out, - plant physiologymethyl)aminomethane (tris), 0.020 m in ethylenedia-mine tetraacetic acid, and...

SOME EFFECTS OF TONICITY ON LUPINE SUCCINOXIDASE1S. I. HONDA & ANNE-MARIE MUENSTER

UT. S. PLANT, SOIL, & NUTRITION LABORATORY, AGRICULTURAL RESEARCH SERNICE, U.S.D.A. ITHACA, NEW YORK

The integrity of mitochondria is relevant to theintracellular distribution of inorganic ions by Donnanequilibria and active transport processes (cf. 1-4,17, 25, 31, 37, 39, 41, 42, 47). Two related propertiesof mitochondria probably dependent upon their in-tegrity and especially relevant to ion distribution arethe permeabilities to salts, sucrose, and organic acids(2, 6, 9, 20, 34) and the ability of mitochondria toact as osmometers (26, 27, 35, 43, 44). Furthermore,since respiratory activity is influenced by the os-molarity of the media used in both preparation and as-say procedures (8, 13, 14, 18, 21-23, 40, 45) the os-motic properties of mitochondria also must be con-sidered with the effects of inorganic ions not onlyupon ion uptake but also with the effects of ions uponthe oxidation of cytochrome c (18, 28, 36,46), re-duced diphosphopyridine nucleotide (18) and variousorganic acids (30).

As Tyler (45) pointed out, few investigators haveconsidered tonicity itself to affect the activity ofmitochondrial enzymes, apart from any osmotic in-fluence on permeability or swelling of mitochondria.One inadequacy in noting the effect of differentosmotic concentrations on enzymatic activity usuallystems from an arbitrary selection of substrate con-centration since either inhibition or activation mayresult depending upon the substrate concentrationused (15, 16). A more complete characterizationis required, such as the determination of theapparent reaction constants for succinoxidase (cf.41). A complicating factor i,n experiments concern-ing tonicity results from the common use of sucroseas the chief solute for varying osmotic pressure.Lelmniinger, Ray, and Schneider (24) discuss evidencefor the inhibitory action of sucrose on mitochondrialactivities. In common with studies on tonicity ef-fects, however, sucrose effects may be incompletelycharacterized.

In our study concerning the ionic relations of plantmitochondria (cf. 17, 18, 37), information was re-quiredl to chlaracterize the behavior of lupine mito-chon(dria in different osmotic conditions. Resultsare reported of experiments studying the effects ofboth pre-assay and assay tonicities upon the kineticsof lupine succinoxidase under conditions used forstudying mitochondrial swelling in the preseice ofsuceinate ( 16).

1 Received revised manuscript August 30, 1960.

METHODS

Mitochondria were isolated from 4 day-old seed-lings of Lupinus albus (L.) and washed as previouslydescribed (16). The medium used for isolating thelupine mitochondria was 0.165 M in tris(hydroxy-methyl) aminomethane (tris), 0.020 M in ethylenedia-mine tetraacetic acid, and the osmolarity2 adjusted to0.15 M, 0.22 M, 0.40 M, 0.60 M, or 0.74 M. Sucrosesolutions used to wash the mitochondria correspondedin tonicity to those of the isolation media.

Mitochondrial concentrations were measured bytheir nitrogen content assayed by the Nessler method.

In the manometric assay for succinoxidase activitythe mitochondria suspended in sucrose solution wereadded to the main compartment from the sidearm ofWarburg vessels to start the reaction after a tempera-ture equilibration of 10 minutes. The earliest oxy-gen uptake data were recorded 3 to 7 minutes aftermixing and the uptake followed for 60 minutes. Theinitial rates of succinoxidase activity were obtainedby extrapolating to zero time. Activities were re-corded as microliters of oxygen consumed per hourper mg mitochondrial nitrogen [Qo., (N) ]. The finalassay mixture of 3 ml contained succinic acid, tris,Na2ATP (Pabst), horse heart cytochrome c (Sigma)as noted, tris-acetate buffer, magnesium acetate asnoted, and sucrose to adjust the osmolarity. AlthoughATP was not required for activity, it increased andprolonged activity. ATP was therefore addedroutinely in a high saturating concentration. Detailsof concentrations used are given for each experi-ment. The succinoxidase assays were carried outaround pH 7, which was previously determined toresult in maximal activity. Preliminary studiesshowed the effect of ATP can be attributed to thecontamination of ATP by ADP and formation ofADP from ATP. No other 5'-nucleotide waseffective.

