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Oxidative phosphorylation in guinea pig mammary gland mitochondria during various functional states
ARCHIVES
OF
BIOCHEMISTRY
Oxidative
AKD
I,. NELSON,
From
96, 500-505 (1962)
Phosphorylation
Mitochondria WALTER
BIOPHYSICS
the Department
in Guinea
during RONALD
Various
Pig Mammary Functional
A. BUTOW”
of Biochemistry, Received
Cornell July
States’
.iso E;DWARD University,
Gland
Ithacrr,
I. CIACCIO” Ncu: York
27, 1961
Oxidatirc phosphorylation was measured in the mammary gland mitochondria of guinea pigs during pregnancy, at parturition, and during lactation and retrogression. The phosphorylation concomitant with oxidation of succinate, a-ketoglutarate, and @-hydroxgbutyratc was shown to be uncoupled in mitochondria from glands at all functional states except during lactation. The phosphorylative capacity could be restored by the in vitro addition of bovine serum albumin to the reaction system. Mitochondrial and microsomal fractions prepared from retrogressing guinw pig mammary glands completely uncoupled phosphorylation concomitant with oxidation of succinate in a rat liver mitochondrial preparation. The supernatant fraction from these glands had no effect on either the oxidatire or phosphorylativc capacity of the liver mitochondria. Preliminary evidence is presented to suggest that a specific uncoupling factor for oxidative phosphorylation is present in mammary tissue preparations during specific functional states. IKTRODVCTIOK
Although respiratory chain phosphorylation has been clemonstrated with preparations of various animal tissues, the most extensive information has been obtained in studies with the mit.ochondria fraction of rat liver. Some recent observat,ions have indicated that variations in efficiency of oxidatire phosphorylation might be expected of mitochondria from tissues whose metabolism differs from that of liver or indeed from mitochondria prepared from a tissue during its various developmental or functional stat,es. For example, Holt,on et al. (1) have compared the phosphorylating efficiencies of mit’ochondria isolated from liver and heart and have reported that heart, ‘This investigation was supported in part, by a research grant (A-1316) from the National Institute of Arthrit,is and Metabolic Diseases, U. S. Public Health Service. ’ Predoctoral Fellow, National Cancer Institute, U. S. Public Health Service. “Present, address: Merck Institute for Therapcutic Research, Rahway, S. J.
sarcosomes oxidizing succinat,e and p-hydroxybutyratc shorn lower P:O ratios than li\-(11,mitochontlria oxidizing thcw ~mc substrates. Akazama and Beevcrs (2)) in a study of the developing endospcrm tissue of the germinating castor bean, have rcported that,, lvith a-ketoglutarate as substrate, the P:O ratio drops from 2.5 at 2 days to zero at the end of 8 days. Mitochondria freshly prepared from isotonic sucrose homogcnnt,cs of transplanted Kovikoff licpatomas (3)) from insect tissues ( 4-6)) and the so called iiniitocllron~(~” isolated from agctl mitochondria 17) bar-c been shown to have an uncoupled phosphorylation which ran bc reversed in vitro by the addition of crystalline bovine strum albumin to the reaction mixture. Rcccnt reports (6, 7) have clearly shown the uncoupling artivity of “mitochromc” and of insect mitochondria to reside in the unesterified fatty acids of the preparation: an effect similar to that reported previously by Pwssman ant1 1,ardy 181. 500
OSID.~TIVE
In this paper we describe the phosphorylative efficiencies of mitochondria isolated from mammary tissue and some properties of an uncoupling factor (10, 11) isolated from the microsomal fractions of this t,issue. MATERItlL
AND
501
PHOSPHORTLATIO1
METHODS
Multiparous guinea pigs from our stock colony and bo\-ine udders obtained from freshly slaughtered cows were used in this study. Cellular particulates were isolated from guinea pig mammary gland and liver tissue by differential centrifugation. Mitochondrial suspensions used in this investigation were made by suspending the pellet, obtained by differential ccntrifugation, with 1 ml. of cold 0.25 21 sucrose solution pc‘r gram of liver or per 3-4 g. of mammary gland, respectively. Cow udders were obtained immediately after slaughter of the animal and placed in ice for transfer IO the laboratory. The udders wcrc selected on the basis of cxtcnsivc development of the duct and alveolar tissue lvith milky fluid still present within tllrt tissue structure. All udders showed evidence of having been recently in la&t.ion. Alveolar tisSIC was c:~rcEull~ disaectcd from the surrounding supporti\-c Gssuc. Twenty per cent homogenates were made by homogenizing in 0.25 M sucrose for 1 min. in a I,ourdcs multimixer. The microsomal fraction was obtained by differential centrifugation and resuspended in cold distilled water. The Ijartitles were disintegrated by sonication of the suspension in a Raytheon magnetostrict,i\-c oscillator for 30 min. at 10 Bc./sec. The sonicate was centri-
fuged at 100,000 X g for 1 hr., and the supernatant fraction was adjusted to pH 4.2 wit,h 0.06 IV HCL. The precipitate formed was collected by centrifuging at 800 X g for 15 min. and washed three times with distilled water. The fractions obtained were designated as follo\l-s: sonic supernatant (0); its resitluc> fraction (0); the pH 4.2 precipitate (c) : and the sul)crn:Ltant from the pH 4.2 precipitate Cd). Oxidnt i\-c phosphorylaiion was det~~rmined using the glucose-hexokinaee trapping system according to t,he protatdures outlined I,- Hunter (12). Inorganic ~)hoq)lrnte was determined on aliquotp of the reaction mistluc, after deproteinization with colti 5% trichloro:lc,~,tic acid by Yumner~’ motlific*ation of the, Fiske> nncl S~~bl)aRow nrclthod (13).
