Plant Physiology - ARONOFF-BIOGENESIS OF THE RING 357 › content › plantphysiol › 31 › 5 › 357.full.pdf · ARONOFF-BIOGENESIS OF THE PYRIDINE RING LITERATURE CITED 1. BONNER,

ARONOFF-BIOGENESIS OF THE PYRIDINE RING

LITERATURE CITED1. BONNER, D. M. and YANOFSKY, C. The biosyntheses

of tryptophan and niacine and their relationships.Jour. Nutrition 44: 603-616. 1951.

2. DAWSON, R. F. Development of some recent con-cepts in the physiological chemistry of the tobaccoalkaloids. Plant Physiol. 21: 115-130. 1946.

3. DEWEY, L. J., BYERRUM, R. U. and BALL, C. D. Bio-synthesis of the pyrrolidine ring of nicotine. Bio-chim. Biophys. Acta 18: 141-142. 1955.

4. FICHTER, F. and BEISSWENGER, A. Die Reductiondes Glutiirsaure anhydrids zum 6-Valerolactone.Ber. deuit. chem. Ges. 36: 1200-1205. 1903.

5. GRAEBE, C. Ueber die Bildung aromatischer Me-thoxysiuren und von Anisol. Annalen Chem.,Justuis Liebigs 340: 205-212. 1905.

6. GROBBELAAR, N. and STEWARD, F. C. Pipecolic acid inPhascolits vuilgaris: Evidence on its derivationfrom lysine. Jour. Amer. Chem. Soc. 75: 4341.1953.

7. HANKES. L. V. and URIVETSKY, M. Mammalianconversion of C"-carboxyl-labeled 3-hydroxyan-thranilic acid into N'-methylnicotinamide. Arch.Biochem. Biophys. 52: 484-485. 1954.

8. HEIDELBERGER, C., ABRAHAM, E. P. and LEPKOVSKY, S.Tryptophan metabolism. II. Concerning the mech-anism of the mammalian conversion of tryptophaninto nicotinic acid. Jour. Biol. Chem. 179: 151-155. 1949.

9. LEETE, E. Biogenesis of nicotine. Chemistry & In-dustrv 1955: 537. 1955.

10. LEETE, E. Amer. Chem. Soc., Univ. of Minnesotameetings, September 1955.

11. LEETE, E., MARION, L. and SPENSER, I. D. The bio-genesis of alkaloids. XIV. Biosynthesis of da-maseenine and trigonelline. Canadian Jour. Chem.33: 404-410. 1955.

12. Lowy. P. H. The conversion of lysine to pipecolic

acid by Phaseoluts vulgaris. Arch. Biochem. Bio-phys. 47: 228-229. 1953.

13. MITCHELL, H. K., Nyc, J. F. and OWEN, R. D. Util-ization by the rat of 3-hydroxyanthranilic acid asa substitute for nicotinamide. Jour. Biol. Chem.175: 433-438. 1948.

14. MUNIER, R. Partition of microchromatography onpaper of alkaloids and various biological nitrogen-ous bases. IV. Separation of some nicotinic acidderivatives and tryptophan. Bull. soc. chim. biol.33: 857-861. 1951.

15. Nyc, J. F. and MITCHELL, H. K. Synthlesis of a bio-logically active nicotinic acid precursor: 2-amino-3-hydroxybenzoie acid. Jour. Amer. Chem. Soc.70: 1847-1848. 1948.

16. OFFERMAN, H. Zur Geschichte des Antihracens.Annalin d Chem., Justus Liebigs 280: 1-35. 1894.

17. ORECHOFF, A. and MENSCHIKOFF, G. Vber die Alka-loide von Anabasis aphylla L. Chem. Ber. 64:266-274. 1931.

18. ROTHSTEIN, M. and MILLER, L. Loss of the a-aminogroup in lysine metabolism to form pipecolic acid.Jour. Amer. Chem. Soc. 76: 1459. 1954.

19. SPADA, Di ALBERTO, and GAVIOLI, ERMANNO SUII'acido 3-assiantranilico intermedio nella biosintesidell' acido nicotinico. Bull. sci. facolt'a chim. ind.,Bologna 8: 101-103. 1950.

20. Tso, T. C. and JEFFREY, R. N. Paper chir-omatog-raphy of alkaloids and their transformation prod-ucts in Maryland tobacco. Arch. Biochem. Bio-phys. 43: 269-285. 1953.

21. ZACHARIus, R. M., POLLARD, J. K. and STEWARD, F. C.-y-methvleneglutamine and y-methyleneglutamicacid in the tulip (Tulipa gesneriana). Jour. Amer.Chem. Soc. 76: 1961-1962. 1954.

22. ZEIJLEMAKER, F. C. J. The metabolism of nicotinicacid in the green pea and its connection with trig-onelline. Acta Bot. Nee'r. 2: 123-143. 1953.

