necessary conditions for isolation of tightly coupled ... · necessary conditions forisolation...

Plant Physiol. (1970) 45, 773-781

Necessary Conditions For Isolation of Tightly Coupled HigherPlant Mitochondrial

Received for publication December 17, 1969

HIROSHI IKUMADepartment of Botany, University of Michigan, Ann Arbor, Michigan 48104

ABSTRACT

A number of parameters associated with the isolation ofplant mitochondria were carefully examined in the hope ofoptimizing the isolation technique. Dark-grown mung bean(Phaseolus aureus var. Jumnbo) hypocotyls were used as thesource of mitochondria. Necessary conditions for the iso-lation of tightly coupled plant mitochondria include: (a)gentle tissue disruption; (b) rigorous exclusion of contami-nating particles from the mitochondrial fraction; (c) theuse of an optimal grinding medium containing a high con-centration of mannitol, 10 mM phosphate or triethanol-amine buffer to maintain the pH above 7.2, ethylenedi-aminetetraacetic acid at 1 mM level, cysteine at a low con-centration (0-5 mM), and a high concentration of bovineserum albumin (0.1-1.0%); and (d) the use of the proper washand suspending medium consisting of mannitol, buffer,and bovine serum albumin at the same concentration as inthe grinding medium.

Recently, a number of workers have reported the isolation ofmitochondria from various higher plant tissues, which show goodrespiratory and phosphorylative activities (2, 5, 6, 11-17, 21-36,38-45). The method in general consists of tissue disruption andhomogenization in an appropriate medium, isolation of themitochondrial fraction from filtered tissue homogenate bymeans of differential centrifugation, and final washing of theresultant mitochondrial pellet. If the techniques of isolationare compared in detail, however, a wide variety of differences isseen in the method of tissue disruption, in the composition ofisolation media, and in the patterns of differential centrifugation.In fact, the same group sometimes changes the procedure withlittle explanation for the modification. In addition, there areseveral reports indicating that the method successfully employedin one laboratory does not apply to another laboratory (cf.34, 39, 40, 45). These situations may reflect the fact that all theparameters which enter into the conditions of mitochondrialisolation have not been exhaustively studied.Another point which does not appear in the literature fre-

quently is the fact that plant mitochondria age faster than animalmitochondria (cf. 16, 28, 34). In addition, the yield of mito-chondria, calculated on the basis of initial fresh weight, is usuallymuch less from higher plant tissues than that from animaltissues. This necessitates the use of a large quantity of tissue

1 This work was supported by Grant GB4342 from the NationalScience Foundation.

and media, thus lengthening the time involved in isolation.These facts raise practical problems in experimentation (cf. 38).

Studies made with isolated mitochondria must always beinterpreted with caution, because the mitochondria may havebeen changed or damaged during the isolation procedure, andthe preparation may be contaminated with nonmitochondrialmaterial (11, 34). Furthermore, the mitochondria isolated withone method may not show the same respiratory activities asthose with other methods.

In order to minimize the possible introduction of artifactsand to optimize the isolation technique, we have analyzed anumber of parameters associated with the preparation of plantmitochondria. In these studies, time course changes of mito-chondrial activities as affected by each condition have beenfollowed over 24 hr after the completion of isolation. In thiscommunication, therefore, the results of these studies will bepresented and the optimal conditions for the preparation ofplant mitochondria will be discussed.

MATERIALS AND GENERAL METHODSThe hypocotyls of dark-grown mung bean (Phaseolus aureus

var. Jumbo) seedlings, 4 to 5 days old, were used as the mainmaterial for this study of parameters associated with the isola-tion of plant mitochondria. The hypocotyls were separated fromroots and cotyledons + epicotyls by means of scissors, washedwith distilled water at least twice to minimize any contamination,and then stored in the cold room (0-4 C) overnight in colddistilled water. This cold storage of the hypocotyls was in-troduced here for experimental convenience, since it was foundin a number of trials not to affect the quality of mitochondriaupon subsequent isolation. The procedure to follow was carriedout in the cold room, in an ice bath, in a Sorvall RC-2 refrig-erated centrifuge set at 1 C, or with an ice-cold medium.About 1.5 kg of hypocotyls were washed twice with distilled

water, then cut into sections of about 2 cm, and placed in thegrinding medium (0.3 M mannitol, 1 mm EDTA, 0.1%2 BSA,aand 0.5% cysteine, starting pH adjusted to about 8.0). The cutsections and medium (volume ratio of 1:1) were placed in aporcelain mortar (capacity 750 ml) and ground by hand with apestle until about two-thirds of the sections appeared to havebeen crushed. This corresponded to about 15 gyrotary strokes.During these steps, the pH of the tissue-medium mixture wasmaintained at 7.2 to 7.4 by dropwise additions of 10 N KOH.The resultant brei was strained through at least eight layers ofcheesecloth. The residue was re-extracted with about one-thirdvolume of the grinding medium. The introduction of re-ex-traction increased the mitochondrial yield by about 50%. Thecombined filtrate was subjected to differential centrifugation.

