Biochimica et Bioph;sica Acta, 1078 (1991) 56-62 + 1991 Elsevier Science Puhlishers B.V. 0167-4838/91/$03.50 A DONIS 0167483891001892
56
BB~PR( ) 31908
Mitochondrial DNA topoisomerase I from human platelets Marshall J. K o s o v s k y ! and G e r a l d Soslau 1.2 'r
Department of Btologtcal ChemistO, Hahnemann Unicerst(v, Philadelphia. PA ( U,S A ) and : Department o]Neoplostie Diseases, Hahnemunn Unit,~.r~tt3. Phdadegphia, PA (US.A.) (Recei,~ed 15 October 1990) [ Revised manuscript received 27 December 1qg())
Key words: DNA topmsomerase l: M~locnondrion; Human platelet: Camptcrthecin
An anucleated cell system has been used for the first time to study mitochondrial topoisomerase aetMty. Mitochondrial extracts from human blood platclets contained ty.pe I topoisomerase. The type I classification was based on ATP-iodependent activity, inhibition by ATP or camptnthecin, and the lack of inhibition by novobiocin. Platelet mitochondrial topoisomemse I relaxation activi~' was inhibited linearly by increasing concentrations of EGTA. Topoisomerase activity > 90% inhibited by 175 pM EGTA was partially restored to 16 and 50% of the initial level of aetivi~" by the subsequent additien of 50 and 100 pM Caz+, respectively. Additionally, results from studies of partially purified platelet mitochondrial topoisomera~ 1 were consistent with the crude extract data. This work supports the hypothesis that platelet mitochandria contain a type ! topoisomerase that is biochemically distinct from that previously isolated ,and characterized from cell nuclei, Introduction DNA topoisomerases regulate the topological state of DNA by catalyzing the cleavage, strand passage, and rejoining of either a single-strand (topoisomerase 1 (TOPI)) or double-strand (topoisomerase II (TOP2)) of the DNA molecule (see Refs. 1-3 for reviews). TOPI functions in an ATP-independent manner, and has been implicated in DNA metabolic processes such as replication [4,5], transcription [6,7], and recombination [8]. TOP2 catalyzes ATP-dependent reactions which are required for the segre~.ation of DNA replication products in simian vim+ 40 [9] and are required during yeast meiosis [10] and mitosis [11]. While nuclear topoisomerases from eukaryotes have been well characterized, little is known about mitochondrial topoisolnerases.
Mammalian mitochondria contain a closed circular double-stranded DNA genome comprised of approx. 16 500 base pairs [1233]. As studies revealed the mechanisms of mitochondrial DNA (mtDNA) 'displacement-
Abbre~ation:. m*.TOP, milochondna] topoisomerase; DMSO, dimethyl sulfoxide; TPEN. N,N,N'.N'.tetrakis.(2.pyridylmethyb.cthytenedianune. Correspondence: G. Soslau, Departments of Biolo'ocal Chetmstry and NeopLastic Diseases. Hahnemann Universily, Broad and Vine Streets. Philadelphia. PA 19102. U.S.A.
loop' replication [141 and transcription [15,16], the search for mitochondrial topoisomerase (mtTOP) activity commenced. The first reported mtTOP was isolated from rat liver [17], and was later shown to exhibit ATP-independent type I activity 118]. Additionally. mtTOP1 has been isolated from hnman acute leukemia cells [19], calf thymus [20] and Xenopus laet,is oocytes [211. Studies have also provided indirect evidence for the presence of mtTOP2 in rat liver 12223], as well as direct evidence for type il activity in the milochondria of rat liver [24] and human acute leukemia cells [25]. The characterizations of the above mitochondrial topoisomerases indicate considerable variability with respect to characteristics, such as molecular weight, the response to metabolites and drug effects. The observed dissimilaritie, may reflect the difficulty in obtaining well purified mitochondria from nucleated cells or that eukaD'otic mitochondrial topoisomerases exhibit species-specific properties. We report for the first time studies of mtTOP derived from an anuclear cell, the human blood platelet. The use of this novel system has enabled the isolation of a mitochondrial fraction that is devoid of nuclear contamination. Crude mitochondrial extracts have been shown to contain a camptothecin-sensitive type I topoisomerase. Onr studies of these extracts suggest that platelet mtTOP1 may require calcium (Ca 2+) for enzymatic activity. Furthermore, the enzymatic activity of partially purified human platelet mtTOP1 and the
57 crude extract activity were qualitatixel3 idcnth:al `shen exposed to stimulatory and inhibitory agents.
Materials and Methods
Matermls Non-injectab,e. 2-7-day-old human platelet concentrates were obtained from the Pennsylvania-New Jersey Red Cros:;. The plasmid pBR322 served as substrate DNA for all topoisomerase assays, and ',','as prepared according to the alkaline lysis procedure of Maniatis et al. [261. TPEN was purchased from Calbiochem and was dissolved in DMSO and stored at - 2 0 ° C . Topoisomerase inhibitors were prepared as follows: Camptothecin was dissolved in DMSO and stored at --20°C; Berenil and novobkv.:in ,,,'ere dissolved in 1056 glycerol, 100 mM Tris, 2 mM Na,EDTA, 2 mM dithiothriotol (DTT) and 0.15; Triton X-100. pH 7.6 {buffer A) and storm at - 2 0 ° C . Unless otherwise indicated, all chemicals were obtained from Sigma. I n optimize contrast in the gel photographs, reverse images were prepared by utilizing the original photographic negatives for contact printing on Kodak electrophoresis duplicating paper.
Purification of human platelets Platelet concentrates (derived from 4-10 individuals) were pooled, resulting in a heterogeneous mixture of cells. The platelets were centrifuged at 650 × g for 15 min. The plasma was decanted and the platelet pellets were collected and pooled. The platelets were layered over 25 3556 sucrose gradients, centrifuged at 650 × g for 30 rain and the upper 2/3 of each gradient was collected. The purified platelets v, ere examined by light microscopy to confirm the homogeneity of each preparation; each field of view. containing approx. 50000 platelets, was devoid of while blood cells [27] All subsequent steps were carried out at 4 ° C. The platelcts were pelleted at 8000 × g for 10 rain. resuspended in a small vol. of 150 mM sodium chloride (NaCI), 20 mM Tris (pH 7.4) (Iris-buffered saline (TBS))/0.3856 stxiium citrate and pelleted at 6000 × g for 30 rain thrtugh a 0-409/ glycerol gradient for swelling prior to ly~,s. The pellets were resuspended in a 1 2 ml solution of 250 mM sucrose/ 10 mM Tns (pH 7.4) and lysed with an Artek Model 300 sonicator with a micro-tip for 5 ~ with 105 W (3556 power). The vol. was increased 3-fold with cold 250 mM sucrose/10 mM Tris (pH 7 4) followed by cenlrifugation at 3000 x g for 10 rain tu pellet the platelet membrane and other large debris. The supernatant was decanted and centrifuged at 12000 × g for 20 rain, yielding a crude mitochondrial pellet (:he pellet also contains platelet granules, 28).