/ THE INFLUENCE OF SALTS ON THE ACTIVITY OF PARTICULATECYTOCHROME OXIDASE FROM ROOTS OF HIGHER PLANTS12

GENE W. MILLER AND HAROLD J. EVANSNORTH CAROLINA STATE COLLEGE, RALEIGH, NORTH CAROLINA

Recent investigations by Webster (21) have shownthat cytochrome oxidase is widely distributed inhigher plant species and it has been suggested (7)that this enzyme may play a major role in the termi-nal transfer of electrons in plant respiration. Lunde-gardh (13) reported that the absorption spectrum ofbundles of roots from wheat and other cereals re-vealed the existence of a complete cytochrome oxidasewhich was similar to that observed in animal tissuesand many miicroorganisms. He calculated that thissystem was responsible for 50 to 75 % of the totalaerobic respiration. Fritz and Beevers (6) observed

1 Recei-ed April 3, 1956.2 Contribution from the Division of Biological Sci-

ences, North Carolina Agricultural Experiment Stationand published with the approval of the Director aspaper No. 725. This research was supported in part bya grant from the Chilean Nitrate Educational Bureau.

that extracts from etiolated pea, wheat and barleyseedlings contained cytoehrome oxidase at all stagesof development. When the amount of enzyme in thetissues was evaltuated in terms of activity at an in-finite concentration of cytochrome c, it was concludedthat shoots or roots of peas, and shoots of either bar-ley or wlheat contained sufficient amount of the en-zyme to account adequately for the respiration ofthese tissues. The roots of barley and wheat alsocontainedl the enzyme but the amounts extracted werenot capable of mediating all the respiratory oxygenabsorption. The results with wheat were in goodagreement with those reported by Lundeghrdh. Itwould appear that the importance of cytochrome oxi-dase in many higher plant species may approach thatof this enzy-me in the respiration of animals and aero-bic microorganisms.

Several investigators have observed that salts

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PLANT PHYSIOLOGY

stimulated the activity of cytochromne oxidase prepa-rations from animal tissues. According to Smith (17)the activity of the enzyme in particulate fractions ofkidney or heart was markedly increased by salt. Themaximum effect was obtained with NaCl at a concen-tration of 0.11 M. A similar observation was made byRiley (16) who reported that a concentration of0.1 MI NaCl increased the activity of a cvtochromeoxidase preparation from mouise melanomas bv 40 %.Meguimu (15) has reported that univalent cation saltsat a concentration of 0.05 M maximally increased theactivity of cytochrome oxidase from ox heart muscle.Divalent cation salts were stimulatory at lower con-centrations.

There are relatively few experiments reported inthe literature concerning the influence of salt on theactivity of cytochrome oxidase from higher plant tis-sues. Lundegardh (12) added macerated wheat rootsto a solution containing reduced cytochrome c and ob-served that 41 % of it was oxidized in five minutes.In a comparable experiment where the solution con-tained KNO3 or KCl at a concentration of 0.1 MI,73 % of the reduced evtochrome c was oxidized in a5-minute period. Weinstein et al (22) observed thegreatest cytochrome oxidase activity of a homogenateof sunflower leaves in a phosplhate buffer (mixture ofNa2HPO4 and KH2PO4) at pH 6.0 and at a concen-tration of approximately 0.13 MI. The activity wasmarkedly depressed with buffer concentrations greateror less than this value. No attempt was made to dis-tinguish between the effect of anions and cations or todetermine whether or not an osmotic phenomenonwas involved.

It has been well established (14) that the additionof salts to plant tissues containing low salt content,smarkedly increased their respiratory rates. This in-creased respiration is sensitive to heavy metal in-hibitors and carbon monoxide inhibition is reversed byliglht indicating the involvement of cytochrome oxi-dase. In view of the possible significance of these ob-servations in an understanding of the mechanism ofthe salt accumulation and of the brief reports that theactivity of plant cytochrome oxidase per se was in-fluenced by salts, the experiments reported here wereinitiated in an effort to provide more detailed infor-mation on the influence of salts on cytochrome oxidasefrom plant sources. The experimental evidence alsomay have an important bearing, on the role of certaincations in plant metabolism.

-MATERIALS AND MXIETHODSPREPARATIONS: Particulate preparations used in

the various experiments were made from the roots ofsoybean (Glycine Max Merr.), tobacco (NicotiantaTabacum L.), oat (Avenza sativa L.) or spinach(Spiniacia oleracea L.) plants which were grown inflats of sand in the greenhouse. The age of the seed-lings ranged from 6 to 20 days. Extracts of root tis-sue were prepared as outlined by Webster (21) withthe exception that the isolation medium contained0.3 MI sucrose and 0.05 \I tris (hydroxymethyl) ami-

nolmiethane (Tris) buffer instead of plhosp)hate buffer.In the preparation of the extracts 1 gin of roots wasground in a cold nmortar with 20 ml of a col(d solutionof buffer andl sucrose, then homogenized for 1 mintutewith a Ten Broeck homogenizer and( centrifuged at1000 x g for 15 miiinutes. The sul)ernatant was de-cainted and centrifuged alt 20,000 x g for 15 minutes.The particles wsere collecte(d and washed w\itli 10 ml ofthe sucrose-buffer soluition then suspended in a volumeof sucrose-buffer equiv.lent to one fifth the originailvolume of the homogenate. Extracts lprel)ared by thisproce(lire were use( in all exp)eriments except that re-

porte(d in figuire 4. Ini this experiment (fi(r 4) the ex-tract was prel)ared as described tabove, wsith the ex-ception thalt buffer was omitted from the isolationmeditum. Also the suispension of particles used in thisexperiment was dialyzed over night in 2 liters of 0.3 AIsucrose in an effort to remove endogenous salts.

