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Isolation of a membrane-DNA-RNA complex from rat liver mitochondria
.ZRCHIVES
OF
BIOCHEMISTRY
Isolation
AND
RIOPHYSICS
of a Membrane-DNA-RNA Liver
GLENN Department
425-43-L (1972)
149,
C. VAN
TUYLE’
December
from
Rat
Mitochondria
of Biochemistry, Jeferson Philadelphia, lleceived
Complex
a4ixn
GEORGE
F. IidLF”
Medical College, Thomas Jeferson Pennsylvania, 19107
9, 1971; accepted
January
T’niversity,
13, 19i2
A membrane-DNA-RNA complex has been isolated from Sarkosyl lysates of inner membrane matrix preparations from normal and regenerating rat, liver mitochondria. Incubation of inner membrane matrix preparations with a radioactively labeled precursor in vitro under conditions which support nucleic acid and protein biosynthesis in mitochondria results in the labeling of the appropriate component of the complex. Only membrane fragments complex with the Mg2+-Sarkosyl crystals and DNA is found in the complex by virtue of its specific attachment to the membrane fragment. RNA, in turn, appears to be bound to the DNA. The percentage of the total DNA of the lysate which is attached to the membrane fragment in the Sarkosyl crystal is 3- to 4-fold higher in the complexes from 22.hr regenerating and fetal rat liver mitochondria than in normal rat liver, whereas the percentage of membrane protein is the same in both cases. The increased attachment of DNA to membrane in actively proliferating tissues suggests a possible functional significance for the attachment of the mitochondrial DNA to the membrane during the replication of mitochondrial DNB. The membrane-DNA-RNA complex from rat, liver mitochondria appears to resemble similar complexes isolated from several bacterial species by the same method.
The semiaut.onomous nature of mitochondria has been documented by t,he fact’s that, t#he organelle contains two to six copies of a 5~ circular DNA which arc found both free in t,hc matrix and bound to the inner membrane (1, 2), as well as its own unique DNA (3-5) and RNA polymerases (6%) bot#h of which appear to be membrane bound 10-12). 6, These fact,s toget,her with the growing body of evidence indi&ing a bacterialmitochondrial homology (13) and the resemblance of mitochondrial inner membrane-mat rix preparat’ions to bwt’erial
spheroplasts, led us to believe that a membrane-DNA-nascent RnTA complex might be isolated from mitochondria. Similar complexes have been isolated from several bacteria by lysis with t,he det,ergent Sarkosyl in the presence of Jig”+ (14) and T4-phage DNA-membrane complexes have been demor&rated in bacteriophage-infected cells (13). We report here on t’he isolation of a membrane-DNA-RNA complex from rat, liver mitochondria using t,he Sarkosyl technique.
1 These results were taken, in part, from t)he Ph.D. dissertation presented to the Graduate School, Jefferson Medical College of Thomas 1971. Present address : Jefferson University, Department of Biochemistry, State University of New York at Stony Brook, Long Island, New York. 2 To whom reprint, requests should be sent).
Experimental animals. Male Wistar rats (150170 g) were maintained on Purina Rat Chow and were fasted 16 hr prior to decapitation. Chemicals and reagen&. DNase (KC 3.1.4.5, hovine pancreas, free of RNase) and RNase (EC 2.7.7.16, bovine pancreas; stock solutions were heated 10 min at 90” to denature any DNase
M.4TERIALS
125
AND
METHODS
4%
VAN
TUYIZ
present) were purchased from Worthington Biochemical Corporation, Freehold, NJ. Pyruvate kinase (EC 2.7.1.40, rabbit musclej was obtained from Sigma Chemical Company, St. I,ouis, MO; and pronase (Sireptomyces qriseus proleasc, B grade) was purchased from Calbiochem, Los Angeles, C.4. Deoxyribonucleoside triphosphates, ribonucleoside triphosphates, calf thymus DNA, yeast bulk RNA, rat liver tltNA, L-leucine, PEPS, BS.4, and digitollin were obtained from Sigma Chemical Company. IXgitonin was recrystallized from absolute ethanol and ground to a fine powder to increase its sol\lhilit,y in water. Sarkosyl NL30 (sodium lnuroyl sarcosinate, 30fiA) was supplied by Geigy ludustrial Chemicals, Ardsley, NY. Calbiochem was the source of ethidium bromide; puromycin and cytochrome c were obtained from Nut,ritional Biochemicals, Cleveland, OII; and CAP was a gift from the Parke I)avis Company. Radioisotopes. Thymidine-[“HI-methyl 5’.triphosphate (9 Ci per mmole), dcoxy[“B]-:ldenosille 5’-triphosphate (11 .l Ci per mmole), [2-14C]thymine (28.8 mCi per mmole) and I,-[4,5-“II]leucine (5 Ci per mmolc) were purchased from New England Nrlclear Corporat.ion, Boston, MA. Thymidil,e-[“II]-met.h~l 5’.triphosphatc (4 Ci per mmole), [“Cl-\lridine 5’.triphosphatc (101 mCi and [5-“HI-rlridine 5’.triphosphatc per mmolc), (14.8 Ci per mmolc) were obtained from Schwarz Bioresearch Corporation, Orangeburg, NY. Packard Instrument Company. Downers (:rovc>, 2 (5.dil)hellyloxazole IT, was the soIIrce of (PPO) and 1,4-bis-(2-(5-phc~~~loxaz~~lyl)]-bc~~zene(POPOP), All other chemicals were A.C.S. or comparable grade. Prepum/ion of mitochmdria. Mitochondria were prepared from normal adult atld regenerating rat liver by the procedure previously described from this laboratory (16). The yield ranged from 9 to 15 mg mit,ochondrial protein per gram wet. weight of liver. Mitochondria prepared in this manner have been shown to be virtually free of bacteria and contaminating subcelllllar components such as nuclei, nuc1ca.r frngmeuts, and microsorncs (3,16). Digitonin fractionation of mitoc~hondria. Separation of the inner and outer membranes of mitochondria was achieved essentially by the met,hod of Schnaitman and GreenawaIt (17, 18) using a 3 Abbreviations used are: MDR, MembraneDNA-nascent, RNA; PEP, phosphoenol pyruvic acid; BS.4, hovine serum albumin; CAP, uchloramphenicol; TMK, 10 rnM Tris, pH 7.4, at, 4O, 10 mu magnesium acetate, 0.1 Y KCl; TCA, trichloroacetic acid; dNTP’s, deoxyribonucleoside ribonucleoside triphostriphosphates; NTP’s, phates.
ANlt
KAI,F
ratio of digitorrin to mitochondrial protein of 0.125. The resulting pellet, represented the mitochondrial inner membrane-matrix fraction (designated as “inner membranes”). In some cases the decanted supernat ant fluid containing the outer membranes was saved for assay of marker enzymes. Chemical assays. Protein was determined by either the biuret method (19) or the method of Lowry el a/. (20) with cr,vst,alline RSA as t.he reference. DNA was determined by the method of Ceriot,ti (21) as described by Webb and Levy (22) or fluoromet.rically (‘23). Calf thyrnus DNA was the reference. Enzy~~c assays. Monoamine oxidase was assayed as described by Tabor, Tabor, and Rosenthal (24). Samples were activated wit.h 0.3 mg Lubrol per mg mitochondrial protein for 15 min at 4” to reduce changes in absorbance associated with mitochondrial swelling. Succinic dehydrogenase was assaycd spectrophotomet,rically at 400 nm (11) and malate dehgdrogenase was assayed by the method of Mchlcr et al. (25). Samples were activated wi t,h Lltbrol as desnribctl for monoamine oxidase, and 0.02 x sodium nmyt,al was used to inhibit oxidation of NAIIIT by t,he respiratory chain. IINase and I:Nase were assayed by the method of Kunit.z (26); the presence of Sarkosyl had no cffcct on the assay of these nncleascs. Pronasr was assayed for contaminating DNase activity t,y the rapid spcctrophotometric method of Lindljerg (‘27). Collt)arninating I)Nase activit+ was shown to be virtually absent when 20 times the amo1mt of protlase normally used for protein digestion carlscd no hyperchromicit)of the DNA solution at. Al,“,,,,, as recorded on a (;ilford 2000 spect,rophotomet,cr with an expanded absorbancy scale. Standard irrc&aliorc. proiocols. The standard incubation medium for incorporation of radioactively labeled deosyriboand ribonucleoside t,riphosphat,es into inner membranes in vitro contained per ml: Tris-HCl buffer, pH 7.4 (50 pmoles); MgCls (10 @moles), PEP (5.25 pmoles), pyrlwate kinase (10 pg), Smercaptoethanol (5 pmoles), KC1 (150 pmoles), three unlabeled deoxyriboor rihonucleoside (or both in dual-label experiments) triphosphates (15 nmoles each), and the appropriate labeled precursor (sp act, 10 &i per 15 nmoles). The standard incubation medium for [“IIIleucine incorporation into inner membranes i,a vitro contained per ml: Tris-IICl buffer, pH 7.4 (50 pmoles) ; MgCl? (5 pmoles) , PEP (3.75 rmoles), pyruvate kinase (10 pgj, ATP (2 pmoles), EDTA (2 rmoles), KC1 (50 pmoles), and 13H]-leucine (sp act, 1 &i per 4 nrnolesj.
