became greater as the concentration of sucrose was increased. In sucrose plus SSM, the increase in volume caused by adding succinate was approximately the same at all sucrose concentrations (compare Curves 2 and 6). It was obvious that these results were very similar to those in Curves Experimental

Cell Research 19

osmotic properties of A ~oe~a ~i~oc~o~~~iu 3 and 4, where versene was added, however,

the swelling

141

was of less mag-

nitude. Small concentrations of calcium ions (0.0001 M) produced considerable shrinking at all concentrations of sucrose and in distilled water (Curve 7). TABLE

III.

~itoc~o~drial

volumes in distilled water and sucrose wifh individual salts.

added

% Change in volume as compared to the volume in Salt added M

0.001 CaCl, 0.001 MgCI, 0.001 KC1 0.001 Ph. Buf. 0.001 KHCO, 0.01 SSM

Distilled water

0.25 M sucrose

20 % dec. 14% dec. 10% dec. 7 % inc. 5 % inc. 9 y. dec.

19 % dec. 11% dec. 7 % inc. 0 6 % inc.

0.88 M sucrose

12 % dec. 16 % inc. 35 % inc.

Averaged results from 3 experiments with quadruplicate determinations in each. The percentage volume changes express the change in mitochondrial size due to the addition of each salt indicated to distilled water, 0.25 &f and 0.88 &f sucrose. Ph.Buf., phosphate buffer.

In other experiments mitochondrial volume was studied as a function of time (Table II). Mitochondrial volume decreased with time in distilled water and to a greater extent in distilled water supplemented with 0.0001 M CaCl,. Versene (0.001 M) prevented the shrinkage with time in distilled water; 0.88 M sucrose prevented this shrinkage with time in the presence of 0.0001 1M CaCl,. ~~itocho~driul volumes in distilled water and sucrose with added iRdividua1 salts.-VVhen the addition of SSM was shown to alter mit~chondrial volumes, a series of experiments was undertaken to determine the effect of individual salts present in this mixture. The results in Table III indicated that the divalent cations, calcium and to a lesser extent magnesium, caused shrinking up to 20 per cent when added to distilled water. KC1 and SSM caused shrinking in distilled water but swelling in sucrose. Phosphate buffer and KHCO, caused slight increases in volume in distilled water. ~ifoc~ondrial volumes with the addition of ade~osi~e tr~FhosFhate (ATP), adenosine diphosphate (ADP), 2-4-dinitrophenol (DNP), thyroxine, malonate, and succinate to sucrose solutions.-The results in Table IV indicated that ATP, ADP, DNP, and malonate, either in the presence or absence of succinate, did not alter the mitochondrial volumes to any extent. Succinate was previously Experimental

Cdl Research 19

R. L. Klein and R. f, Neff noted to cause a small increase in volume. Thyroxine, when alone, did not change the mitochondrial volume, but in the presence of succinate, a small increase occurred. The valume in sucrose-phosphate buffer-magnesium suspending medium, which was used in metabolic experiments, was similar to the volume in sucrose alone.

% vol. Suspending Medium 0.25 M Sucrose 0.25 M Sm., 0.02 M Ph. Buf., 0.005 M M&I,

Sue-Ph-Mg., Sue-Ph-Mg., Sue-Ph-Mg., Sua-Ph-Mg., Sue-Ph-Mg., Sue-P&M& SW-Ph-Mg.,

Sue-Ph-Mg.,

0.01 0.01 0.01 0.01 0.01

M Succinate M Succ., 0.002 34 ATP M Suet., 0.002 M ADP &I Succ,, 0.001 M DNP M Sncc., 0.001 M Thyroxine 0.01 M Sum., Q.001 M Mafonate 0.001 M Thyraxine 0.005 M Malonate

mitOCh*

% vol. compared to that in 0.25 M Sue-Ph-Mg

4.87 4.96

2 % dec. -

5.15 5.17 5.21 5.16

4 % inc. 4 % inc. 5 % inc. 4 ?& inc.

5.28

7 % inc.

5.08 4.30

2% inc.

4.99

1 y, dec. 1 % inc.