A stock solution of 12 mg,/ml of cytochrome c(Sigma Chemnical Company of St. Louis, 'Missouri) in0.05 MI Tris buffer at pH 7.0 was chemlcally redtucedwitli sodium hvdrosulfite (3) and thoroughly aeratedbefore use. The concentration of the stock solutionwas determined by use of the difference in molar ex-tinction coefficients of oxidized and re(luced cvto-chrome c at 550 m,u which is 1.96 x 104 (8). Thestock solution of cvtochrome c used in the experimentreported in figure 4 and the extract used in this ex-periment were dialyzed overnight against a 0.3 MIsucrose solution before they were used. After dialysisthe reduced cytochrome c concentration was deter-mined by the spectrophotometric procedure. Solu-tions of reduiced( cytochrome c were relatively stablewlhen store(l at - 100 C.

Tris salts of Cl- and phosphate (miixture ofH.PO4- and HPO4=) used in the experiments reportedin figure 3 were prepared by adding sufficient amounitsof appropriate acids to Tris base to adjust the 1H to7.0. Tris buffer used in the various l)rocedures wasprepared by neutralization with HCl uintil the desiredpH value was obtained. The soluition of NaHCO3used in the experiments reported in figure 2, was ad-justed to pH 7.5 by bubbling CO2 into tlhe solution.

In the experiment involving purification of theenzyme extract with ion resins, a mixture of hydroxylsaturated anion resin IRA-400 and hydrogen saturatedcation resin IR-120 (both obtained fromii RohmII andHaas Company, Philadelphia) were mixed together inproportions that resulted in a pH of 6.5 in distille(dwater. The enzyme was dialyzed for 12 hours againsta suspension containing 5 gm of mixed resins in 2liters of cold distilled water.

STANDARD ASSAY: Enzyme activities were assayed(spectrophotometrically at 550 my wvitlh a BeckmanDU spectrophotometer (3). The reaction mixture ina final volume of 1 ml contained the following con-stituents in micromoles: 25 Tris buffer at pH 7.0,0.051 reduced cytochrome c, desired concentration andtype of salt, and enzyme containing 0.1 to 0.2 mgl)rotein. Enzyme activity is indicated as the decreasein optical density per mg protein between 15 and 75

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MILLER AND EVANS-SALTS ON CYTOCHROME OXIDASE

seconds after the reaction was started by the additionof the enzyme. Reduced cytochrome c was not oxi-dized when boiled enzyme was used in the assay pro-

cedure with or without the various experimental addi-tions to the mixture. Under the conditions of theexperiments the oxidation rates remained linear for atleast two minutes which is in agreemnient with the ob-servations of Fritz and Beevers (6). The mixture was

stirred prior to each reading in order to prevent errorsdue to the settling of particles in the euvette. Thevarious experiments were run three times and the re-

sults averaged. Aaximum deviation from the meanwas 8 % but in the majority of experiments the de-viation was much less than this.

OTHER MIETHODS: Aliquots of purified assaIV mix-tures were ashed in a muffle furnace at 6000 C andanaly zed for Na and K by use of a Perkin-Elmerflame photomiieter (23). Protein content of extractswas estimatecl by use of either Folin's phenol reagent(11) or by total N analyses (20). Results with the(lifferent methods were in good agreement.

EXPERIMENTAL RESULTSEFFECTS OF AVARIOUS CATIONS: Extracts of roots

from four plant species were prepared and the effecton the activity of cyrtochrome oxidase of chloride saltsat varying concentrations observed. In figure 1 A theinfluence of chloride salts on cytochrome oxidase ac-tivitv of extracts from soybean roots is shown. Addi-tions of univalent cation salts resulted in a strikingincrease in activity. A maximum stimulatory effectwas exhibited at a concentration near 0.1N andgreater concentrations were inhibitorx. It is of in-terest to note that chlorides of Na' and Li' at opti-mum concentrations activated less than chlorides ofK. and NH4> The degree of activation at optimum

TABLE IEFFECT OF VARIOUS ANIONS AND CATIONS ON THE

ACTIVITY OF CYTOCHROME OXIDASE FROM ANEXTRACT OF SOYBEAN ROOTS *

CONC OF ADDED SALT (N) OR SUCROSE (M)ADDITIONNS

0.00 0.05 0.10 0.20

- O.D. chanige per itmlie per. tig proteinVarilous anions

NaCl 0.23 0.37 0.65 0.45NaBr 0.23 0.35 0.70 0.40NaI 0.23 0.38 0.65 0.43Na3 Citrate 0.23 0.40 0.70 0.45

Various cations or

sucroseNaNO3 0.23 0.40 0.68 0.40KNO3 0.23 0.40 0.70 0.45Ca(NO3)2 0.23 0.42 0.12 0.05Mg(NO3)2 0.23 0.40 0.18 0.05SUcrose 0.23 0.20 0.18 0.15

* The standard assay procedure was used with theaddition of salts or sucrose as indicated. One tenth mlof soybean root extract containing 1.6 mg l)rotein per mlwas uised as the enzymii1e.

