Control of cytochrome P-450 in rat testis mitochondria by human chorionic gonadotrophin

.UK!HIVEH

OF BIOCHI~~MISTltY

Control

;LN,l

JIIOPHYSICS

of Cytochrome by Human

J. L. PUWIS,

Chorionic

J. A. CANICK,’

of Hiochemis/rq,

32-38 (1973)

P-450

ANU

Department

169,

in Rat Testis Mitochondria Gonadotrophin

,I. H. IWSENBATJM, S. A. LATIlc

ITniversily

of 1Zhotle Island,

Received

,J. HOLOCXITAS,

Kings/on,

Rhode Island

02881

Jrme 20, 1973

Cytochrome P-150 cannot be detected spectrophotonlctricitlly in testis mitochorldria of untreated rats because of the high cytochrome aa to cytochrome P-350 ratio. Injection of human chorionic gonadotrophin (IIC(:) causes a large increase in mitochondrial cytochrome P-350. After 14 days injection, mitochondrial cytochrome P-450 levels are increased 15. to 30-fold (from 0.007 to 0.13-l nmoles/mg protein) river control levels. Levels of cytochrome a + aa are not altered by this treatment. Mitochondrial cytochrome P-450 can also be demonstrated by injection of HC(; into rats which were hypophysectomized 24 days previously. I)uring hypophysectomy the mitochondrial cytochromes c + cl, a + a3 and mitochondrial protein decay with halflives of 11, 16, and 15.5 days, respectively. IlC(: t,reatment for 8 days increases mitochondrial cytochrome P-450 (from <0.003 to 0.21 nmolrs/mg protein) without, altering the levels of the other mitochondrial cytochromes. The control of c,yt,ochrome P-450 levels in the mitochondria by HCG suggests that the level of this key romponent of cholesterol side-chain cleavage enzyme may be of irnport,arrcc in the regulat.ion of steroidogenesis in the testis,

Although rat testis mitochondria possc~ss cholrsterc~l side-chain clcavagc> activity (1, 2), the prosthetic group of this enzyme, cytochromc P-450, has not been detected in rat testis mitochondria dcspitcl a thorough search (2, 3). The apparent ahwnw of cytochrome P-450 was most surprising sinw this cytochromc has previously bwn found in t,h(> mitochondria of othw stwoidogenic organs such as t)ho adrenal (4-G), ovary (7, S), and placenta (9, 10). Marc recently Mason et al. (11) haw shown that c?-tochromes a + a, may intcrfcre wit,h t,hc d(btc&on of mitochondria,l cytochromc P-450 by shifting the absorbance maximum of th(L carbon monoxide complex of cytochromcb P-450 to higher wavrlcngths as ~11 as dwreasing the 450-490 nm absorbance dificwncc~ cnntribution of cytochromca 1’.450. 1 Present address: Department and Gynecology, IIarvard Medical ton,

mm.

of Obstetrics School, Bos-

Cytochromc: I’-450 can bc mcasurcd undw these conditions by dtrtrrmining the optical diffrrcncc~ spwtra at 460 minus 490 run using an extinction wc#icic~nt of 52 cm-’ m0 as dcscribcd 1)~ Iiowal et al. (12). The diffcwncc: spectrum of th(b cytorhromc~ oxidasclPcarbon monoxid(b complex has onl!, a small spwtral contribution as t hew ivav(:wcn under t how cow lclngths. Howww, ditions of cytochromc~ P-450 dot,ermination, the combination of lon cytochromc I’-450 lcvcls and high cytochromc~ u + u:i/cgtochrome I’-450 ratio make th(> dctwtion of cytochromc: I’450 uncertain. ,Jefcoatc: (13) has attc>mpt,c:d to circumwnt thcsc: difficulties by titration of t~he mitochondria with aminoglutc~thin?id(: which in oth(sr stwoidogcnic t,issws binds only t,o mit,ochondrial cytocahromcl l’-430. Since Mcnard and I’urvis (14) have: drmonstratcld that rnicrosomal cytochronw P-450 in chick Wt,is is undr,r the> cont’rol of LH and Mrnon rt al. have: shown that