Preparation of platelet mit~wkondrlal extract The mitochondrial fraction was re'suspended with 100 ul of a 300 mM sucrose, 12.5 mM Tris (pH 7.4), 1
mM t ' D I A ,,oluinm: the [inal v¢l. of e:t~_h su,pcn.,ion var!¢d from 300-35{; y I 1he ~o!. of each s~mlpte ~a, increased 1o 500 yl `sith buffer .\. Sa-~ple,, "acre lx,ed b', the addition of 01'i m,nidet P-~,,, 21,) mM NaCI and a protcinase inhibitor cc,,zktail containing 1 /*M leupeptin. 1 aM pepstatin and 2~1 'aM phenylmethylsulfonyl fluoride [PMSFL follo`sed by incubation on ice for 30 min. The mttochondrial membrane and other dcbris were pelleted by centrifugation at 140(X)(I ~ ~ for l h at 4°C. The supernatant v.as remo;ed, assaxcd [o~ protein by the method of Lowry [29]. divided into 50 #1 aliqums and stored at 70~C.
Ammonium sulfate mi~rofracrionation Solid ammonium sulfate was added to a sample of crude mitochondrial extract (120 ~t]) to 55~ ,,aturation (0°CL Following 20 rain of mixing at 0°C. the precipitate '.,.as pelleted b', centrifugation and resuspended ",~,ith 120 #1 of a 10'~ glycerol. 1 ~ mM ~[ri~.. 2 mM DTT solution (pH 7.6. buffer BL The supernatant `sas cdletted and solid ammonium suffate v,as added to 80'7 ~,,aluration. Following mixing and ccntrifugatiol~, the pellet `sa, resuspended as above. The 0-55~. 55-80"; and > 80~ fractions were dialyzed again,,t buffer B. assayed for protein, a d stored at - 7 0 ° C
Topoisomerase aasav Samples containing 25-125 'ag of mitt~hondrial protein :',ere assayed in a solution of 130 mM NaCL 0.5 'ag pBR322, plus buffer B {pH 7.6) to a final ,.ol. of 40-100 #h Samples `'.ere assayed for 30-60 rain at 30°C, extracted once with saturated phenol, once `sith phenol/chloroform isoamyl alcohol {('IAAI and once v,ith CIAA. frozen, lyophilized completely and resuspended in 15 #1 of a 10 mM Yri~ (pH 7.6)/1 mM EDTA solution prior to agarose gel electrophoresis. Samples ,sere analyzed on 11 × 14 cm agarose gels {O.gg), which were run for 16-18 h at 17 19 mA. Gels were stained ,sith elhidium bromide I2 'ag,'ml) for 30 rain and subsequently photographed on Polaroid type 55 film `sith the Fotcedyne Polaroid camera, while illuminated by an ultra-violet light box.
Data anah'sts Photographic nc~atise~, 'acre analyzed b_', scanning densitometry using an E-C densitometer interfaced with an Apple lle computer. The results were quantitated from graphical output generated by the Videophoresis II.PC program {Biom~ Instrument.,,). where band intensity was represented by the area under the corresponding peak. 1-he basal levels of the substrate (pBR322) .~upercoiled and completely relaxed forms were determined by analyzing mock treated pBR322 samples which were carried throu~ the topoisomerase assay, extractions and subsequent preparation. The determination of v6 relaxation of supercoiled DNA was
5g
based ~m the percent conversion of the ~upercoiled form to relaxed topoisomer forms, where q telaxatam- (l (fraction of total DNA in supercoiled form:,,~ 90% inhibited by 175 ,aM EGTA (this concentration of EGTA was chosen by extrapolating the EGTA titration curve in Fig. 2a to 0 units of activity), followed by the addition of 25 ,aM-1 mM Ca > (Fig, 2b). MtTOPI activity was partially restored to approx. 16 and 50g~ ~ the base-line level (activity in the absence of EGTA and Ca-'*) ~ith 50 and 100 .aM Ca:*, respectively. The addition of up to 1 mM Ca -`+ did not restore activity above the level achieved with 100 #M Ca > . When this experiment was repeated with a different mitochondrial extract preparation, qualitatively identical results were obtained (data not shown). Additionally, Mg > was unable to restore the activity of EGTA inhibited enzyme (data not shown).
59 1.2
A
I
1
2
3 4
5
6
7
I.(I
0.8
0.6
0.4
0.2
0 0
20
40
60
80
100
120
140
160
180
EGTA (,~M) 1"0 I B 0.8 it
>, ~ 0
0.6 o.4
02 c.0 [EGTAII~M)
0
175
175
175
[©al¢lum]laM) 0
0
25
50
'TS
175
1(]0 1000
Fig 2. Partial rest.arati(]n()r EGTA-inhihited platelet mtTOPI activll3 b; Ca: ", lepoisomera~.¢ relaxation actt~ll':, v.a~ a~,a'~¢dJ~ dc,cribed ~t* Material:, and Methods. Both assays were carried out for 60 rain al 30°C. One nnil - lhe amounl of topnl,.orqer,t~eacti,.kt'~required t, relax 5/1'~ or the supercoiled substrate in 60 rain at 30°C. IA) Lanes 1 7 cont;li;. 0.5 ~Lt. pBR?22 lhat ~as in~,-ub,~tedwith 60 ug of protein fr~)l':.tthe mitochondfial extract of 6 day-old ptatelets, with ihe following addition~: lane t. no addilion: lane',.2 ". 25.50. 75 l~ll t2~ and I:'q ~.t ~!~]| '~. respecti','ely,Follo'.,,ingan analysis of the photographic negatt~.eby scanning de, sttomelr), the ,data ~a~ ~tati~tlcall~.an~13zcdbT. the iea~l-~quarc~ method ~nd represented graphically. {B) All of the ~amples cnntalned 0.5 ,ug pBR322 that s',as i:lctl'naled v,nh 61) ttg nf proleir: fn*rz~the mitochondr:al extract u~ed for part A of this figure, plu~,the ~ddition~iod~catedFck,,..,each t:olumn.