2Throughout this paper, all per cent concentrations of solutions areexpressed on the basis of w/v.

3Abbreviations: BSA: bovine serum albumin: PCA: perchloric acid.

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Plant Physiol. Vol. 45, 1970

A crude mitochondrial pellet was collected between 5OOg(10 min) and 12,000g (15 min). Discarding the first low speedpellet, the pellet from the high speed spin was homogeneouslysuspended in about 30 ml of wash medium (0.3 M mannitol,0.1 mM EDTA, and 0.1% BSA, pH 7.4), then subjected to onespin each of low (500g, 10 min) and high speed (6,000g, 15 min)centrifugation. The resultant pellet was suspended in a smallvolume of wash medium, and then the suspension was equallydivided into several centrifuge tubes. About 40 ml of an ex-perimental medium were added to each of these tubes. Aftercentrifugation at 6,000g for 15 min, the surface of each pelletwas rinsed twice with the experimental medium to remove yellowmaterial sedimenting on top of the mitochondrial pellet. Thepellet was finally suspended in a centrifuge tube with the samemedium. The final suspension contained 20 to 30 mg of proteinper ml and was placed in an ice bath during the experimentalperiod. For control runs, the final wash and suspension step wasmade with the wash medium. Unless otherwise mentioned, thesame grinding and wash media as described above were used andreferred to as the standard media. The experimental mediumwas a modified wash medium in terms of either a different con-centration of a component in or an addition made to the washmedium. Time course changes of respiratory activities weremeasured from the completion of final mitochondrial prepara-tion.

Biochemical integrity of mitochondria was measured with aconventional Clark oxygen electrode, purchased from theYellow Springs Instrument Co., Yellow Springs, Ohio, in astirred Lucite cuvette of 3 ml capacity (cf. 28). The standardsequence of additions to the cuvette was as follows: reactionmedium, mitochondrial suspension, substrate (2 min aftermitochondria), and repreated additions of ADP (the first ADP,2 min after substrate) until anaerobiosis. Unless otherwisestated, the standard reaction medium was composed of 0.3 Mmannitol, 10 mm potassium phosphate buffer (pH 7.2), 10 mmKCl, and 5 mM MgCl2. The primary substrates used in thestudy were succinate and l-malate. The respiratory rates, respira-tory control ratios, and ADP :0 ratios were calculated accordingto Chance and Williams (8, 9). Protein was determined by abiuret procedure (20) after solubilization of mitochondria withdesoxycholate, with crystalline bovine albumin as the standardand the suspending medium as the blank. Critical pH measure-ments were carried out with a Radiometer pH meter, typePHM 25SE, and a narrow range Hydrion paper was used tomeasure the pH range.

Enzymatic analysis of pyridine nucleotides was carried outwith an Eppendorf photometer with a fluorometer attachment(19) on 3.5% PCA extracts of mitochondria with specific enzymes(3); a lactic dehydrogenase system for NAD and NADH,glucose-6-P dehydrogenase or isocitric dehydrogenase forNADP, and lactic dehydrogenase + glutamic dehydrogenasefor NADPH. Concentrations of NADH and NADPH weremeasured immediately after the preparation of extracts. Theseenzymes for pyridine nucleotide assays were purchased fromC. F. Boehringer & Soehne GmbH, Mannheim, Germany.The presence of NADP and NADPH was not detected (23, 24)in any of the mitochondrial extracts, and the values for tri-phosphopyridine nucleotides are not included in the results.

All chemicals were of analytical reagent and the best com-

mercially available grade. Bovine albumin fraction V powderor BSA was purchased from the Nutritional BiochemicalsCorporation, Cleveland, Ohio, and was of unesterified fattyacid-poor form. The concentrations of NADH and ADP,purchased from the Sigma Chemical Company, were deter-mined optically at 340 nm and 260 nm on the basis of extinctioncoefficients of 6.22 and 15.4 mm-v' -cm7l, respectively. Theconcentrations of other chemicals were measured gravimetrically.

RESULTS

Major Osmoticum. The two most commonly used osmotica in

mitochondrial studies are sucrose and mannitol. The effectsof different concentrations of these osmotica on respiratoryactivities of mung bean mitochondria are shown in Figures 1

and 2. In these experiments the same concentration of a givenosmoticum was used for both incubation and assay.

