STUDIES ON THE BIOGENESIS OF SMOOTH ENDOPLASMIC RETICULUM MEMBRANES IN RATS

STUDIES ON THE BIOGENESIS OF SMOOTH ENDOPLASMIC RETICULUM MEMBRANES IN HEPATOCYTES OF PHENOBARBITAL-TREATED RATS II. The Site f Phsphlipid Synthesis in the Initial Phase f Membrane Prliferatin JOAN A. HIGGINS Frm the Department f Anatmy, Yale Schl f Medicine, New Haven, Cnnecticut 06510 ABSTRACT The specific activity f the acyltransferases f smth micrsmes f rat liver rse threefld by 12 h after injectin f phenbarbital, while the activity f the acyltransferases f the rugh micrsmes rse slightly t peak at 3-4 h, and subsequently fell. The latter rise was ablished by treatment f the animal with actinmycin D r purmycin, while that f the smth micrsmes was unaffected. Incrpratin f [14C]glycerl int phsphlipid f smth micrsmes was elevated 100% by phenbarbital, while that f the rugh micrsmes was elevated 15%, and this culd be accunted fr by exchange between the micrsmal phsphlipids. The phsphlipid/prtein rati f the smth micrsmes rse 1.5 times 3-4 h after injectin f phenbarbital, while that f the rugh micrsmes fell slightly. The specific activity f NADPH cytchrme c reductase and NADPH diaphrase rse first in the rugh micrsmes, and subsequently in the smth micrsmes at a time cinciding with the return f the phsphlipid/prtein rati t the cntrl level. The rise in phsphlipid/prtein rati was unaffected by actinmycin D r purmycin. These results indicate that the prliferating smth membranes are the site f phsphlipid synthesis, and that the phsphlipid/prtein rati f these membranes may change independently. INTRODUCTION In respnse t intraperitneal injectins f phenbarbital, there is a marked prliferatin f smth endplasmic reticulum membranes f rat hepatcytes (1-5). This prliferatin is accmpanied by an increase in the specific activity f the enzymes invlved in drug detxificatin (2, 4, 6 9) and by an increase in the phsphlipid cntent f the liver, presumably due t the frmatin f new membranes (2, 4, 7, 10). This tissue system, therefre, presents a useful and easily cntrlled ne in which t study the bigenesis f smth endplasmic reticulum membranes. Previus cytchemical studies frm this labratry have indicated that the acyltransferases invlved in the first steps f synthesis f phsphlipid during new membrane frmatin are assciated with the smth endplasmic reticulum in the phenbarbital-treated rat, while in the untreated rat liver these enzymes are THE JOURNAL OF CELL BIOLOGY VOLUME 62, 1974 - pages 635 646 635

assciated mainly with the rugh endplasmic reticulum (11). Parallel investigatins f the specific activity f the acyltransferases f rugh and smth micrsmes cnfirmed the cytchemical bservatins. On injectin f phenbarbital, the specific activity f these enzymes rse rapidly in the smth micrsmes, while that in the rugh micrsmes remained relatively cnstant fr 12 h and then fell t a lw level. These results were interpreted as indicating that the phsphlipid cmpnents f the newly synthesized smth endplasmic reticulum membrane are synthesized in situ and that the prtein cmpnent, which is prbably synthesized by bund ribsmes (7, 12-14), is incrprated int the smth membranes after phsphlipid synthesis ccurs. On the basis f this cnclusin, it appeared prbable that the early times after injectin f phenbarbital, when the specific activity f the acyltransferases rises mst rapidly in the smth membranes, might be apprpriate fr a detailed investigatin f the rate f incrpratin f membrane cmpnents. If cmpnents are added separately, r ut f synchrny, these differences pssibly may be detected during the first 12 18 h after injectin f phenbarbital, befre membrane prliferatin becmes well established. In the present reprt, lipid, prtein, and enzymic cmpnents f the membranes have been investigated. MATERIALS AND METHODS Islatin f