In a ,,separate experiment, samples t~i" e~".tract ':,ere pre-incubated in the presence of 200/~M EGTA (~hich inhibits > 90%). followed by the removal of EGTA and EGTA-Ca"- by dialysis against buffer B. A conuo; sample not treated with EGTA was also dialyzed. Topoisomerase activity remained inhibited by approx. 60% following dialysis, while no toss of activity ,,,.as observed in the control sample (data not shown). This supports the conclusion that the specific removal of Ca" + from the extract and not EGTA itself, is responsible for the inhibition. The partial restoration of activity observed with the dialyzed EGTA sample (60% compared to > 90% inhibition without dialysis) probably reflects the reassociation of Ca z+ with the enzyme, since Ca :* is a significant contaminant in the dialysis buffer water and components.
lnhihit~r studtes Table I lists inhibitors used to fu,ther characterize the lopoisomerase activLtx present in plalelet uritochonc.d,(, ,.:ude extract. ('amptothecin is a cvtotoxic plant alkaloid that specifically mhtbtts nuclear [ O P l [33,34]. We report for the first time the inhibition of a mtTOP by this drug. Topoisomerase activity v, as inhibited 15 and 31% b 5 10 and 100 'aM camptotheciu, respectively. Th, • inhibitor 5' effects of berenil, ethidium bromide and DMSO (acti,.ity was inhibited 62, IlY~Iand > 46% by 50 ,aM, 1 ,aM and 20g. respectively) were found to bc consistent with previous work [18,20,21}. Novobiocin is a TOP2 inhibitor that has been shov.n to inhibit calf thymus mtTOPl by 75q: at 300 ,uM [20]. yet did not affect human platelet mtTOP1 at concentrations up to 500 t~M.
6O l XB1 I [ I./tl ; , , m:;lhiP,,~ u,2 ,'~,[,,!l,lntt'r~.~' l ~. ti~,s p~,,: h~m:lltl Fl,:le/l't in
l hi, d,ila ~,a- dcri~cd from etperul.enT, ulilizing crude mitochm]drial c'.lra~t [creels, inhibit!on eq0a[~ the percen', decrease ,n the o,n~er,ion ~1 ,uper¢ofled subxlrate to relaxed Iopiiisolnel iolins, relatl~ e to the amount l*f t~pol~onlcra~e acli~it} exhibited in the absence of inhihitor ~hc ~h,.er,.ed mhihilmn ,aith DMSO ' rcflecp, a minimum ~,;lltle, ~lrlce high concentrations of DMSO in the presence of topoi~onlcrasc ]la~c been sho~n to conxert ~upercoiled DNA I~ nicked
.= >. m I,,,-
O
cirOesIlS] Inhibitor l)rug~: campl~thccin
Concentration
1O 'a %1 lf~lll~.l
bcrcnil
DMSO m,~ o]'iocin
] 5 #M Y~ ,a M II 5 tim l.O ,aNt 205 (~,i'.) 500 p~,|
(al kin ehc!alorv [GI % klYl A TPEN
I(~d **M 3 mM hi mM
elhidium hri/mid¢
Inhibition
15 3] 25 62 36 Io0 .> 46 0
0 1
2
3
4
5
Fig 3 Partially purified TOPI activity from human platelet mitochondria, Topoimmerascrelaxation activitywas assayedas described in Materials and Meth~uls.This assay was carried nut for 60 min at 30°C One Lnit -the amounl of topoisomerascactivity required In relax 50ff of the supercoiledsubstrate in 60 rain at 30°C. Columns 1-5 repr Fe z. > Mti2 ~>> Ca2" > Mg:-. TPEN did not affect plateiel mtTOP1 activi:y at concentrations up to 10 m M FL.rthermore, Iopoisomerase activity was not affected by 1,he addition of Zn z+ or Mn2" to platelet mitochondrial extract Idata nol shown).
Partial punfwafiorJ of IDPI flora human plateh't mito~hondria Crude mm~chondrial extract was ftaclionated by ammonium sulfate precipitation. The 55-80,e{- saturated fraction coatained approx. 50% of the total topoisomerasc activity. This fraction was purified 3.6-fold relative to the crude extract (estimate with respect to whole platelets is 450-fold) and was devoid of approx. 87~,: of the total mitochondrial extract protein. The small amount of protein isolated by this microfraetionation was utilized for the experiment shown in Fig. 3. While mtTOP1 activity was unaffected by the addition of 1 mM Mg"+ (column 2), activity was stimulated 1.6-fold by 1 mM Ca:' (column 3). MtTOP1 activity
Discussion
The study of mtTOP is formidable for the following reasons: (I) perturbations associated with cell fractionation techniques may cause the release of contaminating protein from the nucleus, which contains an estimated 10s-10 ~ total molecules of topoisomerase [35-37] and (2) the mitochondrion is a very limited source of topoisomerase, containing approx. 1,~ of the total topoisomerase activity in Xenopus laevis oocytes [21] and human acute leukemia [25] cells. While previous work with nucleated cells has involved the skillful preparation and isolation of intact mitoplasts from digitonin-treated mitochondria [18-21,24,25], it is extremely difficult to rule out the possibility of reassociation of nuclear topoisomerase wi:h mitoplasts during the digitonin treatment. We have circumvented the possibility of nuclear contamination by working with anucleated hualan blood platelets. During the final stage of megakaryocyte maturation, the cytoplasm fragments into 2000-3000 platelets [38,391. Thus, the production of platelets, in vivo, naturally segregates mitochondria from the megakaryocyte nucleus. The platelet cytoplasmic fraction exhibited no lopoisomerase activity (data not shown). This supports the conclusion that platelet mtTOPI is free of nuclear TOPI that might have been present in the cytosol during fragmentation of the megakaryocyle.
