THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 6, Issue of February 25, pp. 3436-3439, 1990 Printed in U. S. A.

Phosphatidylcholine and Phosphatidylethanolamine Enhance the Activity of the Mammalian Mitochondrial Endonuclease in Vitro*

(Received for publication, May 15, 1989)

William A. Parks, Christopher L. Couper, and Robert L. Low+ From the Department of Pathology, University of Colorado Health Sciences Center, Denver, Colorado 80262

The purified endonuclease of bovine heart mitochon- dria extensively degrades a variety of DNA templates in vitro but shows a remarkably strong preference to nick within one specific evolutionarily conserved se- quence block of 12 consecutive guanine residues which resides just upstream from the heavy strand origin of mitochondrial DNA replication (Low, R. L., Cum- mings, 0. W., and King, T. C. (1987) J. Biol. Chem. 262, 16164-16170). If the enzyme serves to provide an important nicking function at this site in vivo, then mitochondrial factors likely exist which further en- hance the enzyme’s recognition of this locus and pre- vent cleavage at other less favored sites. In this study, we report that specific membrane phospholipids ap- pear to exert such effects in vitro. In standard endo- nuclease assays, low levels of phosphatidylcholine or phosphatidylethanolamine (0.5 mM) stimulate the pu- rified enzyme activity lo-20-fold. However, at mod- erate levels (20-40 mM), these phospholipids largely inhibit widespread degradation of duplex DNA while still allowing site-specific nicking at the conserved guanine target in the mitochondrial genome. These findings suggest that an interaction of the endonucle- ase with major lipid components of the inner membrane could be an important determinant of the enzyme’s specificity for mitochondrial DNA.

Mammalian heart mitochondria contain a potent and sol- uble endonuclease activity that appears once intact organelles are disrupted (1). Under assay conditions of low ionic strength and in the presence of Mg2+ (or Mn”), this mitochondrial endonuclease extensively fragments either duplex or single- stranded DNA templates both in the acidic and neutral pH range. The activity of bovine heart mitochondria appears to be a homodimer of a 29-kDa polypeptide (1). In some but not all respects, it resembles the endonuclease activities described previously in mouse cell (2), yeast (3-7), and Neurospora (8, 9) mitochondria. Since the heart enzyme avidly degrades undamaged DNA, it clearly differs from an apurinic endonu- clease of mammalian mitochondria which was reported re- cently (10).

The physiologic role of this endonuclease in mammalian heart mitochondria is still not known. The mitochondrial nuclease in lower eukaryotes has been implicated previously in the repair of certain types of mitochondrial DNA damage

* This work was supported by National Institutes of Health Grant GM-37021. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be sent: Dept. of Pathology (B216), University of Colorado Health Sciences Center, 4200 E. 9th Ave., Denver, CO 80262. Tel.: 303-270-8024.

(11, 12). However, a recent study of the yeast mitochondrial endonuclease has challenged this possibility (13). Inactivation of the structural gene for the yeast enzyme fails to show any effect on viability or phenotype; this deletion mutant remains rho+. Of interest, the enzymes of bovine, human, and rat heart mitochondria each attack DNA sequences in the same highly nonrandom fashion in vitro and show a remarkably strong preference to attack an unusual and evolutionarily conserved stretch of guanine residues (called conserved sequence block (CSB)l-II, (14)) which residues just upstream from the heavy strand origin of DNA replication (15). The participation of the endonuclease at this specific site in vivo to serve as the nicking component of a novel swivel is an interesting possi- bility. Remarkably, this pattern of cleavage of the mitochon- drial endonuclease looks similar if not identical to that of a nuclear endonuclease activity called endonuclease G, which in vitro also appears to target specific runs of guanine residues found in the regulatory domains of some nuclear genes (16). Although it has been proposed that endonuclease G may play a role in site-directed DNA recombination in the nucleus, such a role for the mitochondrial endonuclease seems ex- tremely unlikely since mammalian mitochondrial DNAs rarely if ever recombine (17).

If the mitochondrial endonuclease is site specific, then mitochondrial specificity factors must exist which normally protect the mitochondrial genome from widespread degrada- tion. In this study, we report that phospholipid components markedly affect enzyme activity in vitro. Although low levels of phosphatidylcholine, for example, stimulate endonucleoly- tic degradation of duplex DNA, moderate levels prevent ex- tensive DNA fragmentation while still allowing specific nick- ing of the conserved guanine target of the D-loop region.