MITOCHONl>I:IAI,
R1F:~lBRANP:.I)NA-1:NA
Mitochondria were preswollen for 10 mitt at 37” in a hypotonic mediltm containing 0.1 or sucrose and 0.G rnbl Tris-HCl, pH 7.4, at 0” to facilitate entrance of nucleoside triphosphates. The swollen inner membranes (2 mg protein per ml) were preincrtbated in the appropriate standard react iota mixt ttre for 15 min at ST” before the addition of isotope, after which the illcubatiott was cotttinucd for 15 min. The reaction was terminated by cooling to 0” and diluting wit,h 2 vol of ice-cold 8.5(; sucroscTMK buffer. I,abeled inner membranes were separated from the ittcubaliotr mixture by ctntrifugation at 12,500~ for 10 mitt and resuspended in 8.5’;,;, sucrose-TMK bufier at a concent ration of 3.3 mg protein per ml. Preparation of lhe ?rlel~lhrafle-DSA-RSA mmplea (desiynaled “;1IDZGand”). A standard procedure, adapted from the met.hod of Tremblay el ~1. (14) was carried out at 4” it1 which 10 ~1 of Sarkosyl NL-30 were layered on a discontinrtotts sucrose gradient containing 13.5 ml of 15% sucroseeTMK buffer overlayered on 4O”/d sucroseTMK buffer. Three milliliters (10 mg protein) of inner membranes suspended in 8.5% sucrose-TMK buffer were immediately layered over the Sarkosyl and very gently mixed with the detergent tJo effect the lysis of the membranes with minimum shearing of I)XA. The gradient was centrifuged immcdiatcly at 23,000~ for 15 min in a Spinco SW 25.1 rotor. The MDR complex was visible as a sharp, white band at the interface of the 15y0 and 40% sucrose. Two 5.ml fractions were removed from the top of the gradient with a wide-bore pipet,te and the remainder of the gradient, was collected dropwise from the bott,om of the tube into &ml fract,ions with the exception of the XUIL band which was easily collected as a single R-ml fraction. Acid-insoluble material iu each fraction was precipitated by the addition of cold 50”/;, TCA to a final concentration of 5%,, and then diluted with 1 ml of cold 52& TCA. Native calf t,hymus DNA (100 pg), yeast bttlk RNA (100 pg), or 50 pg of each, were added as carrier material t.o the fractions in experiments in which the labeled precursor was incorporated into DNA, RNA, or both I)NA and RNA, respectively. BSA (100 pg) was added as carrier to fractions coutaining protein labeled with [3H]-leucine. was dc14C and “H unaZ~sis. Radioactivity termined with a Nuclear-Chicago Mark I Liquid Scintillation systetn. The background was 12-18 cpm. Counting efficiency was determined by t)he channels ratio method with an external standard. The counting efficiency for lrC was 82-84y6 and for 3H was 27-297;. Zso2atiorL of nucleic acids. Mitochondrial nucleic acids were prepared hy t,he phenol method of Nass (zsj. t)NA, labeled with 2-[‘G-thymine, was
(‘O?rIPLI~:X
isolated from E. coli by the proccdttre of Martttrtr (29). Techt~iqu~ for l’urlic~l Hepukc~io~,q. Partial hepatectomy was performed as described by Higgins and Anderson (30). Sham operations were performed by the same procedrtre, but ligatttre aud excision of the liver were omitted. Oprrat iotis were performed between !I and 11 AlI, atld the rats were killed between 8 and 9 AM on a subscqltent da.v. The rats were given food and water trd lihitual before and after the operation, but the food was removed 16 hr prior to killing the animals.
preparatio?r . Thrw-timw washt~d rat liver mitochondrin of the: appropriate type’ I\-care trwtcd with digitonin and the outer mcmbrane WZLSseparated from th(l innw mcmbrctrwmatrix fraction as dcwribcd in 1\Icthods. Thc~ completcacss of the rcmowl of the outer mcmbmne was monitowd by thci :ws:~y of various markw cnzymw. Thaw data are prcwnted in Tablo I. Monoaminc~ oxid:ase WASvirtjually abwnt from the inner mcbmbrune preparations, and values :~ppro:whing 100 ‘% wfr(L roof tht: enzymn cnzymc activity covcrcd in the: supernatant fluid. Retention of mal:~tc dehydrogcww suggwts that the innw mitochondrinl mtmbrnnc~ rcmnins largely intact during the prcp:w:tt ion OF the
-~~
-
-~~~~
!--
~
,~
~~
100 100 Whole mitochon100 1 100 dria ! Inner membranes 49.6 ) i8.5 2.2 60.2 i41.2 -1 93.7 Inner membrane 30.0 I supernatant I fraction I , 90.8 I 78.5 ( 95.9 , 90.2 Recoverya ~__. a Based on whole mitochondria. h All percent,ages are reported as mean values from four experiments.
42x
VAN
TUYLE
inner membrane matrix fraction. Inner mcm branrs prepared in this mnnncr and showing these cxharacteristics were used in all experiments reported here. Formation of MDR bards. The addit,ion of Sarkosyl t.o a suspension of inner membranes causes lysis of the membranes and clearing of the suspension. Intcracton of the dctergent, with the magnesium ions of the TAIli-buffer forms white, hydrophobic crystals which prwipit.atc from t.hc aqueous buffer. Portions of the inner membrane adhere to t,he hydrophobic surface of the cryst’als and the ent,ire complex sediments on t,he gradient t,o the interface between 15 and 40% sucrow. In t,he absence of t,he deWgent., t,he inner membranes remain at t,he top of the gradient at, the low speed of centrifugation used with these gradient,s. Quantitative sediment,ation of the crystals into a sharp band at, the 1540% sucrose interface is not, dependent upon the presence of inner membranes and is independent, of caentrifugal forces in the range of 2,500Z5,OOO~.Any particulate debris sediment#s through the 40 % sucrose cushion to t,he bottom of t,hc t,ube and remains as an undist,urbcd pellet during the fract8ionation of the gradient. Figure 1 shows the profile of a
Bottom
FIG. 1. -42~4~~profile of a discontinuous sucrose gradient used for preparation of the MDR band from normal rat liver mitochondrial inner membranes. Inner membranes (6 mg protein) were layered with Sarkosyl on the discontinuous sucrose gradient and the MDR band was isolated by centrifugat,ion at 23,OOOg for 15 min. Fractions (1.5 ml) were collected from the bottom of the tube and were warmed to 45°C prior to reading in a Beckman DB Spectrophotometer at A z6dnrnagainst the corresponding fractions of a duplicate tube which contained no inner membranes.
ANT)
KALF
typical discontinuous grad&t, in which thr: MDR band was easily collectBed as IL 15ml fract#ion which contained the whit,e Mg2+Sarkoxyl crystals. The crystals were dissolved by httating at 40°C and the presewe of inner mentbranc mntcrial was monitored by measuring t’hc absorption at’ 264 rm. However, mwh of the d 2sl,,-absorbing material remained at) t’he top of the gradient. Characterixafion of the d fDR cumple.~:. The observat.ions by >I. Kass that rnfbrnrane association of mitochondrial DNA occurred more frequently in rapidly growing cells (J, cells and ascitcs tumor cells) cwmpared to normal adult, tissues such as rut, and chicken liver (31) suggested a possible functional role for membrane attarhmc,nt of DKA and led us to the isolat’ion of an MDR complex from regenerating rat, live. Most of the ctxperiments to bc discussed were performed 1vit.h Z-hr regcnrr;tt.ing rat, liver since previously published st’udies b> S. Nass (32) indicated that the rate of sgrlt,hesis of both mitochondrial and nuclear DNA iu viva reached a rnaximum 20-24 hr after partial hepatcctomy. Inner mitochondrial membranes from 22. hr regenerating rat liver were incubated wit’h [14C]dATP and [“H]UTP ,Zj/ ~jitw and an MDR comples was prepared which was found to cont,ain inner membrane prot tin and a large portion of the total lab&d DXh and RNA of the Sarkosyl lysatc. Figure 2 presents a typical sucrose gradient profile of this MDR complex. Th(l profiles for prot,cin, labeled DNA and RXA show coincident peaks at, the position of tht> Llg”+-Sarkosyl crystals. To show that, the labehbd DNA and RXA precursors were incorporated int,o int,ernuclcot,ide linkage in th(lir rwpcctivc mitochondrial polymers, experiments ww: carried out in which MDR, complexes ww: isolated from inner membrane-matrix prcpnrations appropriately labeled in t,he presence and absence of low levels of cthidium bromide, a specific inhibitor of nucltG acid SJW thesis in mitochondria. Ethidium bromide (2 pg/mg mitochondrial prot,ein) inhibited t,he incorporatjion of [14C]dATl’ int#o the DNA of the complex by hO% and t.hc incorporat,ion of [3H]UTP into thtt RNA b> grcatw than 80 %.
MITOCHONDRIAL
iLIEMBBANE-DNA-ILNI 7
Bottom
hact,m
No.
TOP
FIG. 2. Isolation of au JlDI1 complex from 22-1~ regenerating rat liver mitochondrial inner membranes. Inner membranes (2 nrg per ml) were incubated for 40 min at 37°C in vitro iu a mixture containing per ml: Tris-HCl buffer, pH 7.4 (50 rmoles) ; MgClz (10 pmoles), PEP (5.25 *moles), pyruvate kinase (10 figj, 2-mercaptoethanol (5 @moles), KC1 (150 wmoles), three unlabeled dNTP’s and NTP’s (15 nmolcs each), and both [WI-dATP (10 &i per 15 nmoles) and [WI-UTP (1 pCi per 15 nmoles). The inner membranes were isolated from the incubation medium by cent,rifugat.ion at 12,500~ for 10 min and suspended in 8.5’& sucrose-T?VIK buffer at a concentjrat,ion of 10 mg protein per 3 ml. hliquots of 3 ml were layered with Sarkosyl ou discontinuoits sucrose gradients and the RIDR band was isolat,ed by centrifugation at 23,OOOg for 15 min. The acidinsoluble material iu the 2.ml fractions was precipitated with TCA at a final concentration were washed for of 5’76, and the precipitates protein determinations (Lowry- et al.) and duallabel (W and “I~) analysis as described in Methods.
I’rofiles similar to t how prcsonted in I’ig. 2 also were obt,:tincd when Surkos~-1 lysates of labeled normal rat. liver mit ochondrial inner membranes were centrifuged on disconGnuous sucrose gradients. Treatment, of the individual fract,ions of duplicate gradients containing t,he MDR complex from normal rat liver mitochondria with Di\;nse and RNase, respectively, converted the bulk of the labeled DXA and RSA into an acidsoluble form, t,hus confirming t,hat# the labcled precursor wts in internucleot ide linkage in tjhc respective polymers. Analysis of ;IIDR bands from inner membrancs Iabclcd with [“H]lcwinc i/r ~ifw (Fig.