61 Our studies have demonstrated that human platelel mitochondria contain type 1 topoison, era~e. The t)pe I classification is supported by the lack of an ATP requirement, the inhibition of activity by ATP or camptolhecin and the lack of inhibition by Novobioein. Since topoisomerase assay buffers typically contain the nonspecific cation chelator, EDTA [17.30]. il was intriguing 1o disco'~er that the presence of this agent in our assay buffer resulted in a decreased level of mtTOP activity (see Table i). Tins observation led to an analysis of ptatelet mITOP activity in the presence of various cations and cation chelators. EGTA completely inhibited mtTOP activity by the specific depletion of Ca ~* from the mitochondrial extract (Fig. 2a). Interestingly, platelet mtTOP exhibited a g,'eater sensitivity towards inhibition by EGTA, compared to EDTA. These results suggest that Ca-"+ may be critical for enzymatic activity, in vivo. While Ca 2. and Mg"+ have been shown to substitute for each other to stimulate both mitochondrial [201 and nuclear [40] TOPI activity, platelet mtTOPI appears to respond uniquely to the presence of these cations. EGTA-inhibited mtTOPl activity was parlially restored by Ca -`+ (Fig. 2b), but not Mg :*. The failure to fully restore activity suggests that EGTA inhibits mtTOPl by extracting Ca -'* from the enzyme, which may adversely affect the protein conformation. Dialysis studies demo:tstrate that EGTA itself does not inhibit the enzyme, The altered enzyme conformation may bind Ca-'" less efficiently, resulting in sub-optimal activity. The importance of the enzyme conformation is also implied by the inhibitory effect of ,8-mercaptoethanol (Fig. 3). Partially purified platelet mtTOPl was stimulated by Ca-" '. but not Mg ~'~ (Fig. 3). Additionally, mtTOP1 activity was unaffected by TPEN. which pref~+ erentially chelates Zn- , Fe '-+ and Mn '~ . These results support the hypothesis that Ca ,+ is specifically required for platelet mtTOP1 activity. While our studies suggest that mtTOPl is biochemically distinct from nuclear TOP1, this point can only be rigorc,usly proven by an analysis of highly purified enzyme. Furthermore, we have shown for the first time that camptothecin inhibits a mtTOP. If mtTOP1 proves to be truly distinct from nuclear TOP1, it may be an effective target in tumor chemotherapy. The inabilily to detect TOP2 activity in platelet mito.chondrial extracts suggests that this protein may not be present in mitochondria. The putative lack of TOP2 in mitochondria may be due to the following possibilities: (1) the amount of TOP2 present is too low for detection by the re'axation assay, yet sufficient to carry out functional requirements: (2) terminal cells lack TOP2, which may contribute to cell senescence: or (3) the !ack of mtTOP2 may be characteristic of all eukaryotic cell types; therefore, previous reports of mrTOP2 activity may be due to the presence of nuclear TOP2. If mitochondria do not contain TOP2. the
topoi~,omerase and toptfisomera,,e-like acto,'ityt~,) required during mtDNA replication and transcription may be accomplished by TOPI in conjunction v,ith other mitochondrial protein(s). An adequate understauding of the role of TOP1 within the mitoehondrion await.,, the functiomd analysis of this protein. The characlerizalion of a mitochondrlal prolem usually begins with its purification from a large amount of starting material (e.g., > 1 kg of tissue). Unfortunateb. the limited quantity and unpredictable availabilily of human platelets necessitated an alternative approach. Therefore. the initial phase of this study has involved the use of crude mitoehondrial extracts and a partially purified mitochondrial fraction isolated by ammonium sulfate pre~,~pitation. The pamally purified fraction, ~,,hich was devoid of approx. 87~ of the mitechondrial exmJet protein, exhibited topoisomerase activity that responded comparably with the crude extract aclivily to stimulator and inhibitory agents (Fig. 3). This data supports the conclusion that the mitochondrial extract TOPI activity does not involve other extract protein(s). We are currently addressing this point and the proposed Ca'-" requirement by the further purification and charucterization of this eezyme. References 1 Vosberg.H-P. (1985)Curr. Top. MicmNol.ImmunoL114.19-10L 2 Wang.J.C. (1985) Annu Roy.Biochem.54, 665-697. 3 Wang.J.C. (1987) Biochim.Biophys.Acta 909. 1-9. 4 Brill, S.J.. DiNardo. S.. VoelkeI-Meiman.K. and Sleruglaruz, R. (1987) Nature 326. 414416. 5 Champoux.JJ. (1988)J Virol 62, 3675-3683. 5 Zhang,H., Wang.J.C. and Liu. t,F. (i988) Pr~. Natl. A~:ad.K'i U.S.A. 85. 1060 lu64. 7 Stewart, A.F.. i~errera, R.E and Nordhcim. A [ltlgO) Cell 60.141-149. g Chnstman. M.F.. Dietrich. F.S. ~nd Fink, GR (1988) Cell 55. 413-425. 9 Yang, L, Wold. M.S.. Li. J,J.. Kelly. TJ and Liu. L.F. (19871 Proc, Nad, Acad. Sci. USA 84. 950 954. 10 Ro:,e. D.. Thomas.W. and Holm.C. 0990) Cell 61),1009-11H7. 11 Vemura,T., Ohkura. H., Adachi.Y.. Monno, K.. Shiozaki.K. and Ya.~aglda, M. (1987)Cell 50. 917-925. 12 Ander~',n. S., Bankier. A.T, Barrdl, BG.. De Brmjn, MH.L, Coulson, A.R. Drovin,J.. Eperon. I C. Nierlich. DP, ltt~. B~t.. Sanger. F. S,,:hreier, P H., Smith. A.J.H. Staden. R and Young, I.G. 11981)Nature 290. 457 465. 13 Bibb, M.J.. Van Etten. R.A.. Wright, C.'I.. Walbet-g. M.W. and Clayton, DA. (19811Cell 2¢J.167-180 14 Cla}ton, D.A. (1982) Cell 28, 693-705, 15 Aloni. Y. and Anardi. G. (1971) Prt',,c.Natl, Acad. S~i. USA 68. 1757-1"/61. 16 Murphy, W.. AUardi. G., Tu. L. and Anardi, G. 119751J. Mol. Biol. 99, 809=814. 17 Fairfield. F.R.. Bauer. WR. and Simp,,,on.M.V. (19791 J. Biol. Chem. 254. 9352-9354. 18 Fairfield. F.R.. Bauer. W.R. and Simpson,M.V. (1985) Biochim. Binph}~.Acta 824, 45-57. 19 Ca~,tora, F.J. and Lazarus. GM. (1084) Biachem Biophys Res. Ct~mmun.121.77-86.
