Tightly coupled mitochondria from human early placenta

Placenta (1982), 3, I97-2IO

Tightly Coupled Mitochondria from Human Early Placenta S.ZOLNIEROWICZ, J.SWIERCZYNSKI& L. ZELEWSKI Department of Biochemistry IBM, Medical School, Gdaf~sk 80--211, Dcbinki i, Poland

INTRODUCTION The studies of Schreiner and Villee (1965) have shown that human early placental mitochondria are able to oxidize ~-ketoglutarate plus nicotinamide adenine dinucleotide (NAD), L-glutamate plus NAD and isocitrate plus nicotinamide adenine dinucleotide phosphate (NADP) and NAD. These mitochondria exhibited a low level of oxidative phosphorylation and high adenosine triphosphatase (ATPase) activity. High concentration of progesterone found in human placental mitochondria led Schreiner and Villee (1965) to the conclusion that this steroid may act as an activator of ATPase. Although isolated heart sarcosomes (Packer, 1957; Chao and Davis, 1972), rat kidney mitochondria (Gmaj, Nowicka and Angielski, 1974) and rat spleen mitochondria (Vijayakumar and Weidemann, 1976) were described as loosely coupled due to the presence of Mg 2+-stimulated ATPase activity they were tightly coupled if incubated in the medium containing ethylene diamine tetra-acetic acid (EDTA) without magnesium ions added. Since addition of Mg 2+ has a marked stimulatory effect on ATPase activity in the human early placental mitochondria (Schreiner and Villee, I965), in these mitochondria, too, ATPase activity might be responsible for a low phosphorylation capacity. To test this possibility the present experiments were carried out. The effect of added Mg 2+ on ADP:O (adenosine diphosphate:oxygen) ratio and on the ATPase activity in mitochondria isolated from human early placenta was estimated. The results presented in this paper indicate that mitochondria from human early placenta incubated in the absence of added Mg 2+ are able to oxidize Krebs cycle intermediates, pyruvate,/3-hydroxybutyrate, glutamate, glutamine, ~-glycerophosphate and palmitoyl-carnitine with ADP:O ratios corresponding to the theoretical values. The addition of magnesium ions to the incubation medium resulted in a decreased ADP:O ratio with all substrates tested. It is concluded that Mg 2+-stimulated ATPase activity is responsible for a low ADP:O ratio in human early placental mitochondria.

MATERIALS AND M E T H O D S Placental tissue was obtained from pregnancies terminated for social reasons between 8 and 12 weeks of gestation. The connective tissue (from five placentae) was removed by blunt dissection, parenchyma was collected and rinsed twice in o. 9 per cent NaCl plus 2 m i EDTA plus IO mM Tris-HCl (pH 7.8). Then the tissue was rinsed two times with o.2 5 M sucrose plus 2 mM EDTA plus IO m i Tris-HCl (pH 7.8). The rinsed tissue was suspended in the sucrose-EDTA-Trisoi43-40o4/82/o3o2-197 $02.0o

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S. Zotnierowicz, J. Swierczynski, L. Zelewski