4’“) -,
COMPLEX
3) indicated that a substantial amount of the “H-labeled protein appeared in the band. Furthermore, a comparison of the specific a&vitg of the protein in the gradient, fractions indicated that the protein of highest specific* radioactivity appeared in the band (not showy in Iig. 3). l’uromycin and especially C’Al suppressed the incorporation (Fig. 3). The possibility existed that, lysatcs of inner membranes c~ontained labeled ma terial not int~rinsically bound to the MDR complex, but found in t’he band because it, sedimcnt,cd t8hrough t’he 15 (;c sucrose and was retained by the 40% cushion. Therefore, it, was necessary to demonst#rate the lack of such material by showing the distribution of protein and labcled material in linear sucrose grad&us. Inner membranes were incubated separately with [3H]TTP and raH]UTP, and tht I\IDR complex prcparcd on a 15-40 7;) limar sucrose gradient. Representative profiles for protSrin, labeled DNA and RX:\, showed coincident, peaks at’ t’he position of
Boltam
iract1on NO.
Toll
FIG. 3. The cf’fect of puromyriii and chloramphenicol ou [3H]-leucine incorporation by rat, liver mitochondrial iuuer membraues irl Gtro. The staudard incubation mixture contained per ml: inner membranes (2 mg protein), Tris-HCI buffer, pH 7.4 (50 pmoles); MgCl2 (5 pmoles), PEP (3.75 pmoles), py-ruvate kinase (10 pg), ATP (2 pmoles), EDTA (2 hmoles), KC1 (50 rmoles), and [31-I]-leucine (1 PCi per 4 nmoles). Puromycin (150 rg per ml) and CAP (100 pg per ml) were added to separate, additional incubation mixtures. After incubating 40 min at 37” the inner membranes were collected by centrifugation and MDR bands were prepared as described in the legend to Fig. 2. Fractions (2 ml) were collected and the acid-insoluble material was analyzed for radioact.ivity as described in Methods.
430
VAN TUYLE
that Mg2+-Sarkosyl sharply
at
30
5% sIlcrosc.
crystals No
hXD
which banded 0th
p&s
KALF
‘T----n
n-PIT
6
17I 9
obscrvcd in the region of tlw gradicwt b+ twm 15 and 30 7:’ sucrose.
not, bc isolated from Sarkosyl lysabes of inner membranes in t’h(L absence of Mg2+ ions. Entrainment studies JV(W carried out in whicah suspensions of native and denatured calf t,hymus DNA, bovine liver tRNA, yeast bulk RNA, and a mixture of rat liwr mitochondrial DNA and RX\T;Z-all in TJIIly lxycred with Sarkosyl on swrosc gradients; only 2% of t,he t,otal prot& laycwd on each of the gradients \vas found in the band. It, WLLS possible that in the presenw of some mctmbranous matwial, DKA fragments shcarcd during isolation or lysis might spuriously bind to the mclmbrane fragments and thus appear in the complex. Thcrclforc, 14Clab&d DNA was IayPred on a typical sucrow gradient’ with Sarkosg-I and unlab&d innor membranes. The unlabeled membrane prot,cGl bound to the Mg2+-Sarkosyl crystals, but> no entrainment of free-lying [‘“ClDNA owurrcd in thr JIDR band (Fig. 4). Conscyuentjly, the DSA normally found in :\lDR bands is probably intrinsically bound to an inner membrane fragment. JIDR bands labclcd in t(he DKJ4 with [3H]TRIP were t’rcatcd with DKuse-free pronase (100 pg per ml; 1 and 2 hr at 36’C) in order t’o digcnt away t,hosc portions of the mcambrane fragment to which t,he labeled DNA was bound. Control bands wit,hout’ prorinse were treated in an identical manner. ,4ftw digestion with pronase, the bands were dilutcd wit’h T&II< buffer to lower t#hesucrose conacntrat~ion below 15 %, and rebanded
1 Bottom
rrac,,on
NO.
Toll
FIG. 4. The degree of entrainment. of [W-UK;A into the MDR band by unlabeled mitochondrial inner membranes. 9 3-ml suspension of unlabeled mitochondrial inner membranes (10 mg protein) in S.57csucrose-TMK buffer containing 26 ~g of X. coli [14C]-DXA (specific radioactivity, 0.15 PC’i per mg) was layered wit.h Sarkosyl on a discontinuous sucrose gradient. The MDIL band was isolated by cent,rifugation at 23,OOOgand the gradient was collected in 5-ml fractions except for the MDR band which was collected as a simple Z-ml fraction. The acid-insoluble material in the fractions was precipitated with TC,4 at a final conce,Itration of 5%,, and the precipitates were washed for protein determination (Lowry el a!.) and ‘*C analysis as described in Methods.
with Sarkosyl on second gradient8s. In a typexperiment, digest’ion wit,h pronnw for 1 and 2 hr, rrspectively, released ‘B.5 and 3s % of the lab&d DNA, and virtually all (91-100 5%)of the rclessrd DNA n-as rwovered as TCA-insoluble matwial at thcb top of the gradient. Other rebanding experiments ww JNTformed to determine whether t,he RKh was present in the XDR complex by virtue of its attachment to DNA. Two l\IDR compk~xes were prepared, one of which was labeled in DKA with [“H]TMP and the ot8her was labeled in RNA wit,h [3H]UhlP. The lab&d complexes were treated m&h RSnsci-free DIVase (13 pg per ml for 1 hr at’ 86°C) and rebanded as described in the pronase c>xperimerits. Controls which contained no added DNase were treated in a similar manner. DBase digestion removed approximately 44 5%of the DNA and 62 o/cof the RKA, suggesting that the RNA might’ be bound to the complex through its physical attachment, to DSA.
id
Complexes frou~ Xomal am! 2R-Hr RecJe7leratiwj Rat TiiEer Xifochh-ia Table II compares the proportions of the various component,s of t’he 1\1DR bands prepared from normal and 22-hr regenerating rat, liver mit80chondrinl inrler membranes. The percentage of tot,al protein in t.hc bands from 2%hr regencrat,ing liver approximated the amount8 in normal bands, but, the pcrcentagc of labeled DKA in the bands 22 hr after part,ial hepatectomy was consistently 34 tdmes higher than in normal livers; valucs as high as 6.i ‘4 wre obtained. However, t,he specific aativit,y (dpm per mg) of the DNA in the band was idcnt,ical to that, of the DXA in the top fractions. The proport’ion of total labeled RISA found in the MDR complex from normal and regenerating liver mitochondria was variable bet wecn cxperiment.s (Table II), but, duplicates or triplicat,es wit,hin any single experim& agreed within &tj % of each other. hkperiments to Demolrstrate a Possible Fwctional Role for the XDR-Complex In order to prow that t.hc increased percentage of DNA present’ in the hIDR complex from 2%hr regenerating liver might, be fun&onally related to the active proliferation of t,he tissue, two types of expcrimrnts were carried out,. JIDR complexes wrc prcpared from sham-opcrat.cd animals and t hc perccnt,ages of the various components found TABLE
II
Percent content
of total lysate” in MDR bands 30% Partial hepatectomy P7
Prot.ein(Lowry) : 13-16 DNA ([$H]TTP, dpm) or 16-21 (Ceriotti indole) : RNA (13H]UTP, dpm) , 22-34
14-24 50-65 15-75
21 18.5
i
34
o The Sarkosyl lysate of inner membranes which is layered on top of the gradient. b The ranges given are for five or more experimen&.
in t ho band wore idcntiwl to t,hose found t’or normal liwr \vit hin thr ranges reported in Table II. Bucher (33) has demonstrat’ed t,hat the iwrcase in DKA synthesis in liver subycquent to pnrtial hcpatwtomy is a function of t hc mass of the liver rcmovcd in the opwation; wmoval of 130% of the totSal liwr yklds only a barely diswrnible inc*nt:tsc in nwlwr Di?A synthwis, and \vith removal of 10 % of the t&w the rwponw is one-third of that, observed aftar a GS‘2 hepat,wtomy. Thwefor to determine whether the high perwnt,age of labeled DSA prcs& in the ;\IDlt c*ompl(hxaftw (is ‘Z. hepatectomy \vas directly related to tht> h!p(>rplasia of the, wmnining rclls, partial hr~patwtomics wrc pc~rformc~d in which onlg the left lateral lobe n-:w wmoved (approximatclly 30 “: of the total liwr mass). Under thew conditions, t ho wmnining lobes should ret nin t hc growth ch:~ra(:t(lrist its of normal liver (33). Twent,y-two hours after t,he operat8ion, inner mcmbrarw ww prepared from tho mitochondria of th(l remaining lobes and labeled with [3H]TTP itr ~‘tr.0 in thr presence and abscnw of c+hidium bromidc and the I\IDR complcxcs \VNY~ prcpared. The MDK band labeled in the’ absence of ct,hidium bromide contaiwd IS.,5 5& (Table II) of the labeled DSA of tht> l>.satt>, whit-h was cyuivalrnt to the mwn V:LILWof DNA in thci 11DR complex from normal liwr. The pcrcentagc of labelrd RNA in the 3iDR band also was similar to that’ found for normal liver mitochondria. The inwrporation of radioactvity into both DKA and RNA was significantly inhibited b\- the ct hidium bromide. The prows of restoration of the liver weight, to kc preopcrativc value owns during a “l-day period (30). Thewfore, to obtain further evidence for a functional rc$tionship between the perwntagc of DS.4 att’ached to the mcmbranr in the ctompl~~sand t hc degree of ccl1 proliferation, cxperimcnt8s wre carried out to determine the pcrwnt.age of DNA in the c+omplcx at int’crvals of time up to 3 weeks after partial hepat.ectomy. These data arc presented in Fig, ;j and show that the prrccntage of lab&d DNA present, in the complex rises without, any apparent timct lag from a normal value of approximat(4y 20 % prior to partial hepatwtomy to
432
VAN
TUYLE
about 55 % at, 22 hr. The amount of labeled DNA dropped to 40 % 44 hr after the operation, but by 72 hr it had again approached the maximal value. It is of considerable int,erest that 21 days after the operation (the point at which the liver mass is restored) the percent,age of DNA in the band returned to preoperative levels. DISCUSSION