62 2i) [.,~,'i!rtr., G.M. Henrich. J.P.. Kcll',, W G . Schmit,'. and (~,tt!ra. } J i19k7) Biochcm~qr) 26. 619§. 620~ 21 Brun, ( i Vunnler P. Sc,+:a,~l, I .rod (allen..t( (1!181~Eur J Bluchcm llS. 4ii" zlS. 22 (';l~lilra 1: J ;Ind Simp~,tm. 3.1V. (197t;1 J. Biol (2hem. 254 11193 11195. 23 ( a',l~ra. F.J., Vis>ering, F.F. and Simpson. M.V 11983) Bx~chim Bioph3s Acla 7411.417 42"L 24 (a~tora. F J, Sternglani'. R and Simps/!n, M.V 11982) in Miu',chondrial Ger.es tSl.:mimski, P.. Borst. P and Altardi. G , edsh pp. 143 154, Cold Spring Harbur Lniver~ity Pre,~, Cold Spring Harbor, New York. 25 Ca~tora, FJ., Lazarus. G.M. and Kunes, D. (19851 Biochem Biophys. Re~. (ommun. 130, 854-866. 26 Maniati,,. T., Friisch. E.F. and Sambrook, J. (1982j Molecular Cloning: A Laboratory Manual, Cold Spring Harbor University Press. Cold Spring Harbor. Ne~ York. 27 Soslau G. and Giles. J ~19821Thrt~mbc~si~ Res 26. 443+455. 28 Soslau. G. 119821 Arch. Biochem Biophys. 215. 532-538 29 Lov,r~,. O.H, Ro~ebrough. N.J.. Farr. A.L. and Randall. R.J. (19511 J Biol. Chem 193. 265-275
31! (a,tora, FJ. and Kelly. 9.+G. d986,1 Prt~. Nalh Acad. Sci USA 83. 1681J-16~4. 3', Liu. L F . [.iu. C-C. aml Alberts. R M 119g0) ('ell 19. 697 7U7. .;2 Schmid. R.W and Reillev, C.N (19571 Anal. Chem. 29. 264-268. 33 tl~iang. Y-H., llertzberg. R. itecht, S, and Liu, L. (19851 J, Biol. Chem. 260. 14873 14878. 34 Nitiss. V. and Wang. J.C 0988) Proc. Nail Acad. Sci. USA 85. 7501 7505. 35 Boocock. MR., Brown, J.L. and Sherratt. D.J. 119851 Biochem. Soc. Trans. 14. 214-217 36 Champoux. J.J. and Dulbecco, R. (19721 Pr.~. Natl. Acad. Sci. USA 69, 143-146. 37 Miller, KG., Liu. LF.. Englund, P.T. O9811 J. Biol. Chem. 256+ 9334-9339. 38 Wright. J.H (1910) J. MorpboL 21. 263-278. 39 Chernoff. A., Levine, R.F. and G(xx:lman. DewS. 119801 J. Clin. Invest. 65, 926-930, 40 (Join. ].. Laipi~. P. and Wang, .IC (19841 J. Biol. Chem 259. 10422-10429.
56
BB~PR( ) 31908
Mitochondrial DNA topoisomerase I from human platelets Marshall J. K o s o v s k y ! and G e r a l d Soslau 1.2 'r
Department of Btologtcal ChemistO, Hahnemann Unicerst(v, Philadelphia. PA ( U,S A ) and : Department o]Neoplostie Diseases, Hahnemunn Unit,~.r~tt3. Phdadegphia, PA (US.A.) (Recei,~ed 15 October 1990) [ Revised manuscript received 27 December 1qg())
Key words: DNA topmsomerase l: M~locnondrion; Human platelet: Camptcrthecin
An anucleated cell system has been used for the first time to study mitochondrial topoisomerase aetMty. Mitochondrial extracts from human blood platclets contained ty.pe I topoisomerase. The type I classification was based on ATP-iodependent activity, inhibition by ATP or camptnthecin, and the lack of inhibition by novobiocin. Platelet mitochondrial topoisomemse I relaxation activi~' was inhibited linearly by increasing concentrations of EGTA. Topoisomerase activity > 90% inhibited by 175 pM EGTA was partially restored to 16 and 50% of the initial level of aetivi~" by the subsequent additien of 50 and 100 pM Caz+, respectively. Additionally, results from studies of partially purified platelet mitochondrial topoisomera~ 1 were consistent with the crude extract data. This work supports the hypothesis that platelet mitochandria contain a type ! topoisomerase that is biochemically distinct from that previously isolated ,and characterized from cell nuclei, Introduction DNA topoisomerases regulate the topological state of DNA by catalyzing the cleavage, strand passage, and rejoining of either a single-strand (topoisomerase 1 (TOPI)) or double-strand (topoisomerase II (TOP2)) of the DNA molecule (see Refs. 1-3 for reviews). TOPI functions in an ATP-independent manner, and has been implicated in DNA metabolic processes such as replication [4,5], transcription [6,7], and recombination [8]. TOP2 catalyzes ATP-dependent reactions which are required for the segre~.ation of DNA replication products in simian vim+ 40 [9] and are required during yeast meiosis [10] and mitosis [11]. While nuclear topoisomerases from eukaryotes have been well characterized, little is known about mitochondrial topoisolnerases.
Mammalian mitochondria contain a closed circular double-stranded DNA genome comprised of approx. 16 500 base pairs [1233]. As studies revealed the mechanisms of mitochondrial DNA (mtDNA) 'displacement-
Abbre~ation:. m*.TOP, milochondna] topoisomerase; DMSO, dimethyl sulfoxide; TPEN. N,N,N'.N'.tetrakis.(2.pyridylmethyb.cthytenedianune. Correspondence: G. Soslau, Departments of Biolo'ocal Chetmstry and NeopLastic Diseases. Hahnemann Universily, Broad and Vine Streets. Philadelphia. PA 19102. U.S.A.
loop' replication [141 and transcription [15,16], the search for mitochondrial topoisomerase (mtTOP) activity commenced. The first reported mtTOP was isolated from rat liver [17], and was later shown to exhibit ATP-independent type I activity 118]. Additionally. mtTOP1 has been isolated from hnman acute leukemia cells [19], calf thymus [20] and Xenopus laet,is oocytes [211. Studies have also provided indirect evidence for the presence of mtTOP2 in rat liver 12223], as well as direct evidence for type il activity in the milochondria of rat liver [24] and human acute leukemia cells [25]. The characterizations of the above mitochondrial topoisomerases indicate considerable variability with respect to characteristics, such as molecular weight, the response to metabolites and drug effects. The observed dissimilaritie, may reflect the difficulty in obtaining well purified mitochondria from nucleated cells or that eukaD'otic mitochondrial topoisomerases exhibit species-specific properties. We report for the first time studies of mtTOP derived from an anuclear cell, the human blood platelet. The use of this novel system has enabled the isolation of a mitochondrial fraction that is devoid of nuclear contamination. Crude mitochondrial extracts have been shown to contain a camptothecin-sensitive type I topoisomerase. Onr studies of these extracts suggest that platelet mtTOP1 may require calcium (Ca 2+) for enzymatic activity. Furthermore, the enzymatic activity of partially purified human platelet mtTOP1 and the
57 crude extract activity were qualitatixel3 idcnth:al `shen exposed to stimulatory and inhibitory agents.
Materials and Methods
Matermls Non-injectab,e. 2-7-day-old human platelet concentrates were obtained from the Pennsylvania-New Jersey Red Cros:;. The plasmid pBR322 served as substrate DNA for all topoisomerase assays, and ',','as prepared according to the alkaline lysis procedure of Maniatis et al. [261. TPEN was purchased from Calbiochem and was dissolved in DMSO and stored at - 2 0 ° C . Topoisomerase inhibitors were prepared as follows: Camptothecin was dissolved in DMSO and stored at --20°C; Berenil and novobkv.:in ,,,'ere dissolved in 1056 glycerol, 100 mM Tris, 2 mM Na,EDTA, 2 mM dithiothriotol (DTT) and 0.15; Triton X-100. pH 7.6 {buffer A) and storm at - 2 0 ° C . Unless otherwise indicated, all chemicals were obtained from Sigma. I n optimize contrast in the gel photographs, reverse images were prepared by utilizing the original photographic negatives for contact printing on Kodak electrophoresis duplicating paper.
