cytochrome system in neurospora … · coarse porosity, fritted glass buchner funnel, ... gooch...

STUDIES ON PYRUVATE METABOLISM\ AND CYTOCHROMESYSTEM IN NEUROSPORA TETRASPERMA 1,"2

RAYMOND W. HOLTON3DEPARTMENT OF BOTANY, UNIVERSfTY OF MICHIGAN, AN-N- ARBOR

Although it has been known for more than 30years that dornmancy in Neurospora ascospores can bebroken by heat treatment (30), the mechanism is notyet understood. Goddard (10) concluded from hisstudies that the metabolic block overcome by the heattreatment was concerned with the enzyme car-boxylase. Studies of Sussman et al (36) demon-strated that both the apoenzyme and coenzyme werepresent in dormant and heat-activated ascospores innearly equivalent amounts. They concluded that ac-tivation induices the formation of an oxidative system,possibly operating through the TCA cycle, that isdifferent from that in the dormant ascospores. Re-cent work from this laboratory (20, 38) on the natureof the endogenous substrates in dormant and activatedascospores supports this idea of a qualitative changein metabolism after activation.

Because pyruvate appeared to be a key inter-mediate in the suggested development of an oxidativesystem, it was thought that pyruvic oxidase might bepresent in the activated ascospores but not in thedormant ascospores. Since anions of the size of py-ruvate do not penetrate dormant spores (37), it isnecessary to study pyruvate oxidation in cell-free ex-tracts. The studies reported here were originallyundertaken in an attempt to demonstrate the presenceof this enzyme system in cell-free extracts of theascospores and in other stages of the life cycle in-cluding conidia and mycelia of Neurospora tetra-spern.a. A preliminary report of this work has beenpublished (16).

MATERIALS AND METHODS

Ascospores of Neurospora tetraspermna Shear andDodge were produced from a cross of strains 394.4and 394.5 originally obtained by Dr. A. S. Sussmanfrom Dr. B. 0. Dodge. The ascospores were grownand harvested using the procedure of Goddard (9)and freed of contaminant conidia by the technique ofSussman (35). Heat activation was accomplished

1 Received revised manuscript March 28, 1960.2 Most of the data reported here were taken from a

thesis submitted to the Horace H. Rackham School ofGraduate Studies of The University of Michigan in partialfulfillment of requirements for the degree of Doctor ofPhilosophy, 1958.

3 Present address: Flint College, University ofMichigan, Flint 3.

by placing a suspension of ascospores in a centrifugetube in a 580 C water bath for 10 minutes.

Good nmycelial growth in liquid shake culture wasobtained by inoculation of a suspension of conidia ofstrain 394.5 into the complete medium of Horowitz[cited by Ryan (26)]. Sixty-five ml of the mediumwere added to 250 ml Erlenmeyer flasks, inoculatedwith 5 ml of a conidial suspension and incubated on areciprocal shaker at 21° C. The mycelium washarvested by filtration on a suction funnel and themycelial mat obtained was washed with distilled waterand used for enzyme preparations. In determiningdry weights for the growth curve of the mycelium,the flask contents were washed into a tared, 25 ml,coarse porosity, fritted glass Buchner funnel, washed,and the mat and funnel dried to constant weight at1050 C.

Conidia were produced on 50 ml of Horowitz'smedium solidified with 1.5 % agar in wide-mouth 500ml Erlenmeyer flasks. After inoculation of the mediawith a suspension of strain 394.5 conidia, the funguswas allowed to grow for 2 days at 210 C under ap-proximately 500 foot-candles of light. Conidia for-mation was stimulated by removal of the cotton plugsovernight (23). The conidia were harvested withdistilled water and passed through glass wool in aGooch crucible to remove mycelial fragments (26).The resulting suspension of orange conidia was wash-ed several times in distilled water by centrifugation.

Gas exchange was measured with a conventionalWarburg apparatus using standard techniques and a260 C water bath. Gas exchange was placed on anitrogen basis by determining the nesslerizable nitro-gen present in an aliquot of the cell-free extract afterdigestion with 1: 1 concentrated sulfuric acid (17).

Acetaldehyde analyses were carried out by themethod of Stotz (33). A slow stream of air waspassed through the Warburg flask in which the re-action was carried out and the effluent containingacetaldehyde was trapped in 6 ml of cold, freshly pre-pared 2 % sodium bisulfite. This bisulfite solutionwas used directly for the acetaldehyde determinationsusing paraldehyde for preparation of the standardcurve.

Cell-free extracts of ascospores are difficult toobtain because of the ascospores' exceedingly toughcoat and the procedure used did not bring aboutbreakage of all spores present. Thirty to 75 mg ofdormant or activated ascospores were added to themortar of a Ten Broeck tissue homogenizer. Threeor 4 ml of 0.05 M phosphate buffer pH 6.0 or otherhomogenizing medium, were added and the teflonpestle attached to a stirring motor. The motor was

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started and the cup raised and lowered around thepestle so that the spores were constantly agitated ver-tically as well as horizontally. After 10 minutes ofhomogenization, the unbroken spores were centrifugedoff at 800 X G and the operation repeated. Thesupernatants were combined to give a faintly yellowand turbid cell-free preparation. All operations werecarried out at 40 C.