HCG* incrcawd cholwtcrol sidechain (*IvRv- 100 mM phosphate buffer or 50 ml1 Tris buffer pH age: activity in testis mitochondria, VX~:C’I i- 7.4. Cytocahrome 1’.450 was determined from t,he carbon monoxide plots dit.hionite minus dithionite mctnts wrct d(~signctdto SW if th(%cytwhromc~ difference spectra at 400-490 nm using an extinca + aJcytochromc P4.50 ratio could lw tion cocficient of 5% as described by Kowal el al. altwcd by (1) incwasing cgtochromc 1’.4.50 (12). Cytochrome ~1 + aa was determined by the 1~~~1s in th(l mitochondria with HCG in- redllccd minus oxidized difference spectra at 445jwtion, (f~) incwaning cgtochronw I’-450 465 nm or at, 605Mi30 rim IIsing extinction coeffiIwt~ls wlatiw to cytocliromc~ a + a3 hJ cient of 91 and 16, respectively (18 ). Cytochrome l&ting the individual cytochromcls dwa) c + cl was determined in a similar way r:sing the aftw hypophywctomy and suhsrqwntl~ wavelength pair 550-540 nm and an caxtinrtion coefficient of 20. injwting HCG to incrww mitochondrial caytochromc> I’-4-50.

Maierials. Aminoglutethemide (Rlipten phosphate) was a kind gift of I)r. J. J. Chart of Ciba Pharmaceutical Co., Summit, New Jersey. NAI)PII and human chorionic gonadotrophin (HCG) were obtained from Sigma Chemical Co. Preparatory procedures. Intact and hypophysectomized Sprague-I)awley rats were purchased from Charles River Co. and were maintained on laboratory rat chow diet and water ad lib. Animals were killed by decapitation and the testis and adrenals rapidly excised. Homogenates of testis were either prepared in 0.15 M KC1 or 0.25 M sucrose rising a Teflon-glass homogenizer at a ratio of 100 mg w-et wt of tissue per ml of KC1 or sucrose. In Method 1, rnitochondria were prepared either from the SIIcrose or KC1 homogenates by differential centrifugation at 1OOOgand 9OOOg. Tn Method 2, thr mitochondria were prepared and were homogenized in 0.25 M sucrose and the nuclei sedimented at, 1OOOg.The nuclei were resuspended in sucrose and resedimented at 1OOOg.The combined supernatants u-ere centrifuged at !lOOOg to bring down the heavy mitochondria. These fractions were resuspended in 0.25 M sucrose and recentrifuged at 9OOOg. Iiicrosomes were prepared as described b> Purvis ef al. (15). Protein concentration was determined by the billret method of (:orwall e( ctl. (l(i). Completeness of hypophysectomv was checked RS described by Purvis e( al. (15). HC(; was inject,ed SC every 12 days at a dose level of 50 or 100 IU/irljection. Spectruiohotonletr!/. I)iRerence spectra of turbid suspensions were recorded at room temperature in a Gary Model 15 Spectrophotometer equippet with a high intensity light source as described b) Menard and Purvis (17). Xlitochondria were diluted to the desired protein concentration with . --.___ * Abbreviations: HC(;, human chorionic gonadotrophin; LH, luteinizing hormone; FSH, follicle stimulating hormone; izG, aininogllltethemide; Tris, 2 amino-2 (hydroxymethyl).1.3 propanrdiol; pregnenolone, 5-pregnen-3 /i ol-20-ottc.

-

HCGl4-

-.-

-041

: s : :

I t

I

410

1

I

430 WAVE

,

‘.:’

HCG

4

-HCG

4

----.

HCG

-

CONTROL

2

’ : : : ,

450 LENGTH

I

,

470 (nm)

I

490

FIG. 1. A spectrophotometric comparison of cytochrome P-450 levels in testis mitochondria in control and HCG treated ra.ts. Difference spectra of trlrbid suspensions were recorded at room tempera.ture in a Cary Model 15 Spectrophotometer eqrlippetl with a high intensity light source as described by Menard and Purvis (14). The carbon monoxide + dithionite minus dithionit,e difference spectra of testis mitochondria from control and HC(+ treated rats from 400~490 run are illustrated in Fig. 1. The mitochondria were prepared inO. M sucrose and were isolated from the 9OOOg pellet. Protein was determined by the biuret method of (~orwall e( al. (IG). The mitochondrial mg protein/ ml in each experiment was: Control 5.9; HCG 2 days, 8.7; 1ICC; 4 days, 6.4; HCG 9 days, 6.5; and IIC(: 11 days, 4.8. HCG treatment consisted of f-lC(+ 100 ITT twice a day for t.he number of days indicated.