EXPERIMENTAL PROCEDURES

Chemicals and Nucleic Acids-Chemicals and other reagents were obtained from commercial sources cited previously (18) unless oth- erwise indicated. All phospholipids were purchased from Sigma and stored at -20 “C. Fresh 100-~1 aliquots of phospholipid were dried in VCKUO (Savant SpeedVac concentrator) to remove the organic solvent. The residue was then resuspended in an equal volume of 10 mg/ml n-octyl /3-D-glucopyranoside by vortexing 5-10 min at ambient tem- perature. Phosphatidylcholine (Sigma, Type III-H from bovine heart) was further dialyzed against 20 mM Hepes (pH 7.5), 50 mM NaCl, 1 mM EDTA for 2 davs at ambient temperature to provide a preparation of unilamellar vesicles (19).

The sunercoiled nlasmid desimated Bovl contains a 4.9-kilobase EcoRI-Cl& restrictibn fragmentif bovine mitochondrial DNA which encompasses all the D-loop region as described previously (18). Bovl, the Ml3 replicative form RF1, and pBR322 DNAs were each purified on two successive CsCl-ethidium bromide gradients.

Enzymes-The bovine mitochondrial endonuclease was purified about lO,OOO-fold to near homogeneity from heart mitochondria,

1 The abbreviations used are: CSB, conserved sequence block; D- loop, displacement loop; Hepes, 4-(2-hydroxyethyl)-l-piperazineeth- anesulfonic acid.

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Phospholipids Affect the Mitochondrial Endonuclease

using a modification of the purification protocol that was published previously (1). In this modified scheme, the Fraction II (20 ml, 20 mg/ml of protein) was dialyzed against 2 liters of 40 mM Hepes (pH 7.5), 1 rni EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 rnh dithi- othreitol. 10% (v/v) elvcerol (Buffer A) for 4 h at 2 “C. and followine

. - I

a 5-lo-fbld dilution using Buffer A’ plus 1% (w/vj n-octyl 0-D: glucopyranoside (Buffer B) to lower the conductance to an equivalent of 40 mM NaCl, the enzyme was applied to a 124-ml (13 X 0.95 cm*) phosphocellulose column equilibrated in Buffer B plus 40 mM NaCl. Following a 300-ml wash with Buffer B plus 160 mM NaCl, activity was eluted using 300 ml of Buffer B plus 260 mM NaCl and then concentrated by ammonium sulfate precipitation (0.4 g of ammonium sulfate/ml). The ammonium sulfate concentrate was further purified on a 50-ml Sephacyl S-200 column followed by a 1.5-ml single- stranded DNA-cellulose column as detailed previously (15). The most purified fraction (the single-stranded DNA-cellulose pool, Fraction V (3 X lo9 units/mg)) was used throughout this study. One unit of activity nicks 250 ng of the input Bovl DNA (500 ng) in 30 min at 37 ‘C.

The HpaI and DraI restriction endonucleases and T4 polynucleo- tide kinase were purchased from New England BioLabs and used as the supplier recommended.

Agarose Gel and Sequencing Gel Electrophoresis-The extent of DNA template degradation was assessed on 150-ml O&2% (w/v) agarose gels as described previously (18). Sequencing gels (12% poly- acrylamide (acrylamide-bisacrylamide) 19:l) were cast and run as described by Maxam and Gilbert (20). DNA samples were prepared as detailed (18).

Other Assays-Protein concentrations were measured by the Coo- massie Brilliant Blue method of Bradford (21) using bovine serum albumin as a standard. Phospholipid concentrations were measured by phosphate determinations (22) using sodium phosphate as a stand- ard.

