A c t a P a t h . Jap. 28(2): 215-223,
1978
MECHANISM OF T H E FORMATION OF MEGAMITOCHONDRIA BY COPPER-CHELATING AGENTS
V. FURTHER STUDIES O N ISOLATED MEGAMITOCHONDRIA Takashi WAKABAYASHI, Masahisa ASANO,Kumiko ISHIKAWA, and Hidemasa KISHIMOTO Department of Pathology, Nagoya City University Medical School, Nagoya (Received on May 28, 1977)
Effect of cuprizone has been studied on some biochemical properties of megamitochondria obtained from the mouse liver. (1) Contents of Caa+,Mg8+ and Cu2+ in the blood o r the liver homogenates were not altered by cuprizoneintoxication, whereas those in liver mitochondria were significantly altered : after 3-4 days’ intoxication, content of Ca2+was decreased and was remarkably increased after 14-15 days’ intoxication. Content of Mg2+behaved contrarily. (2) Both cytochrome oxidase and ATPase activities were unchanged in the liver megamitochondria, but monoamine oxidase (MAO) activity was significantly decreased. Value of I,, (50% inhibition) for MA0 was determined to be 0.33 mM using the control liver mitochondria. Cuprizone had almost no effect on MA0 activity of kidney o r heart mitochondria both in vivo and in vitro. (3) The amount of lysolecithin was increased in the liver megamitochondria. These results were discussed in the light of membrane fusion phenomenon which plays a key role in the mechanism of megamitochondrial formation. ACTA PATH. JAP. 28: 215-223, 1978.
Introduction
Previously we have demonstrated some biochemical aspects of megamitochondria It has turned out that isolated from cuprizone- or DDC-fed mouse phosphorylating efficiencies of megamitochondria, specified above, are moderately preserved and activities of various enzymes including monoamine oxidase, ATPase and cytochrome oxidase are not much affected by the noxious reagents28,29. However, contents of Ca2+ and Mg2+ in such preparations were remarkably d i m i n i ~ h e d ~ ~ , ~ ~ . Recently we have explored an experimental condition by which megamitochondria are induced in mouse hepatocytes within a short period of time such as 40 hours4. ~ , ~have ~, Considering turnover rates of various components in m i t o ~ h o n d r i a ~ J , ~we concluded that membrane fusion phenomenon is actually involved in the formation process of megamitochondria, although a possibility can not be excluded that suppression of the dividing process of mitochondria may partly play a role in the megamitochondrial formation4. Z# E9838 iEA ZJll
< k F 9B*
Department of Pathology, Nagoya City University Mailing Address: Takashi WAEABAYASHI, Medical School, Mizuho-ku, Nagoya, Japan. 215
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CATIONS IN MEQAMITOCHONDBU
A& Pdh. Jap.
The central role of divalent metals, especially of Caa+, together with phospholipids in fusion phenomenon in biological membrane systems has been well documented1,2, 15,18~24~25,s1,38. If the membrane fusion plays a key role in the formation of megamitochondria, then imbalance of Ca2+/Mg2+ratios in the mitochondrion would be intimately related to the mechanism of megamitochondrial formation. Thus, the present communication aimed to analyze further the contents of divalent metals in preparations of various stages of megamitochondria. Since cuprizone or DDC is a copper-chelating agent, activity of monoamine oxidase which is a copper-containing enzyme in mitochondria has been also re-examined. Phospholipid analysis o f megamitochondrial preparations was also performed in the present study to correlate their constituents to the fusion phenomenon.
Materials and Methods Male mice of the ddY strain aging 4 4 weeks were used in the present study. They were fed on a powdered complete diet containing 0.5% cuprizone (biscyclohexanone oxaldihydrazone) for various intervals up to 15 days. The control animals were fed on the same powdered complete diet without containing the noxious reagent. After feeding with the noxious &gent for viious periods, the animal was sacrificed by the dislocation of the neck and the blood was obtained from the cervical arteries. Liver was then replaced and a part of it was processed for electron microscopy as described previouslysO. After fixation with glutaraldehyde-formaldehyde,the specimens were post-fixed in osmium tetroxide. Dehydration was initiated with 25% ethanol containing 1% uranyl acetate. They were finally embedded in Epon, and thin sections were cut with a diamond knife on a Porter-Blum ultramicrotome, stained with lead citrate. They were examined in a Hitachi 12 or llDS electron microscope operated a t 75 KV. Preparcltbn of mitochondria: Preparation of liver mitochondria is described in full in a previous communications. Isolation medium contained 0.25 M sucrose (Caa+-free),10 mM TrisCI, pH 7.4, and 0.05% bovine serum albumin (BSA). Mitochondria were prepared from one liver or from 4-5 livers at a time, For lipid analysis described below, the outer membrane fraction was obtained from the liver mitochondria according to the method of Schnaitman and Greenawaltua. Mitochondria were also isolated from the kidney and the heart. In both cases, mitochondria were prepared from a t least 8-10 animals a t one time to carry out electron microscopic and biochemical analysis. Biochemical aasays and probes Yeaeurevnent of oxygen uptake: The rate of consumption of oxygen was measured a t 30% with a Clark-type electrode (Beckman, Co., Fullerton, California). Details in the assay system are described in full in a previous communications8. Enzyme msays: Activities of cytochrome oxidase, ATPase and monoamine oxidaae were measured according to the methods as described previouslya8. .Measurement of divdent c a t h a : For metal analysis, mitochondria were isolated from either the experimental or control animals in the absence of EDTA as described before. Slices of liver suspended in distilled water were homogenized thoroughly. A suspension of mitochondria, the liver homogenatea thus obtained and the blood were dissolved in 10% trichloroacetic acid (TCA) and were oentrifuged. The supernatant fluid thus obtained was used for the measurement of Cup+, Ca*+and Mga+ using a Parkin-Elmer type 303 atomic absorption spectrophotometer as described previouslyga. Lipid extractwn am? analysis: Whole mitochondria or the outer membrane fraction were extracted with chloroform-methanol (1 :1, v/v) solutions essentially according to the method of Bligh and Dyer*: a mitochondria1 suspension (0.8 ml, 12-15 mg of protein per ml) was sonicated thoroughly, and 3 ml of chloroform-methanol was added to the mixture. The mixture was shaken by the Vortex and 1 ml of chloroform was added to the mixture. The sample was
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then centrifuged for 5 minutes a t 3,000 rpm in a Kubota KR-200B ultracentrifuge. The chloroform layer was washed several times with methanol. The lipid extract was evaporated to dryness under a stream of nitrogen. The residue was then made to volume with chloroform-methanol (2:1, v/v) and the lipids were fractionated into neutral and phospholipids by thin-layer chromatography on Silica Gel H. Phospholipid phosphorus was determined according to the method of Eibl and Lands" in a 3 ml system using Triton X-100. Protein was determined by the method of Lowry et al?5. Sources of chemicals: Cuprizone (biscyclohexanone oxaldihydrazone) was purchased from G. Frederich Smith Chemical Co., Columbus, Ohio. Bovine serum albumin (Fraction V, essentially fatty acid free) and cytoohrome c (type V) from Sigma Chemical Co., Kynuramine was obtained from Nakarai Chemical Co., Kyoto, Japan. All other reagents were of analytical grade.
