Formation and disappearance of an endogenous uncoupling factor during swelling and contraction of mitochondria

442

BIOCHIMICA ET BIOPHYSICA ACTA

FORMATION AND DISAPPEARANCE

O F AN E N D O G E N O U S

UNCOUPLING FACTOR DURING

SWELLING

AND CONTRACTION OF MITOCHONDRIA LECH \VOJTCZAK AND ALBERT L. LEHNINGER Department o~ Physiological Chemistry, The Johns Hopkins University School o/ 3Iedicine, Baltimore, Md. (U.S.A.)

(Received January 3Ist, 1961)

SUMMARY During swelling of rat-liver mitochondria which occurs spontaneously or is induced by Ca ~+ or thyroxine, direct analytical measurements show a parallel intramitochondrial formation of U factor, a heat-stable, isooctane-soluble uncoupling and swelling agent of f a t t y acid nature. On the other hand, mitochondrial swelling induced by glutathione or by phosphate is not accompanied by increased formation of U factor. Serum albumin, which is a " t r a p " for U factor by virtue of its ability to bind fatty acids, prevents swelling induced by Ca 2+ and thyroxine, but not that produced by GSH and phosphate. These findings therefore indicate that Ca 2+ and thyroxine accelerate mitochondrial swelling indirectly, by accelerating the enzymic formation of U factor from a precursor lipid. Stimulation of U factor formation is given bv Ca 2+ in sonicated mitochondria, but not by L-thyroxine. When swollen mitochondria are contracted again by ATP, U factor rapidly disappears at a rate which parallels the rate of contraction and ceases when contraction ceases. While there is an enormous increase in the level of U factor during swelling, experiments with !llC]oleate as an indicator of U factor show that oxidative removal of oleate occurs simultaneously. On addition of ATP, which produces contraction of mitochondria and disappearance of U factor, [14C]oleate rapidly disappears. Most of the !14C]oleate is rapidly incorporated into the phosphatidic acid and cephalin fractions of mitochondrial lipids. These findings thus show that enzymic formation and removal of U factor are closely geared to mitochondrial swelling and contraction and that accumulation of U factor m a y be the cause of swelling in the presence of Ca 2+ and thyroxine.

INTRODUCTION This paper describes an exploratory study of the role of an endogenous uncoupling and swelling agent (U factor 1) in the energy-linked swelling and contraction of ratliver mitochondria (for reviews see refs. 2, 3). It has been shown by LEHNINGER AND REMMERT 1 that incubation of either intact or sonic-disrupted rat-liver mitochondria causes enzymic formation of a heat-stable, Biochim. Biophys. Acta, 51 !1961) 442 456

UNCOUPLING FACTOR IN MITOCHONDRIA

443

isooctane-extractable factor (U factor) capable of uncoupling oxidative phosphorylation, inhibiting the ATP-3*Pt exchange, stimulating ATP-ase activity, and causing the swelling of mitochondria. Such a factor is also found in aged preparations of cytochrome a derived from heart mitochondria ~. Since these effects of U factor can be reproduced b y f a t t y acids 1,5 and can be prevented by serum albumin (which binds f a t t y acids) it was concluded U factor is a f a t t y acid or a mixture of f a t t y acids 1. WOJTCZAK AND WOJTCZAK showed that U factor formation is especially prominent in mitochondria of wax-moth larvae and found preparations of the uncoupling factor to contain a series of higher f a t t y acids 6. The enzyme which forms U factor in rat-liver mitochondria was found to be soluble and presumably derives from the soluble matrix, whereas the precursor of U factor, presumably a lipid, was found to be present in the lipid-rich membrane fraction 1. These studies also showed that enzymic formation of U factor, presumably by a lipolytic reaction, is one of the causes of the inactivation of oxidative phosphorylation during aging of mitochondria, and accounts for the ability of serum albumin to increase or restore oxidative phosphorylation in fresh or aged mitochondrial, 4-9 While U factor is mitochondrial in origin, PRESSMAN AND LARDY demonstrated some years ago that the pronounced uncoupling and respiration-stimulating activity of ratliver microsomes is due to their content of free f a t t y acids 1°. Enzymica]ly formed U factor separated from rat-liver mitochondria has been found to be one of the most potent agents known in causing swelling of mitochondria 1. Both the swelling and contraction of liver mitochondria require the participation of enzymes concerned in phosphorylating respiration (see refs. 2, 3,) and are characteristically affected by uncoupling agents ~, 3,11. These relationships suggested the possibility that endogenous enzymic formation and removal of U factor in the mitochondria could be a factor in their structural state and stability and in the activation of swelling and contraction. In this paper we have examined the role of U factor by direct analytical measurement of its level in mitochondria during swelling and contraction, as well as by examining the effect on these reactions of bovine serum albumin, an effective "trapping" agent for f a t t y acids. The metabolic fate of [14C]oleate, an indicator of U factor, was also studied during the swelling and contraction cycle. The results indicate that U factor m a y be a major intramitochondrial element involved in the mechanochemical mechanisms of swelling and contraction. EXPERIMENTAL DETAILS Preparative Rat-liver mitochondria were isolated as described before 12. Sonic-treated mitochondria were prepared by suspending fresh, washed mitochondria from 25 g liver in 25 ml 0.03 M KC1 + o.oi M Tris buffer, p H 7.4, and subjecting the mixture to sonic radiation for 30 rain at 0-4 ° in a 9-kc Raytheon apparatus. Protein was determined according to GORNALLel al. 1~. Crystallized bovine serum albumin was obtained from Armour Pharmaceutical Co., ATP was a product of Pabst Laboratories and L-thyroxine sodium (Elthrin Sodium), of Smith Kline & French Laboratories, Philadelphia. Ei-14Cloleic acid, (specific activity 7 mC/mmole), was obtained from California Corporation for Biochemical Research. Lipid-free serum Biochim. Bioph3s. Acta, 51 (1961) 442-456

444

L. W O J T C Z A K , A. L. L E H I g I N G E R

albumin was prepared b y extracting crystallized serum albumin with ethanol and ether.