The terminology of Friedenwald and Maengwyn-Davies (10, 11) is used for classifying the order ofreactions. First order reactions concern the com-plexing of only one molecule each of substrate, acti-vator, or inhibitor with each enzyme site. Secondorder theory is concerned with cases in which twomolecules of any one reactant per site are involved.

2 Osmolarities of the media were computed on the basisof the following degrees of dissociation (12): 0.86 forR+R-; 0.72 for R++ (R-)2 and (R+)2R--; and0.45 for R+ +R- -.

105

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PLANT PHYSIOLOGY

The following equations used to characterize lupinesuccinoxidase have been taken essentially as fromFriedenwald and Maengwyn-Davies (10, 11). TheMichaelis-Menten equations have been extended to in-clu(le the effect of an activator and the quantitativerelationships between the formation of the variouscomplexes of enzyme, substrate, inhibitor and acti-vator.

The following equations were used as bases forthe mathematical representations of the apparent firstorder reaction of the succinoxidase systemn in thepresence of a dissociable inhibitor malonate:

k 3 k2+ k'

I Fl+-S ES -E+Prodluct KSk., k,

kis k3 [E] [l1F+I *± El , K1 = - ;

k- ~~~~~[El][ES] [I]

ES+I -± EIS , aK, =[EIS]

[EI] [S]EIT+S EIS , aKs=

[EIS]

where E is succinoxidase, S is the substrate succinate,I is the inhibitor malonate, the combinations of lettersare the various complexes, a is a dissociation factor(10) for the dissociation of EIS to ES, EI, S, and I,and the bracketed letters represent concentrations.If a equals one, the inhibition is non-competitive.The complexes of S and I do not affect the complex-ing of the other on E. The inhibition, thus, affectsthe enzymatic rate, i.e. influences the enzyme, ratherthan the affinity of enzyme with substrate (5). Ifa is infinite, the inhibition is competitive and, inessence, S and I compete for the same site on E.That is, the complex EIS cannot be formed. Fromsteric considerations all values between unity and in-finity may be possible for a depending on how closethe sites for S and I are together.

In the visual estinmation of a from the intersectionof the lines of regression, 1/v versus l/[S] (see later)for [I] and [112, values of a around 100 are noteasily distinguishable from infinity. An intersectionof the lines very near the ordinate may be erroneouslyassigned to occur on the ordinate. Consequently,values of a and all other constants were calculatedfrom the Lineweaver-Burk double reciprocal plots.

The Michaelis-Menten convention is used inwhich:[E]t equals the sum of [E], [ES], [El] and [EIS]v the reaction rate is proportional to [ES]; and Vmthe nmaximal activity is proportional to the totalamount of enzyme [E] ,. The general equations rep-resenting the annarent first order reaction then follow:

VmIIa

v

1 1IIb -=

v Vm

KS [I]KS [I]1 + + +

[S] [S]K1aKK[I] KS [I] 1

(1 + ) + (1±+ ) ;aK, Vill K1 [S]

where v is the reaction rate for any given concentra-tions of S and I, and Vm is the extrapolated reactionrate at infinite S concentration in the absence of I.If 1/v as a function of 1/[S] is deternmined at two ormore different constants [I], all the constants whichoccur in line 2b (Lineweaver & Burk form of lineIIa) can be calculated froml the lines of best fit (leastsquares method). Line IIb yields only apparentvalues for the constants Ks and K1 if a dissociable acti-vator in the system is not accounted for.

Friedenwald and Maengwyn-Davies (10) haveshown that the line lIb has the form following forthe first order reaction system involving both a dis-sociable inhibitor anid activator when certain simplify-ing assumptions ar-e imia(le (see 10):

Vm KS [I]Ks II] KAI-- = (1 + -- + + ) (1 + -) .

v [S] [S]KI aKj [A]On comparison of line III with line IIa, it is seen

that if the effect of a dissociable activator is neglectedthe values of the various constants from line II areunderestimated by 1/(1 + KA/A). Owing to thepractical limitation of the number of assays for anymitochondrial preparation, the routine estimaticof the apparent first order reaction constants we:carried out in the presence of a constant high ATPconcentration without any attempt to account for theunderestimate of values.