Results Previously we have demonstrated some biocemical aspects of megamitochondria of cuprizone-fed mouse In most of these experiments, mitochondria were isolated from 4-5 animals to obtain enough amount of protein since both morphological and various biochemical aspects of megamitochondria had to be correlated in some depth. However, it would be more accurate to correlate biochemical data with each other using mitochondria obtained from individual animal. Thus, most of the data of the present experiments were obtained from mitochondria isolated from each animal except when the heart or the kidney was investigated.
Effmtof Cuprizone
on
Liver Mitochondria in Vitro
We have already demonstrated that cuprizone does not affect coupling efficiencies of mitochondria in vitro3P'. This was again confirmed in the present study (Talbe 1, Pig. 1). Since cuprizone does not dissolve much in aqueous media, the maximum concentration of the reagent a t which the experiment was carried out was 0.14 mM shown in the second communication of this series28. However, it has been Table 1. Effect of Cuprizone on Mitochondria1 Coupling Efieiencies in vitro'
Substrate Succinate no addition Cuprizonea 0.1 mM 0.3 mM 1.0 mM Glutamate no addition Cuprizonea 0.1 mM 0.3 mM 1.0 mM
RCI
RCI (I")' ADP/O
4.0 3.7 4.0 4.5
4.5 4.4 4.5 4.8
1.90 1.99 1.90 1.73
3.8 3.3 3.6 3.3
4.0 3.9 3.8 3.7
2.93 2.78 2.70 2.61
Liver mitochondria were isolated in sucrose-Tris medium. The basic incubation medium contained 3 mM MgCIB,0.26M sucrose and 10 mM Tris-C1, pH 7.4. Oxidizable substrates were succinate (K+-salt) and glutamate (K+-salt). Cuprizone wm dissolved in alcohol ;at a h a 1 concentration of 20 mM. The final concentration of dinitrophenol (DNP) was 0.46 mM.
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RC1=4.0 ADP/O=1.88
01
.-
5
loo
z
ADP 0=1.90
E
30
t
lb0
300
10'00
Concentration of Cuprizone (phi)
Fig. 1 Fig. 2 Fig 1. Effect of cuprizone on mitochondria1 respiration. The assay system is the same m that described in Table 1. Cuprizone (final concentration, 1 mM) w m added to the reaction mixture a t the point indicated by the arrow. Fig. 2. Effect of cuprizone on mitochondrial monoamine oxidase activity. The semi-logarithmic plots of concentration of cuprizone vs. yo activity. The assay system is the same as that described in Table 3. Table 2. Effects of Cwprdzone on Mitochondria1 Cytochrorne Oxidase and ATPase Activities in vitrol
Condition
Cytochrome Oxidasea
-L.L. S.A. no addition Cuprizone 0.1 mM 0.3 mM 1.0 mM
* *
113.86(100.0) 105.67 ( 92.8) 106.46 ( 93.5) 90.62( 79.5)
+L.L.8 S.A. 396.22(100.0) 356.99( 90.1) 413.68 (104.4) 395.83( 99.9)
ATPasel
S.A. 597.7(100.0) 645.7( 91.3) 524.9 ( 87.8) 5.4.1( 84.3)
Data are expressed in natoms oxygen/mg protein/min for cytochrome oxidase and in nmoles inorganic phosphatelmg protein/min for ATPase. Data are the averages of three different animals. Lysolecithm (L.L.) waa added to the reaction mixture a t a concentration of 1.0-1.2 rng/mg mitochondria1 protein as a solubilizing agent in order to obtain full activity.
found that cuprizone dissolves easily at maximum concentration of about 20 mM in 60% ethanol, and it became possible to examine the effect of the reagent on mitochondria a t its higher concentrations. As shown in Table 1, coupling efficiencies of mitochondria obtained from the control mouse liver was not affected so much by cuprizone a t concentrations as high as 1.0 mM. Similarly, activities of both ATPase and cytochrome oxidase remained almost a t the same level after treatment with cuprizone (Table 2). Monoamine oxidase activity, however, was suppressed by the reagent beyond certain levels of concentration (Fig. 2).
E f f d of Cuprizone on Liver Mitochondria in Vivo Both ATPase and cytochrome oxidase activities of mitochondria obtained from animals fed on a diet containing cuprizone for 6-8 days were well preserved: ATPase,
28(2): 1978
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al.
Table 3. Effect of Cuprizone on Monoamine Oxidase Activity of Liver Mitochondria in vivo'
___
~
S.A.*
Condition
_ _ _ _ ~
Control Duration of feeding on cuprizone-diet 2 days 3 days 4 4 days 7-8 days
2
12.61(100.0)
-
9.28( 73.6) 8.71 ( 69.1) 7.58( 60.1) 5.21( 41.3)
_-__
Mitochondria were isolated in the presence of albumin from the livers of mice fed with 0.5% cuprizone-diet for up to 8 days. Mitochondria were obtained from one liver in each experiment. Data are the averages of three different experiments. Specific activity (S.A.) is expressed in nmolcs kynuramine/mg protein/min.
Table 4. Contents' of Divalent Cations in the Blood and Liver Homogenates of Cupizone-Fed Mouse
Blood (pmoles/ml) Cation Ca2+ Mg*+ cu*+ 1
2
Liver Homogenates (nmoles/mg)
Control
Cuvrizone2
Control
Cuvrizonc2
2.01f0.07 1.58f0.03
2.00f0.07 1.40f0.08
2.2f 0 . 6 34.2f 2 . 4 0.29f0.06
2.6* 0.7 33.9f 4 . 0 0.30*0.09
Data are the averages (mean&S.D.) of 10 different experiments obtained from individual animals. Mice were fed with a 0.5% cuprizone-diet for 14-15 days.