The mycelial mat was easily homogenized in aServall High Speed Omni-Mixer with a top-drivestainless steel rotor and a 50 ml stainless steel cup.The mat (approx. 0.5 gm dry wt) and 15 ml "Super-brite" glass beads, grade No. 110 (Minnesota Miningand Manufacturing Co.) were suspended in 20 ml0.05 M phosphate buffer pH 6.0 precooled in an icebath. Homogenization was accomplished in 1 minuteat a rotor speed of 14,000 rpm and the resultinghomogenate was centrifuged at 800 X G for 20 min-utes at 40 C to give a colorless, turbid cell-free ex-tract.

Conidia were also homogenized in the ServallOmni-Mixer. They were suspended in about 10 mlof 0.06 M phosphate buffer at pH 6.1 and enough fineglass beads were added to form a slurry. Afterhomogenization and centrifugation, an orange cell-free extract was obtained.

In some experiments, the cell-free extract wasfractionated by centrifugation in a Servall AngleCentrifuge, Model SS-1, in a 40 C room for 10 min-utes at 16,000 X G.

Cytochrome oxidase activity in cell-free extractswas determined by both manometric and spectrophoto-metric means. The manometric technique used wasthat of Fritz and Beevers (8) with p-phenylenedia-mine (PPD) as the reducing agent which Slater (31)found to be effective in reducing both endogenous andexogenous cytochrome c. The PPD was added atzero time and readings were made every 5 minutes for30 minutes. The initial rate of oxygen uptake was

estimated graphically and autoxidation rates of cyto-chrome c were estimated and subtracted from the ob-served rates (31). In the spectrophotometric methodused, the decrease in optical density at 550 my due tooxidation of the cytochrome c (Sigma Chemical Co.)was followed in a Beckman Model DU spectrophoto-meter (6, 8).

Slater (31) showed that for the manometricmethod, the rate of oxygen uptake depended not onlyon the cytochrome oxidase activity but also on theconcentration of cytochrome c. Thus, to determinethe maximal activity of cytochrome oxidase by bothprocedures, it was necessary to measure the rate ofoxidation at different cytochrome c concentrationsand to extrapolate to infinite substrate concentration.For the extrapolation the method of Lineweaver andBurk (19) was used. The theoretical maximumvelocity of the enzyme action is expressed as the Vmaxand the values obtained in the spectrophotometricmethod were converted to the units used in the mano-

metric method by use of the equation derived by Fritzand Beevers (8).

The absorption bands of the cytochromes were ob-served with a Zeiss pupillary spectroscope mountedon a microscope as described by Hartree (13).Mycelial extracts were prepared using the procedureof Boulter and Derbyshire (4). In order to getenough particulate material to observe cytochromebands, at least 10 g (wet wt) of mycelia were re-quired. For the observations, the particles in bufferwere packed into small cells and the cytochromes re-duced with sodium hydrosulfite. Quantitative meas-urements were made using the wedge trough pro-cedure of Hartree (13).

RESULTS

CARBOXYLASE: It has been earlier shown bySussman et al (36) that the carboxylase content ofcell-free extracts of ascospores did not change quan-titatively after activation. The presence of car-boxylase in these tissues was confirmed in the presentwork and carboxylase shown to be present in themycelium also. In all experiments, approximately90 % of the stoichionietric amount of carbon dioxidewas formed from the pyruvate added whether the ex-periment was carried out aerobically or anaerobically.

Evidence that acetaldehyde is produced frompyruvate by carboxylase in the mycelial extracts ispresented in table I. In flasks 16 and 32 after carbondioxide evolution in the flasks was essentially com-plete, air was passed through the flasks and into atrap containing cold 2 % sodium bisulfite for 50 min-utes. Analysis of the bisulfite solution for acetalde-hjyde showed that on a molar basis approximately halfthe pyruvate was converted to acetaldehyde. In flask37 air was passed through the flask and into a bisulfitetrap throughout the experiment and on a molar basisall of the pyruvate added could be accounted for asacetaldehyde.

PYRUVIC OXIDASE SYSTEM: Several differentprocedures were tried in attempts to demonstrate thepresence of a pyruvic oxidase system in differentstages of the life cycle of Neurospora. The generalprocedures followed were those of Walker andBeevers (40) and Lieberman and Biale (18), both of

TABLE IPRODUCTION OF ACETALDEHYDE BY EXTRACTS OF

MYCELIA OF N. TETRASPERMA

SODIUM ACETALDE-FLASK PYRUVATE HYDENo. ADDED PRODUCED THEORY

,uM AM

16* 1.98 1.08 5532* 1.98 0.92 4637** 1.98 2.04 103

* Air passed through flask and NaHSO3 trap aftercarbon dioxide evolution was essentially complete (86 %of stoichiometric amount of carbon dioxide obtained).