Purification of human platelets Platelet concentrates (derived from 4-10 individuals) were pooled, resulting in a heterogeneous mixture of cells. The platelets were centrifuged at 650 × g for 15 min. The plasma was decanted and the platelet pellets were collected and pooled. The platelets were layered over 25 3556 sucrose gradients, centrifuged at 650 × g for 30 rain and the upper 2/3 of each gradient was collected. The purified platelets v, ere examined by light microscopy to confirm the homogeneity of each preparation; each field of view. containing approx. 50000 platelets, was devoid of while blood cells [27] All subsequent steps were carried out at 4 ° C. The platelcts were pelleted at 8000 × g for 10 rain. resuspended in a small vol. of 150 mM sodium chloride (NaCI), 20 mM Tris (pH 7.4) (Iris-buffered saline (TBS))/0.3856 stxiium citrate and pelleted at 6000 × g for 30 rain thrtugh a 0-409/ glycerol gradient for swelling prior to ly~,s. The pellets were resuspended in a 1 2 ml solution of 250 mM sucrose/ 10 mM Tns (pH 7.4) and lysed with an Artek Model 300 sonicator with a micro-tip for 5 ~ with 105 W (3556 power). The vol. was increased 3-fold with cold 250 mM sucrose/10 mM Tris (pH 7 4) followed by cenlrifugation at 3000 x g for 10 rain tu pellet the platelet membrane and other large debris. The supernatant was decanted and centrifuged at 12000 × g for 20 rain, yielding a crude mitochondrial pellet (:he pellet also contains platelet granules, 28).
Preparation of platelet mit~wkondrlal extract The mitochondrial fraction was re'suspended with 100 ul of a 300 mM sucrose, 12.5 mM Tris (pH 7.4), 1
mM t ' D I A ,,oluinm: the [inal v¢l. of e:t~_h su,pcn.,ion var!¢d from 300-35{; y I 1he ~o!. of each s~mlpte ~a, increased 1o 500 yl `sith buffer .\. Sa-~ple,, "acre lx,ed b', the addition of 01'i m,nidet P-~,,, 21,) mM NaCI and a protcinase inhibitor cc,,zktail containing 1 /*M leupeptin. 1 aM pepstatin and 2~1 'aM phenylmethylsulfonyl fluoride [PMSFL follo`sed by incubation on ice for 30 min. The mttochondrial membrane and other dcbris were pelleted by centrifugation at 140(X)(I ~ ~ for l h at 4°C. The supernatant v.as remo;ed, assaxcd [o~ protein by the method of Lowry [29]. divided into 50 #1 aliqums and stored at 70~C.
Ammonium sulfate mi~rofracrionation Solid ammonium sulfate was added to a sample of crude mitochondrial extract (120 ~t]) to 55~ ,,aturation (0°CL Following 20 rain of mixing at 0°C. the precipitate '.,.as pelleted b', centrifugation and resuspended ",~,ith 120 #1 of a 10'~ glycerol. 1 ~ mM ~[ri~.. 2 mM DTT solution (pH 7.6. buffer BL The supernatant `sas cdletted and solid ammonium suffate v,as added to 80'7 ~,,aluration. Following mixing and ccntrifugatiol~, the pellet `sa, resuspended as above. The 0-55~. 55-80"; and > 80~ fractions were dialyzed again,,t buffer B. assayed for protein, a d stored at - 7 0 ° C
Topoisomerase aasav Samples containing 25-125 'ag of mitt~hondrial protein :',ere assayed in a solution of 130 mM NaCL 0.5 'ag pBR322, plus buffer B {pH 7.6) to a final ,.ol. of 40-100 #h Samples `'.ere assayed for 30-60 rain at 30°C, extracted once with saturated phenol, once `sith phenol/chloroform isoamyl alcohol {('IAAI and once v,ith CIAA. frozen, lyophilized completely and resuspended in 15 #1 of a 10 mM Yri~ (pH 7.6)/1 mM EDTA solution prior to agarose gel electrophoresis. Samples ,sere analyzed on 11 × 14 cm agarose gels {O.gg), which were run for 16-18 h at 17 19 mA. Gels were stained ,sith elhidium bromide I2 'ag,'ml) for 30 rain and subsequently photographed on Polaroid type 55 film `sith the Fotcedyne Polaroid camera, while illuminated by an ultra-violet light box.
Data anah'sts Photographic nc~atise~, 'acre analyzed b_', scanning densitometry using an E-C densitometer interfaced with an Apple lle computer. The results were quantitated from graphical output generated by the Videophoresis II.PC program {Biom~ Instrument.,,). where band intensity was represented by the area under the corresponding peak. 1-he basal levels of the substrate (pBR322) .~upercoiled and completely relaxed forms were determined by analyzing mock treated pBR322 samples which were carried throu~ the topoisomerase assay, extractions and subsequent preparation. The determination of v6 relaxation of supercoiled DNA was
5g
based ~m the percent conversion of the ~upercoiled form to relaxed topoisomer forms, where q telaxatam- (l (fraction of total DNA in supercoiled form:,,~ 90% inhibited by 175 ,aM EGTA (this concentration of EGTA was chosen by extrapolating the EGTA titration curve in Fig. 2a to 0 units of activity), followed by the addition of 25 ,aM-1 mM Ca > (Fig, 2b). MtTOPI activity was partially restored to approx. 16 and 50g~ ~ the base-line level (activity in the absence of EGTA and Ca-'*) ~ith 50 and 100 .aM Ca:*, respectively. The addition of up to 1 mM Ca -`+ did not restore activity above the level achieved with 100 #M Ca > . When this experiment was repeated with a different mitochondrial extract preparation, qualitatively identical results were obtained (data not shown). Additionally, Mg > was unable to restore the activity of EGTA inhibited enzyme (data not shown).
