Differentiation of electron transport systems in mitochondria and microsomes during embryonic development

AR(‘HIVER

OF

BIOCHEMISTRY

Differentiation

AND

BIOPHYSICS

of Electron Microsomes

lo!&

Transport during

CORKELIIJS

(1963)

293-3~5

Systems

Embryonic

in Mitochondria

and

Development

P. STRITTAIATTER

Activities of various particnlate-linked osidative enzyme activities and of cytochrome component,s were examined in well-defined mitochondrial anti microsomul fractions oht,ained from 2Ghr. chick embryos and from heart, and liver of chick embryos and chicks. Changes in relative as wet1 as absolute levels of ttlese components are considered in terms of t,he differentiation of organized electron transport chains in different tissues nnd within a given tissue in t.tre course of emt)r,vonic development.

(‘retain components of the multienzyme clrct,ron trausport system within the mitocholldria are believed to be associated in an orgatlized particulate form (I -8). Simple cluaut,itativc relationships in the molecular proportions of some enzymic components have been observed with mitochondrial mcmbralle preparat,ions, suggesting that the clcctroii transport system may exist in definite structural units with specific and constant composition (2). It is not known to what degree the electron transport enzymes firmly bound to the microsome particles derived from the endoplasmic reticulum may be similarly organized. To learn more of the possible existence and variability of “standard” (~lcctron transport units with specific alld constant composition, it seemed pcrtiileut to examiuc the rrlat’ivr activities of various rlectrou traiisport enzymes in mitocho~ldrial and microsomal preparations obtaiwtl from several Gssues at difierent stages of rmbryollic development. Differ(‘Iices or chaiiges in these relative activities 1 This work (;iXl-930-l frc~m 1‘. S. I’ubtic(;-(il:S fronr :mtl a gr:tnt, t~~~~m:tn (iray

w:ts supported by resexch grant ttle Sntiomd Institutes of Health, Health Service, resexrctr grant tile National Science Foundation, front the Fluid Research Fund of School of Medicine. 293

among the various preparations may iudicate differentiation of the multienzyme systems with which the enzymes are associated. Changes in the levels of both soluble and particulate-linked oxidative enzyme activities (4~8), as well as of nonoxidative enzymes (O), have been demonstrated to occur during embryonic drvclopmcnt~. The observations on palticulatc-linked oxidative activitirs have beeu concerned largely with the initial appearance or marked increases of activit-y that occur at specific stages of cmbryouic developmeut, but a few recent studies have also specifically considered changes in the relative conccntrat,ions of mitochoildrial enzyme compoueut,s as a11 indication of mitochondrial diffcreutiation iI1 the swf clam (7) aud iI1 the amphibiau Xeuopus (8). The present paper considers differences iu both absoliite alit1 relative activity levels of pal.t’iculatc-t)ouIld enzymes associated with mitochoudrial alld microsomal preparat,iolls at, se\wal st’ages in the embryonic developmellt of chicken liver alld heart,. Relatively “pllre”, well-defined particulat,c preparations and specific cllzyme markers are emphasized to minimize difficult,ies of interprctatioll arising from adniisturc of particles with cluai~titativcly different enzymic compositiotl or from variation in the concciitratioii of ,211

active particle t,ype in a preparation. Both mitochondrial and, to a lesser degree, microsomal enzyme components showed changes ill relat,ive concentrations that suggest the differelkiation of the organized electron transport, systems within these cytoplasmic particulate fractions in the course of embryonic development.

Fertile eggs of the J-an Tress-Leghorn strain were obtained from a local hatchery and incuhat,ed at, 38” and 65’:; humidity in the laboratory. The &age of development of embryos at the time of harvest was determined by the scale of Hamburger and Hamilton (lo), and only embryos of :I specific stage (&l) were pooled for a preparation. Chicks after hatching were provided with water and Purina Starter Chow atl libitlcnl until sacrificed. The standard t)issues used in the study were (a) t,he whole chick embryo of Stage G, hereafter rem ferred to as “24-hr. embryo”; (bj the liver and heart of the Stage 29 embryo, lrereafter referred to as “(i-day embryo”; and (c) the liver and heart of Y-day chicks. The 24.hr. embryos were closely trimmed to include essentially only the area pellucida, without extraenlbryonic material; the livers and hearts were trimmed free of ext,rancous connective or adipose tissue. Tissues were excised immediately aft,er sacrifice, trimmed, and rinsed in ice-cold 0.25 !I2 sucrose-O.001 Al EDTA,’ pH 7.4 (sucrose-EDTA medium). The tissue was honlogenized in 9 vol. of the same medium in a Potter-Elvehjem type homogenizer fitted with a Teflon pestle; a moderate speed and duration of homogenization were chosen to disrupt the majority of the ~11s without appreciable breakage of the mit~ochondris. These homogenates were fractionated by different,ial centrifugation at O-4”, essentially as previous13 described (ll), to obtain relatively “pure” samples of mitochondria and microsomes. To remove nuclei and debris, tile homogenate was centrifuged at 600 X q for 10 min. and the supernatant solution was rerentrifuged. The resulting supernatant fluid was centrifuged at 3000 X y for 10 min. to obtain a pellet of mitorlrondria, which was purified by tvSce dispersing in sucrose-EDTA medium and __.~___..___~ __~ * Ahbrevixt ions used : El)TA, sodium salt of ethylenediaminetetraacetic I>l’_l; and acid; I)PNH, oxidized and reduced forms, respectively, of tlipl”)sptlopyridirle nucleotide; TPSH, reduced triphosphopyridine nucleotide; and Tris, tris (t~~droxymet~~~l):~minc~n~et,ll:~nc.