These results are the average of two similar experiments with quadruplicate determinations in each. Sue-Ph-Mg, 0.25 M sucrose, 0.02 M phosphate buffer and 0.005 M MgCl,.

Optical density and volume studies af varying concentrations

of KC1 and su-

crose.-The volumes and optical density readings recorded in Figs. 3 and 4 were determined at comparable time periods, approximately 45 minutes after the mitochondria were added to the various suspending sob&ions. In KC1 (Fig. 3) ~tochond~al volumes were related inversely to optical density throughout the concentration range used. In Fig. 4 it was seen clearly that the results in sucrose were different from those in KCl. Optical density was related inversely to actual mitochondrial volume only up to 0.25 Msucrose, but above this concentration the optical density became related directly to the mitochondrial volume. The optical density changes reflected volume changes inversely only at the lowest sucrose concentrations.

Effect of fime on optical densifg of ~~~a~~v~d~~as~~~nd~d in a variefg of solutions.---The data presented in Table V represented one large experiment. Equal numbers of mitoch~~dria were suspended in each solution and readings of optical density were taken at intervals after mixing. The behavior in various concentrations of sucrose, KC1 and thyroxine, as well as distilled water was verified in other experiments, Experimenfai

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Osmotic properties of Amoeba mitochondria

143

The great variability of optical density readings with time was demonstrated. Generally, when no chelating agent was present the optical density increased with time. When any chelating agent (thyroxine, versene, succinate and citrate) was present, whether or not it also served as a substrate (succinate and citrate), optical densities decreased with time. Some optical densitiesremained rather fixed over the four hours tested.

SUCROSE

SUCROSE MCLARITY

Fig. 4.-Volumes and optical densities of a mitochondrial preparation sucrose concentrations. D , optical density of suspended mitochondria, an equal number of mitochondria in the same suspending medium. Fig. 5.-Mitochondrial 0, respiration during minutes.

MOLARITY

Fig. 5.

Fig. 4.

size and rate of succinate oxidation in various the first 60 minutes (during logarithmic phase),

suspended in a series of 0, percentage volume of sucrose concentrations. A , respiration after 140

The incomparability of volumes calculated by the optical density method to actual mitochondrial volumes by the centrifugal packing method was again seen in Table VI. In some cases the volume changes indicated by the optical density method were as much as ten times too great; in others swelling was indicated where shrinking had actually taken place. Sabstrate oxidation

and mitochondrial

size

This series of experiments was designed to determine whether mitochondrial size affected respiration of added substrate. Conflicting reports in the literature [7, 11, 13, 30, 381 regarding substrate oxidation in swollen mitochondria have shown both inhibition and stimulation of respiration. In Fig. 5 it was shown that the rate of mitochondrial respiration was directly proportional to the Experimental

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R. L. Klein

144

and R. J. Neff

size of the particle between 0.05 M and 0.88 M sucrose when 0.01 M succinate was being oxidized. Initially the respiration rates were high. With time dilute solutions had deleterious effects on mitochondrial respiration. The addition of 0.001 M versene, which increased mitochondrial volume more than did distilled water, inhibited oxidation of 0.01 M succinate 40 per cent. Greater than equimolar concentrations of magnesium (0.002 to 0.004 M) completely reversed the versene inhibition. Magnesium ion (0.004 Ail) by itself, when added to mitochondria washed free of magnesium, increased the rate of 0.01 M succinate oxidation by more than 200 per cent. Calcium TABLE

V. Changes in optical density with time in a single mitochondrial preparation in a variety of suspending media. Optical

density

readings

medium

30 min.

60 min.

120 min.

180 min.

240 min.