A membrane DNA-RNA complex has been isolated from Sarkosyl lysates of inner membrane matrix preparations from normal and regenerating rat, liver mitochondria. Incubation of the inner membrane matrix preparation with the appropriate radioactively labeled precursor in vitro under conditions which support nucleic acid and protein biosynthesis in mitochondria results in t,he labeling of t,he appropriate component of the complex. Att’achment of DNA or RNA to the Mg*+-Sarkosyl crystals is not the result of occlusion during t’he formation of the cryst,als for entrainment st.udies and rebanding experiments suggest that only membrane fragments complex with Mg2+-Sarkosyl crystals (Fig. 4), and that these fragments are drawn through the gradient at a rate commensurate with the properties of t,he Mg2+-Sarkosyl crystals themselves. DXA is found in t,he band by virtue of it,s specific attachmentj to the membrane fragment; and RNA! in turn, appears to be bound to the DNA. Evidence supporting this sequential attachment was obtained from the facts that RWase-free DNase released 44 % of t.he DNA and 62% of the RNA from the complex, lvhereas RNase released radioactivity only from RNA. Furthermore, digestion of the membrane fragment of the complex with DNascl-free pronase released acid-insoluble, labeled DNA (38 %, 2 hr) from t’he complex into t,hc t,op fract.ions. RIDR complexes isolated from inner membranes incubated wit’h [3H]leucine in vitro contain labeled protein in the membrane fragment, of the complex. The inhibition of incorporation of [3H]leucine into the complex by the mitochondrial inhibit,or CAP (Fig. 3) suggest)s that the [3H]leucine was incorporated into peptide linkage in the proteins of the membrane fragment. It has been well documcntjed that bot’h intact mitochondrin
AND
KALF
(34) and inner membrane-mat,rix preparations incorporat’e [3H]leucine predominant,ly into the insoluble proteins of the inner mitochondrial membrane, and this incorporation is inhibited by puromycin and CAP (11, 35). The MDR complex from normal adult. rat liver mitochondria was comprised of a small piece of inner membrane c:ont,aining 14 % of the total protein of the lysate to which was attached 20% of t,he total DNA of the organelle. The remainder of the DNA was located at the t,op of t,he gradient in acid-precipitable form, suggesting that only one out of possible five DNA copies in each organelle was attached to the membrane irz vivo. The percent’age of total DNA found in t’he complex from 22-hr regenerating mitochondria was three to four times higher than in t.he normal complex, whereas the percentage of protein is approximately the same in both cases. An increased binding of DNA t,o the inner mitochondrial membrane would be expected in act,ively proliferating tissues if t,he attaehment of D,NA to membrane is of functional significance in the replication and/or segrcgation of DNA during biogenesis of the organelle. Preliminary experiments in our laboratory have demonstrated that a similar RIDR complex can bc isolated from another actively proliferating tissue, fetal rat liver. The percentage of DNA at,tached to t#he membrane fragments of Dhecomplex from 19day fetal rat, liver mitjochondria was found to be 63 % of the total DlSA of the lysate. (Van Tuyle and Kalf, unpublished observat,ions) _ This is the same pcrcent.age as found in t,hc complex frotn 22-hr regenerating rat, liver. It is not known w-h&her the value of ;LP)proximat,cly 60% of the DNA bound to t’he MDR complex from rcgc~ncrating rat’ liver represents the maximal amount of DNA :wtually bound to the inner membranes of the mitochondria in t,he total population of dividing cells. However, part’ial hepatectomy is followed by a period of synchronization of the hcpatocytes such that 60% of t,he cells will ent’er mitosis (36). The pcrcent’age of D,NA in the band appears t.o vary, depending upon the length of regeneration time (Fig. 5). For example, the percentage of DNA in t,he band increases to a maximum during the period of maximal nuclear DNA synthesis
FIG. 5. The percentage of labeled DNA in the MDlt band as a function of the time after hepateccorny. Regerating rat livers were removed 11, 22, 46, 70, and 504 hr after partial hepatectomy and the mitochondrial inner membranes were radioactively labeled in the standard incubation protocol as described in the legend to Fig. 2 with [W]-TTP at a level of 10 PCi per 15 nmoles. The MDR bands were prepared and analyzed for radioactivit,y as described in Methods. The values designated represent the percentage of t,otal acidinsoluble radioactivity found in the band as compared to the total radioactivity recovered in the entire gradient. The values are reported as mean percentages with average deviations from four or more experiments with the exception of the Sweek value which was obtained from the average of triplicat,es in a single experiment. The perrcnt,aze of labeled DNA in MDR bands from normal, adult rat liver mitochondria is shown as “zero hours after partial hepatectomy.”
prior to mitosis at 28-X hr, t’hen decreases slight.ly at 46 hr after hepatectomy, but, again increases at TO hr presumably conwmit~ant~ with a second wave of nuclear I>SA synt,hcsis which is lmon-n Taooccur in n-wnling rats undergoing liver regcncration :md also in synchronized cultures of mammalian wlls (36). The period of rapid growth which (wsues aft’cr part’ial hepatectomy abates in approximately 21 days, at’ which time the t’otal liver mass has been rcstorcd. As notJeclhere, the pewentage of DNA found in the complex 21 days post,hepatectomy has also returned to the normal level. Furthermore, neit,hcr sham-operated rats nor anmals subjcct#ed to 30 ?I hepatect,omy showed an incwased pcrcent,age of DNA in the complex (Table II). 1litocshondria, prepared as described in Methods, contain minimal eont,aminat,ion by bacteria (3, 16) and t hc prcsenw of thrsc
b:wt (CL docwnot sign&ant l\- influence the incorporation of (lither nucl& acid or protcin precursors into the mitochondrial suspensions. Incrcascd numbers of bacteria, howwr, might havcl bwn present in mitochondrial suspwsions obtained from rcgenerating livrr as a result of the surgical opcrat,ion, and a wmplcx derived from bat*twia might c.ontaminatc the MDR band. Sewral picxcesof evidcncc argue against this. (1) So visible signs of gross bact,crial (‘ontamination were observed in the host animal at the time of pwparxtion of mitochondria. (2) Bacterial cells arc not subject to lysis by Sarkosyl urlless the ~11 \\-a11has been r(wdcred fragile by t,reatment wit,h EDTA and lysozyme. (3) Sham-operat,ed animals showed no increase in t’he amount of con~pl~~x obt)ained or in the percentage of various constituent,s found in t,he complex. (4). The amount of complex and pcrwntage of DSA in the band returned to normal levels in animals 21 days after part’ial hc>pat,c:ct’omy. (5) Thch complex, obtaiwd from animals subjertcd to the operation and to the same incrcawd risk of bacterial caontaminatJion by removing one lobe of t,ht>liver, contairwd cxactly the perccntagc of mcmbrann-bound DKA as normal complexes. Thus, the MDR complex isolated from rat liver mitochondria appwrs to reprewnt :1 small fragment of the inner mc>mbranctwhic*h contains an att~achmcnt site(s) for mitochondrial DNA. RN\‘,4 transcripts appear to bc bound to the DKA of t’he complex on thrk basis of their relcaw by RKasc-free DSaw. Whether t,he complex is biologically meanful is unknown. Howewr, a possible fruwtional significance for the at8tachmcnt, of the DNA to t#hc mrmbrann i? suggestcld by the 3- to -l-fold irwrcnw in the at~t~:rcl~n~~ntof DNA to the membrane component of the complexes deriwd from mitochondri:t of activel!- prolifemting tissues such as f(ktal and 22-hr regenerating rat liver. The fact. that neither sham operation nor 30 ‘Z partial hcpatcct,omy rcs&s in incwascd attachment over that found in normal liver mitochondria also lends support, to t’his point. It is of intorcst, in view of the developing bacteria-mit,ocshondrial homology, t,hat, the MDR complex from rat, livrr mito~hondria rawmblcs similar cwmplcxcs isolatcld from
484 several bxterial (14).
VAN
TIJYLR
specks by t,he sarnc’ mclthod REFEIII~NCJSS
1. Nass, M. M. K. (1969) Science 166, 25. 2. k\BINO\VITZ, &I., AND S\\'IPT, H. (1970) Phlysiol. Rev. 60, 376. 3. KALF, (+. F., am CH’IH, .J. J. (1968) J. Niol. Chem. 243, 4904. 4. MEYER, 1~. R., AND SIMPSON, M. V. (1968~ Proc. :\;at. Acad. Sci. C.S.A. 61, 130. 5. &IEYER, It. R., AND SIhCPdON, M. v. (1970) J. Biol. Chem. 245, 34. 6. WINTERSUERGER, E. (1970) Biochem. Biophys. Res. Commun. 40, 1179. 7. KUNTZEL, H., AND S~H.\FER, K. (1971) ,Vutzue ,Vew Biol. 231, 265. 8. TSAI, M., MICHAELIS, (+., .\ND GRIDDLE, R. S. (1971) Proc. Xat. Acad. Sci. 7T.S.A. 68, 473. 9. REID, B. D., I\ND PARSONS, I'. (1971) Proc. *Vat. Acad. Sci. L:.S.A. 68, 2830. 10. SCHULTZ, S. Ii.., AND Nass, S. (1969) Pd. Eur. Bidl. Sot. Lett., 4, 13. 11. KALF, G. F., AND FAUST, A. S. (1969) Arch. Biochenz. Biophys. 134, 103. 12. WINTERSBERGER,E.,.\NDWINTERSBER(:ER,U. (1970) Fed. Eur. Biol. Sot. Lett. 6, 58. 13. NASS, 8. (1969) Int. Rev. Cutol. 26, 117. 14. TREMUIIAY, G. Y., DANIELS, M. J., AND SCHAECHTER, M. (1969) J. Mol. Biol. 40, 65. 15. EARHART, C. I?. (1970) Virology 42,429. 16. O'BRIEN, T. W., .\XD KALF, G. F. (lQ67) b. Biol. Chem. 242, 2172. 17. SCHNAITMAA-, C. V., J:II\VIN, G., AND (&EEXASALT, J. W. (1967) J. Cell Viol. 32, 719. 18. QCHN.UTILZ.IN, C., ASD GREENAMTALT, J. W. (1968) J. Cell. Hiol. 38, 158.