HC1 (pH 7.8) solution and homogenized manually in a glass Potter-Elvehjem homogenizer with a Teflon pestle. The homogenate (2o per cent) was centrifuged at 6oo g for io min and the pellet discarded. The supernatant obtained was centrifuged at 8ooog for lO min. The mitochondria were suspended in 0.25 M sucrose, 2 mM EDTA and io mM Tris-HCl (pH 7.4). The suspension was centrifuged at 6oog for IO min, the pellet was discarded and the supernatant was centrifuged at 7ooog for 1o min. The pellet was suspended in sucrose-EDTA-Tris-HCl (pH 7.4) medium and centrifuged at 7ooog for xo min. The final pellet was suspended in the isolation medium to obtain about 30mg of mitochondrial protein per i ml. The method yielded about i mg mitochondrial protein from I g of wet placental tissue. Mitochondrial protein was estimated by the biuret method. Electron m i c r o s c o p y This was kindly performed by Dr Teresa Wrzotkowa of the Laboratory of Electron Microscopy, Medical School Gdafisk. The mitochondria were pelleted in isolation medium and fixed in 3 per cent glutaraldehyde. Further operations were carried out as described previously (Popinigis, Wrzotkowa and Swierczyfiski, I97I; Aleksandrowicz, Swierczyfiski and Wrzotkowa, 1973). M e a s u r e m e n t o f mitochondrial respiration Oxygen consumption was measured polarographically with Clark oxygen electrode in the medium containing: 25 mM KC1, 5o m.M Tris-HC1 pH 7.4, 5 mM potassium phosphate buffer pH 7-4, 2 rnM EDTA and o.5 per cent bovine serum albumin (BSA). Final volume was 2.5 ml. All polarographic recordings were made at 3o °C. The respiratory control index and ADP:O ratio were calculated as described by Estabrook (1967). ADP was determined spectrophotometrically in an assay which couples the pyruvate kinase and lactate dehydrogenase reaction (Jaworek, Gruber and Bergmeyer, 1974). ATPase (or ADPase) activity was determined by following the liberation of inorganic phosphate from ATP (or ADP). About I mg of mitochondrial protein was incubated at 3o °C in i ml of the incubation medium containing: 25 rnM KCI, 5o rn~ Tris-HCl pH 7.4, 2 mM EDTA and 5 mM ATP (or other nucleotides). Other additions were as indicated in the corresponding Tables and Figures (see 'Results and Discussion'). The reaction was stopped with o.5 ml xo per cent trichloroacetic acid. Inorganic phosphate was estimated by the method of Gomori (I953). Chemicals All substrates, nucleotides, inhibitors and BSA (essentially fatty acid free) were obtained from Sigma Co., St Louis (USA). Sucrose-AnalaR was from BDH, England. Ficoll was from Pharmacia (Sweden). All other compounds were of the highest purity available from P.O.Ch. Gliwice (Poland).

R E S U L T S AND D I S C U S S I O N Figure 1A shows electron micrographs of the mitochondria prepared by the method described above. It may be seen that the preparation was rich in mitochondria but was contaminated with other subcellular structures and non-specific membranous material. Thus we have tried to pass this mitochondrial fraction through a discontinuous Ficoll gradient as described by Clark and Nicklas (197o) for purification of rat brain mitochondria. This operation did not substantially improve the purity of the mitochondrial fraction as judged by electron microscopy examination (not shown). Figure IA reveals also that the population of mitochondria was heterogenous, and

Tightly Coupled MitochondriaJ?om Human Early Placenta

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two principal morphological forms may be seen, as has been reported previously by Swierczyfiski, Scist"owski and Aleksandrowicz (I976a) and Klimek et al (I97Z) in human term placenta. The majority of the isolated mitochondria were in the so-called condensed state, characterized by large electron-transparent intracristal space and a highly compact electrondense matrix. Some of the isolated mitochondria were in the orthodox configuration characterized by well-defined cristae, small intracristal space and relatively dilute matrix. As mitochondria in situ (Figure IB) were in the state resembling the orthodox configuration it seems likely that during preparation procedure most of the mitochondria underwent conformational changes. A typical polarographic tracing for human early placental mitochondria is shown in Figure 2. In the reaction medium containing EDTA and no added Mg z+ ions, respiration of these mitochondria utilizing ~-ketoglutarate amounted to 7.7 ngatoms O x min-1 per mg protein before ADP was added (Figure 2A) and 3o.9 ngatoms O x min- t per mg protein after addition of ADP (state 3 respiration). Upon depletion of ADP, oxygen consumption returned to the initial stage (state 4 respiration) attained before ADP addition. The addition of a second aliquot of ADP yielded respiratory transitions similar to those obtained with the first ADP addition; the respiratory control ratio in these experiments was 4 and the ADP:O ratio was 2.7. It should be pointed out that the return to state 4 was sharp, indicating a high affinity for ADP. Although M g 2+ had no substantial effect on the respiration when no ADP was added (not shown), after a second addition of ADP and its subsequent depletion Mg 2+ stimulated respiration to the same extent as ADP did in the absence of Mg 2+ ions (Figure 2A). While ot-ketoglutarate was oxidized by the mitochondria in the presence of added Mg 2+ ions, the addition of ADP greatly stimulated the rate of oxygen consumption; however, no return to the state 4 respiration was observed under these conditions (Figure 2B). The results of experiments presented in Figure 2 indicate that human early placental