Distilled Water 0.05 M Sucrose 0.125 M Sucrose 0.25 M Sucrose 0.50 M Sucrose 0.88 M Sucrose 0.0005 M KC1 0.125 M KC1 0.44 M KC1 0.25 M Sucrose, 0.0005 M KC1 0.50 M Sucrose, 0.0005 M KC1 0.88 M Sucrose, 0.0005 M KC1 0.125 M KCI, 0.01 M Succinate 0.44 M KCl, 0.01 M Succinate 0.01 M Succinate 0.25 M Sucrose, 0.01 M Succinate 0.25 M Sucrose, 0.01 M Citrate 0.25 M Sucrose, 0.001 M Versene 0.0002 M Thyroxine 0.25 M Sucrose, 0.01 M Succinate, 0.0002 M Thyroxine 0.25 M Sucrose, 0.0001 M CaCI, 0.25 M Sucrose, 0.0001 M CaCI,, 0.0005 M KC1 0.25 M Sucrose, 0.0001 M CaCI,, 0.001 M Versene 0.25 M Sucrose, 0.02 M Phosphate buffer, 0.005 M MgCl, 0.25 M Sucrose, 0.0001 M CaCl,, 0.01 M Succinate

0.079 0.100 0.114 0.133 0.096 0.074 0.074 0.089 0.091 0.136 0.092 0.074 0.062 0.079 0.064 0.086 0.078 0.087 0.079 0.075 0.120 0.123 0.090 0.082 0.073

0.094 0.119 0.127 0.150 0.103 0.081 0.091 0.096 0.093 0.144 0.100 0.079 0.054 0.052 0.057 0.078 0.057 0.084 0.076 0.068 0.124 0.129 0.086 0.068 0.070

0.118 0.129 0.139 0.159 0.111 0.088 0.109 0.113 0.098 0.143 0.107 0.084 0.058 0.062 0.056 0.074 0.046 0.076 0.068 0.047 0.130 0.138 0.081 0.068 0.066

0.121 0.136 0.137 0.158 0.112 0.090 0.113 0.118 0.100 0.138 0.108 0.086 0.057 0.073 0.053 0.067 0.043 0.067 0.063 0.038 0.127 0.138 0.073 0.074 0.063

0.126 0.135 0.130 0.153 0.110 0.090 0.115 0.119 0.101 0.134 0.105 0.087 0.075 0.078 0.054 0.065 0.044 0.064 0.057 0.030 0.121 0.132 0.060 0.079 0.068

Suspending

Experimental

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Osmotic properties

145

of Amoeba mitochondria

VI. A compilation of volume changes in mitochondria for comparison the centrifugal packing and optical density methods.

TABLE

Suspending Medium

Per cent change in mitochondrial volumes in A. as compared to B.

media Medium

A.

B.

Optical

0.125 M Sue, 0.01 M SSM M Sue, 0.01 M SSM 0.50 M Sue, 0.01 M SSM 0.88 M Sue, 0.01 M SSM

0.125 M Sue. 0.25 M Sue. 0.50 M Sue. 0.88 M Sue.

0.125 M 0.25 M 0.50 M 0.88 M

0.125

0.25

Sue, 0.01 M Versene Sue, 0.01 M Versene Sue, 0.01 M Versene Sue, 0.01 M Versene

M M 0.50 M 0.88 M 0.25

Sue. Sue. Sue. Sue.

58.7 % inc.

6 % inc.

75.3 % inc.

6 % inc. 17 % inc. 36 % inc.

66.7 % inc.

117.0 Y0 inc. 69.5 % inc. 72.4 % inc. 88.7 % inc. 53.8% inc.

0.125 M Sue. 0.25 M Sue.

28.2%

DW, DW, DW, DW, DW, DW,

DW DW DW DW DW DW

20.0 % 10.0 Y. 7.4 % 53.8 % 49.2 %

M Sue. 0.25 M Sue. 0.25 M Sue.

16.2 y0 inc. 7.5 Y. inc. 53.8 % inc.

CaCl, MgCI, KC1 SSM Ph. buffer KHCO,

0.125 M Sucrose 0.50 M Sucrose 0.88 M Sucrose

4 % inc. 4 % inc.

13 Y0 inc. 30 % inc.

inc. 16.3 % inc.

37.0%

0.25

Centrifugal packing

density

0.125 M Sue, 0.01 M Succ. 0.25 M Sue, 0.01 M Succ. 0.001 M 0.001 M 0.001 M 0.01 M 0.001 M 0.001 M

of

2 % inc. 3 % inc.

dec. dec. dec. inc. inc. inc.

dec. 14 % dec. 10 y0 dec. 9 % dec. 7 % inc. 5 % inc.