96.1% of the control; cytochrome oxidase, 98.3% of the control. Monoamine oxidase activities, however, were again lowered just as i n vitro (Table 3). As is clearly shown in the table, the degree of suppression of monoamine oxidase activity is in parallel with the duration of feeding the animal on the noxious reagent. Metal contents in cuprizone-fed mouse are summarized in Tables 4 and 5. Contents of Ca2+,Mg2+and Cu2+in liver homogenates or in blood of cuprizone-fed mouse are not different from those of the control mouse, but contents of these metals in liver mitochondria of cuprizone-fed mouse are much different from those of the control: after 3-4 days of cuprizone-intoxication, content of Ca2+ is definitely decreased whereas that of Mg2+ is slightly increased; after 14-15 days of intoxication, content of Ca2+ significantly increased whereas that of Mg2+now decreases lowering the Mg2+/Ca2+ratio from 1.80 to 1.08. Contents of phospholipids in megamitochondria are summarized in Table 6. The amount of iysolecithin in mitochondria of cuprizone-fed mouse liver increased compared to that of the control. However, there was no appreciable difference in
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CATIONS IN MEGAMITOCHONDRIA
Table 5. Contents of DivaZent Cationa in Cuprizone-Fed Mowre Liver Mitochondria1
Cation
Ca2+ Mg8+ Cu*+ Mg2+/Ca2+ ratio 1
Duration of feeding on cuprizone-diet
Control
17.0k 2.4 30.6f 4.6 0.41f0.04 1.80
3 4 days
14-15 days
12.8% a.4*
20.0* 4.0* 21.6k 6.9*** 0.48f0.10 1.08
34.6k 2.9
0.47fO. 20 2.70
Mice were fed with a 0.5% cupriaone-dietfor certain periods specified in the table. Data are the averages (meanff3.D.) of 10 different experiments. Data are statistically different by Student's t-test from the control at *: 0.05>p>0.01, **: 0.01>p>0.001 and ***: O.Ool>p. Data are expressed in moles/mg protein.
Table 6. Contents of Phospholipids in Cuprizone-Induced Megantitochondria' (yoof total P h s p h l i p i d in Each Fraction)
Control
Cuprizone
~
Phospholipid
Whole Outer Whole Outer Mitochondria Membrane Mitochondria Membrane
Total (Pi nmoles/mg prot.)
188.0
366.8
170.2
344.9
Cardiolipin Phosphatidylethanolamine Phosphatidylinositol Phosphatidylcholine Sphingomyelin Lysolecithin
14.6 34.7
4.9 2s. 3 9.3 49.6
16.9 32.5 6.3 36.6 4.3 6.9
5.8 31.1 9.1 46.3 7.8 1.0
1
6.0
41.7 2.8 2.4
6.0 2.1
Mitochondria were isolated from the livers of mice fed with a 0.5% cuprizonediet for 8-10 days. Mitochondria were obtained from the livers of 10-20 animals in one experiment.
contents of phospholipids between the outer membrane fraction obtained from cuprizone-fed mouse liver mitochondria and that of the control. Discussion
Recently we have demonstrated that fusion phenomenon is involved in the formation process of megamit~chondria~.We have also demonstrated that nialamide, which is one of the well known inhibitors of monoamine oxidase, induces megamitochondria in mouse hepatocytes simply by feeding the animal with a diet containing the noxious reagent6. The above described two lines of evidence compelled us to examine further biochemical aspects of cuprizone-induced megamitochondria. The present study clearly demonstrated that content of Ca2+ in cuprizone-fed mouse liver mitochondria has an intimate correlation with the duration of cuprizoneintoxication. Previously we have shown that it takes a t least a week t o induce
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et al.
megamitochondria in mouse hepatocytes by feeding the animal with a diet containing c ~ p r i z o n e ~After ~ ~ ~ .3-4 days of intoxication with cuprizone, content of Ca2+ is decreased. This is the stage in which megamitochondria have not been induced yet in the liver. The role of Ca2+ on membrane fusion has been repeatedly described. Recently POSTE and ALL IS ON^^ have proposed a scheme for membrane fusion stressing the displacement of Ca2+ from the membranes together with ATP: displacement of Ca2+and ATP from the membranes results in increased motional freedom and a structural transformation of the membrane lipids. A t 1615 days of cuprizone-intoxication, content of Ca2+ in liver mitochondria is raised significantly. At this stage, megamitochondrial formation is complete and megamitochondria are often devoid partially of the outer membrane and most of cristae are located peripherally suggesting the swelling and the degenerative proces~es3~.Thus, increment of Ca2+ at a later stage of megamitochondrial formation would be ascribed to disorganization of integrity of mitochondria1 membrane changing its permeability to Ca2+. The increment of lysolecithin in megamitochondria would be explained in the same fashion as in the case of Ca2+. LUCYand his colleagues21 demonstrated that lysolecithin was able to fuse hen erythrocytes in vitro. The cell fusion potential of lysolecithin has since been confirmed by others9, and the requirement of the membrane fusion reaction for Ca2+ could possibly be interpreted as being due to the Ca2+ requirement of phospholipases ~ ~ ~ ~ ~ ~ ~ ~of. the precise mechanism by which that generate l y ~ o l e c i t h i n ~ ~ ,Regardless lysolecithin induces membrane fusion, it is probable that the insertion of wedgeshaped molecule such as lysolecithin into the membrane would produce substantial disorder22. Although the present experiment shows the increment in the amount of lysolecithin in megamitochondria, the degree of increment was variable from experiment to experiment, and a definite conclusion awaits further detailed investigation. The present study revealed that activity of cytochrome oxidase, which is a coppercontaining enzyme, was unaffected by cuprizone-treatment, but the activity of monoamine oxidase, which is another copper-containing enzyme in the mitochondrion was suppressed both in vitro and in vivo. Then, how is the inhibition of monoamine oxidase activity correlated to megamitochondrial formation Z How is it correlated to the membrane fusion ? In in vitro experiments, the value of I,, (50% inhibition) of monoamine oxidase was the lowest in mitochondria of the liver compared to that of the kidney or the heart, and the activity of the enzyme in vivo was suppressed remarkably only in mitochondria of the liver (Table 7 ) . These data are interesting Table 7 . Effect of Cuprizone on Monoamine Oxidase Activities of Mitochondria from Various Tissues Both in vitro and in vivo ~
Tissue
in vitro (160)
(yoinhibition)
in vivo
Liver Kidney Heart
0.33 mM 6.60mM 11.2 mM
39.3% 21.0% 1.6%
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Acda Path. Jap.