Friedenwald and Maengwyn-Davies (10) pointedout that the values of the various constants are onlyapproximations if the dissociation factors a, x, q,and t are not equal to unity. x, q, and A are analo-gous to a in the dissociations of the various complexesof S, I, A, and E in EAS, EAI, EIS, and EAIS.

For evaluation of the second order reaction con-stants with respect to substrate KJ/K2 and vm thefollowing was used:

vm so S= 1 + k ( l )

V S SO

where k e(quals (K,/K2) ¼, S is the succinate concen-tration giving the activity V, S. is the succinate con-centration giving the highest experimlental activity,and vm is the extrapolated maximal activity. K1and K. are the apparent dissociation constants forsuccinate in the apparent activating and inhibitingpositions, respectively, in the relation:

vm K1 [S]= 1 + - +

V [S] K2

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HONDA & MUENSTER-TONICITY EFFECTS ON SUCCINOXIDASE

EXPERIMENTAL RESULTSINITIAL OBSERVATIONS. Representative data on

the effect of the assay osmolarity on succinoxidaseactivity of lupine mitochondria isolated in hypertonicsolutions are shown in figure 1 A. Succinoxidaseactivity decreased as the osmolarity was progressivelylowered below 0.40 M.

Another type of response to osmotic variation isshown in figure 1 B where a peak of maximal activityaround 0.40 M was found. In an incomplete surveyof experimental conditions, all lupine succinoxidasewhich characteristically showed the peaked typeactivity-tonicity curves of figure 1 B also simultan-eously displayed second order reactions with respectto succinate concentrations. The peaked typeactivity-tonicity curve and second order reactionsmore frequently occurred under hypotonic conditionswhere the mitochondria may be relatively less intact.It appears that these associated behaviors are relatedto mitochondrial damage.

Either inhibitions or activations by high osmolaritycompared vith low osmolarity may result dependingupon the substrate concentration used (fig 2). Itwas apparent that to obtain less equivocal effects of

tonicity a more complete characterization was requiredthan that obtained with an arbitrary single concentra-tion of substrate. We therefore determined the ap-parent reaction constants for succinoxidase from theLineweaver-Burk double reciprocal plots of data suchas in figure 2 (cf. 10).

The data of table I indicate that an oxidation ofmalate converted from the added succinate was prob-ably negligible and thus complete tricarboxylic acidcycle activity was also negligible with the additionof succinate only. In contrast to Price and Thimann(32) and Pierpoint (29) who obtained R.Q. valuesof 0.23 to 0.28 with succinate and 1.08 to 1.40 withmalate, we obtained generally little carbon dioxideproduction with succinate even with the system forti-fied with ATP and DPN (table I). The additionof DPN increased the rate of oxidation of addedmalate, in accord with the absolute requirement forDPN found by Price and Thimann (32); but the rateinitially was nil and very low after 60 minutes. Insome experiments an R.Q. up to 0.23 was found withsuccinate. However, in every case the initial rateof oxidation with a mixture of malate and succinatewas equal only to the rate expected from the amount

.Z%-O

CM00

700F

_ 500z

C44

0

0300

°s 0.2 OA 0.6 M 5 10 15 20mMASSAY TONICITY SUCC I NATE CONC.

FIG. 1. Variation of succinoxidase activity with assay osmolarity. Mfitochondria prepared in 0.74 M media andwashed with 0.60M sucrose. Assay media contained succinic acid (20 mM), tris (42 mM) Na2ATP (601 JUM), tris-acetate buffer (36 mM), and sucrose to adjust the osmolarity. Mitochondrial concentrations were 404 and 378 ,agN/3 ml for A and B, respectively.

FIG. 2. Effect of assay osmolarity on the variation of succinoxidase activity with succinate concentration. Con-ditions as for figure 1 except for tris-succinate and assay osmolarity as indicated. Mitochondrial concentration was

477 ug N/3 ml.

2.