20%

8 % inc. 12 % dec. 30% dec.

The results represent more than twenty experiments with quadruplicate determinations in each, where centrifugal packing volumes and optical density volumes were determined simultaneously. Volumes are calculated as explained in the Methods. DW, distilled water, Sue., sucrose, Succ., succinate, Ph., phosphate. TABLE

VII.

Sucrose molarity M 0.10

Effect of sucrose molarity on mitochondrial and succinate. Comparative mitochondrial size

0.25

1.10 1.00

0.88

0.70

0s uptake in succinate first 60 min Micro-L/Min 11.0 8.9 5.0

These results are taken from two experiments, dria were intially suspended in 0.02 ni’ phosphate 0.01 Jf citrate. 10 - 603701

respiration

0, uptake in citrate first 60 min Micro-L/Min

of citrate

Ratio of rates Succ./Citrate

7.8

1.4

2.1 0.9

4.2 5.6

with duplicate samples in each. The mitochonbuffer, 0.005 Jlf RfgCI, and 0.01 ,lf succinate or

Experimental

Ccl1 Rescurch 19

R. L. Klein

and R. J. Neff

ions (0.004 M), which caused a dramatic volume decrease, reduced the rate of succinate oxidation by approximately 90 per cent. Malonate inhibited both citrate and succinate oxidation. That oxygen tension and enzyme concentrations were not limiting in the above studies was shown by large increases in rate of oxygen consumption when the substrate concentration was doubled. In an effort to discover the localization of the succinic and citric acid oxidizing enzymes, several experiments were performed at different sucrose concentrations (different mitochondrial size) using succinate and citrate as the substrates. The results in Table VII indicated that citrate behaved like succinate at different sucrose concentrations, that is, there were increases in respiration with increases in mitochondrial size. As the particles became smaller, the ratio of succinate rate to citrate rate increased. Thus, as the size of the mitochondria decreased, their ability to utilize succinate was maintained to a greater extent than was their ability to utilize citrate. In all experiments where citrate was used as the substrate there was an obvious initial lag in its oxidation as compared to succinate. DISCUSSION

The results confirm the fact that Acanthamoeba sp. mitochondria possess many properties in common with mitochondria isolated from other animal and plant cells. Such evidence requires only brief comment. However, we feel that the results dealing particularly with the mechanism of volume change, the use of optical density and centrifugal packing methods to measure volume change, and the correlation of mitochondrial volumes with metabolic regulation add new information and deserve discussion and interpretation. Properties of Acanthamoeba sp. mitochondria to those from other sources

compared

The size, percentage water content, permeability to water, sucrose and various salts, and the metabolism of substrates by amoeba mitochondria suggest that they are similar in structure and function to those from other cells [24]. Electron micrographs substantiate the presence of a dense concentration of irregularly folded membranes within the particles as previously shown in other mitochondria [28, 341. The particles also stain with Janus Green. Having substantiated these similarities, we feel that the findings to be discussed can be assumed to have general applicability. At this point it is appropriate to mention those properties of amoeba mitochondria which may differ from others. Experimental

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Osmotic properties of Amoeba mifochondria

147

(1) They are tolerant to low tonicities for long periods and may be washed in distilled water apparently without deleterious effects. (2) They contain a rather large percentage of dry matter, approximately 30 per cent. It is possible that the large fraction of dry weight represents structural components and that the “toughness” of the particles may be due to a larger number of structural bonds per unit volume of mitochondria. This property may have survival value for the organism in that a wide range of tonicities are apt to be encountered in its natural habitat. Mechanism