AND
KALF
19. ROIUNSON, II. W., AND HO~DEX, C. G. (1940) J. Biol. Chem. 136, 727. 20. r,o\\.~I., \ND LEVI-, H. B. (19%) io “Methods of Biochemical Analysis” (( ilick, David, ed.) Vol. 6, lx 14. Tntersciencc, New York. 23 KISSANE, J. M., ASD ROHINS, E:. (1958) d. Biol. Chem. 233, 184. 24. TAIIOR, C. W., TABOR, II., .\ND ROSENTM.U,, S. M. (1954) J. Biol. Chem. 208, 645. 25. MEHLEH, A. I~., KORNI~ERC:, A., GIUSOLIA, S., .AXD OCHOA, 8. (1948) J. Biol. Chem. 174, 961. 26. KUXITZ, M. (1950) J. Grn. Physiol. 33, 349, 363. 27. T,INDF~EEO, U. (1964) Biochim. Biophjys. .+I~tu 82, 237. 28. NASS, M. M. K. (1969) J. Mol. Biol. 42, 529. 29. MARMUR, J. (1961) J. .IloZ. Biol. 3, 208. 30. FhGGINS, (;. hr., .\ND .kNDEltSON, 1~. M. (1!)31) Axh. Puthol. 12, 186. 31. N.\ss, M. M. K. (1969) J. .Uol. Biol. 42, 521. 32. Nass, S. (1967) Biochin!. Biophys. Acta 146, 60. 33. BITC:IIER, ?C. L. Jt. (1963) Inl. Rev. Cytol. 15, 245. A., .\ND WORIC. T. 8. (1970) A/Ln. 34. kH\VELL, 12ec. Biochem. 39, 251. ~5. BEATTIE, D. S., BASFORD, 1:. JS., AXD Ko~trvz, S. B. (1967) Hiochemistrg 6, 3099. 36. BRE~XICIC, 1~:. (1971) .Ile/hotJs Cancer lies. 6, 347.
OF
BIOCHEMISTRY
Isolation
AND
RIOPHYSICS
of a Membrane-DNA-RNA Liver
GLENN Department
425-43-L (1972)
149,
C. VAN
TUYLE’
December
from
Rat
Mitochondria
of Biochemistry, Jeferson Philadelphia, lleceived
Complex
a4ixn
GEORGE
F. IidLF”
Medical College, Thomas Jeferson Pennsylvania, 19107
9, 1971; accepted
January
T’niversity,
13, 19i2
A membrane-DNA-RNA complex has been isolated from Sarkosyl lysates of inner membrane matrix preparations from normal and regenerating rat, liver mitochondria. Incubation of inner membrane matrix preparations with a radioactively labeled precursor in vitro under conditions which support nucleic acid and protein biosynthesis in mitochondria results in the labeling of the appropriate component of the complex. Only membrane fragments complex with the Mg2+-Sarkosyl crystals and DNA is found in the complex by virtue of its specific attachment to the membrane fragment. RNA, in turn, appears to be bound to the DNA. The percentage of the total DNA of the lysate which is attached to the membrane fragment in the Sarkosyl crystal is 3- to 4-fold higher in the complexes from 22.hr regenerating and fetal rat liver mitochondria than in normal rat liver, whereas the percentage of membrane protein is the same in both cases. The increased attachment of DNA to membrane in actively proliferating tissues suggests a possible functional significance for the attachment of the mitochondrial DNA to the membrane during the replication of mitochondrial DNB. The membrane-DNA-RNA complex from rat, liver mitochondria appears to resemble similar complexes isolated from several bacterial species by the same method.
The semiaut.onomous nature of mitochondria has been documented by t,he fact’s that, t#he organelle contains two to six copies of a 5~ circular DNA which arc found both free in t,hc matrix and bound to the inner membrane (1, 2), as well as its own unique DNA (3-5) and RNA polymerases (6%) bot#h of which appear to be membrane bound 10-12). 6, These fact,s toget,her with the growing body of evidence indi&ing a bacterialmitochondrial homology (13) and the resemblance of mitochondrial inner membrane-mat rix preparat’ions to bwt’erial
spheroplasts, led us to believe that a membrane-DNA-nascent RnTA complex might be isolated from mitochondria. Similar complexes have been isolated from several bacteria by lysis with t,he det,ergent Sarkosyl in the presence of Jig”+ (14) and T4-phage DNA-membrane complexes have been demor&rated in bacteriophage-infected cells (13). We report here on t’he isolation of a membrane-DNA-RNA complex from rat, liver mitochondria using t,he Sarkosyl technique.
1 These results were taken, in part, from t)he Ph.D. dissertation presented to the Graduate School, Jefferson Medical College of Thomas 1971. Present address : Jefferson University, Department of Biochemistry, State University of New York at Stony Brook, Long Island, New York. 2 To whom reprint, requests should be sent).
Experimental animals. Male Wistar rats (150170 g) were maintained on Purina Rat Chow and were fasted 16 hr prior to decapitation. Chemicals and reagen&. DNase (KC 3.1.4.5, hovine pancreas, free of RNase) and RNase (EC 2.7.7.16, bovine pancreas; stock solutions were heated 10 min at 90” to denature any DNase
M.4TERIALS
125
AND
METHODS
4%
VAN
TUYIZ
present) were purchased from Worthington Biochemical Corporation, Freehold, NJ. Pyruvate kinase (EC 2.7.1.40, rabbit musclej was obtained from Sigma Chemical Company, St. I,ouis, MO; and pronase (Sireptomyces qriseus proleasc, B grade) was purchased from Calbiochem, Los Angeles, C.4. Deoxyribonucleoside triphosphates, ribonucleoside triphosphates, calf thymus DNA, yeast bulk RNA, rat liver tltNA, L-leucine, PEPS, BS.4, and digitollin were obtained from Sigma Chemical Company. IXgitonin was recrystallized from absolute ethanol and ground to a fine powder to increase its sol\lhilit,y in water. Sarkosyl NL30 (sodium lnuroyl sarcosinate, 30fiA) was supplied by Geigy ludustrial Chemicals, Ardsley, NY. Calbiochem was the source of ethidium bromide; puromycin and cytochrome c were obtained from Nut,ritional Biochemicals, Cleveland, OII; and CAP was a gift from the Parke I)avis Company. Radioisotopes. Thymidine-[“HI-methyl 5’.triphosphate (9 Ci per mmole), dcoxy[“B]-:ldenosille 5’-triphosphate (11 .l Ci per mmole), [2-14C]thymine (28.8 mCi per mmole) and I,-[4,5-“II]leucine (5 Ci per mmolc) were purchased from New England Nrlclear Corporat.ion, Boston, MA. Thymidil,e-[“II]-met.h~l 5’.triphosphatc (4 Ci per mmole), [“Cl-\lridine 5’.triphosphatc (101 mCi and [5-“HI-rlridine 5’.triphosphatc per mmolc), (14.8 Ci per mmolc) were obtained from Schwarz Bioresearch Corporation, Orangeburg, NY. Packard Instrument Company. Downers (:rovc>, 2 (5.dil)hellyloxazole IT, was the soIIrce of (PPO) and 1,4-bis-(2-(5-phc~~~loxaz~~lyl)]-bc~~zene(POPOP), All other chemicals were A.C.S. or comparable grade. Prepum/ion of mitochmdria. Mitochondria were prepared from normal adult atld regenerating rat liver by the procedure previously described from this laboratory (16). The yield ranged from 9 to 15 mg mit,ochondrial protein per gram wet. weight of liver. Mitochondria prepared in this manner have been shown to be virtually free of bacteria and contaminating subcelllllar components such as nuclei, nuc1ca.r frngmeuts, and microsorncs (3,16). Digitonin fractionation of mitoc~hondria. Separation of the inner and outer membranes of mitochondria was achieved essentially by the met,hod of Schnaitman and GreenawaIt (17, 18) using a 3 Abbreviations used are: MDR, MembraneDNA-nascent, RNA; PEP, phosphoenol pyruvic acid; BS.4, hovine serum albumin; CAP, uchloramphenicol; TMK, 10 rnM Tris, pH 7.4, at, 4O, 10 mu magnesium acetate, 0.1 Y KCl; TCA, trichloroacetic acid; dNTP’s, deoxyribonucleoside ribonucleoside triphostriphosphates; NTP’s, phates.
ANlt
KAI,F
ratio of digitorrin to mitochondrial protein of 0.125. The resulting pellet, represented the mitochondrial inner membrane-matrix fraction (designated as “inner membranes”). In some cases the decanted supernat ant fluid containing the outer membranes was saved for assay of marker enzymes. Chemical assays. Protein was determined by either the biuret method (19) or the method of Lowry el a/. (20) with cr,vst,alline RSA as t.he reference. DNA was determined by the method of Ceriot,ti (21) as described by Webb and Levy (22) or fluoromet.rically (‘23). Calf thyrnus DNA was the reference. Enzy~~c assays. Monoamine oxidase was assayed as described by Tabor, Tabor, and Rosenthal (24). Samples were activated wit.h 0.3 mg Lubrol per mg mitochondrial protein for 15 min at 4” to reduce changes in absorbance associated with mitochondrial swelling. Succinic dehydrogenase was assaycd spectrophotomet,rically at 400 nm (11) and malate dehgdrogenase was assayed by the method of Mchlcr et al. (25). Samples were activated wi t,h Lltbrol as desnribctl for monoamine oxidase, and 0.02 x sodium nmyt,al was used to inhibit oxidation of NAIIIT by t,he respiratory chain. IINase and I:Nase were assayed by the method of Kunit.z (26); the presence of Sarkosyl had no cffcct on the assay of these nncleascs. Pronasr was assayed for contaminating DNase activity t,y the rapid spcctrophotometric method of Lindljerg (‘27). Collt)arninating I)Nase activit+ was shown to be virtually absent when 20 times the amo1mt of protlase normally used for protein digestion carlscd no hyperchromicit)of the DNA solution at. Al,“,,,,, as recorded on a (;ilford 2000 spect,rophotomet,cr with an expanded absorbancy scale. Standard irrc&aliorc. proiocols. The standard incubation medium for incorporation of radioactively labeled deosyriboand ribonucleoside t,riphosphat,es into inner membranes in vitro contained per ml: Tris-HCl buffer, pH 7.4 (50 pmoles); MgCls (10 @moles), PEP (5.25 pmoles), pyrlwate kinase (10 pg), Smercaptoethanol (5 pmoles), KC1 (150 pmoles), three unlabeled deoxyriboor rihonucleoside (or both in dual-label experiments) triphosphates (15 nmoles each), and the appropriate labeled precursor (sp act, 10 &i per 15 nmoles). The standard incubation medium for [“IIIleucine incorporation into inner membranes i,a vitro contained per ml: Tris-IICl buffer, pH 7.4 (50 pmoles) ; MgCl? (5 pmoles) , PEP (3.75 rmoles), pyruvate kinase (10 pgj, ATP (2 pmoles), EDTA (2 rmoles), KC1 (50 pmoles), and 13H]-leucine (sp act, 1 &i per 4 nrnolesj.