TABLE IIEFFECT OF COMBINATIONS OF SALTS ON CYTOCHRONIE

OXIDASE ACTIVITY FROM AN EXTRACT OFSOYBEAN ROOTS *

TOTAL SALT CONC (N)SALTS

0.00 0.05 0.075 0.10 0.15 0.20

- O.D. change per min per mg proteinNaCI 0.33 0.52 0.73 0.77 0.67 0.37KCI 0.33 0.55 0.70 0.79 0.67 0.401/2 KCI + ½ 1NaC 0.33 0.47 0.70 0.80 0.67 0.37CaCl2 0.33 0.50 0.30 0.20 0.10 0.08'/2 CaCI + '/, KCl 0.33 0.50 0.45 0.35 0.15 0.13

* The standard assay pirocedu1ire was used with theaddition of salts as indicated. The assay included 0.10ml soybean extiact containing 1.4 mg protein per ml.

univralent salt concentration in this particular experi-ment was greatest with the least hvdrated cationspossessing, greatest relative mobilities (9). Divalentcation chloridles activated slightl up to 0.05N, butincreasedl concentrations cauisedI a sharp dlecline in ac-tivitv resultingf in almost complete inhibition at 0.2 N.CaClo at concentrations below 0.05 N stimulated en-zyme activity more than \IgCl, at comparable con-centrations. At all except low concentrations, uini-valent cation salts were more effective than thedivalent ones. The influence of nitrate salts of Ma,Ca++ K+ and Na+ on cytochrome oxidase activity ofsoybean extract was similar to that observed with therespective chloride salts (table I). These results werenot included in figure 1 because the effect of the twoanions were so similar that an overlapping of manycurves woould have resuilted. The data in this tablealso show that variouis concentrations of sucrose hadlittle effect on the activity of the enzyme.

The effect of chloride salts of variouis cations onthe activity of cvtochrome oxidase from roots of to-bacco, spinach and oat is shown in figures 1 B, 1 C and1 D and the results are generally comparable withthose obtained with soybean. In all experiments withthe exception of those where extracts of oat roots wereused, KCI at optimum concentrations stimulated en-zyme activity slightly more than NaCl. The effect ofdivalent cation salts on cytochrome oxidase activityfrom oat and spinach extracts was similar to that ob-served with soybean, but with extracts from tobacco,MgCl2 was considerably more effective than CaCl2 atlow as well as at high concentrations. This is particu-larlv noticeable at the concentration of 0.05 N.

It is apparent, from figure 1 that univalent cationsalts have siiiiilar effects on enzyme activity. The ac-tivating effect of combinations of salts as comparedwith single salts is shown in table II. A mixture con-taining equal concentrations of NaCl and KCI be-haved in a manner similar to the same total concen-tration of either of the single salts indicating an addi-tiv-e effect of univalent cation salts. The activation ofcytochrome oxidlase by an equal mixture (N) of CaCl2an(cl KCI was similar to that noted with the single

359



360 PLANT PHYSIOLOGY

CONCENTRATIONS OF SALTS IN ASSAY (NORMALITY)

E 080aoo0

070

Z 060w

0

CL

0 50

2ia04w

z 0 30i

. 0.20wz< 0.10

00

0C05 0.10 0.150I20 0.05 0.10 015 020CONCENTRATIONS OF SALTS IN ASSAY (NORMALITY) CONCENTRATIONS OF SALTS IN ASSAY (NORMALITY)

FIG. 1. Effect of various concentrations of salts on the cytochrome oxidase activity of extracts from roots ofseveral plant species. Procedure was that of standard assay with variations in concentration and type of salt as

indicated. The enzyme preparations were from the following species: A-0.10 ml soybean extract containing 1.2mg protein per ml; B-0.10 ml tobacco etxract containing 1.3 mg protein per ml; C-0.10 .ml spinach extractcontaining 1.7 mg protein per ml; D-0.10 ml oat extract containing 1.4 mg protein per ml.

In this figurie and the subsequent figures the O.D. changes are negative.

salts up to a total concentration of 0.05 N. At greaterconcentrations an interaction between Ca++ and K+salts was apparent as indicated by the difference inenzyme activities obtained from combinations of saltsas compared with single salts. Where Ca++ was atmaximum stimulatory level, the addition of univalentcation salts suppressed activity.