Measurement of swelling and contraction The experiments on the effect of oleate and serum albumin on mitochondrial swelling (Figs. i and 2) were carried out optically in a Beckman B spectrophotometer at 520 mF, using dilute mitochondriat suspensions in 15 × IOO m m test tubes exactly as described earlier 1~. ~T4

0.6

A

OA

Mp.MOLES

0.4

OLEATE 0

02

B

I

I

I

MIN. I0

20

30

2

I

J

*0 20 ~ PHLORtDZIN

I

I

I

I0

20

50

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4 6 8

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E MIN.

0.2

IO

0.; CCI4

20

I

I

I0

20

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20

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0

I

I

5

10

J

Min

15

MIN 10

20

30

I

I0

I 20

I

I0

F i g . 2. E f f e c t of s e r u m a l b u m i n o n m i t o c h o n dria] swelling induced by different agents. Medium: o . 1 2 5 M KC1 + o . 0 2 M T r i s - H C 1 , p H 7.4, t e m p e r a t u r e 20 ° . - , without albumin; ..... , i mg/ml bovine albumin. Swell i n g a g e n t s a d d e d a s f o l l o w s : A, s p o n t a n e o u s at 20 ° s w e l l i n g ; B, 1 . 2 . i o - s 2 l / / L - t h y r o x i n e ; C, 2- IO -5 .]I s o d i u m o l e a t e ; D, o . o o 2 M CACI~; E, o , o o i M p h l o r i d z i n ; F, o . o i M G S H ; G , s a t u r a t e d CCI~; H , o . 0 o 2 ~V/ p h o s p h a t e ; T, h y p o t o n i c I m e d i u m = 0.02 JTI T r i s , p H 7.4).

F i g . 1. E f f e c t o f v a r i o u s c o n c e n t r a t i o n s of s o d i u m o l e a t e o n s w e l l i n g o f m i t o c h o n d r i a . The m e d i u m c o n s i s t e d of 5 . 0 m l o . 1 2 5 M K C 1 0 . 0 2 M T r i s , p H 7.4. T h e a m o u n t s of o l e a t e shown were added, and the reaction followed

Monitoring of the swelling and. contraction of mitochondria in relatively dense suspensions, as in the experiments of Figs. 3-7, was also carried out optically in the same manner and the data expressed in absorbancy at 520 m/~ for a i.o-mm light path. Within the zone used, the absorbancy is inversely proportional, approximately, to the water content. In these experiments an aliquot of mitochondria containing about 40 mg protein (i.e. the mitochondria derived from 1.6-2.o g fresh liver) were added for each 50 ml of the medium, which contained either o.125 M KCl-o.o2 M Tris IffC1 pH 7.4 or 0.25 M sucrose-o.o2 M Tris pFI 7.4, as shown. Swelling agents were added as indicated. The suspension was incubated at 230-24 ° and samples of 5 ° ml were removed at different time intervals for the extraction of U factor. The large volumes were necessary to obtain large enough samples for analysis ot U factor, which occurs in rather small amounts. Biochim. Biophys. Acta, 5 i (1961) 4 4 2 4 5 6

UNCOUPLING FACTOR IN MITOCHONDRIA

445

Extraction of U Jactorfrom mitochondria The extraction was based on a procedure used earlier for insect mitochondria 5 and was carried out as follows. To a 5o-ml sample of mitochondrial suspension, 50 mg of lipid-free bovine serum albumin was added as a IO ~o water solution. The sample was quickly mixed during 20-30 sec and then heated rapidly in a boiling water bath for 5 rain. The mixture was cooled, the coagulated proteins centrifuged off, washed with water, and then extracted with three 3-ml portions of ethanol, which remove U factor completely. The extracts were combined and made up to IO.O ml. Since U factor is tightly bound to mitochondria and is not released to the medium when mitochondrial suspensions are heated at ioo °, serum albumin could be omitted and U factor simply extracted from heat-coagulated mitochondria.

Assay of U factor On the assumption that U factor consists of non-esterified fatty acids 1, a microtitration method 14 was used in some earlier experiments. However, this method was not sensitive enough and was subject to a number of errors. Therefore a quantitative test based on the ability of U factor to cause mitochondrial swelling 1 was developed. It proved to be far more sensitive than the micro-titration since as little as 3 m/~moles of oleate could be detected in this way. The test consisted of comparing the rate of swelling of rat-liver mitochondria suspended in a standard medium which is produced by the unknown sample of U factor, with the rate of swelling caused by known amounts of sodium oleate, a standard fatty acid which mimics all known effects of U factor 1. Freshly prepared rat-liver mitochondria were added to 15 × IOO mm tubes containing 5.0 ml of o.125 M KC1 + 0.02 M Tris buffer pH 7.4, to give an initial absorbancy of 0.50-0.60 at 520 m~. Swelling was measured at 5-min intervals over a period of 15 min at 2o °. The "standard" tubes contained known amounts of sodium oleate in the range from 2 to 8 m/~ moles per tube. U factor was added at three levels to three identical tubes, dissolved in no more than 0.2 ml ethanol. The concentration of ethanol was kept equal in all tubes, including those containing standard amounts of oleate. The amount of U factor in the aliquot was expressed as equivalent~ of oleic acid producing the same extent of swelling. A typical example of the swelling response to known amounts of oleate is shown in Fig. I.