The cLt:i prcwntecl in Table I illustrate the relationship between the tfficiency of osiclative phosphorylation in the mitochondria and the functional state of the gland. During early pregnancy lvhcn thv gland is in :I state of comparative quiesccncc. there is little or no pllo~phorvlation concoiuitwt with oxitlation of succinatc in these Illitochontlria. However, tllc I’: 0 ratios of the mitochontlria from tlic livers of those anini:ilS approach 2.0. During l:rt’e period is pregnancy, n-hcrc a proliferative starting, the P : 0 ratio of mammary gland mitochondria arcrage 0.2 while those from
Each flask contained in the main compartment 10 ~.moles potassium phosphate buffer pH i.4, 5 pmoles ATP, 0.01 pmole cytochrome c, 30 pmoles succinate, 30 pmoles fluoride, 30 pmoles MgCI2 , and G.4 ml. mitochondria. Each side arm contained 60 pmoles glucose and hexokinase (65 H&I units). Final volume made up to 3.0 with 0.25 M sucrose. Incubation period 30 min.; temperature 37.5”. The figures are averages for each case with ranges indicated in the parcnt,heses.
Uptake
Functional
state
Number of determina. _ ~~~~~~~~~Phosphate Oxygen tions pmoles 30 min. patoms/30 min.
of gland
P:O
ratio
Liver control P:O ratio
-
Early pregnanq (30-48 days)
11
3.2 (1 (i-6.3)
0.1 (0.0-1.0)
0.0 (0.0-0.1)
1.7 (1.4-2.0)
Late pregnanq ((i3S65 days)
1
5.1 (4.5-5.0)
0.7 (0.5-0.9)
0.2 (0.1-0.2)
1.8 (1.7-1.8)
I3
5.5 (l.W.5)
7.9 (2.1-16.5)
1.4 (1.1-1.8)
1.8 (1.5-2.1)
10
Ii.7 (3.1-8.i)
2.1 (0.0-5.2)
0 3 10. O-O. 6)
1.7 I I .Jp2.0)
Lactation (2-8 days postparturicnt Regression (8-W d:lys postpartrlrient
)
)
502
NELSON, TABLE
BUTOW
hr. 3
5.0 (1.7-5.6)
0 0 (5.2-7.8)
1.3 (1.2-1.1)
1.6 (1.5-l.:)
14-2-l
4
8.0 ifi.%9.6)
4.2 (2.1-5.7)
0 ti (0.34.8)
1.6 (1.3-1.8)
25-65
6
7.6 (6.1-9.5)
0.0
0.0
1 .B
p.2-1.8)
a The components and conditions as indicated in Table I.
are t,he same
liver controls average 1.8. Mitochondria from actively lactating glands show P: 0 ratios approaching the theoretical value for succinate oxidation (average 1.4, range l.l1.8). During the regression period (8 or more days post partum) the efficiency of phosphorylation associated with oxidation decreases at a rapid rate (P:O ratio av. = 0.3, range = 0.0-0.6). Nelson et al. (14) have reported that lactation in the female guinea pig ceases when the young are removed from the cage for periods longer than 24 hr. Therefore, it TABLE OXIDAT~V~~;
1’1ioS~HoRYf~hTI0~ 1s
THE
PRESIGNCE
BY OF
CIACCIO
was decided to determine the efficiency of oxidative phosphorylation in mammary mitochondria after withdrawal of the young for various time periods. The P: 0 ratios of mitochondria from such glands are presented in Table II. It can be seen that up to 13 hr. after withdrawal of young, the phosphorylation concomitant with oxidation of succinate is st,ill intact (P:O ratio av. = 1.3). Between 14 and 24 hr. after suckling has been prevented, some uncoupling occurs. Here the P: 0 ratios ranged from 0.3 to 0.8 for succinate. After 25 hr., the uncoupling of oxidation from phosphorylation is complete. Thus, in these forced regression studies, there is also a positive correlation between lactation and oxidative phosphorylation of the mitochondria, i.e., a correlation between the need for energy for intense extracellular synthesis and the efficiency of the energy-trapping mechanism. In Table III data are presented which confirm t,hc lack of phosphorylation coupled to oxidation in nonlactating gland mitochondria with substrates other than succinate, namely P-hydroxybutyrate and CCketoglutarate. Using the method of withdrawal of young, the young were taken from one animal while another, wit,h young, was used as a posit,ive control. In the case where the gland was not suckled, the P:O ratios with succinate, P-hydroxybutyrate and CXketoglutrate as substrates were all zero, whereas the P: 0 ratios approached theo-
II
O-13
AND
III hI.4m*1.4aY
IIIFFERE~T
GLIIND
?vlrTocffosuRla
SUBSTRATES
The components and condit,ions are the same as indicated in Table 1. The concentrationof butyrate and or-ketoglutaratc w’:ts 30 PJ!!, and in each case diphosphopyridinc nrlcleotidc the reaction flasks.
@-hydroxy~:ts added to
U,,take Condition
of mammary gland
Suckled 4 days suckled 3 days
Suckled
3 days
then
Substrate
not
oxygen *atoms,‘30 min. ~.
Phosphate rmolesj3Omin. ~~
Succin:tt,e &Hydrosybutyrate cu-Ketoglutamte
7.i 2.8 3.2
0 0 0.0 0 0
Succirwte P-Hydroxybutyrste wKetoglutarate
0.9 7.2 13.2
7.8 11.9 42.4
P:O
ratio
~~_
0 0 0 1.1 1.7 3.2
OXIDATIVE
TABLE OXII)ATIVE
PHOSPIIORYLATION
(SLJCCISATE)
and conditions
Functional state of gland Mid.