Figure IA clearly indicates that mannitol concentrations

higher than 0.3 M are needed to maintain good respiratory and

phosphorylative activities -with succinate as substrate duringthe first few hours after isolation. The ADP:O ratio in this

range of mannitol concentrations remained at 1.6 + 0.1. After

A. 4- 5 Hours after Isolation

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FIG. 1 (left). Effect of mannitol concentrations on mitochondrial2activity and aging. A: Succinate oxidation 4 to 5 hr after isolation; B:succinate oxidation 8 to 9 hr after isolation; C: malate oxidation 1.5 to2.5 hr after isolation; D: malate oxidation 8.5 to 9.5 hr after isolation.The reaction medium contained the same mannitol concentration as

that used for mitochondrial suspension. For assay, 9 mM succinate,35 mM malate, and 222 gM ADP were used. Qo = nmoles O2/min- mg P. In this and subsequent figures, Roman numerals (III andIV) denote respiratory states 3 and 4, and subscripts (1, 2, and 3)refer to the first, second, and third.

FIG. 2 (right). Effect of sucrose concentrations on mitochondrialactivity and aging. Grinding and wash media contained 0.25 M sucroseinstead of 0.3 M mannitol. A: Succinate oxidation 4 to 5 hr after isola-tion; B: succinate oxidation 8 to 9 hr after isolation; C: malate oxida-tion 2.5 to 3.5 hr after isolation; D: malate oxidation 9 to 9.5 hoursafter isolation. Oxygen electrode assays were performed with the samesucrose concentrations as those used for mitochondrial suspension.For assay 9 mm succinate, 35 mM malate, and 180 pM ADP were used.

774 IKUMA

C).

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ISOLATION OF HIGHER PLANT MITOCHONDRIA

an aging period of 8 to 9 hr (Fig.1B), the respiratory controlis preserved only in the mitochondria isolated at concentrationshigher than 0.3M mannitol; also, the higher the concentrationof mannitol, the better the preservation of the respiratory control.Although the results at 20 hr after isolation are not given, 0.6Mmannitol was found to be capable of maintaining 30% of originalstate 3 rate and 50% of original phosphorylative activity ofsuccinate oxidation. The ability of higher concentrations ofmannitol to maintain biochemical integrity of mitochondriais also shown with malate as substrate (Fig. 1, C and D).

Figure 2 shows that sucrose concentrations higher than 0.35Mreduce respiratory activity, while phosphorylative efficiencyremains more or less constant throughout the range of con-centrations tested. The ADP:O ratio in sucrose is 1.1 -+- 0.1,which is about 70% of that in mannitol. It is also noteworthythat the state 3 respiration at 0.25M sucrose compares wellwith that at 0.6M mannitol, but the state 4 rate in sucrose isabout 50% higher in succinate oxidation and about 100% higherin malate oxidation than that in mannitol. These data suggestthat sucrose is capable of partially uncoupling mitochondria byincreasing the mitochondrial ATPase activity. It should beadded that the content of pyridine nucleotide (NAD + NADH)was found to be 2.5, 2.8, 1.1, and 0.45 nmoles/mg protein fortreatments with 0.25, 0.35, 0.5, and 1.0M sucrose, respectively.This suggests further that the mitochondria in high sucroseconcentrations lose soluble components, thus resulting in de-creased respiratory activity (cf. 1). These effects of sucrose onmung bean mitochondrial activity are similar to those on skeletalmuscle mitochondria (10, 18).

In an attempt to examine the combined effect of sucrose andmannitol on the respiratory properties of mung bean mito-chondria, the sucrose concentration was kept constant at 0.25M,and mannitol concentration was varied at 0, 0.3, and 0.6M.A combination of 0.25M sucrose and 0.3M mannitol was foundto be optimal for state 3 rate, but the state 4 rates were high,as in the case of sucrose alone. Similarly, the ADP:O ratioswere essentially the same as those of sucrose alone.

These comparisons of the effects of two osmotica lead to aconclusion that preparations made in sucrose medium are ofinferior quality and that mannitol at high concentrations ishighly preferable for the isolation of mung bean mitochondria.pH. Though the pH of isolation media is considered to be

an extremely critical factor (6, 43), few studies on this topicare reported in the literature. On the basis of respiratory controlratios, Verleur concluded that the pH of 6.5 was optimal forisolation of potato tuber mitochondria (40). In general, how-ever, the pH range of 7.0 to 7.5 appears to be the preferred pHfor the isolation of plant mitochondria. The following experi-ments were carried out to examine the pH effects both on respira-tory activities and on aging.

Figure 3 illustrates the effect of pH of the assay medium onthe oxidation of both succinate and malate. Prior to the additionof mitochondria, the pH of the reaction medium was adjustedto a given value with 5 mm phosphate buffer, 30 mm tris-maleatebuffer, and 10 mm succinate or 30 mm malate. The addition ofmitochondria started the reaction from the first state 4, and 4min later, 220 ,M ADP was added for the measurements of thefirst state 3 and second state 4 rates. Change of pH during thereaction was less than 0.15 pH unit. All the experiments werecarried out within 4 hr after isolation to minimize the agingeffect.