** Air passed through flask and NaHSOQ trap through-out course of experiment.

758



HOLTON-PYRUV'ATE METABOLISM AND CYTOCHROME SYSTEM

whom used particulates isolated from higher planttissue. Phosphate buffers of pH 6.0 and 7.4, and0.5 M sucrose solutions were used in the enzyme ex-traction procedures. A number of different sub-stances tried in various combinations failed to stim-ulate oxygen uptake in the presence of pyruvate.These included yeast extract, liver extract, diphos-phopyridine nucleotide, adenosine triphosphate, ade-nosine monophosphate, cytochrome c, diphosphothia-mine, coenzyme A, glucose, Mg+ +, flavine mono-nucleotide, 2. 4-dinitrophenol, succinate, malate, fuma-

rate, and oxalacetate. Both the whole brei and theparticle fraction were used, as well as particles treatedwith deoxycholate, which removes cytochrome c (2)and probably much lipid material, since fatty acidshave been shown by Scholefield (27) to inhibit py-ruvate oxidationi in rat kidney mitochondria.

PYRUVATE AND P-PHENYLENEDIAMINE: Owens(24) reported that he was unable to obtain pyruvateoxidation with cell-free extracts of the conidia ofNeeurospora sitophila until he added p-phenylenedia-

w OO0

Li

A

-i

0 20 *0 *o so @to Ito 0 No 20 too

TIME IN MINUTES0 to 40 s0 so too Io

TIME IN IMNUTES

50

2

0 40

0

30

0

10

00 0 100 120

00,.° .so .

0 40 -

8 ., Jo _

4,.e-i 20

TIME IN MINUTES 0 so so M4TTME IN MINUTES

FIG. 1 (uipper left). Oxygen uptake by dormant ascospore extracts with added pyruvate, PPD, and cytochrome c.Each vessel contained 0.5 ml cell-free extract which contained 107 Ag nitrogen in phosphate buffer at pH 6.0, givinga final concenitration of 0.05 M. Addenda were 0.1 ml sodium pyruvate (10 pM), 0.1 ml PPD (10 pM), 0.05 mlcytochrome c (0.03 pM), and water to total 1.0 ml. If a component was omitted, an equal volume of water wassubstituted. The center well contained 0.2 ml 20 % potassium hydroxide.

FIG. 2 (upper right). Oxygen uptake by activated ascospore extracts with added pyruvate, PPD, and cyto-chrome c. Each vessel contained 10.5 ml cell-free extract of ascospores incubated for 2 hours containing 92 pg nitro-gen in phosphate buffer at pH 6.0, giving a final concentration of 0.05 M. Addenda were the same as in figure 1.

FIG. 3 (lo.wer left). Oxygen uptake by mycelial extracts with added pyruvate, PPD, and cytochrome c. Eachvessel contained 0.5 ml cell-free extract of a 2½2-day-old mycelial culture containing 352 pg nitrogen in phosphatebuffer at pH 6.0, giving a final concentration of 0.05 M. Addenda were the same as in figure 1.

FIG. 4 (lozer right). Oxygen uptake by conidial extracts with added pyruvate, PPD, and cytochrome c. Eachvessel contained 0.5 ml cell-free extract containing 68 pg nitrogeni in phosphate buffer at pH 6.1, givinig a final con-centration of 0.03 M. Addenda were the same as in figure 1.

759

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0

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14 ISO go



PLANT PHIYSIOLOGY

mine (PPD) with the pyruvate. He also noted thatad(le(l cvtochronme c increased the oxygen uptake fur-tlier. Therefore. experiments were carried out to de-termine if the sanme pyruvate oxidation system waspresent in extracts of N. tetraspermiia. These experi-mients vere carrie(l out with extracts of dormant andactivate(d ascospores, Mycelium, and conidia of N.tctrasperiiia. In all cases, it wvas found that if pyru-vate was addle(i with cytochrome c and PPD, it stim-ulate(l oxygen uptake over that found with the cyto-chrome c alnd PPD alone. Results of these experi-mlents are shown in figures 1, 2, 3, and 4.

None of the extracts showed significant oxygenuptake in the presence of pyruvate plus cytochrome cor with cvtochrome c alone. With PPD alone, thedormant spore extract showed no oxygen uptake butthe activated one did consume a small amount of oxy-gen. In the mycelial andl conidial extracts, there wasa considlerable amounit of oxygen uptake with PPDalone. In all cases, the oxygen uptake with PPDwas enhanced wvhen pyruvate was added. All ex-tracts showed considerable oxygen uptake in the pres-ence of PPD and cytochrome c, in contrast to Owens'(24) results with conidial extracts. Oxygen uptakein the presence of cytochlrome c and PPD is generallyattributedl to cytochrome oxidase and on this basis theoxygen uptake in this experiment is ascribed to thisenzyme. Adding pyruvate always enhanced oxygenuptake over those systems with only PPD and cyto-chrome c added but the added uptake was never morethan 10 to 15 % of the theoretical expected for theconmplete oxidation of pyruvate.

Several conclusions can be derived from these

dalta. On a nitrogen basis, the mycelial extract wasgenerally less active than those of the ascospores andconi(lia whose specific activities were almost equiva-lent. An inmportant difference involved the uptake ofoxygen in the presence of PPD alone. \While thiswvas ver- low with the ascospore extracts, it wasnearly as great as that of the cytochrome oxidaseassav system in mycelial extracts. In this respect theconidlia were intermediate. This aspect of the com-parative metabolisimi of the cell-free extracts ofNeurospora tetraspernma was studIied further and theresults are reporte(d below.