Isotopic experiments The fate of E~4C]oleic acid, as an indicator of U factor, was studied in a system of o.125 M KC1 + 0.02 M Tris pH 7.4, containing about 15o m~moles sodium [I-14C~oleate (lO 5 counts/rain)/25 ml of the medium. Mitochondria corresponding to 250 mg liver/25 ml of the medium were added at zero time and the mixture was incubated at 23°-24 °. After 30 min a 25-ml sample was removed, chilled quickly in ice water and centrifuged at i o o o o × g for 5 min at o °. The supernatant fraction and pellet were held for isotopic analysis. ATP and MgCI~ were added to the remainder of the test system to final concentrations of 0.005 M and 0.003 M respectively, to contract the mitochondria, and additional 25-ml samples of the reaction medium were taken after 5 min and after 3 ° min of incubation and treated as described before. The clear supernatant fractions were analyzed for total water-soluble ~*C and for total dissolved 14CO~. The total ~*CO2 in the supernatant fraction was separated by gaseous diffusion from acidified samples into I N NaOH. The mitochondrial pellets Biochim. Biophys. Acta, 51 (1961) 442-456

446

L. WOJTCZAK, A. L. LEHN1NGER

were subjected to extraction with chloroform-methanol (3:I) mixture. The extract was evaporated to dryness, dissolved in petroleum ether, and fractionated on silicic acid columns according to FILLERUP AND MEAD15. The fraction containing fatty acids was chromatographed on paraffin-impregnated W h a t m a n No. 3 paper in 93 o,,o acetic acid according to KAUFMANN1~. The phosphatide fraction was chromatographed on silicic acid-impregnated W h a t m a n No. 3 paper in di-isobutyl ketone-acetic acidwater (40:30:7) according to MARINETT1 AND STOTZ17. Spots of phosphatides and f a t t y acids were located with Rhodamin 6G (see ref. 18) and alkaline permanganate 1~ respectively and counted with a windowless tube scanner. All other counting was performed with a Micromil window counter (Nuclear-Chicago Co.) and corrected for self-absorption. RESULTS AND DISCUSSION

Effect of serum albumin on mitochondrial swelling induced by different agents Serum albumin, presumably because of its ability to bind f a t t y acids tightly19, 2° can reverse or prevent the uncoupling and swelling effects 1 of U factor or oleate when added to mitochondria. Although a number of high molecular weight solutes such as serum albumin, egg albumin, serum 7-globulin, and polyvinylpyrrolidone, inhibit spontaneous or thyroxine-induced mitochondrial swelling when present in high concentrations 1~, presumably through colloid osmotic effects, serum albumin is unique since it is effective in much lower concentrations than the other agents and its effects are much more striking. As little as 0.2 mg serum albumin/ml is effective in blocking spontaneous swelling, for example, whereas the other solutes are completely ineffective at this or even much higher concentrations, a finding which agrees with the relative inability of the other proteins to bind f a t t y acids. Serum albumin is thus an effective and specific " t r a p " of oleate or U factor. Any mitochondrial process dependent on endogenous formation of U factor could be therefore expected to be blocked by serum albumin. In the following experiments the effect of bovine serum albumin on the swelling of rat-liver mitochondria induced by several different swelling agents was studied. Fig. 2 illustrates the effect of bovine serum albumin on mitochondrial swelling induced by nine different agencies. I t is seen that spontaneous swelling as well as swelling induced by oleate, thyroxine, Ca ~+, and phloridzin are strongly inhibited by serum albumin at very low concentrations. On the other hand the mitochondrial swelling induced by the agents phosphate, GSH, CC14, and by a hypotonic medium is essentially unaffected by the presence of bovine serum albumin. These results with serum albumin as f a t t y acid " t r a p " therefore suggest that spontaneous mitochondrial swelling is caused by formation of an endogenous swelling agent, having the properties of a f a t t y acid. They also suggest that the swelling agents thyroxine, Ca 2+, and phloridzin cause swelling of mitochondria in such a way that presence or formation of U factor is a critical component of the swelling reaction. On the other hand the formation of U factor in the mitochondria is apparently not involved in the swelling action of phosphate, GSH, CC14, and in hypotonic swelling, since swelling caused by these agents is not inhibited by serum albumin. It is unlikely that the inhibition of swelling by serum albumin noted with Biochim. Biophys. Acta, 51 (1961) 442 456

UNCOUPLING FACTOR IN MITOCHONDRIA

447

thyroxine could be caused entirely by the binding of thyroxine by serum albumin and prevention of attack of the added thyroxine on mitochondrial structure. Thyroxineinduced swelling of rat-liver mitochondria can be nearly completely inhibited by molar concentrations of serum albumin equivalent to only o.I of the added thyroxine. Under these conditions it has been found that the binding equilibrium between serum albumin and thyroxine is such as to leave a large portion of the thyroxine in the free unbound form. The experiments to follow, which employ direct analysis ot U factor iormation, confirm the tentative conclusions drawn from these experiments with serum albumin as a U factor "trap".