Liver Micrsmes Ttal, rugh, and smth micrsmes were prepared as described previusly (11) frm livers f adult male albin rats (200-300 g bdy wt), either untreated, r after a single intraperitneal injectin f phenbarbital (11 mg per 100 g bdy wt). As diurnal rhythms have been demnstrated in the enzymes f the smth endplasmic reticulum and in the quantities f these membranes (15), rats were sacrificed during the quiescent perid frm 9:00 t 4:00 pm and given injectins f phenbarbital at apprpriate times befre this. Fr acyltransferase assay, micrsmes remved frm the gradient were washed nce by resuspensin in 0.25 M sucrse and recentrifugatin (105,000 g, 45 min) in the case f rugh micrsmes, and by centrifugatin (105,000 g, 45 rain) f the fractin remved frm the gradient after dilutin with distilled water 1:5, vl/vl, in the case f smth micrsmes. Fr phsphlipid/ prtein determinatins, the ttal, rugh, and smth micrsmes were washed fr a secnd time by resuspensin in 0.25 M sucrse and recentrifugatin (105,000 g, 45 rain). Assay f A cyhransferases Unless therwise stated in the text, micrsmes (0.3-3.0 mg prtein) were incubated in media cntaining sn [14C]glycerl-3-phsphate (1.8 ram) (Internatinal Chemical and Nuclear Crpratin, Burbank, Calif.) and palmityl CA (75 um) (Sigma Chemical C., St. Luis, M.) in cacdylate buffer, ph 7.2, cntaining 4.5% dextrse, at a final vlume f 1.0 ml, at 37 C fr a range f times. Cacdylate buffer was used in these experiments t maintain the cnditins f parallel cytchemical investigatins (1 I). Hwever, preliminary experiments were perfrmed t cmpare this buffer with Tris-HCl and phsphate buffers at the same ph. The same results were btained in all cases. At the end f the incubatin perid, the reactin was stpped by additin f ice-cld chlrfrm-methanl (2: I, vl/vl) and the lipids were extracted by the methd f Flch, Lees, and Slane Stanley (16). Aliquts f the ttal lipid extract were cunted in a Packard Tricarb scintillatin cunter (Packard Instrument C., Inc., Dwners Grve, Ill.). In sme experiments the lipids were separated in thin layers f silica gel G as described previusly (11). Assay f NA DPH Cytchrme c Reductase and NA DPH Diaphrase The specific activities f NADPH cytchrme c reductase and NADPH diaphrase f micrsme fractins were determined by the methd f Ernster, Siekeritz, and Palade (17), by the spectrphtmetric determinatin f the appearance f reduced cytchrme c at 550 rim, and the disappearance f the absrptin f 2-6 dichlrindphenl at 600 nm, respectively. Inhibitin f Prtein Synthesis Prtein synthesis was inhibited in viv by intraperitneal injectin f actinmycin D (Sigma Chemical C.) r purmycin (Sigma Chemical C., r Nutritinal Bichemicals Crp., Cleveland, Ohi), and the extent f inhibitin expressed as inhibitin f incrpratin f [3H]leucine (2 mm sp act 50 Ci/mml, Internatinal Chemical and Nuclear Crpratin) int trichlracetic acid-insluble material. In ne series f experiments, the inhibitr (actinmycin D, 20 ~g/100 g bdy wt r purmycin 2 rag/100 g bdy wt) was injected at hurly intervals fr 2 h befre and 3 h after injectin f phenbarbital (11 mg/100 g bdy wt) tgether with [3H]leucine (5 ~Ci, 0.1 nml). The rats were then sacrificed at 4.5 h after phenbarbital injectin. In a secnd series f experiments the inhibitr (actinmycin D, 60 #g/100 g bdy wt, r purmycin 10 mg/100 bdy wt) was injected at the same time as phenbarbital (11 rag/100 bdy wt) and [3H]leucine. These rats were sacrificed 12 h after injectin. In bth series f experiments, the livers were remved and hmgenized in distilled water. Aliquts were taken fr prtein determi- 636 THE JOURNAL OF CELL BIOLOGY. VOLUME 62, 1974