RESULTS

Phosphatidylcholine Inhibits the Endonuclease Activity Identified in a Crude Fraction of Mitochondrial Protein but Stimulates the Activity of the Purified Enzyme--The large majority (>90%) of the bovine mitochondrial endonuclease resides in a crude concentrate of proteins called Fraction II, which is prepared by ammonium sulfate fractionation of the mitochondrial lysate (1). In a standard agarose gel assay for endonuclease activity, 10 rg of the Fraction II of bovine heart mitochondria extensively degrades the 500 ng of input super- coiled plasmid DNA substrate in 30 min at 37 “C to yield a digest of small DNA fragments that readily migrate off the end of the gel. Addition of increasing levels of phosphatidyl- choline to this reaction in the range of 0.1-4 mM markedly inhibits substrate fragmentation in a concentration-depend- ent manner. At 2-4 mM phosphatidylcholine, the main DNA products are the nicked circular and full-length linear (1) species (data not shown). Addition of the detergent n-octyl /3- D-glucopyranoside from 0.02 to 1% (w/v) had no detectable effect on the extent or pattern of inhibition.

In contrast, addition of the same low levels of phosphati- dylcholine sufficient to cause inhibition of the endonuclease activity in Fraction II significantly stimulates the activity of the purified endonuclease (Fraction V). In standard agarose gel assays, maximal stimulation occurs between 0.1 and 0.5 mM phosphatidylcholine, whereas levels above 1 mM are increasingly inhibitory (see below). Identical results were observed in assays carried out with the Bovl DNA substrate at pH 5 (not shown). A titration of the purified mitochondrial endonuclease at pH 7.5 in the absence and presence of 0.5 mM phosphatidylcholine is shown in Fig. 1. Significant en- hancement of activity is seen at each level of enzyme. The similarity between the pattern of nicked circular and linear DNA products seen in lane 4 (+ phospholipid) and lane 11 (- phospholipid) suggests that the phosphatidylcholine stimu- lates endonuclease activity lo-20-fold. Identical results are obtained with other DNA substrates, e.g. pBR322 DNA and

1-

n lane: 1 2 3 4 5 6 7 3 9 10 11 1:

FIG. 1. A low level of phosphatidylcholine (PC) the purified mitochondrial endonuclease (endo). . . . .

stimulates Each 40-~1

reaction was carried out m the absence (odd numbered lanes) or presence (euen numbered lanes) of 0.5 mM phosphatidylcholine and contained 25 mM Hepes, pH 7.5, 25 mM sodium glutamate, 10 mM MgAc*, 1 mM dithiothreitol, 4% (v/v) glycerol, 500 ng of Bovl DNA, and 0, 0.04, 0.1, 0.2, 0.4, or 0.8 ng of purified bovine heart mitochon- drial endonuclease, as indicated. Reactions were run 30 min at 37 “C, then terminated and analyzed as described (18). A photograph of the agarose gel is shown. Nicked circular (nc), linear (1), and supercoiled (SC) forms of the DNA are indicated.

the Ml3 replicative form RF1 DNA; 0.5 mM phosphatidylcho- line also increases the rate of template degradation at least lo-fold.

The purified endonuclease shows an unusually strong pref- erence to attack the evolutionarily conserved sequence block of 12 guanine residues (CSB-II) which resides in the D-loop region of the bovine mitochondrial genome (15, 18). To ex- amine the effect of phosphatidylcholine on the enzyme’s recognition of this site, the purified endonuclease was given a singly 32P 5’ end-labeled HpaI-DraI restriction fragment sub- strate of the Bovl DNA that encompasses the CSB. Digestion was carried out in the absence and presence of phosphatidyl- choline at 0.5 mM. As indicated in the autoradiogram of the Maxam-Gilbert sequencing gel (Fig. 2), 0.04 ng of the purified endonuclease introduces a small but significant amount of specific nicking in the guanine tract only in the presence of the added phospholipid. As seen, this phosphatidylcholine dependence is even more dramatic using 0.4 ng of the enzyme where the effect appears to be at least lo-fold. Using the restriction fragment substrate singly end labeled not at the HpaI terminus but at the DraI terminus, it was found that 0.5 mM phosphatidylcholine also enhances specific nicking of the complementary tract of cytosine residues (not shown).

Some Other Phospholipids Fail to Stimulate the Purified Mitochondrial Endonuclease-A preliminary survey of some other membrane phospholipids indicates that the stimulatory effect of phosphatidylcholine is equally shared by phosphati- dylethanolamine. In endonuclease assays that follow the re- lease of [3H]DNA fragments that are acid soluble, increasing the concentration of either of these two phospholipids from 0.05 to 0.5 mM similarly enhances the rate of enzyme activity in a concentration-dependent fashion (Fig. 3). In contrast, the effects of phosphatidylinositol and phosphatidylserine (Fig. 3) and of phosphatidyl DL-glycerol and cardiolipin are negligible (not shown).