recentrifuging at 3000 X q. The 3000 X 9 supernatsnt fluid was centrifuged twice at, 15,000 X q for 10 min., and the final supernat,ant fluid was centrifuged at 110,000 X 9 for 60 min. to obt,ain a pellet of “microsomes.” The microsome pellet was washed by dispersing in sucrose-EDTA mediurrl and recentrifuging at 110,000 X 8. Finally, the washed particulate preparations were dispersed in suitable volumes of sucrose-EDTA medium. As intact, freshly prepared mit~ochondrial preparations do not react maximally with certain externally supplied substrates, including DI’NH, the particulate preparations were routinely frozen and t.hawed four times, using a solid carbon dioxide-ethanol bath, and stored at -15” until assayed. This freezing-and-thawing procedure rt:sulted in preparations with high osidative enzyme activities similar to those obtained by osmotic rupture or controlled aging. The enzyme activities studied were not significantly altered by storage at -15” for 24 hr., and assa,vs were carried out witllin this period. .kiSAYS Electron transport activities were assayed at 25” in a Beckman DU spectrophotometer with aerobic microcells of l-cm. light path, essentiall? as previously described (11). These assay condtions were somewhat arbitrary and not necessarily optimal, but preliminary studies showed tllat these conditions were favorable for high reproducible activities in the type of preparat,ions studied. Precise adherence to st,andardized preparative procedures and assay conditions was critical for reliable, reproducible measurements of these enzymic activities in part,iculate prep:tr:ltions. The reaction mixtures contained 0.05 M sucrose-0.0002 ‘41 EDTA-0.05 JI phosphat,e buffer, pH 7.4, with other components added as not,ed below-. Routinely the reaction was initiated by addition of enzyme after preincubation of the otherwise complete mixt,urr for 5 min. at 25”. I>PNH-, TPNH-, choline-, and succinate-cyt,ochrome c reductase activities were determined by following increase in absorbancy at 550 mp with 1.5 X 10Y1 M DPNH, 1.5 x 1V M TPNH, 3 x 10m3 M choline chloride or 3 X 1OW M sodium succinate, respectivel,v, as electron donor and 5 X 10-S M oxidized cytochrome c as electron acceptor in each case; 1 X lo-” ;II KCN was added to prevent. reoxidai.ion of cytochrome c. The “I)PN-st,inrlllated TPNH-cytochrome c reductase” activity was obtained hy determining TPNH-cytochronre c reductnse activity in the presence of added 1 X 10-s M l)l’N, and subtracting from this valur the TP~H-cZtoclll*orllc c reductase activity obtained wit,hout added l)PS; the possible relation of tllr

wfu::intler, designated “t)PN-stimulated TPNIIc~ytwtrronlP (’ rcduct ase” wfivity, to trnnshydrogen:tsc activity is considered in the Disctrssio~r. Cytoctrrome c oxidase was dctmuined hy foilowiug at 550 rnp ttic oxidation c)f 1.5 X 10-j :11 reduced c~~tocllrf,llle (‘. To rr~easure I )I’KH-fcrricyxnide rrtiuc~ase iwtivity, the decrease in :thsorhancy iti, NO nip WQS determined with 1.5 X IO-’ JI I)l’n’H as elwtron donor and 5 X 1W” Jf ferricytnide as rlcct rf,n acceptor. I )I’NH oxid:tse wtts measured 1)~. tlir drrrease of absorh;tncy :it 340 mp wit11 1.5 X 1OV’ JI I>PNH as elect ran donor and :tt,mosplwric cjsq’gen as wreptc)r. Ill all assays, readings were taken at 15- or :I()-SW. intervals, and reaction rates were calcnt:~ied from :t segment of the initial period of 1inra.l :rc*t ivity. usuall,v for the period 2-C min. after addtion of enzyme. Ttw v:ttucs reported were cartwted for t.tle rates of any non-enzyrnir xtivit,y. l’t~c csorrwted rates \wl’e c:tIcul:rted in units of rtlittirl~icrolrlc,Ics of elect rc,ns tr:tnsport,cd/rllin.! rrrg. yraltein in the eneynx preparatjions, using wiues for nlillimotar ahsorhancy indcs of 6.22 fol I )t’NH and 18.5 for cytcwtironre c.