* 060M TONICITY

0.22M TONICITY

I a IIIAft --1

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PLANT PHYSIOLOGY

TABLE IOXIDATION OF SUCCINATE & MALATE BYMITOCHONDRIA*, WITH & WITHOUT

INITIAL CONCENTRATION'S

Suc- MALATE DPNTCINATE

20 0

20 0

16 416 40 100 10

0

0.5 mg/ml0

0.5 mg/ml0

0.5 mg/ml

021IINITIAL TAR

Qo, (N) mg60 t

344 26380 25290 20274 170 90 1'

LUPINEDPN

Up-

:E/ R.Q.N 60 min

min

6 0.0;3 0.1)6 0.077 0.0

1.29 0.8

* Mitochondria prepared in solutionis of 0.60 am osmo-

larity transferred to 0.60 mi solutionis for gas exchangemeasurements at 300 C. Mitochondrial concentration was596 ug N/3 ml. Tile assay media contained in additiontris (42 mr for 20 mnI organic acid concentration, & 21m-\ for 10 mM malate conc.), Na.,ATP (601 gM), mag-nesium acetate (33 ,um), tris-acetate buffer (18 mM), andsucrose to adjust the osmolaritv.

of succinate adde(d in the mixture andl the oxidationof malate was initially negligible. It seems likely,then, that the initial rates of oxidation measured uponsuccinate additioni to lupine mitochondria can be at-tributed to succinoxidase activity.

FIRST ORDER KINETICS. Characterization oftonicity effects are shown in table II for relativelysimple variations of experimental treatments in pre-paring the lupine mitochondria and in assaying forsuccinoxidase. Data are from selected experimentsshowing first order kinetics. General trends inchanges of values considered more important thanactual values wlhiclh vary from experiment to experi-ment.

Table II slhows that, regardlless of the assay tonici-ty, progressive lowering of the osmolar concentra-tion of the medium used for preparing the lupinemitochondria progressively decreased a. That is,malonate inhibition of succinoxidase changed fromone with essentially competitive characteristics (aequals infinity) towards a non-competitive type (aequals unity). Low assay tonicity in comparisonwith high also decreased a except for mitochondriafrom 0.15 Ai preparations for which a was alreadynear unity.

Lowering the osmolar concentration in the prepa-ration increased both K. and K, (table II). Lowassay osmolarity for succinoxidase from any prepara-tion, however, decreased Ks. The ratio Ks/K, was

more consistently affected in that lowering eitherassay or preparation osmolarity decreased the ratio.

The data of table III were obtained by extendingthe assumptions and methods of Slater and Cleland(40) to the lupine succinoxidase system. Calcula-tions of k', a composite rate constant which is alsoa function of substrate accessibility to enzyme (Slater& Cleland, 40), are attempts to characterize more

closely the changes in Ks brought about by changes intonicity. Since succinoxidase is a particulate multi-enzyme system and not a simple single enzyme, k' isonly an apparent rate constant which is affected bythe accessibility [i.e. permeability & diffusion of sub-strate to enzyme (cf. 40)]. It is assumed here thatk' can be used to follow the effect of tonicity onaccessibility.

Table III shows that enzymatic activity was un-affected by low assay tonicity except for mitochondriaisolated in 0.60 M solutions. In the latter instance theactivity was decreased 36 % relative to activity inthe high assay tonicity. Succinate affinity and ac-cessibility were increased in all cases by low assaytonicity. The changes in Ks brought about by assaytonicity largely can be attributed to changes in suc-cinate accessibility except for mitochondria isolatedin 0.60 M solutions. In the latter case only 60 % can

TABLE IIEFFECT OF OSMOLARITY ON APPARENT 1ST ORDER REACIJON

CONSTANTS FOR SUCCINOXIDASE ACTIVITY OFLUPINE ]MITOCHONDRIA

AsSAY* PREPARATION OSMOLARITY**MEASURE OSMO-

LARITY 0.15 M 0.40 M 0.60 m

KS, mm 0.22 M 5.10±+1.42 2.87±0.50 1.20±0.10succinate 0.60 M 12.34±1.12 5.47±0.82 5.47±0.90