natin, and ther aliquts were precipitated with trichlracetic acid (15%) and the precipitate was islated by centrifugatin, washed twice with trichlracetic acid (15%), nce with ether, slubilized in Prtsl (New England Nuclear), and cunted in a Packard TriCarb scintillatin cunter. In experiments in which the effects f inhibitrs n the acyltransferase activity r the phsphlipid/prtein ratis f micrsmal fractins were investigated, injectin f [~H]leucine was mitted and micrsmal fractins were islated frm the livers, as described abve, at the end f the treatment with prtein inhibitr and phenbarbital. Incrpratin f [14C] Glycerl int Phsphlipids [14C]Glycerl (2.5 ~Ci/100 g bdy wt) was injected intraperitneally int animals either untreated r treated with phenbarbital as abve. After a predetermined time interval the livers were remved and pieces (apprximately 1 g) were hmgenized immediately in chlrfrm-methanl, 2: 1, t extract the lipids, r the whle liver was hmgenized in ice-cld 0.25 M sucrse and the ttal, rugh, and smth micrsmes were islated. The lipids were extracted frm all fractins and separated int the majr lipid classes as described previusly (11), and the specific activities f the phsphlipid and the triglyceride were determined. Assays Prtein was assayed by the methd f Lwry et al. (18) using crystalline bvine serum albumin as a standard. Phsphlipid was determined as rganic phsphrus by the methd f Fiske and Subbarw (19), and triglyceride by the methd f Snyder and Stephens (20). RESULTS Kinetics f Acyhransferase Activity Incrpratin f [~C]glycerl-3-phsphate int lipid by micrsmes was linear fr shrt perids f time (1 5 min) and this was dependent n the micrsmal prtein cncentratin. A plateau f incrpratin was reached mre rapidly with higher prtein cncentratin. Optimum activity ccurred at a 75 #M palmityl CA with 1 mg f micrsmal prtein; cncentratins f palmityl CA abve this caused inhibitin. This has been reprted by thers in the case f a number f enzymes in additin t acyltransferases (21 26), and appears t be related t the detergent actin f palmityl CA abve its critical micellar cncentratin. Because f the cmplex kinetic characteris- tics acyltransferase activity was determined in each micrsmal fractin under fixed cnditins. Three micrsmal prtein cncentratins abut 1 mg were used and at least three incubatin times (1 5 min). In this way the incrpratin f glycerl- 3-phsphate int lipid at cncentratins f micrsmal prtein f 1 mg and 75 #M palmityl CA was determined under cnditins in which incrpratin was linear with time. Under all cnditins, when the labeled lipids were separated int the majr lipid fractins, all f the label was in the phsphlipid fractin at all times and all tissue cncentratins used, and n separatin f the phsphlipids, ver 90% f the label was in the phsphatidic acid fractin while the remainder was at the rigin in the thin-layer chrmatgraphy system used and pssibly crrespnds t lysphsphatidic acid. Tw acylatin steps ccur under these cnditins, therefre, and the first acylatin is apparently rate limiting, as lysphsphatidic acid des nt accumulate t any significant extent. This has been fund by thers in the case f partially purified slubilized enzymes f rat brain (27), rat liver micrsmes (21, 28, 29), and pigen liver micrsmes (30). Lands and Hart (29) demnstrated that the acylatin f l-mnacyl glycerl-3-phsphate is mre rapid than that f glycerl-3-phsphate, supprting the cnclusin that the first acylatin step is rate limiting. Changes in Specific Activities f Acyhransferases f Liver Micrsmes in Respnse t Treatment with Phenbarbital The specific activity f the acyltransferases f rugh micrsmes f untreated rats was slightly higher than that f smth micrsmes (Fig. I). After injectin f phenbarbital, the specific activity Of the smth micrsmes rse rapidly t peak at 12 h after injectin at a level apprximately three times that f smth micrsmes f untreated rats. The specific activity f the rugh micrsmes rse slightly between 1.5 and 4.5 h after injectin and then fell t a level belw that f the untreated micrsmes. This rise, althugh small, was statistically significantly (p < 1% at 1.5 h). The specific activity f ttal micrsmes rse, paralleling that f smth micrsmes with values intermediate between thse f rugh and smth, but clser t the latter in mst cases. Hwever, the methd used fr islatin f rugh and smth micrsmes des nt result in a cmplete yield f J. A. HIGGINS Bigenesis f Smth Endplasmic Reticulum Membranes 637