considering the fact that megamitochondria are induced only in the liver and not either in the kidney or the heart4s21. However, a question of how monoamine oxidase activity is correlated to the depletion of Ca2+ from the mitochondrion or how it is correlated to megamitochondrial formation can not be answered from the present data. We are now studying various types of inhibitors of monoamine oxidase in order t o know if the megamitochondrial formation is a universal effect by the inhibitors, and the results will be reported soon. References FISCHER, D., HOWELL, J.I., TAMPION, W., VERRINDER, M., and LUCY, J.A. : Chemically induced and thermally-induced cell fusion: lipid-lipid interactions. Nat. New Biol. 242: 215-217, 1973. APOSTOLOV,K. and POSTE,G.: Interaction of Sendai virus with human erythrocytes: a system for the study of membrane fusion. Microbios. 6: 247-261, 1972. ASANO,M., WAKABAYASHI, T., KURONO, C., and OZAWA, T.: Mechanism of the formation of megamitochondria induced by copper-chelating agents. 111. Formation and some biochemical properties of megamitochondria induced by diethyldithiocarbamate (DDC). Acta Path. Jap. 25: 125-134, 1975. ASANO,M., WAKABAYASHI, T., ISHIKAWA, K., and KISHIMOTO, H.: Mechanism of the formation of magamitochondria induced by copper-chelating agents. IV. Role of fusion phenomenon on the cuprizone-induced megamitochondrial formation. submitted to Acta Path. Jap. ASANO,M., WAKABAYASHI, T.,ISHIKAWA, K., and KISHIMOTO, H.: Induction of megamitochondria in mouse hepatocytes by nialamide. J. Electron Microscopy (in press). BAILEY,E., TAYLOR, C.B., and BARTLEY, W.: Turnover of mitochondrial components of normal and essential fatty acid-deficient rats. Biochem. J. 104: 10261032, 1967. BEATTIE,D.S, BASFORD, R.E., and KORITZ,S.B.: The turnover of the protein components of mitochondria from rat liver, kidney, and brain. J. Biol. Chem. 242: 4584-4586, 1967. BLIUH,E.G. and DYER,W.J.: A rapid method of total lipid extraction and purification. Canad. J. Biochem. Physiol. 37: 911-917, 1959. CROCE,C.M., SAWICKI, W., KRITOHEVSKY, D., and KAPROWSKI, H.: Induction of homokaryocyte, heterokaryocyte and hybrid formation by lysolecithin. Exp. Cell Rea. 67: 427-435,
1. AEKONU, Q.F., CRAMP, F.C.,
2. 3.
4.
5. 6. 7. 8. 9.
1971. E.A. and ROTHMAN, J.E. : Fusion and protein-mediated phospholipid exchange 10. DAWIDOWICZ,
studied with single bilayer phosphatidylcholine vesicles of different density. Biochim. Biophys. Acta 455: 621-630, 1976. W.E.M.: A new sensitive determination of phosphate. Anal. Biochem. 11. EIBL, H. and LANDS, 30: 51-57, 1969. M.J. and SANADI,D.R. : Turnover of rat-liver mitochondria. Biochim. Biophys. 12. FLETCHER, Acta 51: 356360, 1961. 13. GREEN,D.E.: Membrane proteins. A perspective. Ann. N.Y. Acad. Sci. U.S.A. 195: 150172, 1972. M.: Apparent turnover of mitochondrial 14. GROSS, N.J., GETZ,G.S., and RABINOWITZ,
deoxyribonucleic acid and mitochondrial phospholipids in the tissues of the rat. J. Biol. Chem. 244: 1552-1562, 1969. R.J.: Protein measurement N.J., FARR,A.L., and RANDALL, 15. LOWRY,O.H., ROSEBROUUH, with the Folin phenol reagent. J. Biol. Chem. 193: 265-275, 1951. J., COLBEAU, A., V I U N ~ SP.M. , : Distribution of membrane-confined phos16. NACHSATJR, pholipase A in the rat hepatocyte. Biochim. Biophys. Acta 274: 426-446, 1972. J.D. and WAITE,M.: Phospholipid hydrolysis by phospholipases Al and As in 17. NEWKIRK, nlasma membranes and microsomes of rat liver. Biochim. BioDhvs. Acta 298: 562-576. 1973.
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Y. and MURAYAMA, F.: Requirement of calcium ions for the cell fusion reaction of 18. OKADA, animal cell by HVJ. Exp. Cell Res. 44: 527351, 1966. D.: Studies on the mechanism of action of local anesthetics with 19. PAPAHADJOPOULOS, phospholipid membranes. Biochim. Biophys. Acta 265: 169-186, 1972. D., POSTE, G., and SCHAEFFER, B.: Fusion of mammalian cells by 20. PAPAHADJOPOULOS, unilamellar lipid vesicles: influence of lipid surface charge, fluidity and cholesterol. Biochim. Biophys. Acta 323: 2440, 1973. A.R., HOWELL,J.I., and LUCY,J.A.: Lysolecithin and cell fusion. Nature 227: 21. POOLE, 810-813, 1970. 22. POSTE,G. and ALLISON, A.C. : Membrane fusion. Biochim. Biophys. Acta 300: 42145,1973. 23. SCHNAITMAN, C. and GREENAWALT, J.W.: Enzymatic properties of the inner and outer membranes of rat liver mitochondria. J. Cell Biol. 38: 158-175, 1968. A., YAOIL,G., and YAFFE,D.: Control of myogenesis in vitro by Cae+concentra24. SHAINBERO, tion in nutritional medium. Exp. Cell Res. 58: 163-167, 1969. Chr. and PETTE, D.: Quantitative investigation on Caa+-and 25. VAN DEN BOSCH,J., SCHUDT, pH-dependence of muscle cell fusion in vitro. Biochem. Biophys. Res. Commun. 48: 326-332, 1972. E.J., VAN GOLDE,L.M.G., HOSTETLER, K.Y., SHERPHOF, G.L., and VAN DEENEN, 26. VICTORIA,
L.L.M.: Some studies on the metabolism of phospholipids in plasma membranes from rat liver. Biochim. Biophys. Acta 239: 443-457, 1971. 27. WAITE,M. and SISSON,P. : Utilization of neutral glycerides and phosphatidylethanolamine by the phospholipase A, of the plasma membranes of rat liver. J. Biol. Chem. 248: 7985-7992, 1973. T. and GREEN,D.E. : On the mechanism of cuprizone-induced formation 28. WAKABAYASHI, of megamitochondria in mouse liver. J. Bioenergetics 6 : 179-192, 1974. T.: Mechanism of the formation T., ASANO,M., KURONO,C., and OZAWA, 29. WAKABAYASHI,
of megamitochondria induced by copper-chelating agents. 11. Isolation and some properties of megamitochondria from the cuprizone-treated mouse liver. Acta Path. Jap. 25: 39-49, 1975. T., ASANO, M., and KURONO, C.: 30. WAKABAYASHI,
Mechanism of the formation of megamitochondria induced by copper-chelating agents. I. On the formation process of megamitochondria in cuprizone-treated mouse liver. Acta Path. Jap. 25: 15-37, 1975. A.M. and WIENEKE,A.A.: The accumulation of calcium by the polymorphonuclear 31. WOODIN, leucocyte treated with staphylococcal leucocidin and its significance in the extrusion of protein, Biochem. J. 87: 487495, 1963. 32. WOODIN,A.M. and WIENEKE,A.A. : The participation of calcium, adenosine triphosphate and adenosine triphosphatase in the extrusion of the granule proteins from the polymorphonuclear leucocyte. Biochem. J. 90 : 498-509, 1964. A. and LOYTER,A.: The mechanism of cell fusion. I. Energy requirements for 33. YANOVSKY, virus-induced fusion of Ehrlich ascites tumor cells. J. Biol. Chem. 247: 40214028, 1972.