b’or spwtrttt studies. frrsllly prepared mitoc~tlondria anti mirrosomes u-err wnslled hy dispersion wit It 50 vol. c>f 0.1 J/ Tris-HCI buffer, pH 8.0. and c*ottcctjed by (*entrifug:Lf ion. The cell fractions w’re then dispersed in 25, sc)diuru droxychoiate in (I.1 .If Tris-HCI tmft’er, pH X.0, :ttlowed to stand at 5” for 10 min., and ctarificd hy centrifugation :it 12,000 X q for 10 min. .%ppropriate dilutions c,f t lw c*t:lrifieti prcpar:ltions in 2: ( d~oxy~~tiolate were us(~i for spcc1 rat measurements :rt 25”. Oxidized spwtra wert’ oht:~inetf in open nrirrocetls after gclntlr wration; reduced specttra were ohtaincd in ~losecl mic~rlwetlti hy addition of reducing agent after. r~cluitihration of ttw cc11 wmtcnts with nitrogrn. Spwtr:r wpre read in ritlwr :I B:rusc*t~ and I,ltnlt) model 505 recording sl.‘ec~trc’t)tioto”lel~r or a 13wkm:m I)[. st~~ctro~~t~otor~~~t~r against :i w:ttel t)I:tnk; t.tr oht:tin a difl’crenw spec*trum, ttlc reduced s:~ntplr was rcwi :qqtinst t Ilr oxidized prtpw:ti ion :,s I I1r hl:mk.

ticins with scldiunl tiydrcwutfite until 90-K ( rrduction of the rytoct~roniP was :wliieved, then aerating gently to renlovc rxcess reducing agent.

The fractionation procedures described above yielded what appeared to he fairly “pure” preparatioils of mitochondris aud microsomw lTirstly, rlectroil microscopic examination of fresh preparations showed that the mitochondrial and microsomal fract,ions from heart or liver of either (i-day embryos or :&day chicks were composed primarily of characteristic pwticles of the type in cluestiori, wit,11 only minor cotitaminat,ion hy (Aher cellular compoueirk. Jii preparations from 21-1~. whole embryos, the microsomal fract,ioil appeared to he fairly homogeiieous, while the mitochondrial fraction appeared to coiit,aiii an appreciable quantity of nollmitocholtdrial material. The fractions from heart atld livrr were similar in appearance to the analogous fractiolts from t’he adult chicken &sues (1 1). Seco~ldly, the electron transport activities of tlw fractions, as described helow, suggested only miiror contalninatioti. Several fact,ors, including duratioir of homogcllizat,iotl, n~re critical in obtaining these relati\:ely pure fi,actions. .Zs example, Iieai,t microsomal fractions free of gross coiitamiitatioii witjh mitochotldrial fragments could 1x1 ohtaillcd oiily hy limitiig the homogeiriziiig period to less than that re(Iuired for complete tlisrupt’ion of the lwart~ muscle and maximum yield of mitocholrdrin. Thirdly, ill the fract,ionatioll of li\rcr from both chicks atld emlxvos, a niajol~ portioii of li\.cr gl~iw~c~-~ipliosphatasr was recoverrd iii the microsonial fractiotis, itI which this eiqmc is ktlow1 to he localized iI) mnmmaliatr liver, aiid less thaii 3’; was fouitd iii t)hc mitochondrial fractiolls.

t\s is indicated ilt Table thawd prcparatiolts of microsomes from heart or emlwyos and :S-day chicks

I, t,he f’rozell-all& mitocho~rdria a11t1 livw of both (i-day coirtailted elcctrotl

296

STRITTMATTER

transport enzymes of types kuowu to be associated with the corresponding particulate fractions from adult, chicken or mammalian tissues. However, both absolute levels (Table I) and relabive levels (Table II) differed in specific ways with the type of tissue and the age of the organism, and differed from the levels fouud in mitocholldria or microsomes from 24.hr. whole embryos. ,411heart and liver mitochondria contaiued high absolute levels (Table I) of cytochrome oxidase, succinate-cytochrome c reductase, &sensitive DPXH-cytoand antimycin chrome c reductase (See Table III for inhibition characteristics), which are specifically associated with mitochondria. The presence of a complete, functioning respirat,ory chain is indicated by the high DPKH oxidase activities. Both TPNH-cytochrome c reductase and “DPN-stimulated TP?;Hcytochrome c reductase” activities were

appreciable. Mitochondria also contained significant levels of choline-cytochrome c reductase, which may be specifically associated with mitochondria (14), and a high level of DPKH-ferricyanide reductase, which is found in both mitochondria and microsomes. The levels of some activities were markedly different in heart mitochondria as compared to liver mitochoudria from animals of the same age; the differeuces were more pronounced in the j-day chick. Further, in each tissue the levels of some activities changed with age, most commonly increasing between the G-day embryo and s-day chick. The mitochondria of 24-hr. embryos possessed significant amounts of the typical mitochondrial enzymes assayed, except that no “DPX-stimulated TPXH-cytochrome c reductase” activity could be detected. However, the levels of all activities were markedly lower than iu either heart or live1

TABLE ELECTRON

AWIVIUES

TRANSPORT

I

CJF MITOCHOSDRIA I)EvELoI+~ENT

ANI)