MITOCHONl>I:IAI,
R1F:~lBRANP:.I)NA-1:NA
Mitochondria were preswollen for 10 mitt at 37” in a hypotonic mediltm containing 0.1 or sucrose and 0.G rnbl Tris-HCl, pH 7.4, at 0” to facilitate entrance of nucleoside triphosphates. The swollen inner membranes (2 mg protein per ml) were preincrtbated in the appropriate standard react iota mixt ttre for 15 min at ST” before the addition of isotope, after which the illcubatiott was cotttinucd for 15 min. The reaction was terminated by cooling to 0” and diluting wit,h 2 vol of ice-cold 8.5(; sucroscTMK buffer. I,abeled inner membranes were separated from the ittcubaliotr mixture by ctntrifugation at 12,500~ for 10 mitt and resuspended in 8.5’;,;, sucrose-TMK bufier at a concent ration of 3.3 mg protein per ml. Preparation of lhe ?rlel~lhrafle-DSA-RSA mmplea (desiynaled “;1IDZGand”). A standard procedure, adapted from the met.hod of Tremblay el ~1. (14) was carried out at 4” it1 which 10 ~1 of Sarkosyl NL-30 were layered on a discontinrtotts sucrose gradient containing 13.5 ml of 15% sucroseeTMK buffer overlayered on 4O”/d sucroseTMK buffer. Three milliliters (10 mg protein) of inner membranes suspended in 8.5% sucrose-TMK buffer were immediately layered over the Sarkosyl and very gently mixed with the detergent tJo effect the lysis of the membranes with minimum shearing of I)XA. The gradient was centrifuged immcdiatcly at 23,000~ for 15 min in a Spinco SW 25.1 rotor. The MDR complex was visible as a sharp, white band at the interface of the 15y0 and 40% sucrose. Two 5.ml fractions were removed from the top of the gradient with a wide-bore pipet,te and the remainder of the gradient, was collected dropwise from the bott,om of the tube into &ml fract,ions with the exception of the XUIL band which was easily collected as a single R-ml fraction. Acid-insoluble material iu each fraction was precipitated by the addition of cold 50”/;, TCA to a final concentration of 5%,, and then diluted with 1 ml of cold 52& TCA. Native calf t,hymus DNA (100 pg), yeast bttlk RNA (100 pg), or 50 pg of each, were added as carrier material t.o the fractions in experiments in which the labeled precursor was incorporated into DNA, RNA, or both I)NA and RNA, respectively. BSA (100 pg) was added as carrier to fractions coutaining protein labeled with [3H]-leucine. was dc14C and “H unaZ~sis. Radioactivity termined with a Nuclear-Chicago Mark I Liquid Scintillation systetn. The background was 12-18 cpm. Counting efficiency was determined by t)he channels ratio method with an external standard. The counting efficiency for lrC was 82-84y6 and for 3H was 27-297;. Zso2atiorL of nucleic acids. Mitochondrial nucleic acids were prepared hy t,he phenol method of Nass (zsj. t)NA, labeled with 2-[‘G-thymine, was
(‘O?rIPLI~:X
isolated from E. coli by the proccdttre of Martttrtr (29). Techt~iqu~ for l’urlic~l Hepukc~io~,q. Partial hepatectomy was performed as described by Higgins and Anderson (30). Sham operations were performed by the same procedrtre, but ligatttre aud excision of the liver were omitted. Oprrat iotis were performed between !I and 11 AlI, atld the rats were killed between 8 and 9 AM on a subscqltent da.v. The rats were given food and water trd lihitual before and after the operation, but the food was removed 16 hr prior to killing the animals.
preparatio?r . Thrw-timw washt~d rat liver mitochondrin of the: appropriate type’ I\-care trwtcd with digitonin and the outer mcmbrane WZLSseparated from th(l innw mcmbrctrwmatrix fraction as dcwribcd in 1\Icthods. Thc~ completcacss of the rcmowl of the outer mcmbmne was monitowd by thci :ws:~y of various markw cnzymw. Thaw data are prcwnted in Tablo I. Monoaminc~ oxid:ase WASvirtjually abwnt from the inner mcbmbrune preparations, and values :~ppro:whing 100 ‘% wfr(L roof tht: enzymn cnzymc activity covcrcd in the: supernatant fluid. Retention of mal:~tc dehydrogcww suggwts that the innw mitochondrinl mtmbrnnc~ rcmnins largely intact during the prcp:w:tt ion OF the
-~~
-
-~~~~
!--
~
,~
~~
100 100 Whole mitochon100 1 100 dria ! Inner membranes 49.6 ) i8.5 2.2 60.2 i41.2 -1 93.7 Inner membrane 30.0 I supernatant I fraction I , 90.8 I 78.5 ( 95.9 , 90.2 Recoverya ~__. a Based on whole mitochondria. h All percent,ages are reported as mean values from four experiments.
42x
VAN
TUYLE
inner membrane matrix fraction. Inner mcm branrs prepared in this mnnncr and showing these cxharacteristics were used in all experiments reported here. Formation of MDR bards. The addit,ion of Sarkosyl t.o a suspension of inner membranes causes lysis of the membranes and clearing of the suspension. Intcracton of the dctergent, with the magnesium ions of the TAIli-buffer forms white, hydrophobic crystals which prwipit.atc from t.hc aqueous buffer. Portions of the inner membrane adhere to t,he hydrophobic surface of the cryst’als and the ent,ire complex sediments on t,he gradient t,o the interface between 15 and 40% sucrow. In t,he absence of t,he deWgent., t,he inner membranes remain at t,he top of the gradient at, the low speed of centrifugation used with these gradient,s. Quantitative sediment,ation of the crystals into a sharp band at, the 1540% sucrose interface is not, dependent upon the presence of inner membranes and is independent, of caentrifugal forces in the range of 2,500Z5,OOO~.Any particulate debris sediment#s through the 40 % sucrose cushion to t,he bottom of t,hc t,ube and remains as an undist,urbcd pellet during the fract8ionation of the gradient. Figure 1 shows the profile of a
Bottom
FIG. 1. -42~4~~profile of a discontinuous sucrose gradient used for preparation of the MDR band from normal rat liver mitochondrial inner membranes. Inner membranes (6 mg protein) were layered with Sarkosyl on the discontinuous sucrose gradient and the MDR band was isolated by centrifugat,ion at 23,OOOg for 15 min. Fractions (1.5 ml) were collected from the bottom of the tube and were warmed to 45°C prior to reading in a Beckman DB Spectrophotometer at A z6dnrnagainst the corresponding fractions of a duplicate tube which contained no inner membranes.
ANT)
KALF
typical discontinuous grad&t, in which thr: MDR band was easily collectBed as IL 15ml fract#ion which contained the whit,e Mg2+Sarkoxyl crystals. The crystals were dissolved by httating at 40°C and the presewe of inner mentbranc mntcrial was monitored by measuring t’hc absorption at’ 264 rm. However, mwh of the d 2sl,,-absorbing material remained at) t’he top of the gradient. Characterixafion of the d fDR cumple.~:. The observat.ions by >I. Kass that rnfbrnrane association of mitochondrial DNA occurred more frequently in rapidly growing cells (J, cells and ascitcs tumor cells) cwmpared to normal adult, tissues such as rut, and chicken liver (31) suggested a possible functional role for membrane attarhmc,nt of DKA and led us to the isolat’ion of an MDR complex from regenerating rat, live. Most of the ctxperiments to bc discussed were performed 1vit.h Z-hr regcnrr;tt.ing rat, liver since previously published st’udies b> S. Nass (32) indicated that the rate of sgrlt,hesis of both mitochondrial and nuclear DNA iu viva reached a rnaximum 20-24 hr after partial hepatcctomy. Inner mitochondrial membranes from 22. hr regenerating rat liver were incubated wit’h [14C]dATP and [“H]UTP ,Zj/ ~jitw and an MDR comples was prepared which was found to cont,ain inner membrane prot tin and a large portion of the total lab&d DXh and RNA of the Sarkosyl lysatc. Figure 2 presents a typical sucrose gradient profile of this MDR complex. Th(l profiles for prot,cin, labeled DNA and RXA show coincident peaks at, the position of tht> Llg”+-Sarkosyl crystals. To show that, the labehbd DNA and RXA precursors were incorporated int,o int,ernuclcot,ide linkage in th(lir rwpcctivc mitochondrial polymers, experiments ww: carried out in which MDR, complexes ww: isolated from inner membrane-matrix prcpnrations appropriately labeled in t,he presence and absence of low levels of cthidium bromide, a specific inhibitor of nucltG acid SJW thesis in mitochondria. Ethidium bromide (2 pg/mg mitochondrial prot,ein) inhibited t,he incorporatjion of [14C]dATl’ int#o the DNA of the complex by hO% and t.hc incorporat,ion of [3H]UTP into thtt RNA b> grcatw than 80 %.
MITOCHONDRIAL
iLIEMBBANE-DNA-ILNI 7
Bottom
hact,m
No.
TOP
FIG. 2. Isolation of au JlDI1 complex from 22-1~ regenerating rat liver mitochondrial inner membranes. Inner membranes (2 nrg per ml) were incubated for 40 min at 37°C in vitro iu a mixture containing per ml: Tris-HCl buffer, pH 7.4 (50 rmoles) ; MgClz (10 pmoles), PEP (5.25 *moles), pyruvate kinase (10 figj, 2-mercaptoethanol (5 @moles), KC1 (150 wmoles), three unlabeled dNTP’s and NTP’s (15 nmolcs each), and both [WI-dATP (10 &i per 15 nmoles) and [WI-UTP (1 pCi per 15 nmoles). The inner membranes were isolated from the incubation medium by cent,rifugat.ion at 12,500~ for 10 min and suspended in 8.5’& sucrose-T?VIK buffer at a concentjrat,ion of 10 mg protein per 3 ml. hliquots of 3 ml were layered with Sarkosyl ou discontinuoits sucrose gradients and the RIDR band was isolat,ed by centrifugation at 23,OOOg for 15 min. The acidinsoluble material iu the 2.ml fractions was precipitated with TCA at a final concentration were washed for of 5’76, and the precipitates protein determinations (Lowry- et al.) and duallabel (W and “I~) analysis as described in Methods.