Vm, Qo2 (N) 0.22 M 783±+-64 701±+ 13 1099±+-440.60 M 766±10 744±26 1491±24

K1, mM 0.22 M 0.91 0.19 0.05malonate 0.60 M 0.64 0.16 0.11

a 0.22 M 2 8 360.60 M 1 191 268

KS/K_I 0.22 M 5.6 15.1 24.40.60 M 19.3 34.2 49.7

r, coefficientOe1atiOn 0.22 M 0.981 0.994 0.953

Lineweaver-Burk doublereciprocal 0.60 M 0.879 0.994 0.977onate series

* Activities measured in media of indicated osmolaritiescontaining succinic acid (2-30 mM), tris (4-63 mM),Na.ATP (601 !LM), tris-acetate buffer (18 mM), tris-malonate (0.25 mm for 0.60 M preparations, otherwise0.5 mM), magnesium acetate (50 ,uM for 0.60M prepara-tions, otherwise 33 ,UM), and sucrose to adjust osmolari-ties. Mitochondrial concentrations for the 0.15 M, 0.40 Mand 0.60 M preparations were 376, 457, and 270 /Ag N/3 ml,respectively. Initial activities measured at 300 C.

** 0.40 M preparation assayed in 0.18 M and 0.57 Msolutions.

*** All values significant at P <0.01, except for requals 0.879 which was significant at P = 0.05.

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IIONDA & MUENSTER-TONICITY EFFECTS ON SUCCINOXIDASE

TABLE IIIVARIATION OF EFFECT OF ASSAY OSMOLARITY ON ENZYMES

OF SUCCINOXIDASE, ACCESSIBILITY OF SUCCINATE,& AFFINITIES OF SUCCINATE &

MALONATE TO THEM*

PREPARATION OSMOLARITYMEASURE**

0.15m 0.40M 0.60MEnzyme Activity VmrA/VmB 1.02 0.94 0.64

Succinate Accessibility***k' /k/ 2.47 1.80 2.74A B

Succinate Affinity(1/KsA)/(I/Ks.) 2.42 1.90 4.48

Malonate Affinity(1/KIA)/(1/KI ) 0.70 0.84 2.20

* Calculated from table II.** Constants with subscript A determined in the 0.22 M

assay and those with subscript B determined in the 0.60 Massay.

***kI is the composite rate constants which is also afunction of the accessibility of succinate to the succinoxi-dase system. It is not the simple rate constant k' sinceit not only concerns the concentration effect of reactantsbut also the diffusion of them to the sites of enzymicactivity. k' Was calculated according to Slater andCleland whose assumptions for the cytochrome c oxidasesystem were extended here to the succinoxidase system(40).

be attributed to change in accessibility. In contrastto the increase in succinate affinity, malonate affinitywas decreased by low assay tonicity, except for 0.60 Mmitochondrial preparations whose malonate affinityincreased. It is clear that the effect of tonicity inpreparation and assay are different for malonate andsuccinate acting in the succinoxidase system.

SECOND ORDER KINETICS. In general loweringthe preparation osmolarity decreased the effectivenessof both succinate and malonate in the succinoxidasesystem with first order kinetics (KS & KI increased,table II). This effect may also be related to thesecond order reaction, with respect to succinate con-

centration, shown by certain preparations. Most0.15 M preparations of lupine mitochondria showedsecond order kinetics in assays of low osmolarities.

Table IV shows the effect of osmolarity and cyto-chrome c upon the apparent second order reactionconstants of lupine succinoxidase. In common withthe effect of preparation osmolarity on Ks/K1 in thefirst order reaction, KJ/K2 was decreased progress-

ively with the decrease in preparation osmolarity,where K1 is analogous to Ks and K2 to KI. The ap-

parent maximal velocity, vm, was not affected in a

regular way The effect of low tonicity was consider-ed as possibly related to loss of a cofactor such as

cytochrome c and the effect of added cytochrome c

was studied. The effect of added cytochrome c was

to increase vm and K1/K2, except for 0.60 M prepara-

tions. That is, succinate seemingly became more

readily bound in the apparent inhibitory position.However, it seems unlikely that mere loss of cyto-chrome c during the mitochondrial preparation in low

I osmolar solutions was involved because in the pres-

ence of added cytochrome c low assay osmolarity stilldecreased K1/K2 and vm.