MICROSOMES

DURING

EMBRYOR.IC

Preparations of each type of particulate fraction were obtained and assayed by the standard procedures described under Ezperinrenfal Methods. The activities are expressed in terms of millimicromoles of electrons transported/min./mg. protein in the preparation assayed. The values tabulated are the means of values obtained from three typical preparations. Enzymatic Particulate

__---

fraction

Cytochrome oxidase

Succinatecytochrome reductase

DPSKcyc tochrome reductase

Cholinec cytachrome c reductase

Activities TPNRcytochrome c reductase

- ...-

Mitochondria from : 2-1-h. whole embryo G-day embryo liver 3.day chick liver A-day embryo heart, May chick heart Microsomes f ram : 2&hr. whole embryo &day embryo liver 3.day chick liver B-day embryo heart, 3.day chick heart

DPNstimulated r;yfky reductase

DPNIIierricyanide reductase

oaidax

72

5

r)p.ult

35

5

20

0.4

2.8

567

80

275

2.5

16.5

9.0

1215

115

610 530

86 11-l

321 262

4.2 2.0

26.0 19.0

17.0 11.5

1130 1565

178 !I6

920

215

245

1.5

25.0

16.0

1935

305

0.2

81

-

260

-

0

0.4

0.2

lti

0.5

9.0

0.7

0.2

65

0.3

11.8

0

1.8 1.2

0.8 0.3

203 59

0.8 0.2

50.5 8.0

0.4 0.6

1270 205

-

6.8

4.3

135

0.4

12.5

1.3

1080

-

TABLE

III

INIIIBITIoN OF EI,E;(.TRoN 'Ikassro~~ CYTWHROYE (‘ BY LkN1'IMTCI~

~0 A

Mitochondrial and microsomal fractions were prepared and assayed as described under Ezperi,rlenlal Jfethods for succinltteand I)PNHcytochrome c reduet.:ise activities, with and witllout the addition of 5 X 1OF JI antimycin .4 to the reaction mixture. Exlr value tabulated is the mean of assays cm four to six preparations; individual values fell within f4 of the tabulated mean for eitc h fr:xt,ion. Per rent electron Source

2&hr.

of cell

fraction

whole embryo O-day embryo liver 3-&y chick live1 (i-day embryo heart

From surrinate mitechondria

%i

in

inhlbition transport

From DPNH in mitochondria

75

of From DPNH in mirrosomes

1

mitochondria of B-day embryos; moreover, the factor of difference from the (i-day embryo levels was markedly different for various enzymes. Microsomal preparations of both heart and liver possessed high activities (Table I) for antimycin A4-insensitive Dl’NH-cytochrome c reductase (see Table III for inhibition data), which is the most nearly specific microsomal oxidative activity tested, and also possessed high activities for DI’SHferricyanide reductase and for TPXHcytochrome c reductase. The low activities for cytochrome oxidase and succinatecytochrome c reductase are believed to represent slight cont,amination by mitochondrial fragments. This interpretation is supported by the fact that both of these t,ypically mitochondrial activities increased progressively in roughly parallel manner if the tissues were subjected to progressively prolonged homogenization prior to fract,ionation, whereas no such increase was observed if the final microsomal preparatiolls were subjected to additional homogellizatioll. The low lcvcls of “DI’K-stimu-

lated Tl’SH-cytochromn c reduct.ase” and of cholilie-cytochrome c reductase may also, at least in part, reflect contamitiation by mitochondrial fragments since they too increase, in approximate proportion to succiliate-cytochrome c reductase contellt, whet1 tissues were subjected to excessive homogenization prior to fract,ionation. Of the activities that probably are intrinsic to the microsomes, Dl’IiH-cytochrome c reductase and DPr\;H-ferricyanide reductase levels in B-day embryo liver aud heart microsomes were greater than in Xhr. embryo microc somes, while the TPNH-cytochromr reductase level was similar in 2-l.hr. and G-day embryo preparations; all three activities were increased markedly, though by different factors, in the Sday chick preparations.

Table II summarizes the results of experiments to determine the degree of cow st’ancy in the relative levels of various Nzyme activities within a cell fraction and the degree to which these relative levels change in the course of the embryonic differeutiation of individual tissues. For each individual preparation, the activities of the various oxidative enzymes were compared to the activity of a specific enzymic marker. Cytochrome oxidase was the marker chosen for mitochondrial preparations, as this activity is concentrated solely in the mitochondria; DI’KH-cytochrome c reductase was the best available marker for microsomes, since this activity was almost CIItirely inseiisitive to antimycin A in the microsomal preparations assayed, and the absence of sensitive DI’SH-cytochrome c reductase indicat,es lack of cont,amiuation by mitochondrial fragments (see discussion of Table III below). The results in Table II indicate that the relative activities of the electron transport enzymes intrinsically linked to a cell fraction are constant for preparations obtained at a given stage of development. The standard deviations are of the order of .j-lO ‘;. of the mean vahles for each of the mitochondrial activities assayed, except fol choli~lc~cytochrome c reductase. Similar