A mito

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Figure 2. EffectofMg 2+ i~ns~nthe~t-ket~g~utarate~xidati~nbyhumanear~yp~acenta~mit~ch~ndria.Theresu~tsare presented as oxygen electrode traces showing the effect of added MgCI 2 (final concentration 2 mM) on human early placental mitochondria oxidizing ct-ketoglutarate (ct-kg; iomM). The mitochondria (mito; 2.7mg protein) were suspended in the total reaction volume of 2.5 ml, as given in 'Materials and Methods'. The respiration was stimulated by adding ADP (4oo nmol) or MgClz(2 mM).

S. Zofnierowicz, J. Swierczy/Iski, L. 7.elewski

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mitochondria were tightly coupled while incubated in the presence of E D T A and in the absence of added Mg 2+ ions. It is worth emphasizing that similar effects of both ADP and MgCI 2 on respiration have been observed using mitochondria isolated from 24-weeks--old and term placenta. In order to determine whether E D T A could be replaced by E G T A the experiments presented in Figure 3 were carried out. As can be seen, with isocitrate as substrate, the addition of ADP to mitochondria incubated in the presence of E D T A greatly stimulated the rate of oxygen consumption and the return to state 4 respiration was sharp (Figure 3A). When E D T A was replaced by the same concentration of EGTA, the addition of ADP also caused an increased oxygen consumption; however, no return to state 4 respiration was observed (Figure 3B). These results could have been anticipated in view of the different ability of E G T A and E D T A to bind Mg 2+ ions (Schwarzenbach, Senn and Anderegg, 1957; Funahashi, Yamada and Tanaka, 197 I). The maximum rate of respiration, maximum acceptor control and maximum ADP:O ratio were observed at ~ to z mM E D T A concentration. However, it should be pointed out that in some mitochondrial preparations high respiratory control and maximum ADP:O ratio were observed at o. i mM E D T A concentration.

A EDTA

o~ 2m~ln

B EGTA

\

[O21=O

k----- [Oa]=O

Figure 3. Effect ofEDTA (A) and EGTA (B) on isocitrateoxidation in the presenceof ADP. The mit~chondria (mito; 4.5 mg protein) were incubated in the conditions described in 'Materials and Methods' and Figure z, except that in Figure 3B z mMEGTA wasadded instead of z mMEDTA. Whereindicated, isocitrate(isocitr;2 raM),ADP (4oonrnol) and MgCl2(zmM) were added.

Figure 4 shows that in the absence of added Mg 2+ (Figure 4A) and in the presence of Mg z+ (Figure 4B) the oxidation of'cis-aconitate by human early placental mitochondria was inhibited by oligomycin. 2,4-Dinitrophenol relieved this inhibition both in the presence and in the absence of added Mg 2+. In this respect mitochondria isolated from human early placenta resemble mitochondria isolated from other sources. The apparent loss of phosphorylation efficiency by human early placental mitochondria observed in response to added Mg 2+ ions is similar to that reported for heart sarcosomes (Packer, I957; Chao and Davis, I972), rat kidney cortex mitochondria (Gmaj, Nowicka and Angielski, I974) and rat spleen mitochondria

Tightly Coupled Mitochondria J?om Human Early Placenta

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203

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Figure 4. Effectofoligomycinon ADP-stimulatedcis-aconitateoxidationin the absence(A) and in the presence(B) of MgCI2 (2 mM) in the incubation medium. The mitochondria (mito; 3.3 mg protein) were incubated in the conditions described in 'Materials and Methods' and in Figure 2. Where indicated, cis-aconitate (cis-acon; 5 mM), ADP (4oonmol), oligomycin(oligo; 5 Pg) and DNP (8o/aM) were added.