DISCUSSION

Our study on the effect of tonicity on succinoci-dase demonstrates that reports of simple activation or

L inhibition are insufficient. Characterization of theI tonicity effects by noting the change in the apparent

reaction constants is less ambigious. The practice

3LE IVEFFECT OF OSMOLARITY & CYTOCHROME C ON APPARENT 2ND ORDER REACTION

CONSTANTS OF LUPINE SUCCINOXIDASE

Exp. PREPARATION ASSAY ASSAY* VOSMOLARITY OSMOLARITY TREATMENT Q02 (N)

A 0.60 M 0.15 M -Cyt c 1.31±0.02 982±48+Cyt c 1.16±0.16 1083±281

B 0.40 M 0.15 M -Cyt c 0.073±0.001 779±15+Cyt c 0.386±0.001 1384±53

C 0.15 M 0.40 M -Cyt c 0.125 ±0.003 642±59+Cyt c 0.379±0.012 1357±70

D 0.15 M 0.15 M +Cyt c 0.034±0.003 832±46

*Assay media contained succinic acid (5-37.5 mx, Exp. A; 5-40 mM, Exp. B & C; 5-30 mm, Exp. D), tris(10-79 mm, Exp. A; 10-84 mM, Exp. B & C; 10-63 mM, Exp. D), Na2ATP (601 AM), tris-acetate buffer (9 mM,Exp. A & B; 18 mM, Exp. C & D), and sucrose to adjust osmolarity. Cytochrome c where added was 0.20 uM.Mitochondrial concentrations were 434, 428, 467, and 336 jug N/3 ml for Exp. A, B, C, and D, respectively.

** K1 and K2 are the apparent dissociation constants for succinate in the activating and inhibiting positions, re-spectively, in the relation: vm/V = 1 + K1/S + S/K2, where vm is the apparent maximal activity and V theactivity at S succinate concentration.

109



PLANT PHYSIOLOGY

of correlating simple respiratory activity of mito-chondcria with physiological states in the intact cellis clearly based upon ambigious information if theorder of reactions and the values of, at least, the ap-

parent reaction constants are not determined.In addition to variation of the values of the ap-

parent first order reaction constants for lupine suc-

cinoxidase, we have obtained second order reactionswith respect to succinate w.Ihich may be related to theexposure of lupine mitochondlria to solutions of lowosmotic concentration. Data fronm earlier studiesalso showed inhibition by high succiniate concentra-tion of succinoxidase from rat liver mitochondria (38)and pigeon liver mitochondria (19). These datafollowed second order kinetics but the investigatorsdid not comment upon it. It is significant that boththe rat and pigeon liver mitochondria were subjectedto hypotonic stress in preparation. The proceduredeveloped for the rat liver mitochondria by Schneiderand Potter (38) has been widely used.

Tonicity clearly affected lupine succinoxidase intwo ways, at least, with the properties analogous tothe affinity and accessibility of succinate to the systembeing affected relatively more than the properties in-fluencing the overall enzymic rate. The patternin the effect of lowering the preparation tonicityshowed succinate becoming less and less effective as

substrate (Ks increased) and, indeed, even becominginhibitory as shown by second order reactions. How-ever, succinate functioning as substrate became rela-tively more effective compared with the inhibitoryaction of malonate in the first order reaction andsuccinate as inhibitor in the second order reaction.It may be more helpful to regard the lowering ofpreparation tonicity as decreasing the effectivenessof both substrate and inhibitor, but the latter rela-tively more. This was shown by the decreases in theratios of Ks/KI and K,/K9.

Malonate has been used as the classical example ofa competitive inhibitor (33). It is therefore surpris-ing that values of a were obtained ranging from 270to 1; that is, malonate changed from affecting primari-ly the affinity of succinate (competitive inhibition)to inhibiting the overall rate (non-competitive inhibi-tion, a equals 1, cf.5). Departure of malonate in-hibition from the pattern of competitive inhibition hasnot been previously reported. Examination of otherpublishedl graphs, however, shows that some valuesof a. in fact, were different from infinity althoughhigh. Friedenwald and Maengwyn-Davies (10)noted that somie inhibitions have been called competi-tive when a was as low as 10 and that when a was

as large as 2 some inhibitors have been classified as

non-competitive.No quantitative relation between the osmotic

swelling properties and succinoxidase activity of lu-pine mitochondria have been noted. However, mito-chondria prepared in 0.15 M solutions usually showedsecond order reaction rates in the oxidation of suc-

cinate; and their optically-measured volumes (1/op-tical density of suspension) no longer varied inverselywith osmolar concentration (16).