I’rofiles similar to t how prcsonted in I’ig. 2 also were obt,:tincd when Surkos~-1 lysates of labeled normal rat. liver mit ochondrial inner membranes were centrifuged on disconGnuous sucrose gradients. Treatment, of the individual fract,ions of duplicate gradients containing t,he MDR complex from normal rat liver mitochondria with Di\;nse and RNase, respectively, converted the bulk of the labeled DXA and RSA into an acidsoluble form, t,hus confirming t,hat# the labcled precursor wts in internucleot ide linkage in tjhc respective polymers. Analysis of ;IIDR bands from inner membrancs Iabclcd with [“H]lcwinc i/r ~ifw (Fig.
4’“) -,
COMPLEX
3) indicated that a substantial amount of the “H-labeled protein appeared in the band. Furthermore, a comparison of the specific a&vitg of the protein in the gradient, fractions indicated that the protein of highest specific* radioactivity appeared in the band (not showy in Iig. 3). l’uromycin and especially C’Al suppressed the incorporation (Fig. 3). The possibility existed that, lysatcs of inner membranes c~ontained labeled ma terial not int~rinsically bound to the MDR complex, but found in t’he band because it, sedimcnt,cd t8hrough t’he 15 (;c sucrose and was retained by the 40% cushion. Therefore, it, was necessary to demonst#rate the lack of such material by showing the distribution of protein and labcled material in linear sucrose grad&us. Inner membranes were incubated separately with [3H]TTP and raH]UTP, and tht I\IDR complex prcparcd on a 15-40 7;) limar sucrose gradient. Representative profiles for protSrin, labeled DNA and RX:\, showed coincident, peaks at’ t’he position of
Boltam
iract1on NO.
Toll
FIG. 3. The cf’fect of puromyriii and chloramphenicol ou [3H]-leucine incorporation by rat, liver mitochondrial iuuer membraues irl Gtro. The staudard incubation mixture contained per ml: inner membranes (2 mg protein), Tris-HCI buffer, pH 7.4 (50 pmoles); MgCl2 (5 pmoles), PEP (3.75 pmoles), py-ruvate kinase (10 pg), ATP (2 pmoles), EDTA (2 hmoles), KC1 (50 rmoles), and [31-I]-leucine (1 PCi per 4 nmoles). Puromycin (150 rg per ml) and CAP (100 pg per ml) were added to separate, additional incubation mixtures. After incubating 40 min at 37” the inner membranes were collected by centrifugation and MDR bands were prepared as described in the legend to Fig. 2. Fractions (2 ml) were collected and the acid-insoluble material was analyzed for radioact.ivity as described in Methods.
430
VAN TUYLE
that Mg2+-Sarkosyl sharply
at
30
5% sIlcrosc.
crystals No
hXD
which banded 0th
p&s
KALF
‘T----n
n-PIT
6
17I 9
obscrvcd in the region of tlw gradicwt b+ twm 15 and 30 7:’ sucrose.
not, bc isolated from Sarkosyl lysabes of inner membranes in t’h(L absence of Mg2+ ions. Entrainment studies JV(W carried out in whicah suspensions of native and denatured calf t,hymus DNA, bovine liver tRNA, yeast bulk RNA, and a mixture of rat liwr mitochondrial DNA and RX\T;Z-all in TJII
1 Bottom
rrac,,on
NO.
Toll
FIG. 4. The degree of entrainment. of [W-UK;A into the MDR band by unlabeled mitochondrial inner membranes. 9 3-ml suspension of unlabeled mitochondrial inner membranes (10 mg protein) in S.57csucrose-TMK buffer containing 26 ~g of X. coli [14C]-DXA (specific radioactivity, 0.15 PC’i per mg) was layered wit.h Sarkosyl on a discontinuous sucrose gradient. The MDIL band was isolated by cent,rifugation at 23,OOOgand the gradient was collected in 5-ml fractions except for the MDR band which was collected as a simple Z-ml fraction. The acid-insoluble material in the fractions was precipitated with TC,4 at a final conce,Itration of 5%,, and the precipitates were washed for protein determination (Lowry el a!.) and ‘*C analysis as described in Methods.
with Sarkosyl on second gradient8s. In a typexperiment, digest’ion wit,h pronnw for 1 and 2 hr, rrspectively, released ‘B.5 and 3s % of the lab&d DNA, and virtually all (91-100 5%)of the rclessrd DNA n-as rwovered as TCA-insoluble matwial at thcb top of the gradient. Other rebanding experiments ww JNTformed to determine whether t,he RKh was present in the XDR complex by virtue of its attachment to DNA. Two l\IDR compk~xes were prepared, one of which was labeled in DKA with [“H]TMP and the ot8her was labeled in RNA wit,h [3H]UhlP. The lab&d complexes were treated m&h RSnsci-free DIVase (13 pg per ml for 1 hr at’ 86°C) and rebanded as described in the pronase c>xperimerits. Controls which contained no added DNase were treated in a similar manner. DBase digestion removed approximately 44 5%of the DNA and 62 o/cof the RKA, suggesting that the RNA might’ be bound to the complex through its physical attachment, to DSA.
id
Complexes frou~ Xomal am! 2R-Hr RecJe7leratiwj Rat TiiEer Xifochh-ia Table II compares the proportions of the various component,s of t’he 1\1DR bands prepared from normal and 22-hr regenerating rat, liver mit80chondrinl inrler membranes. The percentage of tot,al protein in t.hc bands from 2%hr regencrat,ing liver approximated the amount8 in normal bands, but, the pcrcentagc of labeled DKA in the bands 22 hr after part,ial hepatectomy was consistently 34 tdmes higher than in normal livers; valucs as high as 6.i ‘4 wre obtained. However, t,he specific aativit,y (dpm per mg) of the DNA in the band was idcnt,ical to that, of the DXA in the top fractions. The proport’ion of total labeled RISA found in the MDR complex from normal and regenerating liver mitochondria was variable bet wecn cxperiment.s (Table II), but, duplicates or triplicat,es wit,hin any single experim& agreed within &tj % of each other. hkperiments to Demolrstrate a Possible Fwctional Role for the XDR-Complex In order to prow that t.hc increased percentage of DNA present’ in the hIDR complex from 2%hr regenerating liver might, be fun&onally related to the active proliferation of t,he tissue, two types of expcrimrnts were carried out,. JIDR complexes wrc prcpared from sham-opcrat.cd animals and t hc perccnt,ages of the various components found TABLE
II
Percent content
of total lysate” in MDR bands 30% Partial hepatectomy P7
Prot.ein(Lowry) : 13-16 DNA ([$H]TTP, dpm) or 16-21 (Ceriotti indole) : RNA (13H]UTP, dpm) , 22-34
14-24 50-65 15-75
21 18.5
i
34
o The Sarkosyl lysate of inner membranes which is layered on top of the gradient. b The ranges given are for five or more experimen&.