BY ~\~ITOCHONI)RIA
are the same as in Table
4 mg.
None
Parturition
B.S.A. 4 mg. Postparturient,
Postparturient
Lactating
(LB.S.A.
(12 hr.)
(24 hr.)
(2-3 days)
= Crystalline
FROM GUINE:A
PIG
~~IAYMARY
I. rmolesi30 min.
P:U ratio
Liver Control I’: 0 ratio ____-
4.0 4.0
0 .7 4.ci
0.2 1.2
1.8 1.8
9.1 11.7
0.2 7.2
0.0 0.G
1.8 1.9
.4ddition to reaction mixture pato~~~~~in, None B.S.A.”
pregnancy
IV
DURING THE RAPID GROWTH PERIOD
GLANDS
The components
503
PHOSPHORYLATION
Phosphate
iYone B.S.il.
4 mg.
4.2 5 .6
0 0 11.0
0.1 1.8
2.-l 2.4
IC’one B.S.A.
7.2 6.0
6.0 11 .o
0.8 1.6
2.1
4 mg.
None B.S.A.
10.0 8.0
15.0 18.0
1.5 2.2
1.5
4 mg.
bovine
serum albumin.
retical values where the gland was continuously suckled. Histological observations by Kuramitsu and Loeb (9) and chemical analysis on tissue DNA, RNA-DNA ratios, total weight, and total nitrogen content in this laboratory clearly indicate that the rapid proliferative growth period in the guinea pig mammary gland occurs immediately after parturition. The data in Tables I and II in this paper indicate that in this period, as bvell as during retrogression, the phosphorylative efficiency of the mitochondria may be very low. More definitive data conccrning oxidative phosphorylation in these mitochondria are given in Table IV. The effect of adding bovine serum albumin to the reaction mixture is also included. It is evident from the data presented that mitochondria from glands in the quiescent and rapid proliferative stages have extremely low phosphorylative capacities. It is also clear that bovine serum albumin is effective in restoring the phosphorylation concomitant with oxidation of the substrate. This uncoupled phosphorylation, therefore, is similar to the type of uncoupling observed in mitochondria from Xovikoff hepatomas (3), insect tissues (4-6), and the mitechrome effect (15). The common denominator in each case is the ability of bovine
serum albumin to reverse the uncoupling of phosphorylation from oxidation. The ability of the particulate fractions obtained from a retrogressed guinea pig mammary gland to uncouple oxidative phosphorylation in a rat liver mitochondrial preparation is shown by the data in Table V. It should be noted that with these preparations the oxidation of substrate by liver mitochondria is unaffected but phosphorylation is completely uncoupled by both the mammary mitochondrial fraction and the microsomal fraction (particles sedimented at, 100,000 X gi. The supernatant, fraction shows no uncoupling effect. Preparation of these particulate fractions in various sucrose solutions (0.25-0.88 M) ; the addition of 0.001 111 ethylenediaminetetracetic acid t,o the preparative or suspending media or thorough washing by resuspending and rccentrifuging several times in 0.25 M sucrose or 0.1 M phosphate buffer or 0.1 Jr Tris buffer pH 7.2 has no effect on the ability of these particles to uncouple phoephorylation. From these observations it is apparent that t,he uncoupler is firmly associated with mitochondrial and microsomal particulates; it has the ability to affect phosphorylation not only of the mammary mitochondria i&elf but of rat liver mitochondria oxidizing succinate as a substrate;
504
NELSON. TABLE
UXCOUPLINC:
OF
(SUVCINATE)
PIG
(;LYNE.~
I%AT
LIVER
TABLE
&IITOCHOXL.URIA
~~AMMARY
(+LASI)
reaction
arc the same
Phosphate rmo,es/ 30 min.
14.0 l-l.3
23.2 0
15.8
0
lo.8
P:O
ratio
1.7 0
1 33.5
2.0
11The mammary gland fractions added to the flasks, as noted, were derived from a lo’,:; 0.25 211 sucrose homogenate. The mitochondria and microsomes were resuspended in 5 ml. of 0.25 211 sucrose. The supernatant is the soluble fraction from the lOc)h homogenate after removal of cellular centrifagation. In particulates 1)~ differential each instance 0.1 ml. NW added per flask. Each value given iII the table is an average of duplicate r,,ns:.
TABIX UK(‘OI~PI.ING
OF IN
(~UTCCINATE)
FRACTIONS
VI
(~SII~B’IIVE
I’MOS~~IORYLII’IION
RAT
~IITOCHOSDRI.~
OBTAINED
LIVER
BO~-~N~
FROM
BY
WI
Is:oocT.~N~;
~+;XTRA(TION
ACTIV-ITE-
mohf
FRACTIOIZ‘S
oxygen ratoms/ 30 min.
None Guinea pig mammary gland mitochondriaa Guinea pig mammary gland microsomes~‘ (iuinen pig mammar) gland supernat~ant:~
0~
UNCOUPLING
my
Uptake to control
CIACCIO
EFFECT
PHC)SPIIORYLATION
The components and conditions as indicated in Table I.