Moderate Levels of Phosphatidylcholine Inhibit the Degra- dative Activity of the Purified Endonuclease-Although 0.5 mM phosphatidylcholine promotes extensive fragmentation of the template, increasing amounts of phosphatidylcholine (and phosphatidylethanolamine) up to 20 mM progressively restrict the reaction to limited nicking of the input DNA. The

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3438 Phospholipids Affect the Mitochondrial Endonuclease

P.C.: --++

FIG. 2. Phosphatidylcholine (PC) enhances site-specific nicking of the conserved sequence block (CSB)-II of the bo- vine D-loop region. The 170-base pair singly 32P 5’ end-labeled &al-DraI fragment was prepared as detailed (18). Each 40-~1 reac- tion contained 25 mM Hepes, pH 7.5; 25 mM sodium glutamate; 10 mM MgAc?; 1 mM dithiothreitoc 4% (v/v) glycerol; 200 ng of unlabeled Bovl DNA: the 3*P 5’ end-labeled HnaI-DruI fragment (8000 dnm): and none, 0.04 ng, or 0.4 ng of purified endonuclease, without (-) dr with (+) 0.5 mM phosphatidylcholine, as indicated. Reactions were run 10 min at 37 “C, and DNA products were purified by phenol extraction and ethanol precipitation. The G and G + A Maxam- Gilbert sequencing ladders (prepared from the undigested 32P 5’- restriction fragment) are shown in leftmost rwo lanes, respectively. The sequencing gel and DNA samples were prepared as cited under “Experimental Procedures.”

choline moiety is not itself responsible for the inhibition since choline in the range of 0.5-20 mM shows no detectable effect. Preincubating the purified endonuclease with 4 mM phospha- tidylcholine in the absence or presence of 2 mM ATP for up to 20 min at 37 “C (minus DNA) was without any additional effect.

At 40 mM phosphatidylcholine, the CSB-II target is still being avidly nicked. In this experiment, the reaction was stopped after about 40% of the input supercoiled Bovl DNA was converted to the nicked circular product. From the total DNA digest, the HpaI-DruI restriction fragment (that harbors the CSB-II site) was prepared, 32P 5’ end labeled at the HpaI terminus, and analyzed on a Maxam-Gilbert sequence gel. The pattern of bands on the autoradiogram of the gel closely resembles the pattern shown in Fig. 2 and indicates that prominent nicking has selectively occurred in the guanine block of the CSB-II locus. Only about 5% of the complemen- tary cytosine tracts are nicked, however (not shown). Al- though in standard endonuclease assays, increasing amounts of a variety of salts such as NaCl and sodium glutamate will progressively and markedly inhibit the widespread fragmen- tation of duplex DNA seen in the presence of 10 mM Mg2 and low ionic strength (l), the nicking activity in the CSB-II locus which still occurs in the presence of 20 mM phosphati- dylcholine (minus salt) is seemingly unaffected by the further addition of 25-75 mM sodium glutamate. The specificity for

I

05 10 15 20 Phosphollvd (mM)

FIG. 3. Strong stimulation of the purified endonuclease by phosphatidylcholine and phosphatidylethanolamine is also ev- ident in acid solubilization assays. Each 40-~1 reaction contained 25 mM Hepes, pH 7.5, 10 mM MgAc?, 1 mM dithiothreitol, 4% (v/v) glycerol, 620 ng of Bovl [3H]DNA (5.3 X lo4 dpm/pg), 2 ng of purified mitochondrial endonuclease (Fraction V), and 0, 0.05, 0.1, 0.25, 0.5, 1, or 2 mM phosphatidylcholine (U), phosphatidylethanolamine (W), phosphatidylinositol (U), or phosphatidylserine (U) as indicated. Reactions were run 30 min at 37 “C and terminated by the addition of 10% (w/v) trichloroacetic acid (1). After 10 min at 0 “C, the precipitates were centrifuged at 10,000 X g for 10 min in a Beckman Microfuge E, and a 20-~1 aliquot of each super- natant was then counted in 5 ml of CytoScint (ICN Biochemicals, Inc.) by liquid scintillation counting to measure the release of acid- soluble DNA fragments. Less than 0.5% of the input Bovl [3H]DNA was acid soluble in the absence of added enzyme.

this guanine site is not exclusive, however; with 40 mM

phosphatidylcholine in the absence or presence of salt, some nicking of both pBR322 and M13RF DNA is also evident and nearly comparable to that observed with the Bovl DNA.