34

PUR VIS lo’

‘rh? dc,monst,ratiorl of c:ytoc*hrornck I’-450 in rat) twtis mitochondria by HC(; injwtion is aho\vn in Fig. 1. No cgtochronw P-450 can be xpwtrally obswvc~d in matuw control rats dw to thcl porturhation by thca cytochrornc~ osidasc: qwctrum whicsh in its carbon monoxide form has ;L large trough at 445 rim. Cytocliromc~ I’-4,50 cm clewI) hc SWII after HCG tJwatrnc~nt for 4 days and incrcwcs up to at, Iwst 14 days. ‘l’h~ pclak of tkw (‘(1 $- dithionitc, minus dithionitc> diffcwnw spc’ctra at 4 days is at 45s IJJJJ and shifts to 452 urn as the, amount of cytochrome &I50 increasw. l’he dwwuse in the trough at) 4-L> nm lvith the incwasing durat,ion of HCG twatmont is cauwd by t 1::s mcrtxsing amount of cytochromc~ I’-450 which pulls up the trough of the wrbon ~norlo~idr~~~tochrom(~ CL:,c:ompl~~s at 445 11111.

‘~ht’

cp%nthtiVc’

~lSS(!SSIll(Wt

Of’

th!

effwt of HCG on cytochromc! a + acl and c\:tochromc~ I’-450 in intact. rat.9 is slice\-JI in Tahlc I. The an1ou1It of c*ytc)c:hronw I’-4.‘0/2 twtis and cytwhronrcl I’-4.50.‘mg protc,in iricwasc~ with time aftctr H(‘(: injwtion. Aftw 14 days, mitochondrial q+ochromc P4.50 Iwc~ls arc incwwcd 1.5. to :30-fold

Treatment

1. Control (3) IICG 4 (3) IICY: 7 (3) HC(: 9 (3) 2. Control (3) 11tx: 2 (3) HCC 4 (3) HCG 9 (3) HCC 14 (3)

,,er 2T

P-450 nmoles/‘2’1’

10.50 !) GO !).30 11 .OO 12.00 11.53 1s .34 13.70 10.65

(a,0 .05 0.15 0.24 0.54 (F&0.0!, 0.14 0 .3‘ 0.85 1.43

mg Protein

A I,.

(from 0.007 to 0.134 nrnol~:s/mg mitechondrial prot,cin) ovw ckstimntcd c*ontrc)k kwc~ks. T%)th th(, :iriiount (Jf lllitoc~horldri;ll protc+l and thcl Iwc~l (Jf c~ytochromc~ a + a:c/mg protc~in aw not :tff(Y*tcd by HCX irijc~ctiori. Tkw dwrcxw in thcl n + as/l’-450 ratio 1s thP :IJllOUJlt 01’ ~~~tOChI-OJll~~ I’-‘$50 iS iJlcr~Wd

iS

ShO\vIl

iI

the’