59 1.2
A
I
1
2
3 4
5
6
7
I.(I
0.8
0.6
0.4
0.2
0 0
20
40
60
80
100
120
140
160
180
EGTA (,~M) 1"0 I B 0.8 it
>, ~ 0
0.6 o.4
02 c.0 [EGTAII~M)
0
175
175
175
[©al¢lum]laM) 0
0
25
50
'TS
175
1(]0 1000
Fig 2. Partial rest.arati(]n()r EGTA-inhihited platelet mtTOPI activll3 b; Ca: ", lepoisomera~.¢ relaxation actt~ll':, v.a~ a~,a'~¢dJ~ dc,cribed ~t* Material:, and Methods. Both assays were carried out for 60 rain al 30°C. One nnil - lhe amounl of topnl,.orqer,t~eacti,.kt'~required t, relax 5/1'~ or the supercoiled substrate in 60 rain at 30°C. IA) Lanes 1 7 cont;li;. 0.5 ~Lt. pBR?22 lhat ~as in~,-ub,~tedwith 60 ug of protein fr~)l':.tthe mitochondfial extract of 6 day-old ptatelets, with ihe following addition~: lane t. no addilion: lane',.2 ". 25.50. 75 l~ll t2~ and I:'q ~.t ~!~]| '~. respecti','ely,Follo'.,,ingan analysis of the photographic negatt~.eby scanning de, sttomelr), the ,data ~a~ ~tati~tlcall~.an~13zcdbT. the iea~l-~quarc~ method ~nd represented graphically. {B) All of the ~amples cnntalned 0.5 ,ug pBR322 that s',as i:lctl'naled v,nh 61) ttg nf proleir: fn*rz~the mitochondr:al extract u~ed for part A of this figure, plu~,the ~ddition~iod~catedFck,,..,each t:olumn.
In a ,,separate experiment, samples t~i" e~".tract ':,ere pre-incubated in the presence of 200/~M EGTA (~hich inhibits > 90%). followed by the removal of EGTA and EGTA-Ca"- by dialysis against buffer B. A conuo; sample not treated with EGTA was also dialyzed. Topoisomerase activity remained inhibited by approx. 60% following dialysis, while no toss of activity ,,,.as observed in the control sample (data not shown). This supports the conclusion that the specific removal of Ca" + from the extract and not EGTA itself, is responsible for the inhibition. The partial restoration of activity observed with the dialyzed EGTA sample (60% compared to > 90% inhibition without dialysis) probably reflects the reassociation of Ca z+ with the enzyme, since Ca :* is a significant contaminant in the dialysis buffer water and components.
lnhihit~r studtes Table I lists inhibitors used to fu,ther characterize the lopoisomerase activLtx present in plalelet uritochonc.d,(, ,.:ude extract. ('amptothecin is a cvtotoxic plant alkaloid that specifically mhtbtts nuclear [ O P l [33,34]. We report for the first time the inhibition of a mtTOP by this drug. Topoisomerase activity v, as inhibited 15 and 31% b 5 10 and 100 'aM camptotheciu, respectively. Th, • inhibitor 5' effects of berenil, ethidium bromide and DMSO (acti,.ity was inhibited 62, IlY~Iand > 46% by 50 ,aM, 1 ,aM and 20g. respectively) were found to bc consistent with previous work [18,20,21}. Novobiocin is a TOP2 inhibitor that has been shov.n to inhibit calf thymus mtTOPl by 75q: at 300 ,uM [20]. yet did not affect human platelet mtTOP1 at concentrations up to 500 t~M.
6O l XB1 I [ I./tl ; , , m:;lhiP,,~ u,2 ,'~,[,,!l,lntt'r~.~' l ~. ti~,s p~,,: h~m:lltl Fl,:le/l't in
l hi, d,ila ~,a- dcri~cd from etperul.enT, ulilizing crude mitochm]drial c'.lra~t [creels, inhibit!on eq0a[~ the percen', decrease ,n the o,n~er,ion ~1 ,uper¢ofled subxlrate to relaxed Iopiiisolnel iolins, relatl~ e to the amount l*f t~pol~onlcra~e acli~it} exhibited in the absence of inhihitor ~hc ~h,.er,.ed mhihilmn ,aith DMSO ' rcflecp, a minimum ~,;lltle, ~lrlce high concentrations of DMSO in the presence of topoi~onlcrasc ]la~c been sho~n to conxert ~upercoiled DNA I~ nicked
.= >. m I,,,-
O
cirOesIlS] Inhibitor l)rug~: campl~thccin
Concentration
1O 'a %1 lf~lll~.l
bcrcnil
DMSO m,~ o]'iocin
] 5 #M Y~ ,a M II 5 tim l.O ,aNt 205 (~,i'.) 500 p~,|
(al kin ehc!alorv [GI % klYl A TPEN
I(~d **M 3 mM hi mM
elhidium hri/mid¢
Inhibition
15 3] 25 62 36 Io0 .> 46 0
0 1
2
3
4
5
Fig 3 Partially purified TOPI activity from human platelet mitochondria, Topoimmerascrelaxation activitywas assayedas described in Materials and Meth~uls.This assay was carried nut for 60 min at 30°C One Lnit -the amounl of topoisomerascactivity required In relax 50ff of the supercoiledsubstrate in 60 rain at 30°C. Columns 1-5 repr Fe z. > Mti2 ~>> Ca2" > Mg:-. TPEN did not affect plateiel mtTOP1 activi:y at concentrations up to 10 m M FL.rthermore, Iopoisomerase activity was not affected by 1,he addition of Zn z+ or Mn2" to platelet mitochondrial extract Idata nol shown).
Partial punfwafiorJ of IDPI flora human plateh't mito~hondria Crude mm~chondrial extract was ftaclionated by ammonium sulfate precipitation. The 55-80,e{- saturated fraction coatained approx. 50% of the total topoisomerasc activity. This fraction was purified 3.6-fold relative to the crude extract (estimate with respect to whole platelets is 450-fold) and was devoid of approx. 87~,: of the total mitochondrial extract protein. The small amount of protein isolated by this microfraetionation was utilized for the experiment shown in Fig. 3. While mtTOP1 activity was unaffected by the addition of 1 mM Mg"+ (column 2), activity was stimulated 1.6-fold by 1 mM Ca:' (column 3). MtTOP1 activity
Discussion
The study of mtTOP is formidable for the following reasons: (I) perturbations associated with cell fractionation techniques may cause the release of contaminating protein from the nucleus, which contains an estimated 10s-10 ~ total molecules of topoisomerase [35-37] and (2) the mitochondrion is a very limited source of topoisomerase, containing approx. 1,~ of the total topoisomerase activity in Xenopus laevis oocytes [21] and human acute leukemia [25] cells. While previous work with nucleated cells has involved the skillful preparation and isolation of intact mitoplasts from digitonin-treated mitochondria [18-21,24,25], it is extremely difficult to rule out the possibility of reassociation of nuclear topoisomerase wi:h mitoplasts during the digitonin treatment. We have circumvented the possibility of nuclear contamination by working with anucleated hualan blood platelets. During the final stage of megakaryocyte maturation, the cytoplasm fragments into 2000-3000 platelets [38,391. Thus, the production of platelets, in vivo, naturally segregates mitochondria from the megakaryocyte nucleus. The platelet cytoplasmic fraction exhibited no lopoisomerase activity (data not shown). This supports the conclusion that platelet mtTOPI is free of nuclear TOPI that might have been present in the cytosol during fragmentation of the megakaryocyle.