Additions
AND
V
OXIDATIVY
IN
BUTOW
PH
OF
RIAMMARY
4.2
ON
FRICTION
>~~(~ROSO.MES
The components and conditions are t,hc same as indicated in Table 1. The pH 4.2 precipit,ate was added at approximately 1.5 mg. protein per flask. The isooctane extract, acid fraction and base fraction \vas a 0.05.ml. aliquot of a concentrated absolute ethanol solut,ion of each per flask as indicat,ed. The acid fraction :tnd base fraction were prepared as follows: 1 ml. of 5 ,V KCJH tind 25 ml. water was added to an rthanol solution of the isooctane extract and then extracted three times with a total volume of 30 ml. petroleiurr ether. The ether extract was rvaporated in oclc~~o and the residue tlcsignated as the base fraction. The aqueous phase H’IIS adjusted to pH 1.5 cvith HCl and extracted three times with a lot al volume of 30 ml. petrolerml ether. The ether extract \vas evaporated in w,c110 and the rcsiduc designat cd as the acid fraction.
-
I Uptake ~
Additions
to control
reaction
P:O
~
1.07 U 1.02 0 1.72 0.46
8.10 4.4 10.74 2.1 1l.i-l 5.12
xonr
pH 4.2 ppt. Extracted pH 4.2 ppt. Isooctane exi,r:tct None Acid fraction of isooct.ane extract Basic fraction of isooctanc extract
ratio
1 10.50
~LIAMMARY
MICRWOMES The components and conditions are the same as indicated in Table I. 911 additions were equivalent, to approximately 1.5 mg. protein per flask. The preparation of the microsomal fractions is described in t.he text (Materials and Methoda). Uptake !-Additions
to control
I P:O
reaction
Kane Sonicat e (n) Sediment (b) pH 4.2 ppt. (c) Supernatant, from 3.2 ppt. (d)
6.98 10.3
4.75 pH
5.66 6.20
10.3 0 0 0 8.52
ratio
1.18 U 0 0 1.38
TABLE COMPOSITIOX
OF ~4cII)
EXTRACT
OF
VIII
FR.4C’TIo.v
PH a.2
.4cid
Saturated Cl, Cl6 Cl8 cm Unsaturated (218 one double bond C,, two double bonds Cl8 three double bonds C:,, one double bond
FROM
1HOO(T4NE
PRECIPITATE Per cent of total
1.5 39 8.5 Trace 40 11 Trace Trace 100.0
acids
OXIDATIVE
cl-0' n
PHOSPHORYLATIOK
and the uncoupling is reversed in CL vitro systems by t,hc addition of bovine strum albumin to the reaction flasks. As shown by the data in Tables VI and VII, the factor responsible for the microsomal effect on oxidat,ive phosphorylation can bc solublized by sonic oscillation. It is readily precipitated at pH 4.2, and by its lipid and protein nature appears to be a lipoprot,ein. It is also apparent from the data that, the specific uncoupling of oxidative phosphorylation is due to the free fatt,y acids associated with the pH 4.2 precipitate. The free fatty acid composition of the pH 4.2 fraction is given in Table VIII. The presence of non-esterified longchain saturated and unsaturated fat,ty acids as effective uncoupling agents is consistent with previous observations (S-8). The data presented above suggest to us that’ a specific uncoupling factor for oxidat,ivc phosphorylation is present in mammary tissue preparations during specific functional states. Although the uncoupling is due to the unestcrified fatty acids associated with a lipoprotein, the fatty acids are not, randomly distributed in the various protein fractions. The effect is obtained only in that fraction precipitating at pH 4.2. The quantitative relationship of the uncoupling factor and its further charact,erization in mammary tissue is presently under investigation.
REFEREKCES 1. HOLTOS, F. A., H~~I,sM.~N, W. C., MYEKR, D. I<., AKD SLATER, E. C., Biochem. J. 67, 579 (1957). 2. AKAZAWVA, T.. AKD BEEPERS, H., Biochcm J. 67, 115 (1957). 3. DEVLIS, T. &I., AKD PRCSS, bl. P., I"c~/wrrlio,~ Proc. 17,830 (1957). 4. SACKTOR, B., O’SEILL, J. J., AXD COCIIR.IS, D. G., J. Biol. Chcnr. 233, 1233 (1958). 5. \T'O.JTCZAI<. L., AlUD %-OJTCZ.%K, d. B. ~~idcim. et Biophys. Actn 31,297 (1959). 6. WOJTCZAK, L., AKD WOJT~ZAK, 8. B., 13iochi~t. ct Biophgs. Artn 39, 277 (1960). 7. Hti~sar.szr, Iv'. c., ELLIOT, J?'. B., .4s1) kh.ATER, E. C., Biochim. ct Biophys. Acta 39, 67 (1960). 8. Pesssnr.4N, B. C., AND L.ARDY, H. A., Biodc im. ct Biophys. Actn 18, 482 (1955).
9.
I
c.,
AND LOED, L.,
ilm. J. I'h!/sid.
56, 40 (1921). 10. CI.4CCI0, E. I., AND KELSON, Proc. 17, 201 (1958). 11. BUTOW,
It.,
AND
h-ELSOS,
R. W.
L..
Fvric,~~tlion
L.,
Fctic~,~fio,z
I'roc. 20, 49 (1961). in Enzymol12. HTXTEH, F. E., JR., in “Methods ogy” Tel. II, (S. P. Colowick and N. 0. IiapIan, eds.), p. 610. Academic Press, New York, 1955. 12 SUMKEIX, J. B., S&we 100, 413 (1944). 14. XELSOK-, TV. L.. Iiak-q A., MOORE, ht., WILLIAMS, H. H., AND HERRWGTON, B. L., J. Zrlltition 44, 585 (1951). 15. POLIO, B. D., .~ND SHMUKLER, H. W., J. Biol. ^.. Ckm. 227, 419 (1957). IV.