It is clear that transfer of lupine mitochondriafrom one solution to another differing in tonicity hasmarked effects upon the succinoxidase system. Thereis uncertainty about the relations between mitochon-(Irial swelling, electron transfer in respiratory path-ways, and the effect of various agenits which influencephosphorylatioin and electron transfer (cf. 7). Wemay anticipate that in somiie cases insufficient char-acterization has contributed to confusion and that amore complete description by determination of eventhe apparent kinetics of the systems will help to pro-vide rational and consistent explanations.

SUMMARYI. Progressive lowering of the osmolar concentra-tion of solutions used both for isolating lupine mito-chondria and enzyme assay progressively decreaseda, a measure of the interaction of the malonate andsuccinate enzyme complexes in the first order reactionof succinoxidase, from values associated with com-petitive inhibition to values associated with non-competitive inhlibition.

II. Both K. and K,, the apparent Michaelis constantsfor succinoxidase activation and inhibition by succin-ate and malonate, were progressively decreased bylowering the osmolar concentration used in preparingthe mitochondria.

III. Low assay tonicity, compared with high, de-creased Ks and progressive lowering of the prepara-tion tonicity decreased the ratio Ks/KI without affect-ing Vnm in a parallel fashion.

IV. The apparent accessibility of succinate to suc-cinoxi(lase was more affected by assay tonicity thanwas succinoxidlase activity. Malonate affinity wasnot affected by assay tonicity in a parallel manner.

V. Lowering the preparation and assay tonicitiesalso affected the order of succinoxidase activity withrespect to succinate concentration. Most mitochon-dlria isolated in 0.15 M solutions showed second ordersuccinoxidase activity while those isolated in higherosmotic concentrations less frequently showed the sec-ondl order reactions.

VI. For those preparations showing the second orderreaction, lowering preparation tonicity decreased theratio K,/K,, where K1 and K, are the apparent dis-sociation constants for succinate in the activating andinhibiting positions, respectively.

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rat-liver mitochondria at 00 to the metabolitespyruvate, succinate, citrate, phosphate, adenosine5'-phosphate and adenosine triphosphate. Biochem.J. 70: 718-726.

2. AMOORE, J. E. and W. BARTLEY. 1958. The per-meability of isolated rat-liver mitochondria to su-crose, sodium chloride, and potassium chloride at0°. Biochem. J. 69: 223-236.

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1llHONDA & MUENSTER-TONICITY EFFECTS ON SUCCINOXIDASE

3. BARTLEY, W. and J. E. AMOORE, 1958. The effectsof manganese on the solute content of rat-livermitochondria. Biochem. J. 69: 348-360.

4. BARTLEY, W. and R. E. DAVIES. 1954. Activetransport of ions by sub-cellular particles. Bio-chem. J. 57: 37-49.

5. BULL, H. B. 1954. The enzyme-substrate complexas an intermediate in enzyme-catalyzed reactions.In: The Mechanism of Enzyme Action, W. D.McElroy and B. Glass, eds. P. 141-153. JohnsHopkins Press, Baltimore.

6. CLELAND, K. WI. 1952. Permeability of isolated ratheart sarcosomes. Nature 170: 497.

7. CORWIN, L. M. and M. N. LIPSETT. 1959. Studieson stability of rat liver mitochondria. II. Relationof the electron transport system to swelling. J.Biol. Chem. 234: 2453-2458.

8. DIANZANI, M. U. 1953. On the osmotic behaviourof mitochondria. Biochim. Biophys. Acta 11: 353-367.

9. DE DUVE, C., J. BERTHET, L. BERTHET, and F.APPELMANS. 1951. Permeability of mitochondria.Nature 167: 389-390.

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11. FRIEDENWALD, J. S. and G. D. MAENGWYN-DAVIES.1954. Elementary kinetic theory of enzymatic ac-tivity. Second order theory. In: The Mechanismof Enzyme Action, WI. D. McElroy and B. Glass,eds. P 180-208. Johns Hopkins Press, Baltimore.