1978
MECHANISM OF T H E FORMATION OF MEGAMITOCHONDRIA BY COPPER-CHELATING AGENTS
V. FURTHER STUDIES O N ISOLATED MEGAMITOCHONDRIA Takashi WAKABAYASHI, Masahisa ASANO,Kumiko ISHIKAWA, and Hidemasa KISHIMOTO Department of Pathology, Nagoya City University Medical School, Nagoya (Received on May 28, 1977)
Effect of cuprizone has been studied on some biochemical properties of megamitochondria obtained from the mouse liver. (1) Contents of Caa+,Mg8+ and Cu2+ in the blood o r the liver homogenates were not altered by cuprizoneintoxication, whereas those in liver mitochondria were significantly altered : after 3-4 days’ intoxication, content of Ca2+was decreased and was remarkably increased after 14-15 days’ intoxication. Content of Mg2+behaved contrarily. (2) Both cytochrome oxidase and ATPase activities were unchanged in the liver megamitochondria, but monoamine oxidase (MAO) activity was significantly decreased. Value of I,, (50% inhibition) for MA0 was determined to be 0.33 mM using the control liver mitochondria. Cuprizone had almost no effect on MA0 activity of kidney o r heart mitochondria both in vivo and in vitro. (3) The amount of lysolecithin was increased in the liver megamitochondria. These results were discussed in the light of membrane fusion phenomenon which plays a key role in the mechanism of megamitochondrial formation. ACTA PATH. JAP. 28: 215-223, 1978.
Introduction
Previously we have demonstrated some biochemical aspects of megamitochondria It has turned out that isolated from cuprizone- or DDC-fed mouse phosphorylating efficiencies of megamitochondria, specified above, are moderately preserved and activities of various enzymes including monoamine oxidase, ATPase and cytochrome oxidase are not much affected by the noxious reagents28,29. However, contents of Ca2+ and Mg2+ in such preparations were remarkably d i m i n i ~ h e d ~ ~ , ~ ~ . Recently we have explored an experimental condition by which megamitochondria are induced in mouse hepatocytes within a short period of time such as 40 hours4. ~ , ~have ~, Considering turnover rates of various components in m i t o ~ h o n d r i a ~ J , ~we concluded that membrane fusion phenomenon is actually involved in the formation process of megamitochondria, although a possibility can not be excluded that suppression of the dividing process of mitochondria may partly play a role in the megamitochondrial formation4. Z# E9838 iEA ZJll
< k F 9B*
Department of Pathology, Nagoya City University Mailing Address: Takashi WAEABAYASHI, Medical School, Mizuho-ku, Nagoya, Japan. 215
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The central role of divalent metals, especially of Caa+, together with phospholipids in fusion phenomenon in biological membrane systems has been well documented1,2, 15,18~24~25,s1,38. If the membrane fusion plays a key role in the formation of megamitochondria, then imbalance of Ca2+/Mg2+ratios in the mitochondrion would be intimately related to the mechanism of megamitochondrial formation. Thus, the present communication aimed to analyze further the contents of divalent metals in preparations of various stages of megamitochondria. Since cuprizone or DDC is a copper-chelating agent, activity of monoamine oxidase which is a copper-containing enzyme in mitochondria has been also re-examined. Phospholipid analysis o f megamitochondrial preparations was also performed in the present study to correlate their constituents to the fusion phenomenon.
Materials and Methods Male mice of the ddY strain aging 4 4 weeks were used in the present study. They were fed on a powdered complete diet containing 0.5% cuprizone (biscyclohexanone oxaldihydrazone) for various intervals up to 15 days. The control animals were fed on the same powdered complete diet without containing the noxious reagent. After feeding with the noxious &gent for viious periods, the animal was sacrificed by the dislocation of the neck and the blood was obtained from the cervical arteries. Liver was then replaced and a part of it was processed for electron microscopy as described previouslysO. After fixation with glutaraldehyde-formaldehyde,the specimens were post-fixed in osmium tetroxide. Dehydration was initiated with 25% ethanol containing 1% uranyl acetate. They were finally embedded in Epon, and thin sections were cut with a diamond knife on a Porter-Blum ultramicrotome, stained with lead citrate. They were examined in a Hitachi 12 or llDS electron microscope operated a t 75 KV. Preparcltbn of mitochondria: Preparation of liver mitochondria is described in full in a previous communications. Isolation medium contained 0.25 M sucrose (Caa+-free),10 mM TrisCI, pH 7.4, and 0.05% bovine serum albumin (BSA). Mitochondria were prepared from one liver or from 4-5 livers at a time, For lipid analysis described below, the outer membrane fraction was obtained from the liver mitochondria according to the method of Schnaitman and Greenawaltua. Mitochondria were also isolated from the kidney and the heart. In both cases, mitochondria were prepared from a t least 8-10 animals a t one time to carry out electron microscopic and biochemical analysis. Biochemical aasays and probes Yeaeurevnent of oxygen uptake: The rate of consumption of oxygen was measured a t 30% with a Clark-type electrode (Beckman, Co., Fullerton, California). Details in the assay system are described in full in a previous communications8. Enzyme msays: Activities of cytochrome oxidase, ATPase and monoamine oxidaae were measured according to the methods as described previouslya8. .Measurement of divdent c a t h a : For metal analysis, mitochondria were isolated from either the experimental or control animals in the absence of EDTA as described before. Slices of liver suspended in distilled water were homogenized thoroughly. A suspension of mitochondria, the liver homogenatea thus obtained and the blood were dissolved in 10% trichloroacetic acid (TCA) and were oentrifuged. The supernatant fluid thus obtained was used for the measurement of Cup+, Ca*+and Mga+ using a Parkin-Elmer type 303 atomic absorption spectrophotometer as described previouslyga. Lipid extractwn am? analysis: Whole mitochondria or the outer membrane fraction were extracted with chloroform-methanol (1 :1, v/v) solutions essentially according to the method of Bligh and Dyer*: a mitochondria1 suspension (0.8 ml, 12-15 mg of protein per ml) was sonicated thoroughly, and 3 ml of chloroform-methanol was added to the mixture. The mixture was shaken by the Vortex and 1 ml of chloroform was added to the mixture. The sample was
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then centrifuged for 5 minutes a t 3,000 rpm in a Kubota KR-200B ultracentrifuge. The chloroform layer was washed several times with methanol. The lipid extract was evaporated to dryness under a stream of nitrogen. The residue was then made to volume with chloroform-methanol (2:1, v/v) and the lipids were fractionated into neutral and phospholipids by thin-layer chromatography on Silica Gel H. Phospholipid phosphorus was determined according to the method of Eibl and Lands" in a 3 ml system using Triton X-100. Protein was determined by the method of Lowry et al?5. Sources of chemicals: Cuprizone (biscyclohexanone oxaldihydrazone) was purchased from G. Frederich Smith Chemical Co., Columbus, Ohio. Bovine serum albumin (Fraction V, essentially fatty acid free) and cytoohrome c (type V) from Sigma Chemical Co., Kynuramine was obtained from Nakarai Chemical Co., Kyoto, Japan. All other reagents were of analytical grade.