PPD-STIMULATIED PYRUVATE OXIDATION-: Es-sentially stoichiometric carbon dioxidle production,lack of oxvgen uptake, and acetaldlehyde formation bythe action of Neurospora extracts on pyruvate sug-gested that the principal enzyme in this extract actingon pyruvate was carboxylase. In an effort to betterunderstand the reason for oxygen uptake w\hen PPDvwas added wvith pyruvate, several experiments werecarried out.

Owens (24) suggested that the PPD-stimulatedoxidation of pyruvate might be independent of the(lecarboxylation and that the product of pyruvate de-carboxylation might be the compound being oxidized.To determine if this suggestion might be correct,pyruvate was tippe(l into a Warburg flask from thesidearm before and after stoichiometric carbon di-oxide evolution was obtained. In figure 5, it can beseen that oxygen uptake did occur after pyruvate hadbeen almost completely decarboxylated suggesting thedlecarboxylation pro(luct and not pyruvate wN-as being

6' 607r' ~ II.IIIIIIII II. .1A_j40~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~4

- ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~EXT CYT AOCETALD

0~~~~~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~~~~- 2

F20 40 *o0o loo1x0140

xygen200 20 240 2o S X as 125 1

016 zis 24 275 305TIME IN MINUTES T IMEIN MINUTES

FIG. 5 (left). Effect of time of additioni of p-phenylenediamine on oxygen uptake in the presence of pyruvate.In the two lower curves, the PPD was tipped in at the arrow; in the other curves, the PPD was tipped in at zerotime. The vessels contained 0.5 ml of a cell-free extract of mycelium suspended in 0.045 M phosphate buffer at pH6.1. Addenda were 0.1 ml cytochrome c (0.03 ,M), 0.2 ml PPD (recrystallized, 4.4 AM), 0.2 ml sodium pyruvate(3.8 puMI), and buffer to give 1.0 ml.

FIG. 6 (right). Oxygen uptake in the presence of p-phenylenediamine and pyruvate or acetaldehyde. Eachvessel contained 0.5 ml of a cell-free extract of mycelium suspended in 0.045 M phosphate buffer at pH 6.1. Addendawere 0.1 ml cytochrome c (0.03 pM), 0.2 ml PPD (recrystallized, 9.3 pM), 0.2 ml sodium pyruvate (10.3 AM), and0.1 ml acetaldehyde (10.0 pM), and buffer to total 1.0 ml.

760

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IIOLTON-PYRUVATE METABOLISM AND CYTOCHROME SYSTEMN7

oxidized. The decarboxylation product, acetaldehyde,was added in the presence of PPD and found to be-have identically to pyruvate (fig 6) after the first 2hours. During the first 2 hours the greater oxygenuptake with acetaldehyde and PPD over pyruvate andPPD is expected if the pyruvate must first be de-carboxylated before oxygen uptake can occur.

That this is an enzymatic oxidation is suggested inthe failure of boiled enzyme to stimulate oxygen up-take when added to PPD and acetaldehyde. Therewas no appreciable oxygen uptake with PPD andacetaldehyde with or without cytochrome c in theabsence of enzyme. A PPD solution autoxidized inair or an enzymatically oxidized PPD solution fromwhich the enzyme was removed did not stimulateoxygen uptake when added to acetaldehyde. Neithersodium 2, 6-dichlorophenolindophenol, potassium fer-ricyanide nor ferrocyanide, nor reduced DPN wouldsubstitute for PPD.

ENDOGENOUS CYTOCHROME C: It was noted abovethat oxygen uptake occurred when PPD alone was

added to conidial and mycelial extracts (fig 3 and 4)but not when PPD was added to ascospore extracts(figs 1 and 2). This oxygen uptake could be due toone, or a combination, of two entirely different oxi-dative systems: A: laccase, an oxidase that oxidizesPPD directly, or B: cytochrome oxidase, an enzymethat oxidizes cytochrome c which is, in turn, reducedby PPD. Several experiments were carried out todetermine which system was operating here.

Because laccase in other organisms has been foundin the supernatant while cytochrome oxidase is in theparticles, fractionation was used as a method ofseparating the two activities. Centrifugation of theextracts at 16,000 X G yielded particulate and super-natant fractions. The first experiment showed that56 % of the total PPD-oxidase activity was in theparticulate fraction, after centrifugation at 16,000 X

G, of the extract of a 61-hour-old mycelial culturewhile a second showed 68 % in the same fraction ofan extract of a 40-hour-old culture. Because a ma-

jority of the PPD-oxidase activity was localized inthe particles, endogenous cytochrome c could be im-plicated in part of the oxygen uptake with addedPPD.

Ball and Cooper (2) showed that in the partic-ulates isolated from heart muscle, 95 % of the PPD-(xidase activity could be removed by homogenizationof the particulate material in buffer containing 0.5 %deoxycholate. The treated particles regained theirability to oxidize PPD when cytochrome c was added.This technique was applied to particulate extracts ofNeurospora mycelia in two ways. In several experi-ments, after the initial centrifugation of the cell-freeextract, the particles were homogenized and resus-

pended in phosphate buffer with 0.1 % or 0.5 % de-oxycholate in this buffer. They were then centri-fuged again for 10 minutes and this time resuspendedin deoxycholate-free buffer. The treated particlesthat came down during this centrifugation had a dif-ferent appearance from the untreated ones in that the