Succinate oxidation shows a broad pH optimum in the rangeof 7.0 to 7.6 (Fig. 3A), while the optimum for malate oxidationis 6.8 to 7.2 (Fig. 3B). In both succinate and malate oxidation,the ADP:O ratio remains high below pH of about 7.2, while athigher pH levels, the ratio decreases. In another series of ex-periments, where 10 mm phosphate buffer was used in the isola-

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FIG. 3. Effect of the pH of assay medium on succinate and malateoxidation. The grinding medium contained 20 mm tris-maleate in addi-tion to the standard constituents, but it was deleted from the wash

medium. The assay medium consisted of 0.3M mannitol, 20 mm tris-

maleate, 5 mm phosphate, 10mrm KC1, 5mm MgC12, and either 10 mmsuccinate or 30mm malate. The pH was adjusted to a given value withKOH or HC1 prior to the start of assay. A: Effect of pH on succinateoxidation; B: effect of pH on malate oxidation.

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FIG. 4. Effect of pH on aging of mung bean mitochondria. Mito-

chondria were prepared with media containing 10 mm phosphate in

addition to the standard constituents, and suspended in a wash mediumof a given pH. The assay medium was the standard medium of pH 7.2.

A: Succinate oxidation 4 to 4.5 hr after isolation; B: succinate oxida-

tion 9 to 9.7 hr after isolation; C: malate oxidation 3 to 3.5 hr after

isolation; D: malate oxidation 10.3 to 11.3 hr after isolation.

tion media, practically identical pH optima were obtained for

respiratory rates, but the ADP:O ratio remained constant in

thepH range of 6 to 8.

In order to study the effect of pH on aging, the mitochondriawere isolated in the presence of 10 mm phosphate buffer in the

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775Plant Physiol. Vol. 45, 1970

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Table I. Effects of the Kintd of Buffer on Aging and Integrity of MitochonidriaMitochondria were prepared and assayed in the presence of a given buffer (10 mm, pH 7.2) in grinding, wash, and assay media in

addition to the standard constituents. Pyridine nucleotide (PN) contents represent NAD + NADH (see "Materials and General Meth-ods"). In this and subsequent tables, Roman numerals (III and IV) denote respiratory states 3 and 4, and subscripts (1 and 2) refer tothe first and second. The ratio of III to IV2 gives the respiratory control ratio of the first cycle.

Succinate Oxidation Malate Oxidation

Buffer Time after Isolation Q02(P) Q02 (P) PN content

ADP:O ADP:OIIII IX, l IX2

hr nrnoles 02,/min wng P nnmoles 02/min tng P nmoles PN/mg P

No buffer 0-0.5 168 79 1.5 ... ... ... ...

4-5 112 71 1.4 ... ... ... 4.29-10 71 61 0.9 ... ... ... ...

K-phosphate 0-0.5 185 101 1.6 154 54 2.24-5 182 99 1.6 128 47 2.3 3.89-10 149 92 1.4 109 40 1.8 ...

Triethanolamine 0-0.5 182 80 1.6 164 41 2.2 ...

4-5 167 78 1.5 128 38 2.2 4.910-11 119 67 1.4 97 44 1.7 ...

Tris-maleate 0-0.5 134 82 1.1 64 28 1.6 ...4-5 103 69 0.9 ... ... ... 3.17-8 54 47 0.9 ... ... ... ...

Tris-HCI 0-0.5 146 68 1.5 114 35 2.14-5 148 75 1.3 107 36 1.9 3.77-8 129 80 1.2 93 41 1.4 ...

standard grinding and wash media; the pH of the final suspensionmedium was adjusted to a given value, and the assay was carriedout at the standard assay pH of 7.2. Figure 4 shows the resultsmeasured at two different periods after isolation for both suc-cinate and malate oxidation. Essentially the same results areobtained with either substrate. Mitochondria quickly lose state 3respiratory activity below pH 7.2, while very little degradationof mitochondrial activity is seen at higher pH values. The ADP :Oratio, on the other hand, stays essentially constant throughoutthe range of pH used during the first few hours, but the de-grading effect of lower pH (<7.2) becomes apparent later.It is thus important to maintain the pH above 7.2 during isola-tion, while an assay medium of pH 7.2 is best for both succinateand malate oxidation.

Buffer. Owing to the presence of large acidic vacuoles inhigher plant cells, the absence of buffer in the grinding mediumnecessitates extra caution in controlling the pH during prepara-tory procedure. A number of laboratories have, therefore, em-ployed various types of buffer in the grinding media. Bonner'sgroup, on the other hand, has felt that the addition of buffer inthe isolation media should be avoided, because of its abilityto extract cytochrome c and to rupture mitochondrial mem-branes (5, 6, 23, 28-30, 35, 43). Here again, there have been nocritical studies available in the literature concerning the degreeof effectiveness or injury due to the presence of buffer in theisolation media.Four different kinds of buffer commonly used by the workers

in the field were employed in the following study to detect theireffects on respiratory and phosphorylative activities of mungbean mitochondria. The results are summarized in Table I,where the contents of PCA-extractable pyridine nucleotide arealso given.