EFFECT OF VARIOUS ANIONS: Univalent and di-valent cation salts differ in their influence on cyto-chrome oxidase activity but the effect is not inde-pendent of the anion present. It can be seen fromfigure 2 that sodium salts of Cl-, N03-, and S04= were

equally effective in activation of the enzyme. Thesodium salts of Br-, 1- and citrate (tri-sodium) influ-enced the activity in a manner similar to the chloridesas shown in table I. From figure 2 it is apparent thatthe addition of phosphate (HP04 + H2PO4) in-

creased the activity of the enzyme from soybean rootsto a greater degree than other anions. The increasedstimulatory effect of phosphate also was observed withextracts of spinach, oat and tobacco roots. The stim-ulation from phosphate salt at most concentrationswas approximately 20 % higher than that of othersalts. The effect of phosphate was manifested at con-

C- SPINACH 0 NoCi

A KCI

0 CoCi2

X MgcI2



MILLER AND EVANS-SALTS ON CYTOCHRONIE OXIDASE

0 0.60

0.0S 0.10 0.15 0.20CONCENTRATIONS OF SALTS IN ASSAY (NORMkALITY)

FIG.. 2. The effect of vrarious concentrations of so-dium salts on the activity of cytochrome oxidase fromsoybean roots. The procedure of standard assay wasfollowed with the exception that all determinations weremade in 0.025 M Tris buffer at pH 7.5. The assay mix-tures contained variable concentration and kinds ofsalts as indicated. The preparation of the bicarbonatesolution is described in Materials and Methods. Assaysincluded 0.1 ml soybean extract containing 1.7 mg prO-tein per ml.

w 0 TRISPHOSPHAT-o 3. A TRIS CHLORIDEe O~~~~~~~~~~SUCROSE

z

z

o 0.10 -

a

a ,~0.501001502

6.05 0.10 0.15 0.20 025 0.30 035 0.40CONCENTRATION OF BUFFERS OR SUCROSE IN ASSAY CM

centrations as low as 0.0005 i\M. Bicarbonate salts atthe same pH and cation concentration as the abovementioned salts resulted in little activation at concen-trations that were optimum for other salts and pro-nounced inhibition at high concentrations. This in-hibition was noted with extracts of various plantspecies.

REQUIRE-MENT OF SALT FOR ACTIV-ITY: In the ex-periments where the effect of various cations andanions on the activity of cytochrome oxidase wasstudied, marked stimulation was noted from addedsalts. Since all these assay mixtures contained0.025 MI Tris chloride as a buffer, it was deemed neces-sary to test the influence of various concentrations ofbuffer on the enzyme activity. The results of such anexeriment are reported in figure 3. Tris salts of Cl-and phosphate (mixture of H2P04- and HP04=) ac-tivated the enzyme in a manner similar to other uni-valent cation salts. The difference in stimulationbetween chloride and phosphate salts was observedagain and indicated a specific action of the phosphateion. Tris phosphate at concentrations of 0.0125 Mresulted in marked increases in activity when com-pared with Tris chloride at the same concentrations.

An experiment was set up to determine enzymeactivity in an assay mixture containing no added Trisor inorganic salts. The preparation of materials isdescribed in 'Materials and MIethods. Enzyme activ-

o 0 NaCIn 0.90 4 KCI

0 CoC12X MgCI2

0.80z

go 0.70

a0.60

iO 0.30

0.20

d

0.05 0.10 0.15 0.20CONCENTRATIONS OF SALTS IN ASSAY (NORMALITY)

FIG. 3. Effect of various concentrations of Tris salts on the activity of cytochrome oxidase from soybean roots.The standard assay procedure was used with variation in buffers (preparation of Tris phosplhate and Tris chlorideis described in Materials and Methods) as indicated. Extracts were prepared in either Tris chloride or Tris phos-phate depending on which buffer was used in the assays. Assays included 0.1 ml soybean extract containing 1.1 mgprotein per ml.

FIG. 4. The influence of salts on activity of cytochrome oxidase from soybean roots in a purified medium(see Materials and Methods). The assay mixture in a final volume of 1 ml contained 0.051 micromoles of dialyzedreduced cytochrome c; 0.1 ml dialyzed extract containing 1.6 mg protein per ml, and the concentration and typesof salts indicated. No buffer was used in the assay mixtures and the pH varied between 5.2 and 5.3 dturing thedeterminations. All salt solutions were adjusted to pH 5.2 before use.

361



PLANT PHYSIOLOGY

ity was present in this purified medium as shown infigure 4, and the salt stimulation was similar to pre-vious experiments where buffers were used. An analy-sis of a sample of the entire purified reaction mediumshowed that concentrations of 1.5 x 10A4 M potassiumand 7.5 x 1O5 M sodium were present in the mixture.

In another experiment, dialysis of the enzymeagainst a mixed resin, as described in Materials andMethods, failed to decrease the cytochrome oxidaseactivity in the reaction mixture containing no addedsalts. An analysis of the entire reaction mixture un-der these conditions showed 1.25 x 104A potassiumand 6.3 x 10-5 MNI sodiuim were present. In another at-tempt to remove salts, the enzyme preparation wasplaced for 30 minutes in a cold Gooch crucible in con-tact with a mixture of the anion and cation resins de-scribed in Materials and Methods. The enzyme wasrecovered by suction and assayed for activity but noabsoltute dlependlence on salt was observed.