of mitochondrial

volume change

Considering the evidence as a whole, one is forced to conclude that Acanthamoeba mitochondria behave like osmometers only under special conditions. Osmotic behavior has been demonstrated in varying concentrations of pure sucrose and pure KCl. Even under these conditions they are at best “leaky” osmometers as both KC1 and sucrose enter the particles quite rapidly. This is also true for other mitochondria [2, 6, 10, 19, 33, 36, 421. The presence of small quantities of chelating agents and salts, especially divalent cations, produce volume changes disproportionately greater than could be accounted for by a simple osmotic effect. We suggest that volume changes by mitochondria under most conditions are primarily non-osmotic. This presents the interesting possibility that we are dealing with a colloidal phenomenon of swelling and shrinking. The large amount of dry weight, the extensive intramitochondrial membrane framework, the large amount of particle water not participating in osmotic volume changes, and the large volume changes produced by small amounts of salts (0.01 M KCl, 0.0001 M CaCl,) and chelating agents are all indicative of effects on biocolloids [4]. As observed here, a small concentration of added salt (e.g. KCl) may cause either shrinking or swelling depending only on the concentration of sucrose in which the mitochondria are suspended. Such reversal is commonly observed in colloidal gels when the predominant charge is removed or replaced. Reversal may also occur with slight changes in pH. The present data alone are not enough to determine the exact nature of charges involved, such as salt or hydrogen bonds, since both are sensitive to small shifts in salt concentration and may respond by loss or gain of water. With divalent cations, Ca and Mg, the most reasonable hypothesis for shrinking would be the formation of bonds between negative groups on two adjacent protein or lipid chains. After subsequent discharge and dehydration of such bonds the particle would be expected to shrink. The shrinking by Ca ions is not reversible with versene unless the chelating agent is added prior to Ca. Experimental

Cdl Research 19

R. L. Klein and R. J. Neff It is interesting in this respect that shrinking due to high sucrose concentration or standing in distilled water also shows only limited reversibility unless a chelating agent is added to the medium. This suggests the availability of Ca ions from within the particle. The availability of divalent ions has been reported in other mitochondria [l, 35,361. The irreversiblity may be due to a close-knit steric arrangement within the mitochondrial framework. The observations on the effects of versene and other chelating agents on mitochondrial volumes suggest that they prevent shrinking by binding divalent cations. It is probable that other anions (e.g. PO; in SSM) behave in a similar manner. The curious lack of volume variation when SSM was present in the concentration range of 0.25 M to 0.88 M sucrose (Fig. 2, curve 2) also indicates a non-osmotic behavior. Further, the increased but constant volume in the same sucrose concen~ation range, when either versene or succinate was added to the SSM (Fig. 2, curves 4 and 6), indicates that chelation of divalent or polyvalent cations prevents shrinking below a critical level. This suggests that dior polyvalent cations may partially control mitochondrial shrinkage at the higher sucrose concentrations. A surprising outcome of these rather simple observations is that one can speak with a fair degree of certainty about non-osmotic volume change, but one is hardpressed to discover a mitochondrial volume change which is unambiguously osmotic (i.e. movement of water across a semipermeable membrane with a concentration gradient). Permeability of mitochondria may ultimately be explained in terms of a hydrated gel into which solutes pass slowly, rather than particles surrounded by functional osmotic membranes through which solutes pass. Judging from the rash of electron micrographs of mitochondria, including our own, the density of mitochondrial membranes (including cristae) in the total particle volume is large. It is probable that the major portion of the dry weight of the particles can be accounted for by these structural membranes. By contrast, the percentage volume of a whole cell occupied by the cell membrane is small, and any changes in actual membrane volume would be expected to contribute only a small amount in comparison to volume changes due to osmotic processes. In view of the possibility that the structural arrangement of lipids and proteins in both cell and mitochondrial membranes may prove to be similar or identical in the future, one is encouraged to hypothesize that in mitochondria we are dealing primarily, although not exclusively, with membrane volumes. The calculations of osmotically active volumes of mitochondria may be more of an academic exercise than the determination of a real quantity. Eventually one may be able to differentiate between osmotic experimental

Cell l&search

19

Osmotic properties

149

of Amoeba mitochondria

and membrane volumes by comparing and disrupted mitochondria.