IaSt

~~~~UlllJl.

(I(JIl-

trol Iwc+ haw a vc’ry high ratio \\hich apprwwhcs 1.0 aftclr HCX injwtion. When norm:Ll rats :w h\~~o~~h~sc~c~tornizc’d t 1~1~ t,c*stw at rt,J,hy :ind t11c>IWC~lSof Init.c)chondrial ])rotclin and the Iliit,oc,hoJldriak c~~toc:lironws I’ + cl arid a + (I:$ dwrcwc,. Half-liws (tl 2) arc cnlculatc~d bv plotting the dccwaw loFS~rithnlic,all~, as a func+ion of time aft,ctr hSI)~)I)ll~s(,c,to~ll!‘, (I’ig. 2). The half-livw of tcbstis \I(+ \vcGght, 1X days; 111g rnit,ochorldrial protcbin, 15.5 days; cytoc*liromc: c + cl, 16 days; and cytwhromct a + u3, 14 daJ,s arc all wry similar and much greater than the dway (of microsomal cytwhromct 1’.430, fl 2 = :3.8 days (l(i). hftw 24 days of h~:)oph~sc,c:torny t,htt mitochondrinl cytcwhromc~s have dwlinc~d to 2.5-30 ‘,? of thclir control I~vc~l. Thr dway 01 nlit,oc:liorldri;tl cytcwtiromc~ I’-4,jO cwmot tJ(b nwasuwd in this cxpwimcwt dw to thc$

a + aa nmoles/2’1‘

P4.50 nmoles/mg

‘2. 1 2.2 1 .5 1 .!I 2.2 2 ‘2 2.5 2.1 2.1

(i7$0.005 O.Ol(i 0.0% 0.040 @0.008 0.012 0.024 O.O(i2 0.134

a + a3 nmolesimg

0.20 0.23 0.16 0.17 0.18 0.17 0.19 0.15 0.20

Ratio a + a3/I’450 a.0 14.7 G.3 3 5 24.2 15.6 8.0 2.5 1.5

Li iYorma1 male rats weighing 290-310 g were injected twice a day with 50 IU (I’kpt 1) and 100 IU (F,xpt) 2) of human chorionic gonadotrophin (HC(:) per injection for different, number of days and were assayed for mg protein, cytochrome P-450, and cytochrome a + no per 2 testis. The nrm~bers after HC(; indic:tt,e the number of days of HC(+ treat.ment. The numbers in parentheses are the nllmber of animals. Mitochoudria were prepared in experiment 1 in 0.15 M KCI, pH 7.2 and in Ikpt 2 in 0.25 M sucrose. Cytochrome P-450 was assayed by the method of Kowal e/ al. (12) at 41iO-490 nm losing an c = 52 cm-’ mu-‘. Cytochrome n + a3 was determined by the reduced minus oxidized difference spectrum at 445-465 nm using an E = 91 cm-1 mM-‘. Where the rytochrome P-450 vallles are inaccurate due to the high nail’-%50 ratio, the values are estimated and designated by @j. Alitochondrial prokin was determined by thrt biriret method of (iorwall et (~1. (10) and is expressed as mg protein/ZT.

hJ\v lrwls in control rats. Injection of HCG brginning 24 days aft’er hypophyscctom> causes a large increase in the lcvcls of cgtochrome 1’.450/mg protAn without altering thca lr~c~ls of c\-tochronw n + ail or cytoc~hronw c + cl;‘riig mitochondrial prottkin (Table II). Mitorhondrial cytochromcb 1’.450 can also bc rcduccd c~nzymstically in the prcwncc: of carbon monosid(l by the c~~~z~rnes of that S;2DI’H-tclstodoxin-~~tochrom~ 1’.450 pathu-ay insknd of sodium dit,hionitcx as thcl wducing agent. Such an c~xpc~rimcnt ia shown in l’ig. 3 which comparw the d&rminntion of mitochondrial cytorhromc I’-450 by the dithionitc wduccd carbon monoxide minus reduwd diffcrf~nw spwtra with the SADPH reduced carbon monoxide con?plw minus SADPH rcduwd diff (wncc qwctra. In the>lnttcr case, the mitochondria prcincubatcd \vith KCN t(J prchave hrcn Frc;. 