DISCUSSIONThe results of these experiments clearly show that

both type and concentration of salts in contact withsuspensions of p'articulate matter from roots of variousplant species profoundly influenced the cytochromeoxidase activity of the particles. When the anions wereheld constant as the chlorides (fig 1 and table I) thevarious cations up to a1 concentration of 0.05 N gen-erally resulted in similar effects on the activity of cyto-chrome oxidase. When concentrations of cationsgreater than 0.05 M were added to assays then uni-valent cations continued to stimulate activity up to aconcentration of 0.12 MI whereas concentrations ofdivalent cations greater than 0.06 M were inhibitory.When the results were plotted on an ionic strengthbasis different effects of univalent and divalent cationswere observed at concentrations greater than 0.07N.The influence of salts on the activity of cytochromeoxidase from soybean roots is not independent of theamion associatecd with salt as indicated by the resultsin figure 2 and table I. The sodium salts of NO3-,S04, C1-, I- and citratc at comparable pH valueswere strikingly similar in their effect on the activity.Sodium phosphate (Na2HPO4-NaH2PO4) on the otherhandI consistently increased activity to a greater de-gree than the other sodium salts. The specific inhibi-tory action of bicarbonate ions on cytochrome oxidasehas been observed consistently with enzyme prepa-rations from various species and is the subject ofanother communication.

In view of the relatively high concentrations ofsalts required for maximum cytochrome oxidase ac-tivity in isolated particulate matter from soybean rootand the relatively low concentration of salts neededfor maximum salt respiration of intact tissues (4) onemight conclude that enitirely different phenomenawere involved in each catse. These differences may beaccounted for by assuming that intact tissues possessa greater accumulatory capacity for salts than theparticulate preparations containing cytochrome oxi-dase.

It seems possible that different concentrations andtypes of salts may influence cytochrome oxidase activ-ity by their effects on the physical structure of theparticles. Stafford (18) has established that cyto-chrome oxidase in higher plant tissue occurs in intra-cellular particles of the size of mitochondria. Farrantet al (5) have shown with the electron microscopethat mitochondria from red beet prepared in 0.45 %KCI appeared as large diffuse discs whereas those pre-pared in 30 % sucrose appearedl as compact spheres.It has been demonstrated that the cytochrome oxidaseactivity of particles isolated from animal livers wasstrongly affected by the physical state of the particlesand that salts markedly influenced the physical ap-pearance of them (2). One might speculate that thetype and concentration of the various salts used inthe experiments reported in this manuscript affectedthe physical properties of the particles and thereforepossibly altered the accessibility of the enzyme toeither oxygen or reduced cytochrome c. A micro-scopic examination of the particles from soybean seed-lings in both water and 0.1 N KCI indicated dimeni-sions of mitochondrial size, but no gross differences inphysical structure were observed. It is necessary toconduct more detailed studies with these particles be-fore a definite conclusion concerning the effect of salton gross structure can be made.

Since it has been clearly established (5) that beetmitochondria possess a membrane and that cyto-chrome oxidase is associated with mitochondria (18),it is necessary to consider the possibility that the vari-ous reactants in the cytochrome oxidase reaction haveto penetrate a membrane during the course of theenzymatic reaction. In this event various species andconcentrations of ions would very likely have differ-ential effects on membrane permeability (22) and(therefore the rates of the enzymatic reaction. Lehn-inger (10) has pictured an "internal" and "external"pathway of electron transport from the substrate tomolecular oxygen in mitochondrial preparations. Ex-ternally added reduced cytochrome c apparently doesnot penetrate to the internal site of electron transportwithin the mitochondrial body unless some alterationsare made in mitochondrial permeability or structure.Apparently it is necessary for electrons to be trans-ported through the "internal" pathway in order toobtain oxidative phosphorylation. Information con-cerning the relationship of salts to the pathway ofelectron transport and to oxidative phosphorylation inplant mitochondria remains for future study.

In an effort to explain the influence of salts on theoxidation-reduction balance of cytochromes in livingwheat roots, Lundegardh (14) proposed that if twomolecules capable of undergoing oxidation-reductionwere separated from each other, apparently in somestructural unit, they may fail to react unless salts arepresent in the surrounding medium. In this proposalthe movement of ions may compensate for the electro-static potential between the reactants involved in theoxidation-reduction. In order to apply this general-ized explanat,ion to the cytochrome oxidase reaction it

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MILLER AND EVANS-SALTS ON CYTOCHROME OXIDASE

seems that particulate cytochrome oxidase would benecessary to meet the structural requirements of a saltdependent system. If cytochrome oxidase were intrue solution and in intimate contact with a solutionof reduced cytochrome c it seems logical to expect thatno salt influence Nould be manifested if the generalmechanism proposed by Lundegardh is operative.

The striking stimulatory action of univalent cationsalts on the activity of cytochrome oxidase may havea bearing on the role of univalent cations in plantmetabolism. It is well established that K+ is themajor cation in plant tissues and that Na+ will par-tially replace K+ in its role as a univalent cation incertain species. Since Na+ and K+ are approximatelyequally effective in the activation of cytochrome oxi-dase it seems that this enzyme may represent a meta-bolic site where replacement could occur. It is ofinterest to note that dry plant tissues containing 2 to4 % K (1) would contain a concentration calculatedon a fresh tissue basis of approximately 0.05 to 0.1 N.As already indicated this range in concentration ofunivalent cation salts markedly stimulated the activ-ity of cytochrome oxidase in vitro. A a.icant

physiological role of univalent cations other than K+and Na+ in the activation of cytochrome oxidase invivo seems unlikely in view of either the low concen-trations or relative toxicities of other such cations thatare normally present.