TABLE IITREATMENT OF MYCELIAL PARTICLES FRO'M N. TETRA-

SPERMA WITH DEOXYCHOLATE (DOC) ANDRESUSPENSION IN PHOSPHATE BUFFER

ML O., UI TAKE/HR*

CYTOCHROME C 0.1 %, DOC 0.5 % DOCCONC

TREATED UN- TREATED UN-TREATED TREATED

0 8 34 1 151 x 10-5 M ... ... 26 178

1.2 x 10-4M 67 163* All rates are the initial rates obtained after tipping

the PPD (10 yM). Extracts were prepared from a 36-hour-old mycelial culture for the 0.1 % DOC treatmentand from a 37-hour-old mycelial culture for the 0.5 %/DOC treatment.

pellet formed was smaller and more compact. Thesupernatant from the treated particles was turbidwhile that from the untreated was clear. As shownin table II, this procedure removed most of the PPD-oxidase activity. An increase in cytochrome oxidaseactivity was obtained by adding cytochrome c but thetotal cytochrome oxidase activity of the untreated ex-tracts was not recovered.

In an attempt to recover maximal cytochrome oxi-dase activity, a second procedure was tried utilizingthe particles obtained after a single 15 minute centri-fugation. After resuspension in buffer plus deoxy-cholate, the particles were used directly. Table IIIshows that all the cytochrome oxidase activity couldbe recovered by adding cytochrome c but complete re-moval of cytochrome c from the particles was not ef-fected.

TABLE IIITREATMENT OF MYCELIAL PARTICLES FROM N. TETRRA-

SPERMA WITH DEOXYCHOLATE (DOC) ANDRESUSPENSION IN DOC

,LL 02, UPTAKE/HR*

CYTOCHROME C 0.2 % DOC 0.5 % DOCCONC UN- UN-

TREATED TREATED TREATED TREATED

0 9 33 9 263.9 X 10-6M 66 ...

4.5 x 10-6 M ... ... 791.6 X 10-5 M 188 2221.8 X 10-5 M ... ... 165 195

1.6 X 10-4 M 224 ...

1.8 X 10-4 M ... ... 170 ...

2.8 x 10-4 M 210 ... ... ...

3.1 X 10-4M ... ... 157 ...

* All rates are the initial rates obtained after tippingin the PPD (35 ,uM). Extracts were prepared in glycyl-glycine buffer from a 67-hour-old mycelial culture for the0.2 % DOC treatment and in phosphate buffer from a36-hour-old mycelial culture for the 0.5 %/, DOC treatment.

761



PLANT PHYSIOLOGY

From the evidence obtained by this technique, itappeared that the deoxycholate treatment did removeenclogenous cYtochrome c from the particles and thatPPD oxidation depen(led upon the presence of cyto-chrome c in the mycelial extracts as in the heartmuscle preparation of Ball and Cooper (2).

The manometric ancl spectrophotometric methodsfor cvtochrome oxidase are based on different princi-l)Ies, as mentionedl earlier. If laccase is present to-gether with cytochronme oxidase, it can be assayed inthe manonmetric metho(d because any oxygen uptake inthe presence of PPD and cytochrome c would be de-tectedl. Such activity could be (lue to either cyto-chrome oxidase, laccase or their combination. Thespectrophotometric method, on the other hand, assaysonly cytochronme oxidase activity because the disap-pearance of reduced cytochrome c is followed direct-lv. Therefore, if laccase were present with cyto-chrome oxidase, the manometric procedure would givehiglher values than the spectrophotometric, while iflaccase were absent the two methods should giveequivalent results.

In order to compare the two assay methods, the re-suilts mlust be expresse(l in commllon units, thereforethe Vnlax. in terms of PI oxygen uptake per hour permlg nitrogen in the enzyme solution was used. Valuesfor a numlber of these analyses are given in table IV.These clata show that for the mvcelial extracts (brei),the mlanometric and spectrophotometric results agreequite wxell for the first four samples. In the last foursamiples, the agreement is not as good, but the activi-ties measured in the spectrophotometer were greaterthan those obtained manometricallv. Inasmluch as the

TABLE INCYTOCHRO-ME OXIDASE IN EXTRACTS OF N. TETRASPER-MA

TISSUE AGE

(HRS) FRACTION

Dormant ascosporesDormant ascospores

MyceliumMyceliumMycelium

MyceliumMyceliumMyceliumMyceliumMycelium

MyceliumMyceliumMyceliumMyceliumMycelium

434764

5668708943

4764666466

BreiBreiBreiBreiBrei

BreiBreiBreiBreiParticles

ParticlesParticlesParticlesSupernatantSupernatant

PROCEDURE

MANO- SPECTRO-PHOTO-

METRICmax V *

max

1,2301,100330

730

380640410340

3,120

3,5904,8305,150140220

1,230940

.

280820

4001,390920

830.2.9.

5,9505,9504,150

80140

reverse woul(l be expected if more than one oxidasewere present, these data are also compatible with thenotion that laccase was absent.

Consequently, data obtained by three differentmethods suggest that the ability of mycelial extractsto oxi(lize PPD without added cytochrome c is not(lue to laccase but to cytochrome oxidase which oxi-dizes reducedl endogenous cytochrome c. The re-strictecl ability of extracts of (lormant and activatedascospores to carry out the oxidation of PPD sug-gests that the) lack endogenous cytochromle c. or haveonly smlall amounts of it.