OF
BIOCHEMISTRY
Oxidative
AKD
I,. NELSON,
From
96, 500-505 (1962)
Phosphorylation
Mitochondria WALTER
BIOPHYSICS
the Department
in Guinea
during RONALD
Various
Pig Mammary Functional
A. BUTOW”
of Biochemistry, Received
Cornell July
States’
.iso E;DWARD University,
Gland
Ithacrr,
I. CIACCIO” Ncu: York
27, 1961
Oxidatirc phosphorylation was measured in the mammary gland mitochondria of guinea pigs during pregnancy, at parturition, and during lactation and retrogression. The phosphorylation concomitant with oxidation of succinate, a-ketoglutarate, and @-hydroxgbutyratc was shown to be uncoupled in mitochondria from glands at all functional states except during lactation. The phosphorylative capacity could be restored by the in vitro addition of bovine serum albumin to the reaction system. Mitochondrial and microsomal fractions prepared from retrogressing guinw pig mammary glands completely uncoupled phosphorylation concomitant with oxidation of succinate in a rat liver mitochondrial preparation. The supernatant fraction from these glands had no effect on either the oxidatire or phosphorylativc capacity of the liver mitochondria. Preliminary evidence is presented to suggest that a specific uncoupling factor for oxidative phosphorylation is present in mammary tissue preparations during specific functional states. IKTRODVCTIOK
Although respiratory chain phosphorylation has been clemonstrated with preparations of various animal tissues, the most extensive information has been obtained in studies with the mit.ochondria fraction of rat liver. Some recent observat,ions have indicated that variations in efficiency of oxidatire phosphorylation might be expected of mitochondria from tissues whose metabolism differs from that of liver or indeed from mitochondria prepared from a tissue during its various developmental or functional stat,es. For example, Holt,on et al. (1) have compared the phosphorylating efficiencies of mit’ochondria isolated from liver and heart and have reported that heart, ‘This investigation was supported in part, by a research grant (A-1316) from the National Institute of Arthrit,is and Metabolic Diseases, U. S. Public Health Service. ’ Predoctoral Fellow, National Cancer Institute, U. S. Public Health Service. “Present, address: Merck Institute for Therapcutic Research, Rahway, S. J.
sarcosomes oxidizing succinat,e and p-hydroxybutyratc shorn lower P:O ratios than li\-(11,mitochontlria oxidizing thcw ~mc substrates. Akazama and Beevcrs (2)) in a study of the developing endospcrm tissue of the germinating castor bean, have rcported that,, lvith a-ketoglutarate as substrate, the P:O ratio drops from 2.5 at 2 days to zero at the end of 8 days. Mitochondria freshly prepared from isotonic sucrose homogcnnt,cs of transplanted Kovikoff licpatomas (3)) from insect tissues ( 4-6)) and the so called iiniitocllron~(~” isolated from agctl mitochondria 17) bar-c been shown to have an uncoupled phosphorylation which ran bc reversed in vitro by the addition of crystalline bovine strum albumin to the reaction mixture. Rcccnt reports (6, 7) have clearly shown the uncoupling artivity of “mitochromc” and of insect mitochondria to reside in the unesterified fatty acids of the preparation: an effect similar to that reported previously by Pwssman ant1 1,ardy 181. 500
OSID.~TIVE
In this paper we describe the phosphorylative efficiencies of mitochondria isolated from mammary tissue and some properties of an uncoupling factor (10, 11) isolated from the microsomal fractions of this t,issue. MATERItlL
AND
501
PHOSPHORTLATIO1
METHODS
Multiparous guinea pigs from our stock colony and bo\-ine udders obtained from freshly slaughtered cows were used in this study. Cellular particulates were isolated from guinea pig mammary gland and liver tissue by differential centrifugation. Mitochondrial suspensions used in this investigation were made by suspending the pellet, obtained by differential ccntrifugation, with 1 ml. of cold 0.25 21 sucrose solution pc‘r gram of liver or per 3-4 g. of mammary gland, respectively. Cow udders were obtained immediately after slaughter of the animal and placed in ice for transfer IO the laboratory. The udders wcrc selected on the basis of cxtcnsivc development of the duct and alveolar tissue lvith milky fluid still present within tllrt tissue structure. All udders showed evidence of having been recently in la&t.ion. Alveolar tisSIC was c:~rcEull~ disaectcd from the surrounding supporti\-c Gssuc. Twenty per cent homogenates were made by homogenizing in 0.25 M sucrose for 1 min. in a I,ourdcs multimixer. The microsomal fraction was obtained by differential centrifugation and resuspended in cold distilled water. The Ijartitles were disintegrated by sonication of the suspension in a Raytheon magnetostrict,i\-c oscillator for 30 min. at 10 Bc./sec. The sonicate was centri-
fuged at 100,000 X g for 1 hr., and the supernatant fraction was adjusted to pH 4.2 wit,h 0.06 IV HCL. The precipitate formed was collected by centrifuging at 800 X g for 15 min. and washed three times with distilled water. The fractions obtained were designated as follo\l-s: sonic supernatant (0); its resitluc> fraction (0); the pH 4.2 precipitate (c) : and the sul)crn:Ltant from the pH 4.2 precipitate Cd). Oxidnt i\-c phosphorylaiion was det~~rmined using the glucose-hexokinaee trapping system according to t,he protatdures outlined I,- Hunter (12). Inorganic ~)hoq)lrnte was determined on aliquotp of the reaction mistluc, after deproteinization with colti 5% trichloro:lc,~,tic acid by Yumner~’ motlific*ation of the, Fiske> nncl S~~bl)aRow nrclthod (13).