.E ~3 c~ 3 c 2 I l l l l l l l l l I I I I I I I I I I ROUGH MICROSOMES I I I I l l l t l l l l l l l l l l l ~ ~ MICROSOMES t S M O O T H prprtin f the rugh micrsmal fractin is lst cmpared with the smth micrsmal fractin. The specific activity f acyltransferases is expressed cnventinally in terms f prtein. Hwever, in such a system in which the micrsmal cmpnents are changing it is difficult t select a standard fr the expressin f enzyme activity. One alternative t prtein is phsphlipid. The phsphlipid/prtein rati f smth micrsmes rises t peak at 3.0-4.5 h after injectin f phenbarbital, while that f the rugh shws a slight fall (Fig. 5). Therefre acyltransferase activity expressed in terms f phsphlipid shws essentially the same pattern as that in terms f prtein in the rugh micrsmes, while the activity f the smth micrsmes rises mre slwly fr 0 4.5 h but reaches a similar peak at 12 18 h after injectin f phenbarbital. I I I J l l l L I I l i l l l l l l l 0 2 4 6 8 0 12 14 16 16 hurs fter injectin ~ phenbrbitl FIGURE 1 Changes in specific activity f acyltransferases f ttal, rugh, and smth liver micrsmes in respnse t treatment f rats with phenbarbital. Ttal, rugh, and smth micrsmes (0.75-3.0 mg prtein) prepared frm nrmal and phenbarbital-treated rats (11 mg/100 g injected at 0 h) as described in Materials and Methds were incubated in media cntaining [~4C]glycerl-3-phsphate (1.8 mm) and palmityl CA (75 um) in cacdylate buffer at 37 C fr 1, 2, and 3 min. The reactin was stpped and the lipid extracted as described in Materials and Methds. At least three prtein cncentratins were used fr each assay, and the acyltransferase specific activity at 1.0 mg prtein was determined as described in the text. The specific activity (nml glycerl-3-phsphate incrprated/min/mg prtein) is pltted against time after injectin f phenbarbital. Each pint is the average f determinatins n micrsmes frm at least fur animals. Standard deviatins are indicated by bars. the micrsmal fractin and the ttal micrsmes prepared in this way are prbably nt cmpsed f rugh and smth endplasmic reticulum in the prprtins fund in the intact cell. In all preparatins, the yield f smth micrsmes per liver exceeded the yield f rugh micrsmes, althugh in the untreated rat liver, rugh endplasmic reticulum accunts fr sme tw-thirds f the ttal endplasmic reticulum (5). It is prbable, therefre, that in the initial centrifugatin f the liver hmgenates t remve mitchndria, a large Effect f Phenbarbital n the Incrpratin f [14C]Glycerl int Lipids f Rat Liver and Liver Micrsmal Fractins Acyltransferases catalyze the first steps in the synthesis f glycerphsphlipids and triglycerides. Hwever, the site f the increased activity in respnse t phenbarbital is nt necessarily the site f phsphlipid synthesis, althugh this is prbable. In rder t examine this mre directly we investigated the incrpratin f [14C]glycerl int rat liver and liver micrsmal lipids. Ttal liver phsphlipids became labeled very rapidly after intraperitneal injectin f [14C]glycerl (Fig. 2). In rats treated with phenbarbital, the final specific activity reached was elevated ver the cntrls, indicating that under ur experimental cnditins there is an increased synthesis f ttal liver phsphlipid in respnse t phenbarbital. Exchange f Phsphlipids between Rugh and Smth Liver Micrsmes It has been demnstrated by a number f investigatrs that exchange f phsphlipids ccurs between cell fractins, when these are incubated in vitr. This presents a majr prblem in experiments in which incrpratin f istpes int phsphlipid f different fractins is determined. If this exchange is rapid, then any differences between rugh and smth micrsmal fractins at the time f sacrifice f the animal wuld be eliminated during the fairly lng times required t separate rugh and smth mi- 638 THE JOURNAL OF CELL BIOLOGY VOLUME 62, 1974