SUMMARYA study has been made of the influence of various

concentrations and kinds of salts on the activity ofcytochrome oxidase associated with particulate prep-arations showing characteristics of mitochondria fromthe roots of certain higher plant species.

The activity of the enzyme from all the speciesstudied including soybean, tobacco, oat and spinachwas markedly influenced by salts. When the anionswere held constant as chlorides and the effect of cat-ions compared on an equivalent basis, increasing con-

centrations of univalent cation salts stimulated ac-

tivity until a maximum effect was obtained at a

concentration of approximately 0.12 N. Concentra-tions greater than this resulted in a progfressive declinein activity. The addition of chloride salts of divalentcations was less stimulatory than salts of univalentcations. The maximum increase in activity was ob-tained at a concentration of near 0.05 N and greaterconcentrations were inhibitory. The concentration ofTris chloride used as a buffer also influenced the ac-

tivity of the enzyme.Experiments were conducted with extracts from

soybean roots where the cation of salts was kept con-stant as Na+ and the influence of equivalent concen-

trations of anions stuidied. Cl-, NO3-, S04=, Br-, I-and citrate influenced the activity in a similar manner

but phosphate (H9P04- + HPO4=) resulted in approxi-mately 20 % greater activity than other anion salts.The effect of NaHCO3 on the activity when comparedwith other anion salts at the same pH values was in-hibitory.

An absolute salt requiremcnt for cytochrome oxi-dase activity was not demonstrated; however, analy-ses of purified assay mixtures indicated the presence

of small concentrations of both Na+ and K+.The influence of salts on the activity of cytochrome

oxidase was not an osmotic manifestation since vari-ous concentrations of sucr- e in the assay medium hadno appreciable effect.

The experimental result- are discussed in relationto the possible mechanism ti the differential action ofvarious salts and also in relation to the possible roleof certain cations in plant metabolism.

The authors gratefully acknowledge the technicalassistance of Mrs. Thea Schostag.

LITERATURE CITED1. BEESON, K. C. The mineral composition of Criops

with particular rieference to the soils in whichthey were grown. U. S. Dept. Agr., Misc. Publ.369. 1941.

2. CLAUDE, A. J. Fractionation of mammalian livercells by differential centrifugation. Jour. Exptl.Med. 84: 51-61. 1946.

3. COOPERSTEIN, S. J. and LAZAROW, A. A microspec-trophotometric method for the determination ofcytochrome oxidase. Jour. Biol. Chem. 189: 665-670. 1952.

4. EPSTEIN, E. Cation-induced respiration in barleyroots. Science 120: 987-988. 1954.

5. FARRANT, J. L., ROBERTSON, R. N. and WILKINS,MARJORIE J. The mitochondrial membrane. Na-ture 171: 401-402. 1953.

6. FRITZ, G. and BEEVERS, H. Cytochrome oxidase con-tent and respiratory rates of etiolated wheat andbarley seedlings. Plant Physiol. 30: 309-317. 1955.

7. HILL, R. and HARTREE, E. F. Hematin compoundsin plants. Ann. Rev. Plant Physiol. 4: 115-150.1953.

8. HORECKER, B. L. and HEPPEL, L. A. The reductionof cytochrome c by xanthine oxidase. Jour. Biol.Chem. 178: 683-690. 1949.

9. KACHMAR, J. F. and BOYER, P. D. The potassiumnactivation and calcium inhibition of pyruvic phos-phoferase. Jour. Biol. Chem. 200: 669-682. 1953.

10. LEHNINGER, A. L. The Harvey Lectures. Pp. 176-215. Academic Press Inc., New York 1955.

11. LoWRY, D. H., ROSEBROUGH, NIRA J., FARR, A. L. andRANDALL, ROSE J. Protein measurement with theFolin phenol reagent. Jour. Biol. Chem. 193:265-275. 1951.

12. LUNDEG.ARDH, H. Controlling effects of salt on theactivity of cytochrome oxidase. Nature 171: 477-478. 1953.

13. LUNDEGARDH, H. A new cytoclhrome in living roots.Nature 173: 939-941. 1954.

14. LUNDEGA'RDH, H. Mechanism of absorption, trans-port, accumulation and secretion of ions. Ann.Rev. Plant Physiol. 6: 1-23. 1955.

15. MEGUMU, I. Studies on the cytochrome oxidase.Sci. Bull. Fac. Agr., Kyushu Univ. 13: 167-170.1951.

16. RILEY, V. Salt inhibition and enhancement of cyto-chrome, succinic and dopa oxidase systems ofmouse melanomas. Proc. Soc. Exptl. Biol. Med.75: 644-648. 1950.

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PLANT PHYSIOLOGY

17. SMITH, F. G. and STOTZ, E. A colorimetric methodfor the determination of cytochrome oxidase.Jour. Biol. Chem. 179: 891-902. 1947.