2. I)ecay of cytochrome o + a;, cytowrit cytochromr a3 from forming the rechrome c + cl, mitochondrial protein and testis duwd cytochromc aa carbon monoxide wet wt from the rat testis after hypophysectomy: calculation of half-life ([I,*). Cytochromes (t + (~3 rompl(>x. As can br swn in I;ig. 3, NADPH c,ffcctiwly rcduccd cytochrome P-450. Caland c + c,! mitochondrial protein and testis wet culation of th(t amount of cytochromc P-450 wt acre measured as a function of time after hywduccd by NADPH compawd to dithionite pophysectomy and plotted on three cycle semilog HCG was injected 24 days after hypopaper. using an cxt.inction coefficient of 5% from phgsectomv twice a day (100 IU/injection) for 8 -l(iO-490 nm revralcd that SADPH n-as days. Cytochrome a + LIP was measured as deSl ‘Z as cffrctive as dithionit,c. The taxtcnt scribed in Table I. Cytoehrome c + cl was measof reduction of cgtochromc P-4!jO in the ured as described by Cammer and Estabrook (18) pnwncc of carbon monoxidr depends on using an t of 20 at 550 minus 540 nm. The straight, thcb CO/O2 ratio (19). Since the CO/O, lines were drawn by least square calculations and ratio is higher in the presenw of dithionite the correlation coefficient r calculated. The correthan with SADPH, more cJtochrome lation coefficient r was 0.98 for each parameter I’-450 is wduwd. measured. The half-life values in three different Thaw rcsul ts on mitochondrial c\,toexperiments varied less t ban =t 10“;

Treatment

Control (3) Hypox 23 (6) Hypox 24 HCG 4 (4) Hypox 24 HCC 8 (4)

hlitochondrial protein mg ‘Z’I’

10.0 3.5 3.2 3.7

~~ 0 + da nmoles:

Cytochromes c + Cl nmoles/

~~ P450 nmoles/ -~~ mg ~_~____

2T

mg

2T

mg

.___ 2T

2 0 0.G” 0.54 0.G

0.20 0.18 0.17 0.18

2.95 1.00 1.05 1.15

0.30 0.29 0.33 0.28

@JO.05 0.01 0.53 0.90

0.005 0.003 0. ltiti 0.243

Ratio (L + PT50

25.0 GO.0 1.0 0.7

a Male rats hypophysectomized (Hypox) for 23-24 days weighing 180-200 g were injected twice a da) with 100 IU of human chorionic gonadotrophin, HCG, for from 4 to 8 days. Rlitochondria were prepared in 0.15 M KC1 pH 7.2 and the mitochondrial cvtochrfjme assays were as described in Table I.

36

PURVIS

chrome P-450 are not corrected for microsomal contamination. Assays of microsomal marker enzymes (i.e., cytochromc P-450 dependent l’icr-hydroxylasr and Cl?-

-0.03L 400

420

WAVE

440

460

LENGTH

460

500

(nm)

FIG. 3. Enzymatic reduction of cytochrome P-450 by TPNH in the presence of carbon monoxide in rats treated with HCG. The rats were treated with HCG 200 IU/day SC every 12 hr for 10 days and killed 12 hr after the last injection. The average body weight and testis wet wt were 368 g and 3.5 g/2 testis. The seminal vesicles weighed 2.72 g/2 testis (full). Mitochondria were prepared by method 2 as discussed in the Methods. One-hundred and fifty milligrams of mitochondrial protein was isolated from 20 testis. Two difference spectra of the reduced cytochrome P-450 carbon monoxide complex minus reduced cytochrome P-450 are presented. (O---O) Carbon monoxide plus dithionite minus dithionite. (O---O) KCN plus carbon monoxide plus NADPH minus KCN plus NADPH. In the latter experiment KCN was added to both cuvettes in 50 mM (final) Tris buffer pH 7.6 to bind cytochrome or. After 3 min carbon monoxide was bubbled through the top cuvette for 1 min and NADPH added to both cuvettes. Final concentrations of KCN and NADPH has 2000 PM and 125 F&I, respectively. Mitochondrial protein was 12 mg/ml in each cuvette in both experiments. Using an extinction coefficient of 52, dithionite reduced 0.036 nmoles and NADPH 0.029 nmoles of mitochondrial cytochrome P-450/mg protein.

Eli’ AL.