Formation of U factor during swelling of mitochondria The formation of U factor in mitochondria in the presence of various swelling agents was followed by direct analysis. Since only extremely low concentrations of U factor or oleate are required to cause profound mitochondrial swelling, direct analysis of U iactor formation in mitochondria requires that relatively large quantities of mitochondria must be extracted and analyzed for U factor formation in successive aliquots, even with the sensitive analytical test described above. For this reason, U factor formation was studied in relatively heavy suspensions of mitochondria, about 5-1o times as dense as those used in the optical tests of Fig. 2. Under these conditions, mitochondrial swelling occurs much more slowly, at a given concentration of swelling agent, than in the more dilute systems used in optical tests. The relatively lower rate of swelling made possible the analysis of aliquots taken at critical points in the swelling curve. Spontaneous swelling: Fig. 3 shows the level of U factor in rat-liver mitochondria at different times during spontaneous swelling in KC1-Tris medium. Spontaneous 0.3

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0

60

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Fig. 4- F o r m a t i o n of U factor during thyroxineinduced swelling. Concentration of mitochondria, 37 m g protein/5o ml; L-thyroxine was added at i. io -s M. The m e d i u m was KC1-Tris.

Biochim. Biophys. Acta, 51 (1961) 442-456

44S

L. WOJTCZAK, A. L. LEHNINGER

swelling (in the absence of added swelling agents) is of course very slow. I t is seen that the fresh unswollen mitochondria contain very small amounts of U factor. However, as swelling proceeded, U factor accumulated, and after more or less complete swelling had taken place, the level of U factor had reached some 2o-fold the oliginal value. In several such experiments the rate of swelling agreed rather closely with the time course of U factor accumulation. At the point of first detectable swelling and at the point of m a x i m u m swelling, approx. I and 5 mFmoles respectively

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I

0

50

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Fig. 6. F o r m a t i o n of U factor during swelling induced b y o.oo2 3 I phosphate, p H 7.4, in KC1-Tris medium. U factor formation plotted on basis of 37 m g mitochondrial protein/5 o ml, to facilitate comparison with Figs. 3, 4, and 7.

of U factor (calculated as oleic acid) was formed/mg mitochondrial protein. These are relatively small amounts and m a y be compared with the molar amount of cytochrome a in rat-liver mitochondria, approx. 0.2 m/~mole/mg protein. Thyroxine-induced swelling: In a number of experiments it was found that greatly increased formation of U factor also occurred during mitochondrial swelling produced b y thyroxine. This formation also paralleled rather closely the thyroxineinduced swelling. Fig. 4 illustrates such an experiment. Fig. 5 shows that thyroxine greatly stimulates the rate of U factor formation above the spontaneous rate, iust as it increases the rate of swelling. This stimulation was rather variable and was not seen to this extent in every experiment, however. I t is clear that direct measurement of U factor formation during spontaneous and during thyroxine-stimulated swelling fully confirms the effects observed with serum albumin as a " t r a p " of U factor. Phosphate-induced swelling: There was no significant increase in the content of U factor in mitochondria during the initial phase of rapid swelling produced b y phosphate (Fig. 6), above that produced in the absence of swelling agent. An increase in Biochim. Biophys. Acta, 51 (1961) 442-456

UNCOUPLING

F A C T O R IN M I T O C H O N D R I A

449

the level of U factor could be observed after more prolonged incubation with phosphate, long after the swelling plateau had been reached, and it is therefore probably a secondary or a spontaneous effect. Clearly the rapid swelling induced by phosphate is not accompanied by U factor formation, contrary to the case of spontaneous and thyroxine-induced swelling. This is in agreement with the lack of protection by serum albumin against phosphate-induced swelling (Fig. 2) and confirms that swelling induced by phosphate is not caused by an activation of U factor production. 0.6

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of U f a c t o r d u r i n g s w e l l i n g i n d u c e d b y C a C ] 2. C o n c e n t r a t i o n 71 m g p r o t e i n / 5 o m l of CaC12, o . o o z M .

of m i t o c h o n d r i a ,

Ca2+ induced swelling: A rapid increase in the level of mitochondrial U factor occurred when swelling was induced with Ca 2+, (Fig. 7). These results were quite reproducible and indicate a clear correlation between Ca2+-induced swelling and the formation of U factor, in agreement with findings using serum albumin as a "trap". GSH-induced swelling: No increase of U factor formation could be found during swelling induced by 5" lO-8 M GSH in confirmation of the experiments with serum albumin. Actually, an inhibition of spontaneous formation of U factor by GSH was occasionally observed. This effect might be related to the observed antagonism of GSH and thyroxine in inducing mitochondrial swelling 21. Formation of U factor in disrupted mitochondria Formation of U factor during spontaneous swelling and the activation of this process by Ca ~+ or by thyroxine, as well as the protective effect of serum albumin on spontaneous, Ca ~+-, and thyroxine-induced swelling suggest that the primary effect of these swelling agents is the stimulation of U factor formation by enzymes in the mitochondria. The enzymic character of this process has already been described 1. To determine whether Ca 2+ and thyroxine stimulate enzymic formation of U factor, a series of experiments with sonic-disrupted mitochondria was carried out. The results are summarized in Table I. It is evident that substantial amounts of U factor are Biochim. 13iophys. dcta, 51 (1961) 4 4 2 - 4 5 6

45 °

L. W O J T C Z A K ,

A. L. L E H N I N G E R

TABLE EFFECT

OF THYROXINE

AND

C a 2+ O N

SONICATED

I THE

FORMATION

OF

g

FACTOR

IN

MITOCHONDRIA

The amount of U factor is expressed in m#moles of oleic acid formed in the mitochondria derived from I.O g liver. The sonicated mitochondria were incubated in o . o 3 . ~ / K C l - o . o i M T r i s - H C 1 p H 7.4 a t i . o g/ml. Expt.