the effects of ions on volumes

Criticism of mitochondrial volume determinations packing and light scattering methods

of whole

by centrifugal

Preliminary microscopic measurements of the diameters of Acanthamoeba mitochondria indicated that they were close to the resolution limits of the light microscope, 0.5-1.0 ,u, and hence accurate measurements could not be made. In centrifugal packing studies, it was considered that the mitochondrial packed volume after centrifugation for 35 minutes at 10,000 g was directly proportional to the absolute mitochondrial volume. This was supported by the observation that repeated centrifugations for 35 minutes or longer did not alter the original 35 minute packed volume measurement. In a few suspending media there were measurable mitochondrial volume changes with time. Such volume changes were considered due to actual particle volume change and not due to insufficient periods of centrifugation. Longer centrifugation times were required for viscous media such as existed at l-3°C in high concentrations of sucrose. Although volumes measured by this method are considered to be directly proportional to absolute particle volume, the two are necessarily not equal due to the fraction of occluded water between particles in the mitochondrial pellet. As the particles apparently remain spherical in all solutions the percentage occluded water volume should remain relatively constant regardless of mitochondrial diameter. A rough estimate of occluded water volume might approximate 20 per cent of the total volume assuming the particles exhibit reasonable pliability. The centrifugal packing method has been taken by us to be the most reliable in determining comparative mitochondrial volumes. The main disadvantage is the time required for measurement, which is at best 40 minutes after suspension in a given solution. Of considerable importance to workers studying volume changes in relation to mitochondrial function is our demonstration of the unreliability of the optical density method under a number of conditions. Only when the effects of a given solution on particle volume has been determined by both methods can one decide whether optical density can be used to approximate true volume change. A number of cases may be cited where opposite variations in volume determined by optical densities were found by different workers. The following are presented to emphasize this point. Ca and Mg ions have been shown to inhibit swelling by mammalian mitochondria in hypotonic solutions [ 14, 311, Experimental

Cell Research 19

others have reported a swelling with the addition of Ca ions under varying conditions [17, 18, 22, 23, 391. Ca and Mg ions cause marked shrinking of amoeba mitochondria under all conditions tested in the present communication. It has also been found that inorganic phosphate and versene prevented swelling [SZ, 23, 391. The latter is in direct contrast to the marked swelling produced by versene and to a lesser extent by inorganic phosphate in amoeba mitochondria under all conditions tested. In addition, the magnitude of mitochondrial volume changes which one must infer from the various optical density determinations are often very large indeed compared with findings in the present study (e.g. in versene and sucrose). The references above are presented only to point out the possibility, if not probability, that physiological conclusions might be altered or reversed depending on the volume variations which actually occurred, From a perusal of any of several recent reviews of light scattering, for example Oster [27], Doty and Steiner 1121, Bender [3] and Stacey [37], it is apparent that a large number of factors contribute to the amount of light scattered by particles suspended in solution. Some of the obvious variables encountered are particle size, concentration, wavelength of incident light, internal orientation (isotropic or anisotropic), refractive indices of solvent and particle, and others. Of particular interest for this discussion is the relationship between particle size and wavelength of incident light. Caspersson (see Oster, [27]) h as shown that the amount of light scattered by a constant weight-concentration of scattering material increases as the particle diameter increases toward the wavelength of light used. The maximal amount of light is scattered when the particle (isotropic sphere) is approximately the same diameter as the wavelength of light. As the particle diameter increases beyond the wavelength of light, the amount of scattered light decreases. Thus, when the particle is smaller than the wavelength of light it scatters light in direct proportion to the diameter of the particle; when larger than the wavelength of light it scatters light as an inverse proportion to the diameter of the particle. The average diameters of mitochondria vary from 0.2 to 2.0 ,u [24]. The wavelength of light used to measure volume changes in mitochondria by Raaflaub [30], Cleland [6], Lehninger [23], in this study, and by others is 520 mp. It is obvious that the diameters of the majority of mitochondria fall precariously close to the wavelength (520 m,u) where slight chages in volume would give a transition from a direct to an inverse (or vice versa) relationship between particle size and light scattered. In the present study, the reversal in optical density seen with increasing concentrations of sucrose may be due to such a transition. Here, as presumably in the other studies mentioned above, Experimenfal