(Vijayakumar and Weidemann, I976). It has been concluded that the effect of Mg 2+ ions on oxidative phosphorylation is accounted for by the Mg z+ stimulated ATPase activity which ensues simultaneously with oxidative phosphorylation. In the experiments shown in Figure 5 human early placental mitochondria were incubated with isocitrate as substrate in the absence of added Mg 2+ . Addition of ATP did not change oxygen consumption. However, on addition of Mg z+ there was a rapid burst of respiration (Figure 5A). If atractyloside (Figure 5B) or oligomycin (Figure 5C) were added to the incubation medium, Mg 2 + did not stimulate oxygen consumption in the presence of added ATP. The results described above are consistent with the presence of Mg2+-stimulated ATPase activity in intact human early placental mitochondria. Table I shows the results of an experiment designed to show the effect of Mg z÷ on ATPase activity measured by release of inorganic phosphate. ATPase activity was stimulated 8 to I o fold by Mg z+. Oligomycin only partially inhibited MgZ+-stimulated ATPase activity, but completely blocked ATPase activity in the absence of added Mg 2+ ions (Table i). As shown in Figure 5, oligomycin completely inhibited the Mg z +-stimulated respiration in the presence of ATP, but inhibited Mg2+-stimulated ATPase activity only slightly (Table I). This phenomenon may be explained as follows. The Mg z +-stimulated respiration was accompanied by a rapid production of ADP catalysed by Mg 2+-stimulated ATPase localized on the outside of the inner mitochondrial membrane. Inhibition by atractyloside of ADP translocation or inhibition by oligomycin of oxidative phosphorylation has led to the inhibition of respiration in the presence of A T P and Mg z+ (Figure 5)- On the other hand, phosphate release from ATP during incubation of mitochondria with ATP is due to the observed oligomycin-insensitive Mg 2+stimulated ATPase localized outside the inner mitochondrial membrane and to oligomycinsensitive ATPase located in the inner mitochondrial membrane. Taking into consideration that mitochondria isolated from some sources contain oligomycin-insensitive MgZ+-stimulated ATPase located in outer membrane fraction (Gmaj, Nowicka and Angielski, 1974; Vijayakumar and Weidemann, x976), it is likely that the mitochondria isolated from human early placenta also

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Figure 5. Effect of added Mg 2+ ions on isocitrate oxidation in the presence ofATP (A). The influence of atractyloside (B) and oligomycin (C) on Mg2+-dependent ATP stimulation of isocitrate oxidation. The mitochondria (mito; 3.6 mg protein) were incubated in the conditions described in 'Materials and Methods' and Figure 2. Where indicated, isocitrate (isocitr; 2 raM), ATP (i mM), MgCI2 (4 mM), atractyloside (atract; 5o#M) and oligomycin (oligu; 5 #g) were added. Table i. Effect ofoligumycin on ATPase activity in human early placental mitochondria in the presence and in the absence of MgCI2