Results Previously we have demonstrated some biocemical aspects of megamitochondria of cuprizone-fed mouse In most of these experiments, mitochondria were isolated from 4-5 animals to obtain enough amount of protein since both morphological and various biochemical aspects of megamitochondria had to be correlated in some depth. However, it would be more accurate to correlate biochemical data with each other using mitochondria obtained from individual animal. Thus, most of the data of the present experiments were obtained from mitochondria isolated from each animal except when the heart or the kidney was investigated.
Effmtof Cuprizone
on
Liver Mitochondria in Vitro
We have already demonstrated that cuprizone does not affect coupling efficiencies of mitochondria in vitro3P'. This was again confirmed in the present study (Talbe 1, Pig. 1). Since cuprizone does not dissolve much in aqueous media, the maximum concentration of the reagent a t which the experiment was carried out was 0.14 mM shown in the second communication of this series28. However, it has been Table 1. Effect of Cuprizone on Mitochondria1 Coupling Efieiencies in vitro'
Substrate Succinate no addition Cuprizonea 0.1 mM 0.3 mM 1.0 mM Glutamate no addition Cuprizonea 0.1 mM 0.3 mM 1.0 mM
RCI
RCI (I")' ADP/O
4.0 3.7 4.0 4.5
4.5 4.4 4.5 4.8
1.90 1.99 1.90 1.73
3.8 3.3 3.6 3.3
4.0 3.9 3.8 3.7
2.93 2.78 2.70 2.61
Liver mitochondria were isolated in sucrose-Tris medium. The basic incubation medium contained 3 mM MgCIB,0.26M sucrose and 10 mM Tris-C1, pH 7.4. Oxidizable substrates were succinate (K+-salt) and glutamate (K+-salt). Cuprizone wm dissolved in alcohol ;at a h a 1 concentration of 20 mM. The final concentration of dinitrophenol (DNP) was 0.46 mM.
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RC1=4.0 ADP/O=1.88
01
.-
5
loo
z
ADP 0=1.90
E
30
t
lb0
300
10'00
Concentration of Cuprizone (phi)
Fig. 1 Fig. 2 Fig 1. Effect of cuprizone on mitochondria1 respiration. The assay system is the same m that described in Table 1. Cuprizone (final concentration, 1 mM) w m added to the reaction mixture a t the point indicated by the arrow. Fig. 2. Effect of cuprizone on mitochondrial monoamine oxidase activity. The semi-logarithmic plots of concentration of cuprizone vs. yo activity. The assay system is the same as that described in Table 3. Table 2. Effects of Cwprdzone on Mitochondria1 Cytochrorne Oxidase and ATPase Activities in vitrol
Condition
Cytochrome Oxidasea
-L.L. S.A. no addition Cuprizone 0.1 mM 0.3 mM 1.0 mM
* *
113.86(100.0) 105.67 ( 92.8) 106.46 ( 93.5) 90.62( 79.5)
+L.L.8 S.A. 396.22(100.0) 356.99( 90.1) 413.68 (104.4) 395.83( 99.9)
ATPasel
S.A. 597.7(100.0) 645.7( 91.3) 524.9 ( 87.8) 5.4.1( 84.3)
Data are expressed in natoms oxygen/mg protein/min for cytochrome oxidase and in nmoles inorganic phosphatelmg protein/min for ATPase. Data are the averages of three different animals. Lysolecithm (L.L.) waa added to the reaction mixture a t a concentration of 1.0-1.2 rng/mg mitochondria1 protein as a solubilizing agent in order to obtain full activity.
found that cuprizone dissolves easily at maximum concentration of about 20 mM in 60% ethanol, and it became possible to examine the effect of the reagent on mitochondria a t its higher concentrations. As shown in Table 1, coupling efficiencies of mitochondria obtained from the control mouse liver was not affected so much by cuprizone a t concentrations as high as 1.0 mM. Similarly, activities of both ATPase and cytochrome oxidase remained almost a t the same level after treatment with cuprizone (Table 2). Monoamine oxidase activity, however, was suppressed by the reagent beyond certain levels of concentration (Fig. 2).
E f f d of Cuprizone on Liver Mitochondria in Vivo Both ATPase and cytochrome oxidase activities of mitochondria obtained from animals fed on a diet containing cuprizone for 6-8 days were well preserved: ATPase,
28(2): 1978
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Table 3. Effect of Cuprizone on Monoamine Oxidase Activity of Liver Mitochondria in vivo'
___
~
S.A.*
Condition
_ _ _ _ ~
Control Duration of feeding on cuprizone-diet 2 days 3 days 4 4 days 7-8 days
2
12.61(100.0)
-
9.28( 73.6) 8.71 ( 69.1) 7.58( 60.1) 5.21( 41.3)
_-__
Mitochondria were isolated in the presence of albumin from the livers of mice fed with 0.5% cuprizone-diet for up to 8 days. Mitochondria were obtained from one liver in each experiment. Data are the averages of three different experiments. Specific activity (S.A.) is expressed in nmolcs kynuramine/mg protein/min.
Table 4. Contents' of Divalent Cations in the Blood and Liver Homogenates of Cupizone-Fed Mouse
Blood (pmoles/ml) Cation Ca2+ Mg*+ cu*+ 1
2
Liver Homogenates (nmoles/mg)
Control
Cuvrizone2
Control
Cuvrizonc2
2.01f0.07 1.58f0.03
2.00f0.07 1.40f0.08
2.2f 0 . 6 34.2f 2 . 4 0.29f0.06
2.6* 0.7 33.9f 4 . 0 0.30*0.09
Data are the averages (mean&S.D.) of 10 different experiments obtained from individual animals. Mice were fed with a 0.5% cuprizone-diet for 14-15 days.