~ 4OO _~. 300 f u 20 t00 ~ 0 ( untreated/." 0 / I I I I I I I 2 3 4 5 6 7 Time After Injectin f[14c]glycerl (h) FIGURE 2 Incrpratin f [14C]glycerl int phsphlipid f rat liver: effect f phenbarbital. Rats were injected with [~'C]glycerl with and withut phenbarbital and were sacrificed at a series f time intervals after injectin. Apprximately 1 g f liver was remved frm the same lbe in each animal, hmgenized in distilled water, and extracted with chlrfrm-methanl (2: 1). The lipids were separated and the specific activity f the phsphlipid was determined as described in Materials and Methds. Pints pltted are averages f at least three determinatins n three animals. crsmes. We therefre investigated the extent f exchange between micrsmal subfractins during ur experimental prcedures. Bth rugh and smth micrsmes cntaining [l'c]phsphlipids were prepared and added separately t hmgenates f liver. Rugh and smth micrsme fractins were islated in each case and the specific activity f the phsphlipids f these fractins was determined (Table I). In bth cases the specific activity f the added micrsmes was cnsiderably reduced and recveries were lw, indicating cnsiderable exchange between micrsmes and ther fractins, as well as lw yields f micrsmal fractins. When the label was initially in the smth micrsmes the specific activity f the phsphlipids f the islated rugh micrsmes was apprximately 50% that f the smth. Similarly, when the label was initially in the rugh micrsmes the specific activity f the phsphlipids f the smth micrsmes was 25% that f the rugh. Cmplete exchange between fractins des nt ccur therefre, but a significant amunt f label mves frm ne fractin t anther and this must be taken int accunt when cnsidering the results f incrpratin f [l'c]glycerl int micrsme fractins. TABLE I Exchange f Phsphlipids Between Micrsmal Fractins Fractin Original rugh micrsmes Fractins islated frm hmgenates t which labeled rugh micrsmes were added Rugh micrsmes Smth micrsmes Original smth micrsmes Fractins islated frm hmgenates t which labeled smth micrsmes were added Rugh micrsmes Smth micrsmes Specific activity f phsphlipid cpm/taml 375 28.3 9.6 362 23.3 46.2 Rats were injected with [t4c]glycerl (2.5 #Ci/100 g bdy weight) and sacrificed 1 h later, and rugh and smth micrsmes were islated. Lipids were extracted frm aliquts f these and the remainder added separately t hmgenates f liver frm untreated rats. Rugh and smth micrsmes were islated frm these and the lipids extracted. The specific activity f the phsphlipids f all fractins was determined as described in Materials and Methds. Incrpratin f [14C]Glycerl int Lipids f Rugh and Smth Micrsmes [14C]Glycerl was incrprated rapidly int the phsphlipid f bth rugh and smth micrsmes. In bth phenbarbital-treated and untreated rats, incrpratin peaked at apprximately 1 h after injectin. In micrsmes frm untreated animals the specific activity f the rugh micrsmes was higher than that f the smth micrsmes at all times studied, whereas in micrsmes frm phenbarbital-treated rats the specific activity f the smth micrsme phsphlipid was higher than that f the rugh micrsmes (Fig. 3). At 30 min after injectin f phenbarbital the specific activity f the smth micrsmal phsphlipid was 197% that f the same fractin frm untreated rat liver, while the specific activity f the phsphlipid f the rugh micrsmes f phenbarbital-treated rats was 113% that f the rugh micrsmes f the untreated rats. This difference was maintained at later times, althugh this was less marked. At 120 min after phenbarbi- J. A. HIGGINS Bizenesis f Smth Endplasmic Reticulum Membranes 639