Addition None + oligomycin, 5#g

Pi released (nmol x min- 1 x rag- 1 protein) 17.o o

+ MgCI2, 4mM

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All values are the means of at least three distinct experiments, each measured in duplicate. For experimental conditions see 'Materials and Methods'. contain such activity. However, one could not exclude the contamination of the mitochondrial preparation with M g 2+-stimulated A T P a s e localized in the endoplasmic reticulum. T h i s could also explain the effect of M g 2÷ on oxidative phosphorylation. T h e studies o f Schreiner and Villee (1965) have shown that i i-weeks h u m a n placental mitochondria exhibit high A T P a s e and A D P a s e activity. W e also investigated this p r o b l e m using A T P , A D P , A M P a n d / L g l y c e r o p h o s p h a t e as substrates. T h e results are given in T a b l e 2. As can be seen, phosphate release from A T P and A D P was very low in the absence o f added M g 2 +. Addition o f i g 2 + caused a several-fold stimulation o f A T P a s e and ADPase activity. T h e amounts o f phosphate released from A T P and A D P were almost the same. T h e phosphate release from A M P amounted to about 3o per cent of that released from A T P or A D P , whereas the phosphate release f r o m / L g l y c e r o p h o s p h a t e was negligible. At present it is difficult to say whether only one or two distinct proteins catalyse dephosphorylation of A T P and A D P . F u r t h e r

Tightly Coupled Mitochondria from Human Early Placenta

205

Table 2. Release ofinorganic phosphate from different substrates by human early placental mitochondria Pi released (nmol x m i n - 1 x r a g - 1 protein)

Substrate

without M g 2+

with M g 2+

ATP

17.o

I56.o

ADP

7.o

I49.o

AMP

20.0

48.0

/~-glycerophosphate

-

5-o

All values are the means of at least three distinct experiments each measured in duplicate. For experimental conditions see 'Materials and Methods'.

experiments are needed to elucidate this problem. Nevertheless, the stimulation of ATPase activity explains why human early placental mitochondria obtained by Schreiner and Villee (I965) displayed a low P:O ratio. Klimek et al (1972) were able to improve the P: O ratio of human term placental mitochondria by the addition of BSA both to the homogenates prior to the isolation and to the incubation medium. Similar results have been reported by Olivera and Meigs (1975). BSA in the incubation medium was also required for optimal phosphorylation of human early placental mitochondria. Figure 6 presents the effect of increasing concentrations of BSA in the incubation medium on the respiratory control ratio of early human placental mitochondria oxidizing isocitrate. Maximum respiratory control ratio was obtained at o.2 to o.4 per cent of BSA concentration. Omission of BSA from the incubation medium resulted in a lack of stimulation of the oxygen consumption by ADP. A possible explanation is furnished by a high progesterone content of the 4. •

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zo6

S. ,~otnierowicz, J. Swierczyfiski, L. Zelewski

human placental mitochondria (Schreiner and Villee, I965). According to the suggestion of Schreiner and Villee (x965) progesterone is responsible for the stimulation of ATPase activity in human placental mitochondria. However, other possibilities of the action of BSA on oxidative phosphorylation should be taken into consideration. The results presented in this paper led us to suppose that two factors could have been responsible for a low P:O ratio obtained by Schreiner and Villee (I965): (i) the presence of Mg 2÷ in the incubation medium and (2) the presence of progesterone or other compounds which may be removed by the addition of BSA. The human early placental mitochondria did not oxidize to a significant rate the externally added N A D H at the final concentration, o.4 raM, in the presence of ADP. Further addition of N A D H increased the oxygen uptake slightly (Figure 7A). Thus these mitochondria behaved in a way similar to the mitochondria isolated from other sources. A subsequent addition of isocitrate evoked an immediate burst of oxygen uptake (Figure 7B). Figure 7A shows also that the addition of cyctochrome c caused a several-fold stimulation of the oxidation of externally added N A D H . We suppose that the major pathway for the oxidation of externally added NADH is via a rotenone insensitive external route which could be artificially linked by exogenous cytochrome c to the cytochrome c oxidase region of the internal electron transport chain, as has been previously reported for term placental mitochondria Scis~owski and Swierczyfiski, 1976). A

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ADP

B

mi~

ADP

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2rain

Figure 7. Oxidation of exogenousNADH by early human placental mitochondria. The mitochondria(mito; 3.8 mg protein) were incubated in the conditionsdescribed under 'Materials and Methods' and in Figure 2. Where indicated, ADP (4oonmol), NADH (I #mol in Figure 7A or 2/lmol in Figure 7B),cytochromec (cyt.c; 2o/~M),isocitrate(isocitr; z mM), MgCI2 (2 raM) were added.