CT, lyase) show that 10 % of the cytochromr P-450 measured in th(> mitochondria is of microsomal origin. The activit,y of 17~ hydroxylase and tho C1,-C20 lyase in t,cstis mitochondria decreases from 14.3 5 to 4.0 ‘% and 9.4 % to 4.6 % of t,hr microsomal levels respect’ively as the mitochondria arc subjected to an increasing number of sucrose washes. Mitochondrial P-450/mg protein in HCG-treated animals remains constant during this washing procedure. Therefore, in the experiments in Tables I and II, about 10% of the microsomal cytochrome P-450 is mcasurcbd in th(t mitochondria. Since control and HCG 14.day microsomes have cytochrome I’-450 lev(~ls of 0.93 nmolrs/gT and 2.13 nmoles/ZT, wspcctively, it is clear that the cstimatcld lcwl of mitochondrial cytochrome P-450 in COIItrol rats of 0.09 nmoles/ZT can be accounted for by microsomal contamination. Howwr, in animals treatchd with HCG for 14 days, a 10 % contamination of the mitochondria (10% of 2.13 nmolcs/ZT) would lo\vcr t,hc mitochondrial cytochronw P-4.50 level only by 13 ‘Z. Thus it, is clrar that the cytochromc~ P-4.50 levels determined in t,he mitochondria after HCG treatment represent for th(h most part true mitochondrial cytochrome P-450. This conclusion is confirmed by aminoTABLE

III

BINDING OF AMINOGLUTETHIMIDE (AC;) TO ;CIITOCHONDRIAL CYTOCHHOME P-450’

Treatment

Control HCG 4 HCG 9 HCG 14

2. tein/ ml

Optical density A 425410/ml

9.0 4.0 4.9 4.8

0.0020 0.0075 0.016 0.020

A AG/mg

0.0002L2 0.00188 0.00327 0.00418

RAT

cytochrome P-450 nmoles/ mg 0.008 0.024 0.062 0.134

0 Mitochondria isolated after HCG treatment (Fig. 1) were used to study the binding of aminoglutethimide (AG). Difference spectra of oxidized cytochrome P-450 plus 500 PM (saturating) aminoglutethimide minus oxidized cytochrome P-250 were used to calculate the A absorbancy change between 425 and 410 nm with each preparation. Cytochrome P-450 values/mg protein are from Table I.

glutethimide expcrimcnts which react mainl! with mitochondrial cytochrome P-450 (Tablcb III). hminoglutcthimidr, an inhibitor of mitochondrial side-chain cleavage activity (20) has been found to bind with mitochondrial cytochromc P-450 in the adwnal (21). .Jcfcoatc (22) has recently usrd the binding of aminoglutcthimidc to testis mitochondria to estimato thtk amount of mitw chondrial cytochromr P-450 und&rctablc b\- spwtral methods in rat testis mitochondria. In Table III the binding of saturat,ing amounts of aminoglutcthimide is compared with the amount’ of cytochrome K4.50 determined in the preparation. *As the amount of mitochondrial cytochromc P-450 is increased by HCG injection, the amount. of aminoglutcthimidr-binding component is also increased. As indicat)ed by the wawlq.$h maximum and minimun+ aminoglutclthimidr gives a type II binding spectra. I’wgncnolonc n-as found t’o induce an invortcld type I binding spectra (not shown). AIason et al. (11) have shown that prcgnc~nolo~w induces type I binding spectra with pig testis microsomal cytochromc I’-450 and an inverted type I wit,h pig testis mitochondrial cytochromc P-450. DISCUSSION

The levels of mitochondrial

cytochromc in adult rats with intact pit.uitaries. This is the caw in the testis of most species examined b\ SIason et al. (11) with the exception of the pig and the dog. In thcsc two species mitochondrial cytochrome I’-450 was found at conccntratlions of 0.07 and 0.04 nmolw/‘mg mitochondrial protein. Cytochromc K4.50 cannot be deteckd spcctrophotomrtricall~ in t&is mit,ochondria of untreated rats brcausc of the high cyt’ochromc uR to cytochrome I’-450 ratio. AIason et al. (11) haw clearly shown the cffrcts of adding cytochrome a3 obtained from h(aart mitochondria on the diff crcnw spwtra of the carbon monoxide complex of wduced cytochromcb I’-450 of liver microsomw. It is also clear from th(b spwtra and data prtwntc~d in Fig. 1 and Tablr I that cytochromc a:$ in testis mitochondria intcrfrrw \\ith thr d(+c>rminat)ion of mitochondrial cytochromcl I’-&50