Examination of the results reveals that phosphate and tri-ethanolamine buffers are more effective in preserving the respira-

tory and phosphorylative activities than tris buffers or no buffer.The use of tris-maleate buffer repeatedly yielded mitochondriawhich lost activities within a few hours. This loss of activity wasparticularly noteworthy in malate oxidation. Table I furtherpoints out that the pyridine nucleotide content is not so greatlyaffected by the kind of buffer as the respiratory activities. Itcan thus be concluded that inclusion of 10 mm phosphate ortriethanolamine buffer in the isolation media is not harmful formitochondrial isolation and does not induce soluble componentssuch as pyridine nucleotide to leak out.Mg2+, ATP, Phosphate, and Oxygen. The addition of ATP

and magnesium to the suspending media has been used suc-cessfully for the preparation of mitochondria from skeletalmuscles (10, 18) and for the preparation of phosphorylatingelectron transport particles from beef heart mitochondria (4).Avocado fruit mitochondria are isolated with a medium con-taining MgCl2 (25, 26, 36, 42, 44). The following experimentswere, therefore, carried out to examine the effects of mag-nesium, ATP, and phosphate, applied singly or in combination,on the biochemical integrity and aging of mung bean mito-chondria.The results (Table II) clearly indicate that magnesium ion

exerts a deleterious effect on the maintenance of mitochondrialactivity, while a combination of ATP and phosphate preservesthe activity better than control. The magnesium effect is partiallyreversed by phosphate. The combination of phosphate and ATPis capable of maintaining about 65% of state 3 activity and about75c%O of ADP:0 ratio after 26 to 28 hr of aging.

In another experiment, 10 mm succinate or malate was in-cluded in the wash and suspending medium of mitochondria inorder to keep the isolated mitochondria essentially under an-aerobic conditions. Under these conditions mitochondria lostrespiratory and phosphorylative activities in a few hours. Ex-cessive aeration by means of stirring the mitochondrial suspen-

776 IKUMA




Table II. Effect of Inorgantic Phosphate, A TP, and Magnesium, Applied Singly or in Combination, on Mitochonidrial Integrity and AgingA crude mitochondrial pellet was obtained with the standard grinding medium; mitochondria were washed and suspended in media

containing the additives in addition to the standard constituents. Phosphate buffer, 10 mm, pH 7.2; ATP, 1 mM; MgCl2, 5 mM. For assay,8.3 mm succinate and 160 ,uM ADP were used.

4 to 5.5 hr after Isolation 26 to 28 hr after Isolation

Addition Respiratory rate Respiratory rateADP:0 ADP:O

_lls IV2 II12lIII IVI 1112

nmoles 02/min-mg P nmoles C2/min-mg P

None 152.5 75 217 1.6 95 ... ... ...

Phosphate 169 74 222 1.6 81.5 72 96 0.95ATP 132.5 75 163 1.7 121.5 92.5 134 1.05MgCl2 88.5 49 107.5 1.6 10 ... ... ...

Phosphate + ATP 176.5 71 187 1.7 113 68 128.5 1.25Phosphate + MgCl2 167 72.5 188 1.7 32.5 ... ... ...

ATP + MgC12 78 44.5 84.5 1.7 12.5 ...

Phosphate + ATP + MgC12 149 56.5 139.5 1.6 53.5 44.5 53.5 1.03

2II-

.:

A. 5-6 HOURS AFTER ISOLATIONo02r

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150;

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FIG. 5. Effect of EDTA concentrations on mitochondrial aging.Mitochondria were prepared with a medium containing 10 mm tri-ethanolamine buffer and assayed for succinate oxidation. For assay,9 mM succinate and 170 Mm ADP were used. A: 5 to 6 Hr after isola-tion; B: 9 to 10 hr after isolation.

sion or bubbling air through the suspension also hastened theloss of activity. Mitochondrial biochemical integrity was bestmaintained by simply standing the suspension in an ice bathwith occasional gentle shaking.EDTA and BSA. In order to remove injurious cations from

environment of mitochondria during isolation, EDTA is com-

monly added to the isolation medium. Various concentrations ofthis chelating agent were thus included in the suspending medium

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FIG. 6. Effect of BSA concentrations on mitochondrial aging.Mitochondria were prepared with media containing 10 mm phosphatebuffer and assayed with the standard medium for succinate oxidation.BSA concentrations in the mitochondrial suspensions were examinedchemically with a biuret method (18).

of mitochondria, and the effect on the maintenance of activitywas examined with time. The results are shown in Figure 5.EDTA at concentrations higher than 0.5 mm reduces state 3respiration rate and ADP:O ratio by 15 to 25% during thefirst 6 hr. Essentially the identical conclusion can be reached


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Table III. Relationship between Centrifugal Force and Degree of ContaminationA crude mitochondrial fraction, obtained between SOOg (10 min) and 13,000g (15 min), was successively fractionated by centrifugation

at 500 g for 10 min, 6,000 g for 15 min, and 25,000 g for 20 min. The resultant pellets were suspended in the wash medium and assayedwith an oxygen electrode. Grinding medium: 0.5 M mannitol, 10 mm K-phosphate, 1 mM EDTA, 0.05%O cysteine, and 0.2%0 BSA. Washmedium: 0.5 M mannitol, 10 mm K-phosphate, and 0.2% BSA, pH 7.4. Reaction medium: 0.5 M mannitol, 10 mm K-phosphate, 10mMKCl, and 5 mM MgCl2, pH 7.2. For assay, 10 mm succinate, 30 mm malate, and 170 Mm ADP were used.