Data collected in Table 3 show that mitochondria from early placentae, when incubated under the conditions described in Figure 2, readily oxidize added tricarboxylic intermediates of Krebs cycle. Data presented in Table 3 indicate that in the presence of Mg 2+ isocitrate was oxidized more readily than cis-aconitate or citrate; the latter was oxidized at a rate of about half of that for isocitrate. We obtained similar results with human term placental mitochondria (Swierczyfiski et al, I976 ). These mitochondria also oxidized added dicarboxylic Krebs cycle intermediates such as malate, succinate, ct-ketoglutarate and some amino acids, such as

Tightly Coupled Mitochondria from Human Early Placenta

207

Table 3. Oxidation of substrates by human early placental mitochondria

Substrate used

Citrate, iomM Isocitrate, lOmM Cis-aconitate, lOmM Malate, lOmM Pyruvate, lOmM Pyruvate, 1omM Malate, 2 mM Palmitoyl-,carnitine, 5o # Palmitoyl--carnitine, 5o #M Malate, iomM

Oxygen uptake (ngatoms O x m i n - 1 x m g - 1 protein)

RCI

ADP:O

28.9 65.i 57.o 18.9 21.6

3.8 3.i 4.1 4.2 3.8

3.0 3.0 3.1 2.9 3.0

State 4

State 3

Mg 2+-stimulated respiration

4-5 ii.o 5.5 3.0 5-7

I7.1 34.6 22.4 12.6 21.6

5.7

24.4

52.9

4-2

3.o

4.5

I6-4

22-3

3.6

2.5

4.5

14.6

27.7

3.2

2-3

a-Glycerophosphate, io mM ~-Ketoglutarate, lOmM Succinate, lOmM Glutamate, lOmM Glutamate, 5 mM Malate, 5 mM

7.6 7.7 i6.i 4.2

i i .8 30-9 42.8 14. 7

24. 7 29.9 45.3 16.8

1.6 4.0 2.7 3.5

i .6 2.7 1.9 3 .o

4.6

23.2

27.8

5.0

2.9

Glutamine, xo mm /~-Hydroxybutyrate, tOmM

3.2 4.3

11.4 8.1

12.2

3.6 1.9

3.0 3.0

All values are the means of at least three distinct experiments, each measured in duplicate. For experimental conditions s e e 'Materials and Methods'.

glutamine and glutamate. Pyruvate was oxidized both in the presence and in the absence of malate (which could serve as precursor of oxaloacetate). Experiments listed in Table 3 also show that human early placental mitochondria similar to the human term placental mitochondria (Swierczyfiski, Scist'owski and Aleksandrowicz, I976c) were able to oxidize palmitoyl-carnitine. We have shown previously that ~-glycerophosphate was oxidized by human term placental mitochondria at a substantially higher rate than all other substrates tested (Swierczyfiski, Scisl'owski and Aleksandrowicz, i976a , b). The oxidation of ~glycerophosphate by human early placental mitochondria was also examined. As can be seen in Table 3, these mitochondria oxidized ~-glycerophosphate at the rate comparable to that for some Krebs cycle intermediates. These mitochondria were also able to oxidize /~hydroxybutyrate. This is interesting, as Olivera and Meigs 0975) observed that ]~hydroxybutyrate was not oxidized by human term placenta. Data presented in this paper indicate that mitochondria from human early placentae have the capacity to oxidize the added tricarboxylic acid cycle intermediates and pyruvate, thus suggesting the presence of the transmembrane systems for tricarboxylates, dicarboxylates and pyruvate translocation. The ADP:O ratios for all the substrates tested, presented in Table 3, did not differ from the theoretical values. However, the respiratory control index was relatively low if calculated, as the state 3/state 4 respiration in the absence of added Mg 2+ ions increased the oxygen uptake over the state 3 respiration in the case of most substrates investigated. From the amount of oxygen uptake by mitochondria incubated in the medium containing Mg 2+ ions in the presence of ADP and in the absence of ADP, a ratio may be calculated which is about twice as high as the respiratory control index. Similar results have been reported by Packer 0957) for rat heart sarcosomes. It is difficult to explain this phenomenon at present. Taking into consideration that the effect of Mg 2+ is especially conspicuous in the oxidation of tricarboxylic acids, one can t