cotlstancy of ratio is secu for DPKHand TI’SH-cyt,ochromc c reductase and 1)1’S H t’crricyanide reductase it1 microsomes. In contrast, the variable levels of the low cvt,ochromc osidase and succinate-cyt~orhrome c rcductasc activities iii microsomal preparations reflect the fact that these are variable cotlt~aminatlt~ activities; this itltcrpretat,iott may also apply to t,hc !ow “I)I’S~stimulated TI’XH-cytochrome c rrductase” and clloline~cytochrome c rethxtase activities itr microsomal preparatiotia. (‘omparisott of the activity ratios ilt diffrrettt types of mitochondrial and microsomal preparatiotts (Table II) indicates that tltc relative activities of ccrtaiti particulatclinked electrott transport enzymes are sigttificant,l.y different in dif%rrttt~ tissues 01 may chattgc within a specific t’issue itt the coutw of developmctit. The significatwe of these changes iti relative activities is coitsidtred i11 the Discussion. INHIHITIOX

STI~DIES

To t#est whet~her the character and iutet*relatiolts of the electron tratisport enzymes change during embryottic development, the ef?‘ccts of several classical iuhibitors were tested. The data in Table II1 itldicat,e that the atltimycin .\ scnsit~ivities of heart atld liver preparations from cmbyyos and chicks arc similar to the sctisitivltics previously f’otmd (11) for adult chicken fractions. Thus, sttccinat,c-cytochrome c reductase was cssetttially completely itthibitrd in both heart atld liver mit~ochondria; the low activity found in microsomal preparations, prcsumablv as contamination, also was sensitive to the “inhibitor (data not tabulated). The I)t’SH-cytochl,ome c reductase of heart mitochondria was almost completely itIhibited by 5 X lOPi ;I/ antimyciu :I, whereas ottly about, 70 “; of the DPr\;HPcytochrome c rcductase associatjed with liver mitochondria was sensitive to the inhibit’or at, Irvels of either 5 X lo-’ .U or (ttot tabulated) I X lo-” JI. The partial inhibition in the livtr indicates t,hat the adult pattern, with botlt sensitive and insensitive systems in rather dcfiiiitje proportions, appears car1y itt embryonic development of the liver.

The partial sensitivity of Dl’XH~~cytochrome c reductase in mitochondria from early whole embryo may reflect the presettcr of various types of mitochottdria, rathet t,han a sitigle type with the proportioti of settsitive and itwttsitive enzymes suggested by t,he degree of inhibition observed. Jlicrosomal I)P~HH~yto~hrollie c I’(‘ductase itt both liver and hcalt was essetttially itisetisit,ive t,o atitimycitt A. ‘I’lw slight inhibition observed, particularly with heart microsomes, may reflect cotit,amitlatioti by sensit,ive mit,ochondrial enzyme; ittdeed, seiisitivity to aiit,iniycitt Ii was iticrcased itt heart nurrosomal preparations that, cotttaitted appreciable cytochrome oxidase or succittate~cytoclltoriie c reduct’ase activit’ics as the result of esccssive honiogetiizatioti of the tissue prior t,o fractionat’iotl (data not t’abulated). The addit’iott of 1 X IO-:’ .I/ KCX produced 100 ‘; itiliibitioti of the cytochronie osidase act’ivitics it1 all mitochondrial fractions and 90-100 ‘4 inhibit,iotl of the DI’NH oxidasc activities of the mitocho~tdt~ial fractions, indicating that the bulk of tcrmital electron transport to oxygen is futuielled through the cyaltidePscttsiti\c cgtocltromc chain (dat,a ttot, tahulat,ed).

Difference spectra of mitochondrial atld microsomal preparations were obtained to determitte whether significant changes it1 the nature of the hemoproteins associated with the respiratory chain occur during devclopmerit. I:igures 1 and 2 present typical difference spect,ra obtained wit*11 freshly prepared heart and li\:er preparatious from !I-dav chick embryos (Stage 36). Both heart and -liver mitochondria (l;ig. 1) show the typical absorptiotl maxima of cytochromcs a + a:t, b, and c + Q. Furthcrmorr, the relative heights of the xrarious praks above the adjacent’ mitlima are approximately the same for heart and liver mitochottdria, indicating that the relative proportiotts of the cytochrome compouents in the mitochondria of these t’wo tissues arc similar. Although thrse spectra do not permit, strictly quatititat~ivc calculations, the cow centration of cytochrome compotirtits prt

300

STRITTNL4TTER

-:::$y, (,, ,,,,I:;:I,(,,,,,,,,,,, 400

450

500 WAVELENGTH

FIG. 1. Difference spectra of mitochondria obtained as described under Experimental and one reduced with sodium hydrosulfite. heart mitochondria (4.5 mg. protein/ml.).