The cLt:i prcwntecl in Table I illustrate the relationship between the tfficiency of osiclative phosphorylation in the mitochondria and the functional state of the gland. During early pregnancy lvhcn thv gland is in :I state of comparative quiesccncc. there is little or no pllo~phorvlation concoiuitwt with oxitlation of succinatc in these Illitochontlria. However, tllc I’: 0 ratios of the mitochontlria from tlic livers of those anini:ilS approach 2.0. During l:rt’e period is pregnancy, n-hcrc a proliferative starting, the P : 0 ratio of mammary gland mitochondria arcrage 0.2 while those from
Each flask contained in the main compartment 10 ~.moles potassium phosphate buffer pH i.4, 5 pmoles ATP, 0.01 pmole cytochrome c, 30 pmoles succinate, 30 pmoles fluoride, 30 pmoles MgCI2 , and G.4 ml. mitochondria. Each side arm contained 60 pmoles glucose and hexokinase (65 H&I units). Final volume made up to 3.0 with 0.25 M sucrose. Incubation period 30 min.; temperature 37.5”. The figures are averages for each case with ranges indicated in the parcnt,heses.
Uptake
Functional
state
Number of determina. _ ~~~~~~~~~Phosphate Oxygen tions pmoles 30 min. patoms/30 min.
of gland
P:O
ratio
Liver control P:O ratio
-
Early pregnanq (30-48 days)
11
3.2 (1 (i-6.3)
0.1 (0.0-1.0)
0.0 (0.0-0.1)
1.7 (1.4-2.0)
Late pregnanq ((i3S65 days)
1
5.1 (4.5-5.0)
0.7 (0.5-0.9)
0.2 (0.1-0.2)
1.8 (1.7-1.8)
I3
5.5 (l.W.5)
7.9 (2.1-16.5)
1.4 (1.1-1.8)
1.8 (1.5-2.1)
10
Ii.7 (3.1-8.i)
2.1 (0.0-5.2)
0 3 10. O-O. 6)
1.7 I I .Jp2.0)
Lactation (2-8 days postparturicnt Regression (8-W d:lys postpartrlrient
)
)
502
NELSON, TABLE
BUTOW
hr. 3
5.0 (1.7-5.6)
0 0 (5.2-7.8)
1.3 (1.2-1.1)
1.6 (1.5-l.:)
14-2-l
4
8.0 ifi.%9.6)
4.2 (2.1-5.7)
0 ti (0.34.8)
1.6 (1.3-1.8)
25-65
6
7.6 (6.1-9.5)
0.0
0.0
1 .B
p.2-1.8)
a The components and conditions as indicated in Table I.
are t,he same
liver controls average 1.8. Mitochondria from actively lactating glands show P: 0 ratios approaching the theoretical value for succinate oxidation (average 1.4, range l.l1.8). During the regression period (8 or more days post partum) the efficiency of phosphorylation associated with oxidation decreases at a rapid rate (P:O ratio av. = 0.3, range = 0.0-0.6). Nelson et al. (14) have reported that lactation in the female guinea pig ceases when the young are removed from the cage for periods longer than 24 hr. Therefore, it TABLE OXIDAT~V~~;
1’1ioS~HoRYf~hTI0~ 1s
THE
PRESIGNCE
BY OF
CIACCIO
was decided to determine the efficiency of oxidative phosphorylation in mammary mitochondria after withdrawal of the young for various time periods. The P: 0 ratios of mitochondria from such glands are presented in Table II. It can be seen that up to 13 hr. after withdrawal of young, the phosphorylation concomitant with oxidation of succinate is st,ill intact (P:O ratio av. = 1.3). Between 14 and 24 hr. after suckling has been prevented, some uncoupling occurs. Here the P: 0 ratios ranged from 0.3 to 0.8 for succinate. After 25 hr., the uncoupling of oxidation from phosphorylation is complete. Thus, in these forced regression studies, there is also a positive correlation between lactation and oxidative phosphorylation of the mitochondria, i.e., a correlation between the need for energy for intense extracellular synthesis and the efficiency of the energy-trapping mechanism. In Table III data are presented which confirm t,hc lack of phosphorylation coupled to oxidation in nonlactating gland mitochondria with substrates other than succinate, namely P-hydroxybutyrate and CCketoglutarate. Using the method of withdrawal of young, the young were taken from one animal while another, wit,h young, was used as a posit,ive control. In the case where the gland was not suckled, the P:O ratios with succinate, P-hydroxybutyrate and CXketoglutrate as substrates were all zero, whereas the P: 0 ratios approached theo-
II
O-13
AND
III hI.4m*1.4aY
IIIFFERE~T
GLIIND
?vlrTocffosuRla
SUBSTRATES
The components and condit,ions are the same as indicated in Table 1. The concentrationof butyrate and or-ketoglutaratc w’:ts 30 PJ!!, and in each case diphosphopyridinc nrlcleotidc the reaction flasks.
@-hydroxy~:ts added to
U,,take Condition
of mammary gland
Suckled 4 days suckled 3 days
Suckled
3 days
then
Substrate
not
oxygen *atoms,‘30 min. ~.
Phosphate rmolesj3Omin. ~~
Succin:tt,e &Hydrosybutyrate cu-Ketoglutamte
7.i 2.8 3.2
0 0 0.0 0 0
Succirwte P-Hydroxybutyrste wKetoglutarate
0.9 7.2 13.2
7.8 11.9 42.4
P:O
ratio
~~_
0 0 0 1.1 1.7 3.2
OXIDATIVE
TABLE OXII)ATIVE
PHOSPIIORYLATION
(SLJCCISATE)
and conditions
Functional state of gland Mid.