S. Zotnierowicz,.7..~wierczyfiski, L. Zelewski

suppose that the effect o f M g 2+ is linked to the activation ofisocitrate dehydrogenase. T h e same applies to the ~-glycerophosphate dehydrogenase (Swierczyfiski, Scistowski and Aleksandrowicz, I976a, b). However, other possibilities are not excluded. It is also interesting that the rate of oxidation of isocitrate was the highest among the various tricarboxylic cycle intermediates. This suggests a high activity ofisocitrate dehydrogenase in human early placental mitochondria. It has been shown previously that isocitrate is the best substrate to support the progesterone biosynthesis in human term placenta (Bogushwski et al, I976; Klimek et al, 1976). Because human early placental mitochondria are also able to carry out the conversion of cholesterol to progesterone (not shown) it is reasonable to suppose that isocitrate dehydrogenase is the main source of N A D P H for steroid hydroxylations.

SUMMARY I. A procedure for the isolation of tightly coupled mitochondria from human early placenta is described. 2. Mitochondria obtained by this method were able to oxidize Krebs cycle intermediates, pyruvate, glutamate, glutamine, palmitoyl-carnitine, g-glycerophosphate and fl-hydroxybutyrate. 3- These mitochondria incubated in the medium containing ethylene diamine tetraacetic acid and bovine serum albumin and no added Mg z + ions exhibited a high respiratory control and adenosine diphosphate:oxygen (ADP:O) ratios corresponding to the theoretical values for all substrates tested. Addition o f M g 2+ ions markedly reduced the respiratory control index and A D P : O ratio. 4. Adenosine triphosphatase (ATPase) activity in the obtained mitochondrial preparation was stimulated about tenfold by Mg 2+. Oligomycin inhibited Mg 2+-stimulted ATPase activity by about 25 per cent, but completely inhibited this activity in the absence of Mg 2+ ions. 5. It is concluded that the effect of M g 2+ ions on the respiratory control and A D P : O ratio reported in this paper is exerted mainly through the Mg2+-stimulated oligomycininsensitive ATPase activity.

ACKNOWLEDGEMENTS The authors would like to thank Dr W. Migdlikowski(from Przychodnia PrzyszpitalnaSzpitala Wojew6dzkiegow Gdafisku) for making availablethe placentaeused in this study. The work was supported by the Ministry of Science Higher Education and Technology within the project R.I. 9.o3.o4. REFERENCES

Aleksandrowicz, Z., Swierezyfiski, J. & Wrzolkowa, T. (I973) Protective effect of nupercaine on mitochondrial structure. Biochimica et Biophysica Acta, 3o4, 59-66. Bogushwski, W., Klimek, J., Tiat'owska, B. & Zelewski, L. (1976) Inhibition by Mn2+ of citrate supported progesterone biosynthesisin mitochondrialfractions of human term placentae.Journal of Steroid Biochemistry, 7, 39-44. Chao, D. L.-S. & Davis, E. J. (i 97z) Studies on the role of Mg2+ and the Mg2+-stimulatedadenosinetriphosphatase in oxidative phosphorylation.Biochemistry, ii, 1943-I952. Clark, J. B. & Nicklas, W. J. (197o) The metabolismof rat brain mitochondria.Journal of Biological Chemistry, 245, 4724-4731. Estabrook, R. W. (I967) Mitochondrialrespiratory control and the polarograpbicmeasurement of ADP:O ratios. In Methods in Enzymology (Ed.) Estabrook, R. W. & Pullman, M. E. Volume X, pp. 4i~7 . New York and London: Academic Press.

Tightly Coupled Mitochondria from Human Early Placenta

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