96.1% of the control; cytochrome oxidase, 98.3% of the control. Monoamine oxidase activities, however, were again lowered just as i n vitro (Table 3). As is clearly shown in the table, the degree of suppression of monoamine oxidase activity is in parallel with the duration of feeding the animal on the noxious reagent. Metal contents in cuprizone-fed mouse are summarized in Tables 4 and 5. Contents of Ca2+,Mg2+and Cu2+in liver homogenates or in blood of cuprizone-fed mouse are not different from those of the control mouse, but contents of these metals in liver mitochondria of cuprizone-fed mouse are much different from those of the control: after 3-4 days of cuprizone-intoxication, content of Ca2+ is definitely decreased whereas that of Mg2+ is slightly increased; after 14-15 days of intoxication, content of Ca2+ significantly increased whereas that of Mg2+now decreases lowering the Mg2+/Ca2+ratio from 1.80 to 1.08. Contents of phospholipids in megamitochondria are summarized in Table 6. The amount of iysolecithin in mitochondria of cuprizone-fed mouse liver increased compared to that of the control. However, there was no appreciable difference in
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CATIONS IN MEGAMITOCHONDRIA
Table 5. Contents of DivaZent Cationa in Cuprizone-Fed Mowre Liver Mitochondria1
Cation
Ca2+ Mg8+ Cu*+ Mg2+/Ca2+ ratio 1
Duration of feeding on cuprizone-diet
Control
17.0k 2.4 30.6f 4.6 0.41f0.04 1.80
3 4 days
14-15 days
12.8% a.4*
20.0* 4.0* 21.6k 6.9*** 0.48f0.10 1.08
34.6k 2.9
0.47fO. 20 2.70
Mice were fed with a 0.5% cupriaone-dietfor certain periods specified in the table. Data are the averages (meanff3.D.) of 10 different experiments. Data are statistically different by Student's t-test from the control at *: 0.05>p>0.01, **: 0.01>p>0.001 and ***: O.Ool>p. Data are expressed in moles/mg protein.
Table 6. Contents of Phospholipids in Cuprizone-Induced Megantitochondria' (yoof total P h s p h l i p i d in Each Fraction)
Control
Cuprizone
~
Phospholipid
Whole Outer Whole Outer Mitochondria Membrane Mitochondria Membrane
Total (Pi nmoles/mg prot.)
188.0
366.8
170.2
344.9
Cardiolipin Phosphatidylethanolamine Phosphatidylinositol Phosphatidylcholine Sphingomyelin Lysolecithin
14.6 34.7
4.9 2s. 3 9.3 49.6
16.9 32.5 6.3 36.6 4.3 6.9
5.8 31.1 9.1 46.3 7.8 1.0
1
6.0
41.7 2.8 2.4
6.0 2.1
Mitochondria were isolated from the livers of mice fed with a 0.5% cuprizonediet for 8-10 days. Mitochondria were obtained from the livers of 10-20 animals in one experiment.
contents of phospholipids between the outer membrane fraction obtained from cuprizone-fed mouse liver mitochondria and that of the control. Discussion
Recently we have demonstrated that fusion phenomenon is involved in the formation process of megamit~chondria~.We have also demonstrated that nialamide, which is one of the well known inhibitors of monoamine oxidase, induces megamitochondria in mouse hepatocytes simply by feeding the animal with a diet containing the noxious reagent6. The above described two lines of evidence compelled us to examine further biochemical aspects of cuprizone-induced megamitochondria. The present study clearly demonstrated that content of Ca2+ in cuprizone-fed mouse liver mitochondria has an intimate correlation with the duration of cuprizoneintoxication. Previously we have shown that it takes a t least a week t o induce
28(2): 1978
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et al.
megamitochondria in mouse hepatocytes by feeding the animal with a diet containing c ~ p r i z o n e ~After ~ ~ ~ .3-4 days of intoxication with cuprizone, content of Ca2+ is decreased. This is the stage in which megamitochondria have not been induced yet in the liver. The role of Ca2+ on membrane fusion has been repeatedly described. Recently POSTE and ALL IS ON^^ have proposed a scheme for membrane fusion stressing the displacement of Ca2+ from the membranes together with ATP: displacement of Ca2+and ATP from the membranes results in increased motional freedom and a structural transformation of the membrane lipids. A t 1615 days of cuprizone-intoxication, content of Ca2+ in liver mitochondria is raised significantly. At this stage, megamitochondrial formation is complete and megamitochondria are often devoid partially of the outer membrane and most of cristae are located peripherally suggesting the swelling and the degenerative proces~es3~.Thus, increment of Ca2+ at a later stage of megamitochondrial formation would be ascribed to disorganization of integrity of mitochondria1 membrane changing its permeability to Ca2+. The increment of lysolecithin in megamitochondria would be explained in the same fashion as in the case of Ca2+. LUCYand his colleagues21 demonstrated that lysolecithin was able to fuse hen erythrocytes in vitro. The cell fusion potential of lysolecithin has since been confirmed by others9, and the requirement of the membrane fusion reaction for Ca2+ could possibly be interpreted as being due to the Ca2+ requirement of phospholipases ~ ~ ~ ~ ~ ~ ~ ~of. the precise mechanism by which that generate l y ~ o l e c i t h i n ~ ~ ,Regardless lysolecithin induces membrane fusion, it is probable that the insertion of wedgeshaped molecule such as lysolecithin into the membrane would produce substantial disorder22. Although the present experiment shows the increment in the amount of lysolecithin in megamitochondria, the degree of increment was variable from experiment to experiment, and a definite conclusion awaits further detailed investigation. The present study revealed that activity of cytochrome oxidase, which is a coppercontaining enzyme, was unaffected by cuprizone-treatment, but the activity of monoamine oxidase, which is another copper-containing enzyme in the mitochondrion was suppressed both in vitro and in vivo. Then, how is the inhibition of monoamine oxidase activity correlated to megamitochondrial formation Z How is it correlated to the membrane fusion ? In in vitro experiments, the value of I,, (50% inhibition) of monoamine oxidase was the lowest in mitochondria of the liver compared to that of the kidney or the heart, and the activity of the enzyme in vivo was suppressed remarkably only in mitochondria of the liver (Table 7 ) . These data are interesting Table 7 . Effect of Cuprizone on Monoamine Oxidase Activities of Mitochondria from Various Tissues Both in vitro and in vivo ~
Tissue
in vitro (160)
(yoinhibition)
in vivo
Liver Kidney Heart
0.33 mM 6.60mM 11.2 mM
39.3% 21.0% 1.6%
222
CATIONS I N MEUAMITOCHONDRIA
Acda Path. Jap.