in t ho band wore idcntiwl to t,hose found t’or normal liwr \vit hin thr ranges reported in Table II. Bucher (33) has demonstrat’ed t,hat the iwrcase in DKA synthesis in liver subycquent to pnrtial hcpatwtomy is a function of t hc mass of the liver rcmovcd in the opwation; wmoval of 130% of the totSal liwr yklds only a barely diswrnible inc*nt:tsc in nwlwr Di?A synthwis, and \vith removal of 10 % of the t&w the rwponw is one-third of that, observed aftar a GS‘2 hepat,wtomy. Thwefor to determine whether the high perwnt,age of labeled DSA prcs& in the ;\IDlt c*ompl(hxaftw (is ‘Z. hepatectomy \vas directly related to tht> h!p(>rplasia of the, wmnining rclls, partial hr~patwtomics wrc pc~rformc~d in which onlg the left lateral lobe n-:w wmoved (approximatclly 30 “: of the total liwr mass). Under thew conditions, t ho wmnining lobes should ret nin t hc growth ch:~ra(:t(lrist its of normal liver (33). Twent,y-two hours after t,he operat8ion, inner mcmbrarw ww prepared from tho mitochondria of th(l remaining lobes and labeled with [3H]TTP itr ~‘tr.0 in thr presence and abscnw of c+hidium bromidc and the I\IDR complcxcs \VNY~ prcpared. The MDK band labeled in the’ absence of ct,hidium bromide contaiwd IS.,5 5& (Table II) of the labeled DSA of tht> l>.satt>, whit-h was cyuivalrnt to the mwn V:LILWof DNA in thci 11DR complex from normal liwr. The pcrcentagc of labelrd RNA in the 3iDR band also was similar to that’ found for normal liver mitochondria. The inwrporation of radioactvity into both DKA and RNA was significantly inhibited b\- the ct hidium bromide. The prows of restoration of the liver weight, to kc preopcrativc value owns during a “l-day period (30). Thewfore, to obtain further evidence for a functional rc$tionship between the perwntagc of DS.4 att’ached to the mcmbranr in the ctompl~~sand t hc degree of ccl1 proliferation, cxperimcnt8s wre carried out to determine the pcrwnt.age of DNA in the c+omplcx at int’crvals of time up to 3 weeks after partial hepat.ectomy. These data arc presented in Fig, ;j and show that the prrccntage of lab&d DNA present, in the complex rises without, any apparent timct lag from a normal value of approximat(4y 20 % prior to partial hepatwtomy to
432
VAN
TUYLE
about 55 % at, 22 hr. The amount of labeled DNA dropped to 40 % 44 hr after the operation, but by 72 hr it had again approached the maximal value. It is of considerable int,erest that 21 days after the operation (the point at which the liver mass is restored) the percent,age of DNA in the band returned to preoperative levels. DISCUSSION
A membrane DNA-RNA complex has been isolated from Sarkosyl lysates of inner membrane matrix preparations from normal and regenerating rat, liver mitochondria. Incubation of the inner membrane matrix preparation with the appropriate radioactively labeled precursor in vitro under conditions which support nucleic acid and protein biosynthesis in mitochondria results in t,he labeling of t,he appropriate component of the complex. Att’achment of DNA or RNA to the Mg*+-Sarkosyl crystals is not the result of occlusion during t’he formation of the cryst,als for entrainment st.udies and rebanding experiments suggest that only membrane fragments complex with Mg2+-Sarkosyl crystals (Fig. 4), and that these fragments are drawn through the gradient at a rate commensurate with the properties of t,he Mg2+-Sarkosyl crystals themselves. DXA is found in t,he band by virtue of it,s specific attachmentj to the membrane fragment; and RNA! in turn, appears to be bound to the DNA. Evidence supporting this sequential attachment was obtained from the facts that RWase-free DNase released 44 % of t.he DNA and 62% of the RNA from the complex, lvhereas RNase released radioactivity only from RNA. Furthermore, digestion of the membrane fragment of the complex with DNascl-free pronase released acid-insoluble, labeled DNA (38 %, 2 hr) from t’he complex into t,hc t,op fract.ions. RIDR complexes isolated from inner membranes incubated wit’h [3H]leucine in vitro contain labeled protein in the membrane fragment, of the complex. The inhibition of incorporation of [3H]leucine into the complex by the mitochondrial inhibit,or CAP (Fig. 3) suggest)s that the [3H]leucine was incorporated into peptide linkage in the proteins of the membrane fragment. It has been well documcntjed that bot’h intact mitochondrin
AND
KALF
(34) and inner membrane-mat,rix preparations incorporat’e [3H]leucine predominant,ly into the insoluble proteins of the inner mitochondrial membrane, and this incorporation is inhibited by puromycin and CAP (11, 35). The MDR complex from normal adult. rat liver mitochondria was comprised of a small piece of inner membrane c:ont,aining 14 % of the total protein of the lysate to which was attached 20% of t,he total DNA of the organelle. The remainder of the DNA was located at the t,op of t,he gradient in acid-precipitable form, suggesting that only one out of possible five DNA copies in each organelle was attached to the membrane irz vivo. The percent’age of total DNA found in t’he complex from 22-hr regenerating mitochondria was three to four times higher than in t.he normal complex, whereas the percentage of protein is approximately the same in both cases. An increased binding of DNA t,o the inner mitochondrial membrane would be expected in act,ively proliferating tissues if t,he attaehment of D,NA to membrane is of functional significance in the replication and/or segrcgation of DNA during biogenesis of the organelle. Preliminary experiments in our laboratory have demonstrated that a similar RIDR complex can bc isolated from another actively proliferating tissue, fetal rat liver. The percentage of DNA at,tached to t#he membrane fragments of Dhecomplex from 19day fetal rat, liver mitjochondria was found to be 63 % of the total DlSA of the lysate. (Van Tuyle and Kalf, unpublished observat,ions) _ This is the same pcrcent.age as found in t,hc complex frotn 22-hr regenerating rat, liver. It is not known w-h&her the value of ;LP)proximat,cly 60% of the DNA bound to t’he MDR complex from rcgc~ncrating rat’ liver represents the maximal amount of DNA :wtually bound to the inner membranes of the mitochondria in t,he total population of dividing cells. However, part’ial hepatectomy is followed by a period of synchronization of the hcpatocytes such that 60% of t,he cells will ent’er mitosis (36). The pcrcent’age of D,NA in the band appears t.o vary, depending upon the length of regeneration time (Fig. 5). For example, the percentage of DNA in t,he band increases to a maximum during the period of maximal nuclear DNA synthesis
FIG. 5. The percentage of labeled DNA in the MDlt band as a function of the time after hepateccorny. Regerating rat livers were removed 11, 22, 46, 70, and 504 hr after partial hepatectomy and the mitochondrial inner membranes were radioactively labeled in the standard incubation protocol as described in the legend to Fig. 2 with [W]-TTP at a level of 10 PCi per 15 nmoles. The MDR bands were prepared and analyzed for radioactivit,y as described in Methods. The values designated represent the percentage of t,otal acidinsoluble radioactivity found in the band as compared to the total radioactivity recovered in the entire gradient. The values are reported as mean percentages with average deviations from four or more experiments with the exception of the Sweek value which was obtained from the average of triplicat,es in a single experiment. The perrcnt,aze of labeled DNA in MDR bands from normal, adult rat liver mitochondria is shown as “zero hours after partial hepatectomy.”
prior to mitosis at 28-X hr, t’hen decreases slight.ly at 46 hr after hepatectomy, but, again increases at TO hr presumably conwmit~ant~ with a second wave of nuclear I>SA synt,hcsis which is lmon-n Taooccur in n-wnling rats undergoing liver regcncration :md also in synchronized cultures of mammalian wlls (36). The period of rapid growth which (wsues aft’cr part’ial hepatectomy abates in approximately 21 days, at’ which time the t’otal liver mass has been rcstorcd. As notJeclhere, the pewentage of DNA found in the complex 21 days post,hepatectomy has also returned to the normal level. Furthermore, neit,hcr sham-operated rats nor anmals subjcct#ed to 30 ?I hepatect,omy showed an incwased pcrcent,age of DNA in the complex (Table II). 1litocshondria, prepared as described in Methods, contain minimal eont,aminat,ion by bacteria (3, 16) and t hc prcsenw of thrsc
b:wt (CL docwnot sign&ant l\- influence the incorporation of (lither nucl& acid or protcin precursors into the mitochondrial suspensions. Incrcascd numbers of bacteria, howwr, might havcl bwn present in mitochondrial suspwsions obtained from rcgenerating livrr as a result of the surgical opcrat,ion, and a wmplcx derived from bat*twia might c.ontaminatc the MDR band. Sewral picxcesof evidcncc argue against this. (1) So visible signs of gross bact,crial (‘ontamination were observed in the host animal at the time of pwparxtion of mitochondria. (2) Bacterial cells arc not subject to lysis by Sarkosyl urlless the ~11 \\-a11has been r(wdcred fragile by t,reatment wit,h EDTA and lysozyme. (3) Sham-operat,ed animals showed no increase in t’he amount of con~pl~~x obt)ained or in the percentage of various constituent,s found in t,he complex. (4). The amount of complex and pcrwntage of DSA in the band returned to normal levels in animals 21 days after part’ial hc>pat,c:ct’omy. (5) Thch complex, obtaiwd from animals subjertcd to the operation and to the same incrcawd risk of bacterial caontaminatJion by removing one lobe of t,ht>liver, contairwd cxactly the perccntagc of mcmbrann-bound DKA as normal complexes. Thus, the MDR complex isolated from rat liver mitochondria appwrs to reprewnt :1 small fragment of the inner mc>mbranctwhic*h contains an att~achmcnt site(s) for mitochondrial DNA. RN\‘,4 transcripts appear to bc bound to the DKA of t’he complex on thrk basis of their relcaw by RKasc-free DSaw. Whether t,he complex is biologically meanful is unknown. Howewr, a possible fruwtional significance for the at8tachmcnt, of the DNA to t#hc mrmbrann i? suggestcld by the 3- to -l-fold irwrcnw in the at~t~:rcl~n~~ntof DNA to the membrane component of the complexes deriwd from mitochondri:t of activel!- prolifemting tissues such as f(ktal and 22-hr regenerating rat liver. The fact. that neither sham operation nor 30 ‘Z partial hcpatcct,omy rcs&s in incwascd attachment over that found in normal liver mitochondria also lends support, to t’his point. It is of intorcst, in view of the developing bacteria-mit,ocshondrial homology, t,hat, the MDR complex from rat, livrr mito~hondria rawmblcs similar cwmplcxcs isolatcld from
484 several bxterial (14).
VAN
TIJYLR
specks by t,he sarnc’ mclthod REFEIII~NCJSS
1. Nass, M. M. K. (1969) Science 166, 25. 2. k\BINO\VITZ, &I., AND S\\'IPT, H. (1970) Phlysiol. Rev. 60, 376. 3. KALF, (+. F., am CH’IH, .J. J. (1968) J. Niol. Chem. 243, 4904. 4. MEYER, 1~. R., AND SIMPSON, M. V. (1968~ Proc. :\;at. Acad. Sci. C.S.A. 61, 130. 5. &IEYER, It. R., AND SIhCPdON, M. v. (1970) J. Biol. Chem. 245, 34. 6. WINTERSUERGER, E. (1970) Biochem. Biophys. Res. Commun. 40, 1179. 7. KUNTZEL, H., AND S~H.\FER, K. (1971) ,Vutzue ,Vew Biol. 231, 265. 8. TSAI, M., MICHAELIS, (+., .\ND GRIDDLE, R. S. (1971) Proc. Xat. Acad. Sci. 7T.S.A. 68, 473. 9. REID, B. D., I\ND PARSONS, I'. (1971) Proc. *Vat. Acad. Sci. L:.S.A. 68, 2830. 10. SCHULTZ, S. Ii.., AND Nass, S. (1969) Pd. Eur. Bidl. Sot. Lett., 4, 13. 11. KALF, G. F., AND FAUST, A. S. (1969) Arch. Biochenz. Biophys. 134, 103. 12. WINTERSBERGER,E.,.\NDWINTERSBER(:ER,U. (1970) Fed. Eur. Biol. Sot. Lett. 6, 58. 13. NASS, 8. (1969) Int. Rev. Cutol. 26, 117. 14. TREMUIIAY, G. Y., DANIELS, M. J., AND SCHAECHTER, M. (1969) J. Mol. Biol. 40, 65. 15. EARHART, C. I?. (1970) Virology 42,429. 16. O'BRIEN, T. W., .\XD KALF, G. F. (lQ67) b. Biol. Chem. 242, 2172. 17. SCHNAITMAA-, C. V., J:II\VIN, G., AND (&EEXASALT, J. W. (1967) J. Cell Viol. 32, 719. 18. QCHN.UTILZ.IN, C., ASD GREENAMTALT, J. W. (1968) J. Cell. Hiol. 38, 158.
AND
KALF
19. ROIUNSON, II. W., AND HO~DEX, C. G. (1940) J. Biol. Chem. 136, 727. 20. r,o\\.~