500

550

600

(m,u)

from May chick embryo liver and heart. Kach spectrum, Methods, represents the difference between an oxidized sample Curve A: liver mitochondria (7 mg. protein/ml.). Curve R:

milligram protein is also seen to be similar in mitochondria of both tissues. The cytochrome components in the embryo preparations appear to be in a functional state, since (a) anaerobic addition of DPKH or succinate resulted in nearly the same degree of reduction as was obtained with sodium hydrosulfite as reducing agent, and (0) aerobic addition of succinate and antimycin A, which blocks electron transport to cytochrome c, resulted in partial reduct,ion of only cytochrome b. Difference spectra obtained with A-day embryo heart and liver mitochondrial preparations (‘i mg. protein/ ml.) did not show sufhciently distinct peaks in the visible region t’o permit certain characterization, but the profiles and relative heights of the (a + aa)y and the by maxima in the near-ultraviolet region resembled those of the !&day embryo preparations. The profiles of difference spectra obtained with heart and liver mitochondria from 3day chicks resembled those shown for the g-day embryos, suggestingthat no significant change in the nature or relative concentrat,ions of the various cytochromc components had occurred. The difference spectrum of a washed liver microsomal preparation from g-day embryos (Fig. 2, curve il) suggeststhat a microsomal

cytochrome b5-like material, with absorption maxima at about 325, 527, and S.57 rnp in the reduced state, is the predominant intrinsic hemoprotein component8.The cytochrome bb-like nature and functional state of this microsomal pigment was further suggested by the fact that it was not reduced by anaerobic addition of succinate but was largely reduced by DPKH. The difference spectrum of liver microsomes from is-day chicks closely resembled that shown for Y-day embryos; dilute preparations (lj mg. protein/ml.) from &day embryo liver also showed a low maximum at Uj mp and minimum at -I12 rnw, but the profile was not sufficiently sharp for further chal,acterizatioii. The difference spectrum of microsomal preparations from !&day embryo heart (I:ig. 2, curve R) suggeststhe possible presenceof a cytochrome bsmlikecomponent in low concentration, but identification is uncertain; more concentrated preparations from ?-day chick heart suggested such a component more strongly, but small amounts of contaminating mit80chondrial cytochromes somewhat obscured the spectral profile. It was essential to wash microsomal preparations with a suitable electrolyte medium in order to remove such obscuring contami-

301

I

400 FIG. 2. Difference obtained as described and one reduced with lieart microsomes (10

I

I

I

I

i

450

I

I

I

I

I

I

I

500 500 WAVELENGTH (my)

spectra of microsomes from under Experimental Methods, sodium hydrosulfite. Curve mg. protein/ml.).

(

I

I

1 I

550

‘3.tiny chick embryo liver represents the difference .Z : liver microsornes ill

nauts as hemoglobin, which is strougly adsorbed to microsomes in sucrose media (1.5, 16) and whose difference spectrum somewhat resembles that of cytochrome b5. In contrast to such adsorbed substances, the cytochrome bs-like component of live1 microsomes was not removed even by multiple washings with Tris buffer.

To det.ermiue t.he validity of enzyme activities chosen as markers for mitochondria, the ratios of various activities were compared in a series of preparations from :%-daychick heart and liver, including whole liomogciiatle, mitocholldria, aud au “iiitcrmediate” fraction (3000-l 5,000 X g), RMl in microsomal preparations grossly coutaminatcd with mitochondrial fragmcnt,s as a result. of excessive homogenization of the tissues. In all preparations, the ratio of succillate-c?itochromr c rrducbase sctivit,y to cytochrome osidase was the same as in the standard mitochondrial prcparatious, within the limits of variability indicated for the standard mitochondrial preparat8ions in Table II ; these markers therefore do specifically reflect mit~ochondritl activity. 111 eont,rast, t,he rat’io of I~l’~H~cyt~)cl~~~(~mec rcductasc activity t,o cytochrome osidase

I

I

I

I

I

600 and heart. Each spectrum, between nn oxidized sample mg. protein/m.), Curve H:

varied in proportion t’o the mixture of microsomes and mitochondria in the prtparations. 111other experiments, t’he ratios of succiliate- and cholille-cytochrome c reduct,ascs to cytochromc oxidase were compared in heart and liver mitochondria prepared from tissues subjected to the normal amount of homogenization or to either one-fifth OI five t,imes this amount, of homogenization, aud ilt mitochottdria subjected t.o further homogenization after isolation. The various activity ratios remained constant under t,hesedifferent preparative procedures, withit1 the limits of variability encount~cred in standard prcparatious. Such constaucy despite varyitig treatment of the t,issues suggests that the enzyme differences conlpiled in Table II reflect, intrinsic differences brtweetl the tissues rather than artifacts of preparative procedures.

The devtlopmcntal stages used in this study were chosctl t’o represent distinctive periods of embryonic differentiation. The Whr. chick embryos represent an early and relat.ively undiffereutiattd state prior t,o emergence of definitive structures and fully differeutiated tissues. ‘The (i-day chick