2.5 to 3 hr after Isolation 9.5 to 10 hr after Isolation

Succinate oxidation Malate oxidation Succinate oxidation Malate oxidationFraction (Range of

Centrifugal Force)Qo2(P) Qo2(P) Qo2(P) Qo2(P)

ADP:O ADP:O ADP:O ADP:OIII IV21iII IV^ III1 IV2 III, 1V2

g nmnoles 02/M in )iig P nmnoles 02/min mg P nmoles 02/inin mg P Pnmoles02/mnfmg0-500 32.5 15.6 1.0 24 8.5 1.2 28.5 15 1.0 16.5 8 1.2

500-6,000 153.5 66 1.3 80.5 22.5 2.0 136 65 1.3 64.5 20 2.06,000-25,000 60 27.5 1.1 33.5 8 1.8 59 37 0.9 26 12 1.5

after 9 to 10 hr of aging. These results suggest that the inclusionof EDTA in the wash medium is unnecessary. In view of theinjurious effect of magnesium ion, however, the inclusion ofthis chelator in the grinding medium is preferable.

Contrary to the EDTA effect, BSA in the suspending mediumis found to be favorable in maintaining the mitochondrial ac-tivity (Fig. 6). With the range of concentrations tested, the high-est concentration, 0.65%, was found to be most beneficial in thepreservation of mitochondrial quality. This effect is only seenin state 3 respiration; both state 4 rates and ADP:O ratios arenot affected by the BSA concentrations employed.

Effect of Contaminating Fractions on Mitochondrial Activity.Contaminating fractions in mitochondrial preparations tendto have an injurious effect on biochemical integrity of mito-chondria (cf. 6, 28). Furthermore, quick aging of plant mito-chondria may be attributed to this effect of contaminatingfractions. In the following experiments, therefore, three fractionswere separated by means of differential centrifugation of acrude mitochondrial fraction, each containing a varying degreeof heavier and lighter fractions, and tested for the maintenanceof mitochondrial activities. For this purpose, improved isolationmedia (see Table III legend) were used and the three fractionswere sedimented successively between Og and 5OOg (10 min),500g and 6,000g (15 min), and 6,000g and 25,000g (20 min).The first fraction obviously consists mostly of the heavier par-ticles with some mitochondria (mostly granular dark graypellet), the second fraction mostly of mitochondrial pellet witha thin layer of light yellow material covering it, and the lastfraction of both tan-colored mitochondria and covering yellowmaterial.

Table III illustrates the results at two different times afterisolation. Both heavier and lighter fractions show much lowerrespiratory activity than the second fraction. The ADP:O ratiosare also low in these fractions. It is noteworthy that in the lighterfraction the decrease of the ADP:O ratio with time is roughlyinversely proportional to the increase in state 4 rates. Theseobservations suggest the presence of ATPase in this fraction,probably due to breakage of mitochondria.

In a number of other experiments, contamination of yellowmaterial in mitochondria induced not only a lowering of theADP:0 ratio, but also a decrease in state 3 rate with time.The yellow material is heterogeneous; in electron micrographsit consists of various sizes of vesicles with smooth single anddouble membranes (cf. 2, 11, 34), and in a discontinuous sucrosedensity gradient it is also heterogeneous with at least four bands(unpublished work of R. M. Tetley; also, Ref. 2).From these observations, it should be concluded that rigorous

A. SUCCINATE OXIDATIONO 0 2

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FIG. 7. Aging property of mung bean mitochondria isolated withimproved media. Grinding medium: 0.6 M mannitol, 10 mm phosphate,1 mM EDTA, 0.1% BSA, 0.05% cysteine. Wash and suspending me-dium: 0.6 M mannitol, 10 mm phosphate, 1%G BSA. Assay medium:0.6 M mannitol, 10 mM phosphate, 10 mm KCl, 5 mm MgCl2. Forassay 8 mm succinate, 33 mm malate, and 170,qM ADP were used.

exclusion of both heavier and lighter fractions from the mito-chondria is highly importantThe heavier fraction can be removed by a 5OOg spin for 5 min,

and the lighter fraction by rinsing the first 6,000g pellet a fewtimes with the wash medium and by lowering the final spin to5500g for 15 min. Further washings of this pellet by suspendingin the wash medium followed by centrifugation tend to lowerthe mitochondrial activity. By using this centrifugation patternand the improved isolation media of the above experiment, themitochondrial preparation is found to be far more stable thanbefore (Fig. 7). Results similar to Figure 7 were obtained withtomato fruits and white potato tubers as starting materials.