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Osmofic properties

of Amoeba mitochondria

151

we were measuring the optical density of transmittedlight, such that an increase in optical density indicated a decreased scattering, whereas a decrease in optical density indicated an increased scattering. Thus, from 0 to 0.25 M sucrose the mitochondria became smaller and progressively scattered less light. Above 0.25 M sucrose the particles became even smaller (presumably smaller than the wavelength of light), but scattered more light. The failure to find such a reversal in KC1 solutions may well be due to the rapid penetration of this salt (followed by water) into the mitochondria. Some mitochondria are undoubtedly small enough or large enough not to be affected. Other explanations are equally plausible at this time. Small changes in ion concentration which cause large volume changes have been interpreted in part as membrane effects. It is quite possible that small changes in the structural orientation of the particle may be reflected by large changes in the amount of light scattered. Also, any degree of clumping [27] would tend to alter the scattering characteristics of the suspension dramatically. This brief discussion is intended to stress the point that reasonable caution should be taken when interpreting physiological phenomena on the basis of mitochondrial volume changes determined by the optical density method. Mitochondrial

size and substrate oxidation

From observations of the rate of respiration at various mitochondrial volumes one can conclude: (1) the intracellular tonicity is a factor which may control the rate of cellular respiration, and (2) some enzymes of the tricarboxylic acid cycle are localized largely, if not completely, in the interior of mitochondria. Evaluations of the conclusions are given below. The role of tonicity in intracellular oxidations.-In general, the rate of respiration is directly proportional to mitochondrial volume except at extremely low tonicities. This result confirms and extends the observations of others [7, 11, 13, 381. If one assumes that external variations in tonicity are rapidly paralleled by the cells’ interior, then it is apparent that a proportional tonicity control of respiration would be advantageous for the survival of cells exposed to a wide variety of external tonicities in their natural environment. Other data [26] show that increases in concentration of sodium chloride above 0.12 M cause a progressive decrease in respiration of whole Acanthamoeba cells. The effects of sodium chloride concentrations below 0.06 M remain to be studied. One would predict that an amoeba displaced to a more dilute environment would respond by increased respiration due to reduction of its intracellular tonicity. The increased energy output could serve to increase water or ion pumping. In reality other adaptive factors, such as decreased Experimental

Cell Research 19

152

R. L, Klein

and R. d. Neff

permeability of the cell membrane to water or the loss of osmotically active particles from the cell, might be active and complicate the story. It is doubtful that such a tonicity mechanism is of particular importance to the majority of cells of higher animals where the tonicity of the internal environment is regulated. It may be of fundamental importance to those regulating cells which face large or abrupt changes in tonicities such as renal tubule or mucosal cells. It is not to be inferred that tonieity would be the only controller of respiration. Substrate concentration, the concentration of activating ions such as magnesium, or competition between ions such as magnesium and calcium would be expected to control respiration even without tonicity changes. Site of citrate and succinate oxidation.-No single datum that we have collected proves by itself the site of localization of citrate and succinate oxidizing enzymes. However, we feel that all of our data on this point are consistent with and provide strong circumstantial evidence for the conclusion that the major portion (if not all) of these enzymes is in the interior of the mitochondria. Although the rate of oxidation of either citrate or succinate is proportional to the mito~hondrial volume over a wide concentration range, succinate oxidation is reduced less (in proportion) than is citrate oxidation as the volume decreases. This is what one would predict if the rate of oxidation depended on the rate of penetration of these two substrates to an interior site. Due to the greater hydrated size of the citrate molecule one would expect its mobility in solution to be less than that of succinate. Further, citrate migration through pores of decreasing size would be more severely restricted than that for the smaller succinate molecule. The initial lag in citrate oxidation by these washed mito~hondria further supports the contention that Fenetration of substrate to an internal site is the rate limiting step in oxidation. That enzyme concentration is not limiting is indicated by the fact that increasing the substrate concentration results in increased oxygen consumption [20]. A further point of support for internal localization comes from the data on different rates of oxidation of equimolar concentrations of citrate and succinate. Since the oxidizing enzymes are not limiting and both substrates are probably oxidized by a common system (both are inhibited by malonate), one would expect that at equimolar concentrations both rate and total oxygen consumption due to citrate addition should be greater than that for succinate. It is suggested that citrate concentration at the site of aconitase localization is the limiting factor which, in turn, is due to a slow rate of penetration of citrate to the interior of the mitochondria. In many of our experiments, decreases in volume were obtained by increasExperimental Cell Research 19