embryo liver represents an emerging organ which has just completed development of characteristic morphologic features and a marked increase of cell size (17) and is about to begin a period of major functional differentiation. On the seventh day, glycogen and, apparently, cholesterol first appear in the liver (18), the respiratory rate of the liver in v&o decreases (lY), and bile secretion apparently begins about this time (20). The (i-day embryo heart, in contrast, has been in a functional state for some time, as the first heartbeats begin after about 30 hr. of incubation and circulation of blood after about T,O-55 hr. (17). The S-day chick liver and heart represent these organs in essentially mature form and functional state; the biochemical characteristics observed with these chick tissues in the present study resemble closely those previously found with the adult chicken tissues (11). In assessing the data obtained on electron transport activities and their variation in the course of development, several factors should be borne in mind. I:irstly, the assay conditions employed are somewhat arbitrary, and the quantitative relationship between the activity level measured in vitro and the precise physiologic capacity for that enzymic activity is therefore uncertain. However, since the preparative procedures and assay conditions were chosen to elicit high activities, with a minimum of uncontrolled variables, and since the assays on all preparations were performed under as nearly identical and controlled conditions as feasible, the activity levels obtained were reproducible and the results appear to be reliable and valid for the comparative purposes of this study. Secondly, most of the activities measured represent operationally defined spans of an electron transport chain involving more than one component, and the pathway of electrons through these spans is somewhat uncertain. As example, the activity here designated TPSH-cytochrome c reductase may reflect electron transport through a pathway analogous to DI’NHcytochrome c reductase, but it is possible that the observed activity also involved some electron flow from TPKH to DPK via a transhydrogenase mechanism involving firmly bound DPX and then from

DI’KH to cytochrome c via DPSH-cytochrome c reductase. The “DPN-stimulated TP?;H-cytochrome c reductase” activity may reflect electron flow from TPKH to DPK via a TPiYH-DPN transhydrogenase utilizing added DPS and then to cytochrome c via DPNH-cytochrome c reductase. Since the DPSH-cytochrome c reductase activity of each preparation studied was always greatly in excess of the observed “DPK-stimulated TPNH-cytochrome c reductase” activity, the latter activity may provide a measure of TPKH-DPK transhydrogenase activity; however, this measure would be inaccurate to the degree that’ TPSH-DI’S transhydrogenation was a component of the observed TPKH-cytochrome c reductase activity without added DPN. Thirdly, the mechanisms and components involved in activity changes may not be precisely definable. An observed change of activity level may not reflect a change in molecular concentration of an enzyme, but rather may result, at least in part, from an altered functional capacity, e.g., an intramolecular or intermolecular alteration of structure that affects the capacity for electron transport. Differentiation in the sense of such functional changes cannot be excluded, but the relative constancy of activities observed in a cell fraction following varied pretreatment does not suggest such a mechanism as a predominant factor. The component whose variation was responsible for an observed activity change cannot be precisely defined, since the activities measured represent a span of the electron transport chain rather than the function of a single molecular species, and the change may be referable to any one or to all of the components involved in that span. The data of Table I suggest that the general levels of particulate-linked electron transport activities in mitochondria and microsomes increase during development. The apparent increases of specific activities are most obvious in comparing preparations from C-day embryo heart and liver with those from 24hr. whole embryos but, particularly in the microsomes, there may also be significant increases in heart and liver duriug subsequent stages of development.

In part the low activities of early whole embryo preparations probably reflect the relative inhomogeneity of these preparations, but the observed changes are not entirely attributable to this factor. It has previously been reported (6) that the specific activities (per milligram protein) of cytochrome oxidase, DI’KH-ferricyanide rcductase, and Dl’KH oxidase in whole chick embryo homogenates increase about sixfold bet)ween the first and fourth days of development. The present data, obtained with wellcharacterized and fairly homogekleous particulate preparations, suggest more specifically that the increased levels of electron t#ransport activities represent increased cow centrations of activities within individual mitochondria rather thall increased amounts of mitochondria in the preparations assayed. In the sense of absolute increasesin osidativr enzyme concentrations within mitochondria and microsomes, there thus appears to be differentiation of these cytoplasmic particulate. I’reliminary attempt,s during the present study to correlate t,he number of christac seen in electron micrographs of mitochondria with the general levels of oxidatiw enzyme activity were inconclusive. The data summarized in Table IT indicate that the wlatiw specific activities of \-arious electron transport enzymes specifically hound to mitochondria and microsomes also change significantly during development, suggesting that, these cytoplasmit particles also differentiate in the senseof a chnngillg relative pattern of part.iculatclinked enzymes. The patterns of relative act’ivit’ies (compared to cytochrome oxidasc activity) seen in mitocholldria from &day embryo heart, and liver are significantly tlifferetlt, ill some respects, and one or hot.h of t,hesr preparations differ in certain respects from t’lie pattern seen in mit,oc*hoiidria from the 24.hr. embryo. Specifically, the (i-day embryo heart’ mitochondria possesssig;llificantly higher relatiw acti\-it,ies of ~~lccillat,c-cyt,ocllromc c l,eductase alld I>I’XH -ferricyanide rrductaw than &her (i-day embryo liver or 24.hr. embryo, alld hot11(i-day embryo tissuesshow significantly lowcr relative levels of TI’NH~cvtochrome e reductasc alld a significantly higher level of “I)I’N~st,itHulat,e(l TI’NII ~cytocliromc (