DISCUSSION

Phospholipids comprise a large and integral part of the inner and outer membranes of the mitochondrion. Among these, phosphatidylcholine and phosphatidylethanolamine are the major constituents, each about 40% of the total phospholipid, whereas cardiolipin (~20% of the total), phos- phatidylinositol (-3% of the total), and trace amounts of phosphatidylserine constitute the remainder (23). The large number of proteins and protein complexes that occupy mem- brane compartments usually requires specific phospholipids or specific combinations of phospholipids for enzymatic activ- ity. Two well known examples are cytochrome oxidase (24) and the reconstituted membrane ATPase (25).

As shown in this present study, phospholipids, in particular phosphatidylcholine and phosphatidylethanolamine, exert major effects on the activity of the bovine heart mitochondrial endonuclease in uitro. Endogenous phospholipids likely con- taminate the crude fractions of the endonuclease which are first prepared using moderate levels of the detergent n-octyl @-D-glucopyranoside. Hence, the failure of 0.1-0.5 mM phos- phatidylcholine or phosphatidylethanolamine to stimulate nuclease activity in the Fraction II is not surprising. Although lower levels of these phospholipids significantly stimulate the purified enzyme (at least lo-fold), moderate levels markedly inhibit activity with the same Bovl DNA substrate and appear to elicit increased specificity of the enzyme for the evolution- arily conserved sequence tract of guanine residues which resides at the boundary of the D-loop region next to the tRNAPh’ gene. This latter finding further supports a model that the preference of the endonuclease to attack this unusual sequence block in vitro is not simply fortuitous but that the

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Phospholipids Affect the Mitochondrial Endonuclease 3439

enzyme serves a specific function at this site in uiuo (15, 18). However, it is still possible that the enzyme assumes an alternative role in the destruction of selected genomes during the remodeling of organelles in uiuo (1,2).

The precise manner in which these phospholipids effect enzyme activity in vitro is still uncertain. At low levels of phospholipid, a direct interaction of the lipid molecule with the enzyme could stabilize a protein conformation that favors either binding of the enzyme to the DNA or the hydrolysis of phosphodiester bonds. Higher levels could greatly retard the diffusion of the enzyme along DNA, actually block access of the enzyme to the DNA, or reduce the enzyme’s processivity so that only initial highly favored sites in the nucleotide sequence would be hit. These higher levels of phosphatidyl- choline (20-40 mM) do not appear to promote the formation of a covalent attachment of the endonuclease to the Bovl DNA.* When supercoiled Bovl DNA is nicked by the mito- chondrial endonuclease in the presence of 40 mM phosphati- dylcholine and subsequently digested with HpaI restriction endonuclease and 32P 5’ end labeled, less than 1% of the [““PI DNA product can be precipitated by sodium dodecyl sulfate in the presence of 70 mM potassium chloride (26). So far, attempts to use low speed centrifugation to help identify a stable complex between the purified endonuclease and a sus- pension of unilamellar vesicles of phosphatidylcholine have also not been successful.3

An effect of phospholipids on the activity of the endonucle- ase in uitro is not altogether surprising since the enzyme is likely bound to the inner membrane in uivo. This localization was suggested from earlier studies of both the yeast (4-7) and Neurosporu (12) mitochondrial endonucleases in which the enzyme was found to be strongly associated with fractions of the inner membrane. For the bovine heart mitochondrial activity as well, detergents such as 1% (w/v) Triton X-100 or n-octyl p-D-glucopyranoside are required to maximize release of the enzyme from disrupted organelles and to enhance recovery of the enzyme activity during chromatography. Whether phospholipids interact directly with other DNA replicative or transcriptional enzymes on the mitochondrial DNA has been less obvious since the mitochondrial RNA polymerase, DNA polymerase-y, and mitochondrial nucleic acids are regarded as components of the matrix, not inner membrane compartment (23). Neither the bovine RNA po- lymerase nor DNA polymerase-y, for example, appears to require exogenous phospholipid for full activity in uitro.4 However, although the mitochondrial genome may reside in the matrix, a local attachment of the DNA to the inner membrane first suggested from early electron microscopic studies (27) seems likely. A single large protein-containing structure, associated with fragments of inner membrane, was