Swelling agent

None Thyroxine 2.

None None Thyroxine Thyroxine

Concentrcaion ( M)

I.O. IO 5

5 . o . lO -5 5.0.10

None None

4.

5.

Temperature Time (rain)

U factor formed (ml*moles)

24 o 24 °

12o 12o

82 90

37 °

6o 12o 6o

zo8 324 96

120

220

37 °

.5

37 °

192 532 692

282

CaCI~ CaCI~

o.oo 4 0.004

None CaC12 CaC1 e CaC12

o.ooi 0.002 0.004

37 37 37 37

° ° ° °

Ioo ioo too Ioo

137 487 63 ° 525

I80 18o 18o

220 200

0.o02

37 ° 37 ° 37 °

None None plus 1 . 7 m g / m l serum albumin CaC12

37 °

6o I2o 6o i2o

7oo

formed on incubation of sonicated mitochondria, in agreement with the earlier observations by LEHNINGER AND REMMERT1. However, it is seen that thyroxine has no stimulatory effect on this process. On the other hand, Ca2+ is a powerful activator of U factor formation. As little as 2. lO -3 M CaCI~ brings about a maximum activation; this is approximately the same concentration which induces a rapid swelling of mitochondria. It is well known that Ca 2+ stimulates enzymic hydrolysis of neutral fats and phosphatides. In some cases, this stimulation is due to removal of products, i.e. fatty acids, which form insoluble calcium salts, rather than to a true activation of enzymic hydrolysis. In the present case, however, Ca~+ seems to be a true activator, since serum albumin, which is also a good acceptor for fatty acids, does not stimulate U factor formation (Table I, Expt. 5).

Disappearance of U factor during A TP-induced mitochondrial contraction It has been observed that the addition of ATP and Mg 2+ to swollen rat-liver mitochondria causes their rapid contractionZ, 3, zz, z3. Experiments were carried out to determine whether the high levels of U factor which accumulate during swelling undergo changes during ATP-induced contraction of swollen mitochondria. Typical results are exemplified by Fig. 8 and Table II. It is clearly evident that a large decrease in the amount of mitochondrial U factor occurs during the contraction of swollen mitochondria induced by addition of ATP and MgC12. It also can be seen that azide Biochim. Biophys..4cta,

5 t (1961) 4 4 2

456

451

UNCOUPLING FACTOR IN MITOCHONDRIA

or sucrose, which block ATP-induced contraction*2, ~8, do not inhibit the removal of U factor. Mere removal of U factor by an ATP-requiring reaction alone does not suffice to contract swollen mitochondria; some other ATP-dependent reaction blocked by azide is necessary to contract mitochondria. Table III shows that when ATP is added to rat-liver mitochondria swollen with added sodium oleate, there is a rapid disappearance of the added oleate, which ATP

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Fig. 8. Effect of ATP and Mg ~+ on the level of U factor formed during s p o n t a n e o u s swelling. The s y s t e m contained 9o m g mitochondrial protein/5o ml. At the t i m e indicated b y the arrow, ATP and MgCI~ were added to the final concentration of o.oo 5 M and 0.003 M respectively.

TABLE II THE LEVEL OF U FACTOR DURING ATP-INDUCED CONTRACTION OF MITOCHONDRIA SWOLLEN BY DIFFERENT AGENTS Mitochondria were allowed to swell at 23 ° (I2O-I6O min) and ATP and MgC12 were t h e n added to a final concentration of o.oo 5 M and 0.oo3 _M respectively. The level of U factor was measured j u s t before and IO to 3o min after the addition of ATP + Mg ~+, and was expressed in m/~mole equivalents of oleic acid/mg of mitochondrial protein. U .factor (re#moles~rag protein) Swelling agent

Spontaneous Spontaneous Spontaneous Thyroxine Thyroxine Thyroxine Thyroxine Phosphate CaC12 CaC12

Medium

KC1-Tris KC1-Tris KC1-Tris KC1-Tris KC1-Tris KCI-Tris Sucrose-Tris KC1-Tris KCI-Tris KCI-Tris o.oo2 M azide

Before ATP

After ATP

13.4 3.7 5.5 18.2 4.3 6-5 11.9 13.8 5,2 5.9

3.2 I.O 1.8 9.5 1.8 3 .6 5.4 7 .8 1.6 2.3

Per cent U factor removed

76 73 67 48 58 45 55 44 69 61

B i o c h i m . B i o p h y s . A c l a , 51 (1961) 442 456

452

L. W O J T C Z A K , A. L. L E H N I N G E R TABLE

III

REMOVAL OF EXTERNALLY ADDED OLEATE BY RAT-LIVER MITOCHONDRIA UPON ADDITION OF A T P AND M g 2+

The m e d i u m c o n t a i n e d o . 1 2 5 M NC1 a n d o . 0 2 M T r i s - H C l , p H 7-4, and m i t o c h o n d r i a from t g l i v e r , in a f i n a l v o l u m e of 4 ° m l . A T P a n d MgC12 were added to final c o n c e n t r a t i o n s of 0 . 0 0 5 M and 0 . 0 0 3 M respectively. Oleate in mitochondrial suspension (mumoles) Expt.