TABLE VMEASUREMENTS OF CYTOCHROME C tONTENT

OF N. TETRASPERMA

SOURCE OF N CONTENT CYTOCHROME CYTOCHROMEPARTICLES ,LG C MOLARITY C ,UM/IAG N

Dormantascospores

26-hour-oldmycelium45-hour-oldmycelium

25 *

1410 2.77 X 10-- .90 X 10-6

1375 4.87 X 10- 1.04 X O-

* No cytochrome c was detected. The lowest conI-centration of the standard cytochrome c solution thatcould be detected was 4.78 X 10- M.

Final evidence of the presence of cytochromiie c inmycelial extracts was obtainecl by dlirect observationof particulate preparations with the spectroscope.Qualitative observations at room tenmperature showedbands at 550. 565. and 610 my presunmed to be the ca,

b,3. and a-a,; cytochrome bands, respectively. Thesewere observ-ed in particles from both 26-hour-oldmyceliuml andcI 45-hour-old mycelium. In addition, a

dliffuse ban(d at 520 to 530 nm was observed in theolder mycelial particles while in the younger particlesan uni(lentifie(d pigment obscured this portion of thespectruml. No dlifferences were observe(d wlheln thesamples were frozen in liquid air except that the dif-fuse ban(d at 520 to 530 my was resolve(d into twodistinct lines in that region. Quantitative measure-

ments of cytochrome c by means of the wedge troughmethod of Hartree (13) are shown in table V.

Although no evidence of cytochromle c was ob-servedl in the particles fronm the dormant ascospores,this extract ha(l such a low nitrogen content comparedwith that of the particles from the mycelia that theresult wNTas not unexpected. Because of the diffi-culties of obtaining ascospores in the (juanitities neces-

sary to obtain an extract with a nitrogen content com-

parable to that of the mycelial extracts, no furtherexperimiienits were carried out with this material.

CYTOCHROMAF OXIDASE: Table IVT also affordIsinformation on the relative cytochrome oxi(lase activi-ties at (liffel-ent phases during the growth of Neuro-spora. Comllparison of the data on the w-hole breis on

W Vmax values are expressed in ,ul 02 uptake per hourper mg N.

7652



HOLTON-PYRUVATE METABOLISM AND CYTOCHROME SYSTEM76a nitrogen basis show nearly four times as much ac-tivity on the average in the dormant spore extractsas in the extracts of the approximately 2-day-old (43-and 47-hour-old) mycelia. The average activity inthe extracts from the approximately three-day-old(64-, 66-, 68- and 70-hour-old) mycelia is twice thatof the 2-dav-old mycelia but still far less than that ofthe dormant spore extracts. In general, good agree-ment for particular extracts is found between the twoassay methods.

Fractionation oni the mycelial brei showed, as ex-pected, that nearly all (97 %) of the cytochrome oxi-dase activity was localized in the particles. As wastrue with the breis, the particles from the 3-day-oldmycelia possessed more activity than their 2-day-oldcounterparts.

This apparent variation with age led to a study ofthe rate of growth of the mycelium of N. tetraspernia.The resulting growth curve is shown in figure 7.From examination of this curve it is apparent that theextracts prepared from the organism after 2-days'growth represent mycelium at the end of the log phaseof growth while the 3-day-old mycelial cultures arewell into the stationary phase. Therefore, the ex-tracts of 2- and 3-day-old mycelia were of quite dif-ferent physiological ages and variations in enzymaticactivity are expected.

DISCUSSION AND CONCLUSIONSAlthough the pyruvic oxidase enzyme system has

been demonstrated in cell-free extracts of many higherplants, it was not possible to do so in extracts ofNeurospora. In the fungi, Bonner and Machlis (3)were able to slhow the presence of this enzyme systemin cell-free extracts of the mycelium of Allomyces andNossal (22) and Linnane and Still (21) found it inyeast. Ramakrishnan (25) demonstrated activity inextracts of Aspergillius niger by following the reduc-

HOURS AFTER INOCULATION

FIG. 7. Growth curve of Neurospora mycelium. Eachcircle represents the dry weight of mycelium obtainedfrom one flask containing originally 65 ml of medium and1 ml of conidial suspension.

tion of 2, 6-dichlorophenol-indophenol, spectrophoto-metrically. The present work, however, is not thefirst failure to demonstrate this enzyme system asother unsuccessful attempts have been reported.Strauss (34) found that cell-free homogenates ofmycelia of an acetate-requiring mutant of Neurosporadid not oxidize pyruvate even when supplementedwith cocarboxylase, DPN, ATP, and fumaric acid.However, he did obtain pyruvate oxidation with in-tact mycelia. Owens (24) was unable to obtain oxi-dation with cell-free extracts of Neurospora conidiaalthough he supplemented the extracts with a numberof different possible co-factors. On the other hand,oxygen uptake did occur when a dye. p-phenylenedia-mine (PPD). was present in the mixture. The addi-tion of cytochrome c further stimulated oxygen up-take. This oxidation of pyruvate will be discussedlater.