BY ~\~ITOCHONI)RIA
are the same as in Table
4 mg.
None
Parturition
B.S.A. 4 mg. Postparturient,
Postparturient
Lactating
(LB.S.A.
(12 hr.)
(24 hr.)
(2-3 days)
= Crystalline
FROM GUINE:A
PIG
~~IAYMARY
I. rmolesi30 min.
P:U ratio
Liver Control I’: 0 ratio ____-
4.0 4.0
0 .7 4.ci
0.2 1.2
1.8 1.8
9.1 11.7
0.2 7.2
0.0 0.G
1.8 1.9
.4ddition to reaction mixture pato~~~~~in, None B.S.A.”
pregnancy
IV
DURING THE RAPID GROWTH PERIOD
GLANDS
The components
503
PHOSPHORYLATION
Phosphate
iYone B.S.il.
4 mg.
4.2 5 .6
0 0 11.0
0.1 1.8
2.-l 2.4
IC’one B.S.A.
7.2 6.0
6.0 11 .o
0.8 1.6
2.1
4 mg.
None B.S.A.
10.0 8.0
15.0 18.0
1.5 2.2
1.5
4 mg.
bovine
serum albumin.
retical values where the gland was continuously suckled. Histological observations by Kuramitsu and Loeb (9) and chemical analysis on tissue DNA, RNA-DNA ratios, total weight, and total nitrogen content in this laboratory clearly indicate that the rapid proliferative growth period in the guinea pig mammary gland occurs immediately after parturition. The data in Tables I and II in this paper indicate that in this period, as bvell as during retrogression, the phosphorylative efficiency of the mitochondria may be very low. More definitive data conccrning oxidative phosphorylation in these mitochondria are given in Table IV. The effect of adding bovine serum albumin to the reaction mixture is also included. It is evident from the data presented that mitochondria from glands in the quiescent and rapid proliferative stages have extremely low phosphorylative capacities. It is also clear that bovine serum albumin is effective in restoring the phosphorylation concomitant with oxidation of the substrate. This uncoupled phosphorylation, therefore, is similar to the type of uncoupling observed in mitochondria from Xovikoff hepatomas (3), insect tissues (4-6), and the mitechrome effect (15). The common denominator in each case is the ability of bovine
serum albumin to reverse the uncoupling of phosphorylation from oxidation. The ability of the particulate fractions obtained from a retrogressed guinea pig mammary gland to uncouple oxidative phosphorylation in a rat liver mitochondrial preparation is shown by the data in Table V. It should be noted that with these preparations the oxidation of substrate by liver mitochondria is unaffected but phosphorylation is completely uncoupled by both the mammary mitochondrial fraction and the microsomal fraction (particles sedimented at, 100,000 X gi. The supernatant, fraction shows no uncoupling effect. Preparation of these particulate fractions in various sucrose solutions (0.25-0.88 M) ; the addition of 0.001 111 ethylenediaminetetracetic acid t,o the preparative or suspending media or thorough washing by resuspending and rccentrifuging several times in 0.25 M sucrose or 0.1 M phosphate buffer or 0.1 Jr Tris buffer pH 7.2 has no effect on the ability of these particles to uncouple phoephorylation. From these observations it is apparent that t,he uncoupler is firmly associated with mitochondrial and microsomal particulates; it has the ability to affect phosphorylation not only of the mammary mitochondria i&elf but of rat liver mitochondria oxidizing succinate as a substrate;
504
NELSON. TABLE
UXCOUPLINC:
OF
(SUVCINATE)
PIG
(;LYNE.~
I%AT
LIVER
TABLE
&IITOCHOXL.URIA
~~AMMARY
(+LASI)
reaction
arc the same
Phosphate rmo,es/ 30 min.
14.0 l-l.3
23.2 0
15.8
0
lo.8
P:O
ratio
1.7 0
1 33.5
2.0
11The mammary gland fractions added to the flasks, as noted, were derived from a lo’,:; 0.25 211 sucrose homogenate. The mitochondria and microsomes were resuspended in 5 ml. of 0.25 211 sucrose. The supernatant is the soluble fraction from the lOc)h homogenate after removal of cellular centrifagation. In particulates 1)~ differential each instance 0.1 ml. NW added per flask. Each value given iII the table is an average of duplicate r,,ns:.
TABIX UK(‘OI~PI.ING
OF IN
(~UTCCINATE)
FRACTIONS
VI
(~SII~B’IIVE
I’MOS~~IORYLII’IION
RAT
~IITOCHOSDRI.~
OBTAINED
LIVER
BO~-~N~
FROM
BY
WI
Is:oocT.~N~;
~+;XTRA(TION
ACTIV-ITE-
mohf
FRACTIOIZ‘S
oxygen ratoms/ 30 min.
None Guinea pig mammary gland mitochondriaa Guinea pig mammary gland microsomes~‘ (iuinen pig mammar) gland supernat~ant:~
0~
UNCOUPLING
my
Uptake to control
CIACCIO
EFFECT
PHC)SPIIORYLATION
The components and conditions as indicated in Table I.