P-450 in rat testis are undetectable

and that mitochondrial cytochrome P-450 is not rasily detected \vhcn the cytochrome a:{ ‘cytochrome I’-450 ratio is greater than 1.5. The results show that cytochromc P-450 in rat testis mitochondria can b(l clcarlj demonstrated in control or hypophyscctomized rats given HCG and that cytochrome P-450 levels in the mitochondria are in fact, under the control of gonadotrophins such as HCG. This is also thcb cast for microsomal cytochromc P-450, thr t\vo microsomal cytochrome 1’.450 dependent enzymePt he 17cr-hydroxylasc and t h(> Cl;C,” lyasr and the microsomal 5a-rcductase (15). ;\Iason et al. (11) have rcwntly r(lported that testodoxin is also under HCG control in rat testis. I’urvis et al. (23, 24) have made similar findings that the lrvc~ls of adrenocortical mitochondrial cytochrome I’-450 and adrcnodoxin arc under ACTH control. Aftrr hypophyscctomy, the mit,ochondria rytochromcs c + cl and a + a:{ decay \vith well-defined half-lives of 16 and 14 da!.*, rcspcctivrly. Thrsc half-livrs arc similar to the half-lives of rnitochondrial protein and testis wet wright and undoubtedly reflect both the decay of interstitial cells and tubular components. However, t,hew halflivw are very much slow-cr than thr mcrosomal cytochromc P-450 (tIj2 = 3.3 days) which is found exclusiwly in interstitial crlls (25). The wry low lrvrl of mitochondrial cytochromc 1’.450 in thr testis of mature rats may be th(l rwult of a rapid half-lift (t, ?). The tl ,2 for mitochondrial cytochromc, 1’.450 decaying from HCG inducrd lwrls has brrn calrulatcd at 1.2 days (“6). This is much more rapid than the fl 2 of mirrosomal cytochrome I’-450 of 3.3 days (15) and suggests that the lcvc~l of mitochondrial cytochronw P4.50 mav br of importance in the wgulation of stc&idogtw+ in thr testis.

1.

~:NON,

Ii.

Ar.

.J.,

I>ROSD~NSKY,

hl.,

r)OI(F-

MAN, It. I., .\ND FORCHIC:I,LI,Ii:. (l!W) Steroids,

Suppl. I, 95.

2. PURWS,J. L., .~NDhIl:S.\llD,

klUST0,

N. o., CANK’K, J. :I.,

Proc. 3rd ht. Congr. Endocrinology, Mexico D.F., Excerpta Xed. Found. Znf. Congr. Ser. 167, Abst.
It.

(1(3(B)

3s

PURVIS

-4., INAX0, H., A1~~ TAMAOKI, B. (I!%%) J. Sferoid Biochenz. 1, 9. 4. H.IKDIN(:, B. W., WILSON, L. I>., WONG, S. H. .\NI) ?rTELSON, 1). H. (1965) ,%roids, Suppl. II, 61. 5. &lUR.I, T., SATO, l<., COOPER, L). Y., l~OSI:NTHAL, O., .\sD ~~ST.\I~ROOI<, It. W. (1965) Fe,/. Proc. 24, 1181. G. CAMMKIL, W. AND ESTAI. V. (1971) Proc. Biochem. Sot., 517 ,Ueeting, Edinburgh p. 13. 11. hI.\SON, I. A., PURVIS, J. L. .1ND ESTAI3ROOK, It. W. (1973) in Multienzyme Systems in l
14. nIKNARD,

n.

H.,

AND

PURVIS,

nology 91, 1506. 15. PURVIS, J. I,., CANICK,

J. L.

&dOC!%

J. A., ROSENBAUM, J. H., LATIF, 8., HOLOGGITAS, J., .&SD MAN.\RD, 1~. H. Arch. Biochem. Biophys. 169, 39.

ET AL. 16. GOI~~ALL, I)AvID, 17. ~II;N.\RD,

A. (;., BARON\{-JILL, C. J., .\NU RI. (1949) J. Biol. Chem. 177, 751. I<. H., .\XD PURVIS, J. I,. [lOi:<) Arch.

nr.

Biochem. Biophys. 164, 8. 18. C.w~im, W.,, \ND I
20.

21. 2%.

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