Failure to extract an active pyruvate oxidase en-zyme system from Neurospora can be explained inseveral ways. Loss of a necessary co-factor mayhave occurred although many known co-factors wereadded to the extract without stimulation of oxygenuptake. Inhibitory compounds may be released fromthe cell during preparation of the extract. Fattyacids in concentrations of 10-3 M and greater havebeen shown to inhibit pyruvate oxidation by ratkidney mitochondria (27). This is interesting inview of the fact that lipid appears to be the en-dogenous substrate for respiration during certainphases of the life cvcle of Neurospora (20).

A TCA cycle acid. oxalacetate, in a concentrationof 0.018 M, has been shown to temporarily inhibit theoxidation of pyruvate by avocado fruit particulates(1). At this concentration, oxalacetate inhibited oxi-dation of 0.02 M1 pyruvate for 80 minutes due, as theauthors suggest, to a reaction in which the oxidationof pyruvate is coupledl to the reduction of oxalacetateinstead of oxygen.

That there is a strong pyruvic carboxylase presentin these cell-free extracts is shown by this work andthat of Sussman et al (36). Because both the py-ruvic carboxylase and oxidase systems are derivedfrom a common intermediate, the aldehyde diphos-phothiamine enzynme complex (12), it appears that acompetition may exist between these two enzyme svs-tems in cell-free homogenates which may not reflectcorrectly their status in the intact cell. Sussman etal (36) detected no acetoin in cell-free extracts sothis system is apparently not functioning here.Schwett et al (28) attempted to separate the oxidativefrom dlismutative activity of the pyruvic oxidase en-zynme system in pigeon breast muscle andl in both thecrude and most purified pyruvic oxidase systems, aconstant ratio of non-oxidized to oxidized productswas always maintained. Thus closely related en-zymatic activities may interfere with measurements ofthe oxidative system.

Owens (24) was unable to obtain oxygen uptakein the presence of pyruvate with cell-free extracts ofNeurospora conidia. However, when he added

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PLANT PHYSIOLOGY

p-phenvlenediamine (PPD), a dye used for assayingcytochrome oxiclase activity, oxygen uptake occurred.WX hen hie added cytochrome c with the PPD andpyruvate, oxygen uptake was even greater but withcytochrome c and PPD there was no oxygen uptake.The results reported here do not agree with this latterobservation of Owens; ve always obtained rapidoxygen uptake wlhein PPD and cytochronme c wereadded to cell-free extracts. Inasmuch as this com-bination has been used in assays of cytochrome oxi-dase (31), the oxygen uptake writh these addendaverifies the xvork of Sussman et al (36) who foundthis enzyme to be present in the ascospores. Owens(24) in the text of his paper indicated "the presenceof active cytochrome oxidase" but the data in his tablecontradict this statenment.

Owens suggestedl that the stimulation of pyruvateoxidation that is indluced by PPD might not be due tothe oxidatioln of pyruvate hut of its decarboxylationproduct. The data present here are consistent withthe suggestion that acetaldehyde is the clecarboxyla-tion product of pyruvate and that oxygen uptake inthe presence of PPD and pyruvate or acetaldehyde isclue, in part at least, to the oxidation of these comI-pounds. Because PPD is oxidized in this system, itcloes not act analogously to ferricyanide or 2, 6-d(ichlorophenolindophenol which may accept electronsfrom the aldehyde-enzyme complex to liberate the car-bon skeleton as free acetic acid (12). Thus the un-equivocal oxidation of pyruvate by cell-free extractsof Neurospora has yet to be demonstrated.

Evidence presented here indicates that oxygen up-take when PPD is added to mycelial extracts is dueto the oxidation of reduced endogenous cytochrome cby cytochrome oxiclase rather than to laccase. Cheng(5) had earlier reported the absence of laccase inmlvcelial extracts onl the basis of slow oxidation ofPPD. This slow oxidation is attributed here to theaction of cytochrome oxiclase on endogenous cyto-chrolmie c. This indirect evidence is backed up by thedirect spectroscopic observation of cytochrome c inmycelial particles. This agrees with earlier observa-tions of cytochrome c in NTeuirospora crassa (4, 39).Haskins et al (14) found that the whole mycelia ofN. cravsa contained 0.15 to 0.22 % cytochrome c on adry weight basis. Unfortunately it is not possible tocompare directly these results with those reportedhere. The very low rates of oxygen uptake whenPPD is addled to ascospore extracts suggests thatthere is a low level of endogenous cytochrome c inthose extracts. A possible alternative explanation isthat the apparent lack of cytochrome c in the extractsdemonstrated by the indirect manometric method doesnot correctly represent the concentration of cyto-chrome c in the intact cell. Unfortunately, the directspectroscopic exanmination of the particles from thespores does not give us any helpful information sincealthough no cytochlrome c was observed, it was notpossible to obtain enough material for a comparablesiample size to that obtained from mycelia.

The endIogelnous Qo., of N. crassa mycelium was

found to be 29.6 (15) while that of dormant ascosporewas 0.25 to 0.59 (11). Thus it would not be sur-prising to find more cytochrome c in the mycelia thanin the ascospores if the cytochrome system is theprincipal oxidative system in this organism. The in-direct manometric data is consistent with this sugges-tion. A parallel in insect tissue was reported byShappirio and Williams (29), who found no detect-able cytochrome c in the heart of dormant pupa of theCercropia silkworm while in the late developing adultand mature adult moderate amounts were found.