osmotic ~roFertie~ of Amoeba ~itoc~o~~ri~

153

ing sucrose concentration. The possibility must be considered that sucrose or a contaminant inhibits enzyme activity. The data on loss of succinate oxidizing capacity with time (Fig. 5) clearly demonstrates that sucrose protects enzyme activity. Percentage loss of activity with time is much greater in low than in high sucrose concentrations. This result is the opposite of what one would expect if sucrose or a contaminant was inhibiting enzymatic activity. Finally, agents which cause an exaggerated swelling (versene) or shrinking (calcium) cause large reductions in the rate of succinate oxidation. These effects may involve more than simple volume changes. The large inhibition of respiration by 0.001 M versene, which is prevented by slightly more than equimolar concentrations of magnesium ions, probably results from two related mechanisms: (1) chelation of magnesium ions which are needed for enzymatic activation and/or reduced mitochondrial volume (Table III), and (2) the subsequent swelling of mitochondria with loss of cofactors and/or stretching of critical enzyme orientations along the membranes. The drastic reduction in succinate oxidation (90 per cent or more) by small ~oncen~ations of calcium ions (0.0001 to 0.004 M) is accompanied by a large reduction in volume. Again one cannot make an easy interpretation of the data since calcium is known to exchange with magnesium in a number of biological systems. It is possible that at least a part of the effect is due to the reduction of the optimal concentration of magnesium needed for activation. Thus, one cannot correlate unequivocally the reduction in rate of respiration with volume change since the magnitude of the non-volume effects of versene and calcium remain to be determined. However, the fact remains that volume and respiratory changes can be related. In tracing out functional relationships or intracellular control mechanisms, the site of localization of the enzymatic machinery is of great interest. The above method of localizing oxidizing enzymes in mitochondria may serve as a model for localizing other functions in other subcellular particles. It is conceivable that microsomes, nuclei, and nucleoli, will prove amenable to the same type of analysis. The data and conclusions on the site of enzyme localization provide grounds for speculation on the structure of the mitochondrial membrane (p, 147, 148 of this discussion). From a functional point of view the external membrane could be equally we11 represented by a number of structural models such as pores in a continuous membrane or tortuous passage-ways in a folded mass of films. The main requirement of the model would be that the openings to the interior be large enough to accommodate hydrated citrate Experimental

Cell Research 19

R. L. Klein and R. J. Neff

154

molecules even when the mitochondrial volume is reduced to half its “normal” size. Electron micrographs of Acanthamoeba mitochondria reveal them to be a spherical mass of film only a small area of which is in direct contact with the external medium. Although discrete channels at the particle surface are not seen, it is possible that there exists a continuous or discontinuous connection between intramitochondrial and cytoplasmic space. The extremely large and at least partially enclosed surface area, as seen in electron micrographs, make interpretations of non-osmotic behavior and substrate penetration to internal enzymatic sites reasonable. From the present study it is not possible to infer more than an indirect relationship between the functions of cellular and mitochondrial membranes. By responding to tonicity changes to provide energy for water and perhaps for ion pumping, the mitochondria can most logically be assigned the role of protector of the cell membrane and thus of cell integrity. SUMMARY

Osmotic properties of mitochondria isolated from Acanthamoeba sp. were studied. Included were volumes of amoeba mitochondria in a wide variety of suspending media, a comparison of actual mitochondrial volumes determined by centrifugal packing with volumes determined by the optical density method, and mitochondrial metabolism at different volumes. Amoeba mitochondria were found to have many properties similar to those from mammalian tissues. Particle volume changes were likened to biocolloid effects rather than to osmotic phenomena. The unreliability of the optical density method in determining mitochondrial volumes was demonstrated. The role of intracellular tonicity and the site of citrate and succinate oxidation were discussed. REFERENCES

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