12. Handbook of Chemistry and Physics. 1947. 30thed.

13. HARMAN, J. W. and M. FEIGELSON. 1952. Studieson mitochondria. TII. The relationslhip of struc-ture and function of mitochondria from heartmuscle. Exp. Cell Res. 3: 47-58.

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PLANT PHYSIOLOGY

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MECHANISMS BY WHICH WIND INFLUENCES TRANSPIRATION'JOSEPH T. WOOLLEY

SOIL & WATER CONSERVATION RESEARCH DIVISION, AGRICULTURAL RESEARCH SERVICE, U. S. DEPARTMENTOF AGRICULTURE, & DEPARTMIENT OF AGRONOMY, UNIVERSITY OF ILLINOIS, URBANA

Although the amount of transpiration from a leafis predominantly a function of the amount of energyreceived by the leaf, wind can influence the mannerin which the leaf loses energy, and thus can affecttranspiration significantly. Wind influences tran-spiration by removal of the so-called "layer" of satu-rated air from the surface of the leaf, and also bychanging the temperature of the leaf. Bange (1)showed that in the isothermal case the stimulation oftranspiration by wind could be accounted for quanti-tatively by theoretical calculations involving the re-moval of the layer of saturated air. Thermal effectsof wind on transpiration are more (lifficult to evaluate,since any change in transpiration rate will usually beaccompanied by a secondary temperature change, butwind does have one direct effect on the leaf tempera-ture. The increased mass of air brought into contactwith the leaf by wind tends to bring the leaf tempera-ture closer to the air temperature, regardless ofwhether the leaf be warmer or colder than the air.

Certain other mechanisms of win(d action appearto be possible or have been mentioned in the litera-ture as possibilities, andI therefore deserve investiga-tion. These mechanisms are:

I. Decrease in air pressure on the lee side ofthe leaf, causing increased evaporation on this side.

II. Ventilation of intercellular spaces, caused byactual passage of air through amphistomatous leaves.

III. Bending of the leaves in the wind, causingcompression of the intercellular spaces and conse-quent pumping of saturated air out of the stomata.

These three mechanisms are the subject of thispaper.

1 Received revised manuscript September 30, 1960.

I. DECREASE IN AIR PRESSURE. Maximov (5)mentioned that wind may cause a reduced pressureon the lee side of a leaf, causing increased evaporationon that side, and Gaumann and Jaag (4) invoke thissame mechanism to account for part of their sub-stomatal transpiration. Putting aside the fact that areduced pressure on one side of the leaf would usuallybe compensated for by an increased pressure on theother, we can estimate the possible magnitude of thiseffect by considering a leaf held normal to a windwhich is blowing 7 meters second-' (ca. 15 miles/hr). Such a wind could cause a maximum pressuredifferential of about 570 dynes cm-2 between thetwo sides of the leaf. This 570 dynes cm-2 wouldbe less than 0.06 % of the difference between atmos-pheric pressure and the vapor pressure of water.Therefore the rate of transpiration could not be in-creased by more than 0.1 % by such a pressure dif-ferential.

II. VENTILATION THROUGH AMPHISTOMATOUSLEAVES. -Measurements made by a modification ofthe Darwin and Pertz (2) porometer technique indi-cate that a pressure of 104 dynes cm-2 may force amaximum of 10-3 cm3 of air per second through 1cm2 of a corn leaf when the stomata are open. Thusa 7 m second-' wind, causing a pressure differentialof 570 dynes cm-' between the sides of the leaf, mightforce as much as 5.7 x 10-6 cm3 of air per secondthrough each square centimeter of a corn leaf.

Miller and Coffman (9) found that corn plantsin the field often transpired as much as 5.5 X 10-6 gof water per square cm of leaf per second (200 gm-2hr-1). If the relative humidity inside the leaf were100 % and that outside of the leaf were 50 %, an ex-change of 3.6 X 10-1 cm3 of air per cm2 of leaf sur-

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