61 Our studies have demonstrated that human platelel mitochondria contain type 1 topoison, era~e. The t)pe I classification is supported by the lack of an ATP requirement, the inhibition of activity by ATP or camptolhecin and the lack of inhibition by Novobioein. Since topoisomerase assay buffers typically contain the nonspecific cation chelator, EDTA [17.30]. il was intriguing 1o disco'~er that the presence of this agent in our assay buffer resulted in a decreased level of mtTOP activity (see Table i). Tins observation led to an analysis of ptatelet mITOP activity in the presence of various cations and cation chelators. EGTA completely inhibited mtTOP activity by the specific depletion of Ca ~* from the mitochondrial extract (Fig. 2a). Interestingly, platelet mtTOP exhibited a g,'eater sensitivity towards inhibition by EGTA, compared to EDTA. These results suggest that Ca-"+ may be critical for enzymatic activity, in vivo. While Ca 2. and Mg"+ have been shown to substitute for each other to stimulate both mitochondrial [201 and nuclear [40] TOPI activity, platelet mtTOPI appears to respond uniquely to the presence of these cations. EGTA-inhibited mtTOPl activity was parlially restored by Ca -`+ (Fig. 2b), but not Mg :*. The failure to fully restore activity suggests that EGTA inhibits mtTOPl by extracting Ca -'* from the enzyme, which may adversely affect the protein conformation. Dialysis studies demo:tstrate that EGTA itself does not inhibit the enzyme, The altered enzyme conformation may bind Ca-'" less efficiently, resulting in sub-optimal activity. The importance of the enzyme conformation is also implied by the inhibitory effect of ,8-mercaptoethanol (Fig. 3). Partially purified platelet mtTOPl was stimulated by Ca-" '. but not Mg ~'~ (Fig. 3). Additionally, mtTOP1 activity was unaffected by TPEN. which pref~+ erentially chelates Zn- , Fe '-+ and Mn '~ . These results support the hypothesis that Ca ,+ is specifically required for platelet mtTOP1 activity. While our studies suggest that mtTOPl is biochemically distinct from nuclear TOP1, this point can only be rigorc,usly proven by an analysis of highly purified enzyme. Furthermore, we have shown for the first time that camptothecin inhibits a mtTOP. If mtTOP1 proves to be truly distinct from nuclear TOP1, it may be an effective target in tumor chemotherapy. The inabilily to detect TOP2 activity in platelet mito.chondrial extracts suggests that this protein may not be present in mitochondria. The putative lack of TOP2 in mitochondria may be due to the following possibilities: (1) the amount of TOP2 present is too low for detection by the re'axation assay, yet sufficient to carry out functional requirements: (2) terminal cells lack TOP2, which may contribute to cell senescence: or (3) the !ack of mtTOP2 may be characteristic of all eukaryotic cell types; therefore, previous reports of mrTOP2 activity may be due to the presence of nuclear TOP2. If mitochondria do not contain TOP2. the
topoi~,omerase and toptfisomera,,e-like acto,'ityt~,) required during mtDNA replication and transcription may be accomplished by TOPI in conjunction v,ith other mitochondrial protein(s). An adequate understauding of the role of TOP1 within the mitoehondrion await.,, the functiomd analysis of this protein. The characlerizalion of a mitochondrlal prolem usually begins with its purification from a large amount of starting material (e.g., > 1 kg of tissue). Unfortunateb. the limited quantity and unpredictable availabilily of human platelets necessitated an alternative approach. Therefore. the initial phase of this study has involved the use of crude mitoehondrial extracts and a partially purified mitochondrial fraction isolated by ammonium sulfate pre~,~pitation. The pamally purified fraction, ~,,hich was devoid of approx. 87~ of the mitechondrial exmJet protein, exhibited topoisomerase activity that responded comparably with the crude extract aclivily to stimulator and inhibitory agents (Fig. 3). This data supports the conclusion that the mitochondrial extract TOPI activity does not involve other extract protein(s). We are currently addressing this point and the proposed Ca'-" requirement by the further purification and charucterization of this eezyme. References 1 Vosberg.H-P. (1985)Curr. Top. MicmNol.ImmunoL114.19-10L 2 Wang.J.C. (1985) Annu Roy.Biochem.54, 665-697. 3 Wang.J.C. (1987) Biochim.Biophys.Acta 909. 1-9. 4 Brill, S.J.. DiNardo. S.. VoelkeI-Meiman.K. and Sleruglaruz, R. (1987) Nature 326. 414416. 5 Champoux.JJ. (1988)J Virol 62, 3675-3683. 5 Zhang,H., Wang.J.C. and Liu. t,F. (i988) Pr~. Natl. A~:ad.K'i U.S.A. 85. 1060 lu64. 7 Stewart, A.F.. i~errera, R.E and Nordhcim. A [ltlgO) Cell 60.141-149. g Chnstman. M.F.. Dietrich. F.S. ~nd Fink, GR (1988) Cell 55. 413-425. 9 Yang, L, Wold. M.S.. Li. J,J.. Kelly. TJ and Liu. L.F. (19871 Proc, Nad, Acad. Sci. USA 84. 950 954. 10 Ro:,e. D.. Thomas.W. and Holm.C. 0990) Cell 61),1009-11H7. 11 Vemura,T., Ohkura. H., Adachi.Y.. Monno, K.. Shiozaki.K. and Ya.~aglda, M. (1987)Cell 50. 917-925. 12 Ander~',n. S., Bankier. A.T, Barrdl, BG.. De Brmjn, MH.L, Coulson, A.R. Drovin,J.. Eperon. I C. Nierlich. DP, ltt~. B~t.. Sanger. F. S,,:hreier, P H., Smith. A.J.H. Staden. R and Young, I.G. 11981)Nature 290. 457 465. 13 Bibb, M.J.. Van Etten. R.A.. Wright, C.'I.. Walbet-g. M.W. and Clayton, DA. (19811Cell 2¢J.167-180 14 Cla}ton, D.A. (1982) Cell 28, 693-705, 15 Aloni. Y. and Anardi. G. (1971) Prt',,c.Natl, Acad. S~i. USA 68. 1757-1"/61. 16 Murphy, W.. AUardi. G., Tu. L. and Anardi, G. 119751J. Mol. Biol. 99, 809=814. 17 Fairfield. F.R.. Bauer. WR. and Simp,,,on.M.V. (19791 J. Biol. Chem. 254. 9352-9354. 18 Fairfield. F.R.. Bauer. W.R. and Simpson,M.V. (1985) Biochim. Binph}~.Acta 824, 45-57. 19 Ca~,tora, F.J. and Lazarus. GM. (1084) Biachem Biophys Res. Ct~mmun.121.77-86.
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