’ R. L. Low, unpublished data. 3 R. L. Low and W. A. Parks, unpublished data. 4 C. L. Couper and R. L. Low, unpublished data.

in fact subsequently identified on a large majority of mito- chondrial DNA circles, isolated from HeLa cell mitochondria (28). Of interest this protein structure was localized to a restriction fragment of the genome that contains the D-loop region. Whether such a structure shares specific interactions with phospholipid and contains mitochondrial endonuclease and/or other membrane-associated enzymatic activities are important and intriguing questions.

Acknowledgments-We thank Nancy Hart and Clairene Mraz for their diligent typing of the manuscript.

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REFERENCES

Cummings, 0. W., King, T. C., Holden, J. A., and Low, R. L. (1987) J. Biol. Chem. 262,2005-2015

Tomkinson, A. E., and Linn, S. (1986) Nucleic Acids Res. 14, 9579-9593

Jacquemin-Sablon, H., Jacquemin-Sablon, A., and Paoletti, C. (1979) Biochemistry l&119-127

Rosamond, J. (1981) Eur. J. Biochem. 120,541-546 Morosoli, R., and Lusena, C. V. (1980) Eur. J. Biochem. 110,

431-437 von Tigerstrom, R. G. (1982) Biochemistry 21,6397-6403 Dake, E., Hofmann, T. J., McIntire, S., Hudson, A., and Zassen-

haus, H. P. (1988) J. Biol. Chem. 263,7691-7702 Linn, S., and Lehman, I. R. (1966) J. Biol. Chem. 241, 2694-

2699 Chow. T. Y.-K.. and Fraser. M. J. (1983) J. Biol. Chem. 258.

12010-12018 Tomkinson, A. E., Bonk, R. T., and Linn, S. (1988) J. Biol. Chem.

263,12532-12537 Foury, F. (1982) J. Biol. Chem. 257, 781-787 Fraser, M. J.. and Cohen, A. (1983) J. Bacterial. 154.460-470 Zassenhaus, H. P., Hofmann, T. J., Uthayashanker, k., Vincent,

R. D.. and Zona. M. (1988) Nucleic Acids Res. 16.3283-3296 Walberg, M. W., and C&ton, D. A. (1981) Nucleic Acids Res. 9,

5411-5421 Low, R. L., Buzan, J. M., and Couper, C. L. (1988) Nucleic Acids

Res. 16,6427-6445 Ruiz-Carillo, A., and Renaud, J. (1987) EMBO J. 6, 401-407 Zuckerman, S. H., Solus, J. F., Gillespie, F. P., and Eisenstadt,

J. M. (1984) Somatic Cell Mol. Genet. 10, 85-92 Low, R. L., Cummings, 0. W., and King, T. C. (1987) J. Biol.

Chem. 262,16164-16170 Mimms, L. T., Zampighi, G., Yasuhiko, N., Tanford, C., and

Reynolds, J. A. (1981) Biochemistry 20,833-840 Maxam, A. M., and Gilbert, W. (1977) hoc. Natl. Acad. Sci. U.

S. A. 74.560-564 Bradford, Ih. M. (1976) Anal. Biochm. 72, 248-254 Petitou. M., Tuv. F.. and Rosenfeld. C. (1978) Anal. Biochem.

91,350-353 "' , ,

Tzagoloff, A. (1982) Mitochndria, Plenum Publishing Corp., New York

Tzagoloff, A., and MacLennan, D. H. (1965) Biochim. Biophys. Acta 99,476-485

Kagawa, Y., and Racker, E. (1966) J. Biol. Chem. 241, 2461- 2466

Liu, L. F., Rowe, T. C., Yang, L., Tewey, K. M., and Chen, G. L. (1983) J. Biol. Chem. 258,15365-15370

Nass, M. M. K. (1969) J. Mol. Biol. 42, 521-528 Albring, M., Griffith, J., and Attardi, G. (1977) hoc. Natl. Acad.

Sci. l% S. A. 74,1348-1352

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W A Parks, C L Couper and R L Lowmammalian mitochondrial endonuclease in vitro.

Phosphatidylcholine and phosphatidylethanolamine enhance the activity of the

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