considering the fact that megamitochondria are induced only in the liver and not either in the kidney or the heart4s21. However, a question of how monoamine oxidase activity is correlated to the depletion of Ca2+ from the mitochondrion or how it is correlated to megamitochondrial formation can not be answered from the present data. We are now studying various types of inhibitors of monoamine oxidase in order t o know if the megamitochondrial formation is a universal effect by the inhibitors, and the results will be reported soon. References FISCHER, D., HOWELL, J.I., TAMPION, W., VERRINDER, M., and LUCY, J.A. : Chemically induced and thermally-induced cell fusion: lipid-lipid interactions. Nat. New Biol. 242: 215-217, 1973. APOSTOLOV,K. and POSTE,G.: Interaction of Sendai virus with human erythrocytes: a system for the study of membrane fusion. Microbios. 6: 247-261, 1972. ASANO,M., WAKABAYASHI, T., KURONO, C., and OZAWA, T.: Mechanism of the formation of megamitochondria induced by copper-chelating agents. 111. Formation and some biochemical properties of megamitochondria induced by diethyldithiocarbamate (DDC). Acta Path. Jap. 25: 125-134, 1975. ASANO,M., WAKABAYASHI, T., ISHIKAWA, K., and KISHIMOTO, H.: Mechanism of the formation of magamitochondria induced by copper-chelating agents. IV. Role of fusion phenomenon on the cuprizone-induced megamitochondrial formation. submitted to Acta Path. Jap. ASANO,M., WAKABAYASHI, T.,ISHIKAWA, K., and KISHIMOTO, H.: Induction of megamitochondria in mouse hepatocytes by nialamide. J. Electron Microscopy (in press). BAILEY,E., TAYLOR, C.B., and BARTLEY, W.: Turnover of mitochondrial components of normal and essential fatty acid-deficient rats. Biochem. J. 104: 10261032, 1967. BEATTIE,D.S, BASFORD, R.E., and KORITZ,S.B.: The turnover of the protein components of mitochondria from rat liver, kidney, and brain. J. Biol. Chem. 242: 4584-4586, 1967. BLIUH,E.G. and DYER,W.J.: A rapid method of total lipid extraction and purification. Canad. J. Biochem. Physiol. 37: 911-917, 1959. CROCE,C.M., SAWICKI, W., KRITOHEVSKY, D., and KAPROWSKI, H.: Induction of homokaryocyte, heterokaryocyte and hybrid formation by lysolecithin. Exp. Cell Rea. 67: 427-435,
1. AEKONU, Q.F., CRAMP, F.C.,
2. 3.
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5. 6. 7. 8. 9.
1971. E.A. and ROTHMAN, J.E. : Fusion and protein-mediated phospholipid exchange 10. DAWIDOWICZ,
studied with single bilayer phosphatidylcholine vesicles of different density. Biochim. Biophys. Acta 455: 621-630, 1976. W.E.M.: A new sensitive determination of phosphate. Anal. Biochem. 11. EIBL, H. and LANDS, 30: 51-57, 1969. M.J. and SANADI,D.R. : Turnover of rat-liver mitochondria. Biochim. Biophys. 12. FLETCHER, Acta 51: 356360, 1961. 13. GREEN,D.E.: Membrane proteins. A perspective. Ann. N.Y. Acad. Sci. U.S.A. 195: 150172, 1972. M.: Apparent turnover of mitochondrial 14. GROSS, N.J., GETZ,G.S., and RABINOWITZ,
deoxyribonucleic acid and mitochondrial phospholipids in the tissues of the rat. J. Biol. Chem. 244: 1552-1562, 1969. R.J.: Protein measurement N.J., FARR,A.L., and RANDALL, 15. LOWRY,O.H., ROSEBROUUH, with the Folin phenol reagent. J. Biol. Chem. 193: 265-275, 1951. J., COLBEAU, A., V I U N ~ SP.M. , : Distribution of membrane-confined phos16. NACHSATJR, pholipase A in the rat hepatocyte. Biochim. Biophys. Acta 274: 426-446, 1972. J.D. and WAITE,M.: Phospholipid hydrolysis by phospholipases Al and As in 17. NEWKIRK, nlasma membranes and microsomes of rat liver. Biochim. BioDhvs. Acta 298: 562-576. 1973.
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Y. and MURAYAMA, F.: Requirement of calcium ions for the cell fusion reaction of 18. OKADA, animal cell by HVJ. Exp. Cell Res. 44: 527351, 1966. D.: Studies on the mechanism of action of local anesthetics with 19. PAPAHADJOPOULOS, phospholipid membranes. Biochim. Biophys. Acta 265: 169-186, 1972. D., POSTE, G., and SCHAEFFER, B.: Fusion of mammalian cells by 20. PAPAHADJOPOULOS, unilamellar lipid vesicles: influence of lipid surface charge, fluidity and cholesterol. Biochim. Biophys. Acta 323: 2440, 1973. A.R., HOWELL,J.I., and LUCY,J.A.: Lysolecithin and cell fusion. Nature 227: 21. POOLE, 810-813, 1970. 22. POSTE,G. and ALLISON, A.C. : Membrane fusion. Biochim. Biophys. Acta 300: 42145,1973. 23. SCHNAITMAN, C. and GREENAWALT, J.W.: Enzymatic properties of the inner and outer membranes of rat liver mitochondria. J. Cell Biol. 38: 158-175, 1968. A., YAOIL,G., and YAFFE,D.: Control of myogenesis in vitro by Cae+concentra24. SHAINBERO, tion in nutritional medium. Exp. Cell Res. 58: 163-167, 1969. Chr. and PETTE, D.: Quantitative investigation on Caa+-and 25. VAN DEN BOSCH,J., SCHUDT, pH-dependence of muscle cell fusion in vitro. Biochem. Biophys. Res. Commun. 48: 326-332, 1972. E.J., VAN GOLDE,L.M.G., HOSTETLER, K.Y., SHERPHOF, G.L., and VAN DEENEN, 26. VICTORIA,
L.L.M.: Some studies on the metabolism of phospholipids in plasma membranes from rat liver. Biochim. Biophys. Acta 239: 443-457, 1971. 27. WAITE,M. and SISSON,P. : Utilization of neutral glycerides and phosphatidylethanolamine by the phospholipase A, of the plasma membranes of rat liver. J. Biol. Chem. 248: 7985-7992, 1973. T. and GREEN,D.E. : On the mechanism of cuprizone-induced formation 28. WAKABAYASHI, of megamitochondria in mouse liver. J. Bioenergetics 6 : 179-192, 1974. T.: Mechanism of the formation T., ASANO,M., KURONO,C., and OZAWA, 29. WAKABAYASHI,
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Mechanism of the formation of megamitochondria induced by copper-chelating agents. I. On the formation process of megamitochondria in cuprizone-treated mouse liver. Acta Path. Jap. 25: 15-37, 1975. A.M. and WIENEKE,A.A.: The accumulation of calcium by the polymorphonuclear 31. WOODIN, leucocyte treated with staphylococcal leucocidin and its significance in the extrusion of protein, Biochem. J. 87: 487495, 1963. 32. WOODIN,A.M. and WIENEKE,A.A. : The participation of calcium, adenosine triphosphate and adenosine triphosphatase in the extrusion of the granule proteins from the polymorphonuclear leucocyte. Biochem. J. 90 : 498-509, 1964. A. and LOYTER,A.: The mechanism of cell fusion. I. Energy requirements for 33. YANOVSKY, virus-induced fusion of Ehrlich ascites tumor cells. J. Biol. Chem. 247: 40214028, 1972.