t 2 3

Before addition z o mid after A T P of A T P addition

870 375 390

Difference

67 ° 280 250

TABLE

--200 --95 --14o

IV

METABOLISM OF EI-14C~ OLEIC ACID DURING SWELLING AND CONCENTRATION OF RAT-LIVER MITOCHONDRIA Incubation

medium:

o . 1 2 5 M KC1 + 0 . 0 2 M T r i s - I t C l , p H 7.4, 2 5 . 0 m l , s o d i u m o l e a t e c o n t a i n i n g

a b o u t lO 5 c o u n t s / m i D 14C i n t h e c a r b o x y l g r o u p , 146 m / ~ m o l e s ; m i t o c h o n d r i a f r o m 2 5 0 m g rat l i v e r . A T P a n d MgCI~ were added after 3 ° miD ( d u r i n g w h i c h s u b s t a n t i a l swelling had t a k e n p l a c e ) to a f i n a l c o n c e n t r a t i o n of o . o o 5 M a n d 0 . 0 o 3 3 / respectively. T e m p e r a t u r e w a s 23 °. Sample 1

I 2 3 4 5

T i m e of i n c u b a t i o n (min) A T P + M g 2+ State of mitochondria TimeofincubationwithATP 14C i n s u p e r n a t a n t (a) T o t a l ( c o u n t s / m i D )

(b) As 14CO2 (%) 6

7

14C i n C H C l s - C H 3 O H (a) T o t a l ( c o u n t s / m i D ) (b) A s f a t t y acids (%) (c) A s p h o s p h a t i d e s (~/o) Recovery (%)

+ M g 2+(miD)

2

o -initial --

.3

3° -Swollen --

4

3° +

30 --

Contracted

Contracted

5

30

9 600

49 700

58 4 ° 0

59 ooo

3

45

58

ii2

84 i o o

17 8 0 0 93 7 68

i t 200 62 38 7°

i i ooo 63 37 7°

ioo o 94

accompanied the contraction. ATP can thus remove either endogenously formed U factor or externally added oleate.

Fate of Ez-laCloleic acid during swelling and contraction of mitochondria In order to study the fate of free fatty acids ( = U factor) during swelling and contraction, mitochondria were allowed to swell in the presence of [IJ4Qoleic acid. It was observed that practically all the oleate added to a suspension of fresh unswollen mitochondria in KC1-Tris was immediately adsorbed or bound by the mitochondria. Only IO °/o of the radioactivity could be recovered in tile supernatant fraction of the zero time sample (Table IV, sample No. i) but this was found not to consist of long chain fatty acids since it could not be extracted by iso-octane. It is conceivable that some oxidative degradation of the labeled oleate had occurred during centrifugation of the zero time sample at o °. Biochim.

Biophys.

A c t a , 51 (1961) 4 4 : . - 4 5 6

UNCOUPLING FACTOR IN MITOCHONDRIA

453

After 3o min incubation, during which the mitochondria had undergone nearly complete swelling, about half of the added 14C was found in the supernatant medium, part of it as CO 2. The amount of 14C extractable from the mitochondria by chloroform-methanol decreased very markedly as compared with the zero time control (Table IV, sample 2), following swelling. Since there is normally a net increase in the amount of fatty acids (U factor) during spontaneous swelling of the mitochondria, as already described above, the rapid disappearance of I14Cloleate indicates that a "turnover" of free fatty acids occurs during mitochondrial swelling. Some of the oleate disappearing was recovered as COs, indicating that it underwent oxidation. It is of course quite possible that the behavior of added oleate is not representative of the behavior of all the f a t t y acids present in U factor (see below). When mitochondrial contraction was now induced by addition of ATP + Mg ++, a further conversion of [14C]oleate into water-soluble compounds was observed, together with an increased total formation of 14C0~ (Table IV, sample 3). The most striking effect of ATP, however, was on the incorporation of E14Cloleicacid into mitochondrial phosphatides. Whereas only 7 % of the total lipid 14C could be found in the phosphatide fraction after 3 ° min of swelling in the absence of ATP and Mg 2+, as much as 38 ~o of the oleate was rapidly incorporated into phosphatides when mitochondria were incubated for only 5 rain with ATP and Mg2+, during which period complete contraction took place. No further incorporation of [14C1oleate into the phosphatides was observed 30 min following the addition of ATP and Mg 2+ (Table IV, sample 4). The only change which was observed on longer incubation with ATP was the conversion of all water-soluble ~4C compounds into CO s. Thus the incorporation of oleic acid into the phosphatide fraction seems to be a very rapid process and it attains its maximum extent very shortly after addition of ATP, parallel to the kinetics of mitochondrial contraction. Paper chromatography of the total phosphatide fraction after ATP-induced contraction yielded two radioactive spots whose RE values corresponded to those found by MARINETTI AND STOTZ17 for phosphatidic acid (or cardiolipin) and for cephalin.