reductase” than the ‘24.hr. embryo. In addition, several changes occur wit,hin heartr and liver between the G-day embryo and ?-day chick stages, and the over-all patterns in the “mature” chick Cssues are more divergent than in the embryo tissues. These relative activity changes include significant’ declines of Dl’SH-- alld choline-cytochromc c ~eductases alld a possibly significant, decline of Dl’~I~~-fert,icyallide reductase iti heart mitochondria: in liver mit,ochondria there are significant iiicreasesiu the relative activities of TI’SH -cyt.ochrome c reduct.asc and “Dl’K-stimulated TI’NH- cytochromr c wductase” and possibly of cholin~~wytochrome c reductase. I II the microsomal fractions, the preparations from (i-day embryo tissues possesssignificaiitly lower relative aet.ivities of Tl’r\;II~c~tochromc c reductase atld DI’KII ~fewicyanide reductasc (compared to DI’SH -cytochrome c reductase) than do %I-hr. embryo prcparatiolls; ill addition thew are possibly aigllificatlt differences iti these ratios between heart and liver microsomcs, particularly in preparations from :&day chicks. The changes IF fcrred to here as “significant” all showed significance at the I’ < 0.001 le\rcl iti the t test (Zl), and those labeled “possibly significant” gave f’ values of < 0.01. With respect to cytochromc compoiieiit~s, heart and liver mitochondria ill May embryos and, apparently, also ill (i-da.v embryos, already contailwd the t,ypical cytochrome complemelit of adult vcltcbrate mitochondria; the relative colwctlt.rat8ions of these components were similar iii t,tie two t,issucsalld not markedly diffrrellt from the patter11 seen in :&day chicks. ‘I’hcsc results are consistent with obser\-atioiis reported for mitochondria of I-L-day chick embryo tissues (2”) and of 4-day and .S-day whole embryos (-5, 22). In additioll, liver microsomes from !&clay embryos alid, apparelltly, (i-day embryos already possesseda cytochrome bs-like component trypica 01 adult liver microsomes. Cert.aill idetltifiratioli of the apparelitly similar absorbing material in embryo heart niicro~omes is not possible at present. The challgcs ill relative uct,ivity levels reported here appear t,o reflect, the diffcre~ltiatioli of orgalrized paiticulatr c~lr~t.ro~~

STRITTMATTER

transport systems in mitochondria and, possibly, in microsomes in the course of embryonic differentiation. With respect to the previously discussed criteria of validity and interpretation of such apparent changes of enzymic activity, it appears probable from the magnitude of the changes observed under carefully controlled corlditions that at least some of these changes reflect significant alterations in the levels of activity or concentration of certain specific segments of these particulate electron transport systems. With these reservations of interpretation in mind, the present results may be cullsidered in terms of current, views (l-3) regarding mitochondrial composition and organization. The organized electron transport, chain of the mitochondria includes pathways for electron transport to oxygen from DP?;H and from succinate. These pathways, despite uncertainty regarding their degree of independence, may be described in terms of several spans or subunits: (a) terminal electron transport from cytochrome c to oxygen, (0) succinate to cytochrome c, and (c) DPXH to cytochrome c. The components comprising these subunits are believed to be present in definite stoichiometric ratios within the organized mitochondrial unit. The mitochondrial unit also contains specific dehydrogenating complexes @-hydroxybutyric, pyruvic, a-ketoglutaric, malic-isocitric, and fatty acid) that feed into the electron transport chain at the DPNH level, and are considered to be present in specific stoichiometric amounts (2). In addition there are present firmly bound particulate capacities for electron transport to oxygen from such substances as choline (l-L), TPKH, and a-glycerophosphate (23); some of t,hese capacities may also represent standard subunits or components of the mitochondrial electron transport unit,. The present results are generally consistent with these views, in that (a) there may be subunits of a definite composition, e.g., the constant ratio of cytochrome components in the terminal span of the chain; and (b) the submlits may be present in a constant ratio characteristic for a given cell type. The present results further suggest

that the relative numbers and/or activity levels of various subunits or spans within an organized mitochondrial unit may vary in different tissues and within a given tissue at different stages in the development of an organism. It is not yet clear in what sense or degree this apparent differentiation of organized particulate electron transport units may involve the differentiation of a heterogeneous population of cytoplasmic particulates. Differentiation of a tissue might involve (a) appearance of a single 01 predominant type of mitochondrion that possesses electron transport units of a composition and activity specific for a cell type, (b) appearance of several such specific mitochondrial types in a ratio characteristic for a cell type, or (c) presence of several mitochondrial types common to many cell types in a ratio characteristic for a given cell type. The possibility of enzymic heterogeneity in the mitochondrial population within a cell type has often been noted, but present evidence does not favor such heterogeneity [cf. (24)]. In concurrence, the present findings that the relative activities of specifically mitochondrial enzymes are very similar in homogenates and in various part,iculate fractions suggest that if enzymitally different types of mitochondria exist in a cell type, these types are not readily separable by the usual differential centrifugation procedures. A(:KNOWLEJ,GPVIE?;TS The aut,hor wishes to thank Dr. Norman Sulkin, Mrs. Dorothy Sulkin. and I>r. Jean-Paul Revel for generously carrying out. Ohe electron microscopic examinations, and also wishes to acknowledge the technical assistance of Mrs. Doris ,4shton and Mr. William Bell in this study. REFERENCES 1.

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