During the course of the present study, possible introductionof artifact was examined on both the ADP:O ratio and the

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I.

80

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FIG. 8. Effect of ADP concentrations on succinate (A) and malate(B) oxidation. Mitochondria were prepared with media containing0.5M mannitol, 10 mm phosphate, and 0.2% BSA, and assayed with amedium containing 0.5 M mannitol, 10 mM phosphate, 10 mni KCl,and 5 mm MgC12. Various concentrations of ADP were added 5 minafter 8 mm succinate or 4 min after 33 mm malate addition. The firststate 4 rates were subtracted from the first state 3 rates and plotted inthis figure.

nature of the ADP titration curve. In all conditions studiedabove, the ADP :0 ratios for both succinate and malate oxidationwere about 75% of the expected values, as reported earlier (28).The nature of the ADP titration curve, on the other hand, changedfrom the previous pattern (Fig. 6 in Ref. 28) to the one shown inFigure 8. The difference between this and the previous results isnow interpreted as being due to the presence of contaminatingparticles in mitochondrial preparations used previously.

DISCUSSION

Grating (14-16, 24-26, 33, 34, 36, 43), hand grinding (5, 6,13, 17, 22, 28-30, 31, 32, 35, 39), and blending (24, 40, 41, 43,45) are the major methods of plant tissue disruption employedfor mitochondrial isolation. These are used singly or in com-

bination, depending upon the type of tissues employed.With mung bean hypocotyls, excessive grinding in a mortar

with a pestle or the use of a Waring Blendor was found to bedetrimental to the retention of good respiratory activities in theisolated mitochondria. The principle of tissue disruption is to

break tissues gently enough to squeeze cell contents and mito-chondria intact out into the medium. With this principle, thequantity has to be sacrificed to some extent, while high integrityof mitochondria can be achieved. The centrifugation pattern isin part dependent upon the major osmoticum used in isolationmedia. Electron micrographs of mitochondrial pellet invariablyshow the presence of nonmitochondrial particles (unpublishedobservation by R. M. Tetley; also Ref. 2, 11, 34). Thus, it ishighly important to minimize the contamination of these particles.For this purpose, inclusion of both low and high speed centrif-ugation is necessary in the wash procedure (28). RecentlyPalmer (38) successfully introduced the use of a nylon fabric(mesh 50 strands/cm) to shorten the time of centrifugation(39). This fabric apparently removes heavier particles, thusrendering the first and the second low speed spins unnecessary.

Most commonly used osmotica in isolation media are sucroseat 0.4 or 0.5M (17, 22, 25, 26, 31-34, 36, 39, 45) and mannitolat 0.3 to 0.7M concentrations (5, 6,13-16, 28-30, 35, 40, 41).Wiskich and Bonner (43), however, successfully employed acombination of sucrose and mannitol, and Sarkissian andSrivastava (39) found that sucrose in the grinding medium andmannitol in the wash and suspending media were most favor-able. With animal mitochondria, Hagihara (7) introduced acombination of 0.21M mannitol and 0.07M sucrose in the isola-tion medium for maintaining mitochondrial integrity for a longtime. Our own results, however, indicated that sucrose prepara-tions were of inferior quality, giving high ATPase activity andpoor respiratory control; and the combination of sucrose andmannitol gave rise to a preparation showing essentially thesame quality as that with sucrose alone. Mannitol at a highconcentration consistently yielded mitochondria of high qualityfrom mung bean hypocotyls. Similarly, Verleur concluded thatthe use of 0.7M mannitol was the best for the preparation ofwhite potato mitochondria (40, 41). Both Verleur's and ourisolation media contain a low concentration (10 mM) of buffer,whereas sucrose preparations are made with media containing50 to 100 mm buffer, and in some cases, MgCl2 and KC1 inaddition. The difference in observations on sucrose and mannitolmay in part be due to the difference in the tissues used and alsoin the composition of isolation media, or else to the degree ofcontamination in the mitochondrial suspension with foreignparticles. It is noteworthy that sucrose preparations are oftenassayed in media containing BSA (25, 26, 34, 36), whereas it isnot necessary for mannitol preparation. It should be added thatmannitol was found to interfere with chemical analysis of phos-phate by a conventional method, and it causes aggregation ofmitochondria upon freeze-thawing. These phenomena do nottake place with a sucrose medium. Though some limitationsexist in the use of mannitol as the major osmoticum of the isola-tion medium, mannitol media are preferred in our laboratory,especially in light of the results presented in this communication.The presence of magnesium in the incubation medium was