The presence of an enzyme capable of oxidizingp-phenylenecdiamine, interpreted here to be cyto-chrome oxiclase and endogenous cytochrome c, wasdetected in Neurospora by Tissieres and Mitchell(39). They found oxvgen uptake in the presence ofI'PD alone (Qo, - 25), while with cytochrome cadded the Qo, increasecl to 450. With ascorbate asthe reducing agent in place of PPD, the values were10 and 472, respectively. The fact that oxidation ofascorbate alone was less intense than that of PPDalone agrees with the statement of Slater that en-dogenous cytochrome c can be reduced rapidly byPPD but not by ascorbic acid.

The Qo2 for cytochrome oxidase of 340 obtainedin the present work with extracts of 4-day-old myceli-um compares reasonably well with the 450 obtaineclby Tissieres and Mitchell (39) with extracts of 4- to5-day-old mycelia of N. crassa at the higher tempera-ture of 350 C. On the other hand, Cheng (5) report-ed 665 using hydroquinone as the reducer with ex-tracts of younger (1.5-day-old) mycelium of NT. tct;-a-sPLrna at 250 C.

The fact that the Vmax values for cytochrome oxi-dase are greater in the brei from dormant ascosporesthan in the mycelial brei suggests that in the formerthis enzyme is not the limiting factor in the electrontransport system. Cytochrome c, in contrast, may bea limiting factor in the dormant ascospores and oneconsequence of the activation of ascospores may beanl increase in the amiount of cytochrome c.

Because the nianometric and spectophotometricmethods of assaying cytochrome oxidase activity arebased on different principles, it was of interest tocompare the two assay procedures on the same en-zyme preparations. The data in table IV demon-strate that quite good agreement between the twoprocedures was obtained with extracts of tissues up to3 days old.

The literature contains several reports of the com-parison between the two assay- methods (6, 7, 8).Except in the experinments of Fritz and Beevers (8),discrepancies were found between the two methods.Because that work and the results reported here werethe only studies comnparing methods in which differ-ent cytochrome c concentrations were used and a Vmaxvalue obtained, it is suggested that the lower valuesof cytochrome oxidase activity obtained with themanometric methocl in the past was due to lower thanoptinmal cytochrome c concentrations.

On the other hand, Smith and Conrad (32) sug-

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HOLTON-PYRUVATE METABOLISM AND CYTOCHROME SYSTEM 6

gested that a reason for disagreement between the twoassay methods was that concentrations of cytochromec often used in manometric assays inhibit cytochromeoxidase. They contended that the method of ex-trapolation to infinite concentration used by Slateractually measured a greatly inhibited cytochrome oxi-dase activity. The fact that results of Fritz andB3eevers and those reported here show good agreementbetween the two methods suggests that at the concen-trations of cytochronmc c used in these studies no in-hibition was occurring. The lack of agreement in theassays of these older mycelia could also be ascribedto the presence of an enzyme which was not presentearlier and which was assayed in the spectrophoto-metric but not the manonmetric procedure.

SUMMARYAttempts to demonstrate the oxidation of pyruvate

by cell-free extracts of ascospores and mycelium ofNeurospora tetraspermiia by any of the commonly usedmethods of extraction and mixtures of co-factors wereunsuccessful. Adding a dye, p-phenylenediamine(PPD) did stimulate oxygen uptake when pyruvatewas added to extracts of dormant and activatedascospores, conidia, and mycelia. Further experi-ments showed that this oxygen uptake is probably duenot to oxidation of pyruvate but to oxidation of thedecarboxylation product of pyruvate, acetaldehyde,formed from pyruvate by the carboxylase in these ex-tracts. Acetaldehyde was oxidized by these extractsenzymatically but only in the presence of PPD.

Oxygen uptake by mycelial extracts in the pres-ence of PPD alone has been shown to be due to theoxidation of endogenous cytochrome c by cytochromeoxidase. The endogenous cytochrome c content -wasmeasured spectroscopically. Lack of oxygen uptakeby extracts of dormant ascospores in the presence ofPPD suggests an absence of endogenous cytochromec in these extracts. On the other hand, the level ofcytochrome oxidase is higher in the dormant asco-spore extracts than in the mycelial extracts. If theextracts represent accurately the status of these en-zymes in the intact cells, it can be inferred that thelevel of cytochrome c and not the level of cytochromeoxidase may be one factor accounting for the low rateof respiration in the dormant ascospores.

ACKNO'WLEDGMENTSThe author is indebted to Dr. A. S. Sussman for

advice and encouragement throughout the course ofthese studies. The aid of Dr. D. E. Bianchi with thespectroscopic cytochrome c analyses is also gratefullyacknowledged. The author is indebted to The Uni-versity of Michigan for a Horace H. Rackham Pre-doctoral Fellowship and an F. C. and Susan EastmanNewcombe Fellowship in Plant Physiology.

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2. BALL, E. G. and OCTAVIA COOPER. 1957. Observa-tions on the function of cytochromes c and cl.Jour. Biol. Chem. 226: 755-763.

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12. GUNSALUS, I. C. 1954. Group transfer and acyl-generating functions of lipoic acid derivatives. In:The Mechanism of Enzyme Action, W. D. McElroyand B. Glass, eds. Pp. 545-580. Johns HopkinsPress, Baltimore.

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