Additions
AND
V
OXIDATIVY
IN
BUTOW
PH
OF
RIAMMARY
4.2
ON
FRICTION
>~~(~ROSO.MES
The components and conditions are t,hc same as indicated in Table 1. The pH 4.2 precipit,ate was added at approximately 1.5 mg. protein per flask. The isooctane extract, acid fraction and base fraction \vas a 0.05.ml. aliquot of a concentrated absolute ethanol solut,ion of each per flask as indicat,ed. The acid fraction :tnd base fraction were prepared as follows: 1 ml. of 5 ,V KCJH tind 25 ml. water was added to an rthanol solution of the isooctane extract and then extracted three times with a total volume of 30 ml. petroleiurr ether. The ether extract was rvaporated in oclc~~o and the residue tlcsignated as the base fraction. The aqueous phase H’IIS adjusted to pH 1.5 cvith HCl and extracted three times with a lot al volume of 30 ml. petrolerml ether. The ether extract \vas evaporated in w,c110 and the rcsiduc designat cd as the acid fraction.
-
I Uptake ~
Additions
to control
reaction
P:O
~
1.07 U 1.02 0 1.72 0.46
8.10 4.4 10.74 2.1 1l.i-l 5.12
xonr
pH 4.2 ppt. Extracted pH 4.2 ppt. Isooctane exi,r:tct None Acid fraction of isooct.ane extract Basic fraction of isooctanc extract
ratio
1 10.50
~LIAMMARY
MICRWOMES The components and conditions are the same as indicated in Table I. 911 additions were equivalent, to approximately 1.5 mg. protein per flask. The preparation of the microsomal fractions is described in t.he text (Materials and Methoda). Uptake !-Additions
to control
I P:O
reaction
Kane Sonicat e (n) Sediment (b) pH 4.2 ppt. (c) Supernatant, from 3.2 ppt. (d)
6.98 10.3
4.75 pH
5.66 6.20
10.3 0 0 0 8.52
ratio
1.18 U 0 0 1.38
TABLE COMPOSITIOX
OF ~4cII)
EXTRACT
OF
VIII
FR.4C’TIo.v
PH a.2
.4cid
Saturated Cl, Cl6 Cl8 cm Unsaturated (218 one double bond C,, two double bonds Cl8 three double bonds C:,, one double bond
FROM
1HOO(T4NE
PRECIPITATE Per cent of total
1.5 39 8.5 Trace 40 11 Trace Trace 100.0
acids
OXIDATIVE
cl-0' n
PHOSPHORYLATIOK
and the uncoupling is reversed in CL vitro systems by t,hc addition of bovine strum albumin to the reaction flasks. As shown by the data in Tables VI and VII, the factor responsible for the microsomal effect on oxidat,ive phosphorylation can bc solublized by sonic oscillation. It is readily precipitated at pH 4.2, and by its lipid and protein nature appears to be a lipoprot,ein. It is also apparent from the data that, the specific uncoupling of oxidative phosphorylation is due to the free fatt,y acids associated with the pH 4.2 precipitate. The free fatty acid composition of the pH 4.2 fraction is given in Table VIII. The presence of non-esterified longchain saturated and unsaturated fat,ty acids as effective uncoupling agents is consistent with previous observations (S-8). The data presented above suggest to us that’ a specific uncoupling factor for oxidat,ivc phosphorylation is present in mammary tissue preparations during specific functional states. Although the uncoupling is due to the unestcrified fatty acids associated with a lipoprotein, the fatty acids are not, randomly distributed in the various protein fractions. The effect is obtained only in that fraction precipitating at pH 4.2. The quantitative relationship of the uncoupling factor and its further charact,erization in mammary tissue is presently under investigation.
REFEREKCES 1. HOLTOS, F. A., H~~I,sM.~N, W. C., MYEKR, D. I<., AKD SLATER, E. C., Biochem. J. 67, 579 (1957). 2. AKAZAWVA, T.. AKD BEEPERS, H., Biochcm J. 67, 115 (1957). 3. DEVLIS, T. &I., AKD PRCSS, bl. P., I"c~/wrrlio,~ Proc. 17,830 (1957). 4. SACKTOR, B., O’SEILL, J. J., AXD COCIIR.IS, D. G., J. Biol. Chcnr. 233, 1233 (1958). 5. \T'O.JTCZAI<. L., AlUD %-OJTCZ.%K, d. B. ~~idcim. et Biophys. Actn 31,297 (1959). 6. WOJTCZAK, L., AKD WOJT~ZAK, 8. B., 13iochi~t. ct Biophgs. Artn 39, 277 (1960). 7. Hti~sar.szr, Iv'. c., ELLIOT, J?'. B., .4s1) kh.ATER, E. C., Biochim. ct Biophys. Acta 39, 67 (1960). 8. Pesssnr.4N, B. C., AND L.ARDY, H. A., Biodc im. ct Biophys. Actn 18, 482 (1955).
9.
I
c.,
AND LOED, L.,
ilm. J. I'h!/sid.
56, 40 (1921). 10. CI.4CCI0, E. I., AND KELSON, Proc. 17, 201 (1958). 11. BUTOW,
It.,
AND
h-ELSOS,
R. W.
L..
Fvric,~~tlion
L.,
Fctic~,~fio,z
I'roc. 20, 49 (1961). in Enzymol12. HTXTEH, F. E., JR., in “Methods ogy” Tel. II, (S. P. Colowick and N. 0. IiapIan, eds.), p. 610. Academic Press, New York, 1955. 12 SUMKEIX, J. B., S&we 100, 413 (1944). 14. XELSOK-, TV. L.. Iiak-q A., MOORE, ht., WILLIAMS, H. H., AND HERRWGTON, B. L., J. Zrlltition 44, 585 (1951). 15. POLIO, B. D., .~ND SHMUKLER, H. W., J. Biol. ^.. Ckm. 227, 419 (1957). IV.