found to be detrimental to mung bean mitochondria (TableII), whereas fruit mitochondria apparently require the presenceof magnesium in the isolation media in order to reduce aggre-gation (25, 26, 34, 36). It is of interest to note that where mag-nesium is included, the EDTA concentration is also high. SinceEDTA lowers both state 3 respiration and ADP:0 ratios (Fig.5), it should be omitted from the wash and suspending media.On the other hand, probable action of detrimental cations, suchas Mg2+, Ca2+, etc., can be prevented if EDTA is included inthe grinding medium.The buffer employed in the isolation of plant mitochondria

is either phosphate (39-41, 45), or tris-HCl (25, 26, 33, 34, 36),or both (17, 22, 31, 32). Recently, Drury et al. (14-16) success-fully introduced the use of Na-barbital buffer. Our results indi-cate that phosphate or triethanolamine buffer was better thantris or no buffer. With the concentrations employed here, it wasconcluded that buffers have little effect on rupturing mito-chondrial membranes or in extracting soluble components.Obviously, higher concentrations of buffer cause deleteriouseffects on mitochondrial activity (34).BSA is a protective agent against deleterious action of fatty

acids and other chemicals released by cell breakage or duringincubation of mitochondria (cf. 11, 12, 34, 36). Exclusion ofBSA altogether from the isolation media results in mitochondriawith little respiratory control (34, 36). Some workers do notuse BSA in the isolation medium, but in the assay medium (17,22, 31, 32, 45), or vice versa (6, 7, 13, 14-16, 24, 28-30, 35, 40,41). From our data, the addition of BSA to the isolation mediumis beneficial, but BSA in the assay medium is not necessary. It





should be pointed out that BSA is not usually added to theassay systems of animal mitochondria (cf. 7-9, 37).

In order to prevent the harmful effect on isolated mitochondriaof polyphenoloxidase and phenolic compounds released upontissue disruption (cf. 27, 40), cysteine is often added to thegrinding medium (5, 6, 13-16, 24-26, 28-30, 33-36, 40, 41).Several workers, on the other hand, do not employ cysteine intheir isolation media atall (17, 22, 31, 32, 39, 45). In our ex-periments with mung bean mitochondria to clarify this discrep-ancy in theliterature, we found that the deletion of cysteinefrom the grinding medium altogether did not damage the mito-chondrial activity (unpublished results). Since cysteine itselfis autooxidizable and the oxidized product harmful(cf. 27, 40),its concentration should be kept minimal (0-5 mM), and thechemical should be deleted in the wash and suspending media.With the exception of Verleur (40, 41) the preferred isolation

pH in the literature is in the range of 7.0 to 7.5. From our data,the optimal pH to prevent aging is above 7.2. A pH higher than8.0 apparently shows a detrimental effect (43). The optimal pHfor assaying mitochondrial activity was found to be 7.2.The wash and suspending media usually contain fewer chemi-

cals than grinding media. Important ingredients are majorosmoticum, buffer to keep the pH above 7.2, and a stabilizingagent such as BSA. The osmoticum and BSA concentrations areusually the same as those in the grinding medium, whereas thebuffer concentration, if added, is 10 mm. Some workers includein the suspending medium other components (25, 26, 33, 34, 36);others delete both buffer and BSA (17, 22, 24, 31, 32, 39, 43, 45).The major constituents in the assay media are an osmoticum,

phosphate, and magnesium. The osmoticum concentration inthe assay medium is often lowered to an isosmolar level from thehyperosmotic level in isolation media. Only Verleur (40) usedthe same osmoticum concentration (0.7M mannitol) throughoutisolation and assay. The concentration of phosphate is in general10 mm. The requirement for magnesium has not been studiedcritically, but a generally used concentration is 5 mm. Verleur(40) reported that magnesium could be omitted from the assaymedium.Comparison of necessary conditions between those reported

in the literature and those obtained in the present study lead tothe following conclusions: (a) tissue disruption should be carriedout gently; (b) differential centrifugation should aim at mini-mizing the contamination of nonmitochondrial particles; (c) ahigh concentration of mannitol is a preferred osmoticum in theisolation media; (d) the pH during the isolation procedure shouldbe maintained above 7.2 but below 8.0; (e) in order to maintainthe pH, phosphate and triethanolamine buffers are more satis-factory than no buffer or tris buffers; (f) EDTA at a low con-centration is needed in the grinding medium but not in the washand suspending media; (g) BSA is beneficial in the isolationmedium to protect the mitochondria during isolation; and (h)the important ingredients in the wash and suspending mediaconsist of osmoticum, buffer, and BSA. In addition, the neces-sary components in the assay medium are osmoticum, phosphatebuffer (pH 7.2), and probably magnesium. If some of thesepoints are lightly treated during isolation procedure, the resultantmitochondria tend to age quickly and may show anomalouscharacteristics.Acknowledgments-The author expresses his appreciation for

the skilled technical assistance of Mrs. Malca Sela and MissEmma J. Zimmerman in preparing mitochondria.

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