18. STAFFORD, HELEN A. Localization of enzymes in peaseedlings. Physiol. Plantarum 4: 696-741. 1951.

19. STEINBACH, H. B. Permeability. Ann. Rev. PlantPhysiol. 2: 323-337. 1951.

20. UMBREIT, W. W., Bur-w. R. H. and STAUFFER. J. F.Manometr ic tech; Ates and tissue metabolism.Pp. 161. Burgess Publishing Co., Minneapolis,Minnesota 1951.

21. WEBSTER, G. C. The occurrence of a cytochromeoxidase in the tissues of higher plants. Amer.Jour. Bot. 39: 729-745. 1952.

22. WEINsTEIN, L. H., ROBBINS, W. R. and WAINIO,W. W. Assay of cytochrome oxidase activity ofsunflower leaf tissue in relation to pH value andcation concentration of the buffer. Plant Physiol.29: 398-399. 1954.

23. WILLARD, H. H., MERRITT, L. L. and DEAN, J. A.Instrutimental Methods of Analysis. Pp. 49-84.D. Van Nostrand Company, Inc., New Yor-k 1951.

XOVERW'INTERING TRENDS OF COLD RESISTANCE AND CARBOHYDRATES

IN IEDIUM RED, LADINO, AND COMMON WHITE CLOVER 1'2

0. CHARLES RUELKE3 AND DALE SMITHDEPARTMENT OF AGRONOMY, WISCONSIN AGRICULTURAL EXPERIMENT STATION,

MADISON, WISCONSIN

Medium red clover, Trifoliumii prateutse L., andladino clover, Trifolium repens L., are used exten-tively in the northern areas of the United States inlhay and pasture mixtures while common white clover,Trifolizim repens L., occurs naturally in most of theclosely grazed pastures. Unfortunately, these trueclovers are often killed or weakened duiring the wintermonths, and maintaining stands over winter is one ofthe important problems related to their culture in thenorth. A better understanding of the overwinteringphysiology of these legumes is needed in order to pro-vide information that will aid in the maintenance ofstands during the winter period.

Several workers have studied the trend and devel-opment of cold resistance in alfalfa during the falland winter months (2, 3, 5, 8, 14). Similar work withred, ladino, and common white clover is limited. Bulaand Smith (2) compared the cold resistance trend inmedium red clover with that in Ranger alfalfa andbiennial sweetelover under Wisconsin conditions. Thetrends in field-grown seedling plants of alfalfa andsweetclover were similar although sweetelover devel-oped resistance more rapidly and reached a higherlevel than alfalfa. Red clover developed cold resist-ance later, more slowly, and did not reach as high alevel as alfalfa or sweetclover. Most of the resistancein these legumes was developed by the time of thefirst sampling following permanent freezing of the soilsurface in late November to mid-December. Cold re-sistance in red clover and alfalfa began to decreaseafter mid-February while it was retained longer insweetclover. Cold resistance was not lost rapidlyuntil after the snow was gone and the soil surfacethawed in late March. Bula et al (3) have shownfurther with alfalfa that certain responses differedwhen the same varieties were grown at diverse lati-

1 Received April 6, 1956.2 Published with approval of the Director, Wisconsin

Agricultural Experiment Station.3 Present address: Department of Agronomy, Uni-

versity of Florida, Gainesville, Florida.

tu(des. Smithi (15) has compared the survival offield-hardened stolons of common white and ladinoclover "*A ien encased in solid ice at non-injurious tem-peratures during winter dormancy. The more severeinjury sustained by ladino appeared to be associatedwith the larger stolons and the higher level of meta-bolic activity during winter dormancy.

The overwintering of such legumes as alfalfat,sweetclover, and red clov-er has been shown to beassociated with a high level of carbohydrate storageand with subsequent changes in certain carbohydratecomponents (2, 3, 7, 8, 9, 10, 16, 17, 19). Wood andSprague (21) at New Jersey have reported that themore cold-hardy clones of ladino clover were higherin most carbohydrate fractions during the winterthan the less hardy ones. Changes in the levels ofsucrose, total sugars, and total polysaccharides in thehardy clones were made at a more gradual rate dur-ing winter. Fall cutting decreased some carbohydratefractions and increased others but had no measurableinfluence on low temperature survival. Tesar andAhlgren (18) at Wisconsin have shown that the per-centages of total available carbohydrates in ladinoclover stolons in November were not significantly dif-ferent even though various heights and frequencies ofcutting were applied during the growing season.

The investigations reported here deal with thetrends of cold resistance and the trends of certaincarbohydrate fractions from fall to spring in the over-wintering parts of medium red, ladino, and commonwhite clover, and the relationship of these trends tothe prevailing weather. The studies were made atMadison, Wisconsin, during the winters of 1952-53and 1953-54.

MATERIALS AND METHODSWisconsin-grown seed of medium red clover, Tri-

folium pratense L., and of ladino and common whiteclover, Trifolium repens L., was seeded in rows andin broadcast plots during June of 1952 and of 1953.Plants grown in rows were used in the cold tests initi-

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Plant Physiology - ARONOFF-BIOGENESIS OF THE RING 357 › content › plantphysiol › 31 › 5 › 357.full.pdf · ARONOFF-BIOGENESIS OF THE PYRIDINE RING LITERATURE CITED 1. BONNER,