Composition of U factor from rat-liver mitochondria A preliminary analysis by gas phase chromatography of rat-liver U factor gave the following composition of free fatty acids. Saturated: C14, IO °/o; CI~, 19 ~o; C18, 30 %. Total saturated 59 ~o. Unsaturated: Cls monounsaturated, 16 ~o; Cls diunsaturated, 12 %. Unidentified, presumably C2o polyunsaturated, 13 ~o. Total unsaturated 41 ~o. This analysis was carried out by Dr. I. SARLOS of the Biochemistry Research Division, Department of Medicine, Sinai Hospital, Baltimore, Md., and was made possible by the courtesy of Dr. D. A. T U R N E R , to whom we are greatly indebted. These data on rat-liver U factor composition may be compared with those of WOJTCZAK AND WOJTCZAK6 on U factor from the mitochondria of Wax-moth larvae, which is much richer in unsaturated fatty acids. ADDITIONAL COMMENTS

These experiments indicate that spontaneous, Ca2+-induced and thyroxine-induced swelling of mitochondria may be caused primarily by the action of U factor formed Biochim. Biophys. Acta, 51 (1961) 4 4 2 - 4 5 6

454

L. W O J T G Z A K , A. L. L E H N I N G E R

enzymically in the mitochondria. This view is supported not only by the fact that the U factor concentration in mitochondria rises greatly during swelling, but particularly by the fact that serum albumin prevents mitochondrial swelling produced by Ca ~+ or thyroxine. On the other hand, swelling induced by phosphate or GSH is not accompanied by an increase in U factor concentration nor is it inhibited by serum albumin; presumably swelling induced by these agents is different in kind and may be a more direct consequence of the action of phosphate and GSH on the membrane itself. Table V shows a summary of the role of U factor in the action of various mitochondrial swelling agents. The complete parallel between stimulation of U factor formation in mitochondria with the inhibitory effect of bovine serum albumin not only supports these general conclusions but also indicates that there may be two major classes of swelling agents. TABLE

V

CLASSIFICATION OF MITOCHONDRIAL SWELLING AGENTS

Swelling agent

Inhibition by serum albumin

Accumulation of U factor

None (spontaneous) Thyroxine Ca 2+ Phloridzin

+ + + +

+ + + +

Phosphate Glutathione CCI~ Hypotonicity

o o o o

o o

The mechanism by which Ca 2+ and thyroxine stimulate U factor formation is not entirely clear. Ca 2+ stimulates this reaction in both intact and sonic-disrupted mitochondria, presumably by a direct activating effect on the enzyme producing U factor. An activation of hydrolysis of mitochondrial lipids by Ca ~+ has also been observed by F. L. CRANE24 (personal communication). These general conclusions agree with earlier findings in this laboratory by TAPLEY AND COOPER25 that Ca ~+ and thyroxine do not uncouple oxidative phosphorylation in digitonin fragments of mitochondrial membranes and with similar findings on sonic fragments in LARDY'S laboratory ~6, whereas these agents readily uncouple phosphorylation in intact mitochondria under specific conditions. It appears possible that Ca 2+ and thyroxine are not themselves directly active as uncouplers but act only by stimulating enzymic production of U factor from a precursor lipid. The precursor lipid is present in the membrane, but the membrane fragments contain relatively little of the enzyme necessary for U factor production, as shown by LEHNINGER AND REMMERT 1, and this deficit could thus account for the failure of Ca ~+ and thyroxine to uncouple phosphorylation in the membrane fragments. The variability of the uncoupling action of thyroxine in different submitochondrial preparations may be caused by variations in the amounts of the U factor producing enzyme present in the fragments. Contraction of swollen mitochondria by ATP + Mg~+ is accompanied by a rapid decrease in the amount of U factor, with essentially parallel kinetics. Removal of B i o c h i m . B i o p h y s . A c t a , 51 (1961) 4 4 2 - 4 5 6

UNCOUPLING FACTOR IN MITOCHONDRIA

455

U factor by ATP accompanies the contraction and may facilitate it, but it is not the primary cause of contraction, since removal of U factor by ATP + Mg2+ occurs even when mitochondrial contraction is inhibited by azide or sucrose. The rapid incorporation of added [14Cloleic acid (as an indicator of U factor) into mitochondrial phosphatides which occurs during contraction, presumably takes place by already known mechanisms (recently reviewed by ROSSITER AND STRICKLAND27). It is difficult to say at the present state of this investigation whether this process has direct molecular or enzymic relationship to the contraction phenomena. However, it is relevant that the membranes, which contain the contractile elements, contain some 40 ~o lipid, most of which is phospholipid. The rapid incorporation of [14Cllabeled oleate into a phospholipid fraction containing cardiolipin, a polymer of a glycerol phosphatidic acid, is of especial interest. Such a polymeric lipid could be expected to be a significant structural element in the lipoprotein membrane. The phosphatidic acid fraction of mitochondrial phospholipids is also of interest in connection with the hypothesis of HOKIN AND HOKIN~ on the function of phosphatidic acids in active transport of electrolytes. Further work is under way to identify more securely the chemical nature of the phospholipids in which the rapid incorporation of oleate and U factor takes place. Preliminary experiments showed that 0.3 M sucrose, which blocks ATP-induced contraction, also blocks the incorporation of [l*Cloleate into the phosphatidic acid fraction. This effect, which is under further investigation, may be involved in the action of sucrose and other polyhydroxylic compounds in inhibiting mitochondrial contraction. While the experiments described in this paper deal with mitochondrial formation of U factor, it must be recalled that a heat-stable factor of f a t t y acid nature present in microsomes (derived from the endoplasmic reticulum) can uncouple phosphorylation 1° and also cause swelling of mitochondria 2°. .AVI-DOR has pointed out that the intimate physical contact between mitochondria and the endoplasmic reticulum which may occur in the intact cell may provide a device for changing permeability of the mitochondrial membrane at specific locations through the action of the free f a t t y acids present in or generated by microsomes 29. ACKNOWLEDGEMENTS

This investigation was supported by grants from the National Institutes of Health, the National Science Foundation, the Nutrition Foundation, Inc. and the Whitehall Foundation. REFERENCES 1 A. L. LEHNINGER AND L. F. REMMERT, Jr. Biol. Chem., 234 (1959) 2459. 2 A. L. LEHNINGER, Ann. N. Y. Acad. Sci., 86 (196o) 484 . 3 A. L. LEHNINGER, Physiol. Revs., in t h e press. 4 W. C. H/3LSMANN, ~V. B. ELLIOTT AND H. RUDNEY, Biochim. Biophys. Acta, 27 (1958) 663. 5 B. C. PRESSMAN AND H. A. LARDY, Biochim. Biophys. Acta, 21 (1956) 458. 6 L. WOJTCZAK AND A. B. WOJTCZAK, Biochim. Biophys. Acla, 39 (196o) 277. 7 M. E. PULLMAN AND E. RACKER, Science, 123 (1956) ILO5. s B. D. POLLS AND H. W. SHMUKLER, J, Biol. Chem., 227 (1957) 419. " B. SACKTOR, J. J. O'NEILL AND D. G. COCHRAN, J. Biol. Chem., 233 (1958) 1233. 10 B. C. PRESSMAN AND H. A. LARDY, Biochim. Biophys. Acta, 18 (1955) 482; Biochim. Biophys. ,4cta, 21 (1956) 458 .

Biochim. Biophys. Acla, 51 (1961) 442-456

456

t. WOJTCZAK, A. L. LEHNINGER

TAPLEY, J. Biol. Chem., 222 (1956) 325. LEHNINGER, B. L. RAY AND M. SCHNEIDER, J, Biophys. Biochem. Cytol., 5 (1959) 97GORNALL, C. J. BARDAWILL AND M. M. DAVID, J. Biol. Chem., 177 (1949) 751. ALBRINK, J. Lipid Research, i (1959) 53. FILLERUP AND J. F. MEAD, Proc. Soc. Exptl. Biol. Med., 83 (1953) 574. t~AUFMANN, Analyse der Fette und Fettprodukte, Springer Verlag, Berlin-G6ttingen-Heidelberg, 1958 , p. 847. 17 G. V. MARINETTI AND E. STOTZ, Biochim. Biophys. Acta, 21 (1956) 168. IS G. V. MARINETTI, J. ERBLAND AND J. KOCHEN, Federation Proc., 16 (1957) 837. 19 p. D. BOY]~R, C. A. BALLOU AND J. M. LUCK, J. Biol. Chem., 167 (1947) 4o7 . 20 D. S. GOODMAN, J. Am. Chem. Soc., 80 (1958) 3892. 21 A. L. LEHNINGER AND M. SCHNEIDER, J. Biophys. Biochem. Cytol., 5 (I959) lO9. 22 A. L. LEHNINGER, J. Biol. Chem., 234 (1959) 2187. 2~ A. L. LEHNINGER, J. Biol. Chem., 234 (1959) 2465 24 V. g. CRANE, personal communication. 25 D. F. TAPLEY AND C. COOPER, J. Biol. Chem., 222 (1956) 341. 26 ~vV. C. MACMURRAY, G. F. MALEY AND H. A. LARDY, J. Biol. Chem., 230 (1958) 219. 27 R. J. ROSSITER AND K. P. STRICKLAND, in IZ. BLOCH, Lipide 21letabolism, John Wiley and Sons, New Yo rk , 196o, p. 69. 2s L. E. HOKIN AND M. i . HOKIN, J. Biol. Chem., 233 (1958) 800. 29 y . AVI-DOR, Biochim. Biophys. Aeta, 39 (196o) 53. 11 D. 12 A. 13 A. 14 M. 15 D. 16 U.

F. L. G. J. L. P.

Biochim. Biophys. Acta, 5 I (I96I) 442-456

MOLECULAR CHANGES IN MYOSIN CAUSED BY METHYLMERCURIC HYDROXIDE D A V I D R. K O M I N Z

National Institute o/Arthritis and Metabolic Diseases, National Institutes o[ Heat,th, Bethesda, Md. (U.S.A.) (Received February 2nd, 1961)

SUMMARY

An ultracentrifugal study has been made on myosin treated with methylmercuric hydroxide. I. Myosin is transformed by titration of its - S H groups with methylmercuric hydroxide to a faster-sedimenting product. This occurs in the same range of methylmercuric hydroxide titration at which the calcium-activated ATPase activity is inhibited. A parallelism can be demonstrated between the loss of ATPase activity and the loss of myosin due to the transformation process. 2. Complete titration of the - S H groups of myosin with methylmercurie hydroxide causes the release of a small subunit with sedimentation rate of 1.6 S, at 25 ° but not at 15 ° . 3. These results suggest that the - S H groups whose titration causes inactivation of ATPase activity are responsible for the configurational integrity of the active site, and that release of the small subunit is not related to titration of these - S H groups.

Biochim. Biophys. .4cta, 5I (t96~'1 456 4~3