tal injectin there was a fall in the specific activity f the phsphlipids f bth rugh and smth micrsmes cmpared with the same fractins frm livers f untreated rats. If exchange ccurs between the phsphlipids f these fractins at a rate cmparable t that in the experiments indicated abve, then the differences between rugh and smth micrsmes wuld be greater at the time f sacrifice f the animal. Hwever, it is extremely difficult t assess the exchange between 6 ~ 50 fractins, as this ccurs cntinuusly, and bth fractins were labeled initially at the time f 3 sacrifice. Nevertheless, these results d indicate :~ 400 that n treatment with phenbarbital there is a = shift in the predminant site f incrpratin f ~ 300 ~k [14C]glycerl int phsphlipid frm rugh mi- crsmes t smth. " z The specific activity f the triglycerides f ; rugh, smth, and ttal micrsmes did nt shw any increase in respnse t phenbarbital but to0 O rather shwed a small decrease at 60 and 120 rain.,~ The specific activity f the triglyceride f the ttal 0 micrsmes was greater than that f bth rugh and smth micrsme fractins, suggesting that there may be mre than ne pl f triglyceride in FIGURE 3 the liver, ne f which was lst frm the ttal.:! micrsmes n subfractinatin (Fig. 4). This pl prbably cnsisted f lipprtein particles r lipid ~ l drplets, many f which flated during the separa- --~ E tin f rugh and smth micrsmes. These 3 c culd remain trapped in the ttal micrsmes, which were subjected t less washing than the subtractins. The triglyceride cntent f rugh and smth micrsmes was lw, and this may represent largely a structural cmpnent f the mem- 400 brane (32). 200 Changes in Phsphlipid/Prtein Ratis f Liver Micrsmes in Respnse t Treatment with Phenbarbital The previus results indicate that the phsphlipids f phenbarbital-induced membranes are synthesized in situ. Reprts frm a number f labratries indicate that prtein is synthesized by bund ribsmes. As the majr cmpnents are synthesized at separate sites, it is pssible that transient differences exist in the rate f incrpratin f these tw majr cmpnents f smth micrsmes. The phsphlipid/prtein ratis f the micrsmal fractins were therefre investigated. This rati was similar in bth rugh and smth micrsmes prepared frm livers f untreated rats (Fig. 5). After injectin f phenbarbital, hwever, there was a rapid rise in the phsphlipid/prtein rati f the smth micrsmes 0 smth micrsmes phenbrbitl A,/ / rugh micrsmes / / ~ phenbarbital / \\ rugh micrsmes I 1 2 Time After Injectin (h) Specific activity f phsphlipid. j ttal micrsmes phenbarbital I/ \\ smth miersmes ~/ ~ untreted smth micrsmes r ~ phenbarbital / ~ ~ rugh micrsmes phenbar bltal. m 0 t 2 FIGURE 4 Time After Injectin (h) Specific activity f triglyceride. FIGURES 3 and 4 Incrpratin f [l'c]glycerl int phsphlipid and triglyceride f rugh, smth, and ttal micrsmes f rat liver; effect f phenbarbital. Rats were injected with [14C]glycerl with and withut phenbarbital and were sacrificed at a series f time intervals after injectin. Livers were remved and rugh, smth and ttal micrsmes islated. Lipids were extracted and separated and the specific activities f the phsphlipid and triglyceride were determined. Pints pltted are averages f at least three determinatins n at least three animals. I 640 THE JOURNAL OF CELL BIOLOGY. VOLUME 62, 1974

. i 1.0 0.8 0.6 1.0 O.S.m cx 0.6 1.0 O.S 0.6 TOTAL MICROSOMES I I I I I I I I I '1 I I I ROUGH MICROSOMES I I I I I I I I I I I I I SMOOTH MICROSOMES I I I I I I I I I I I I I 0 2 4 6 8 I0 12 hurs after injectin f phenbarbital FIGURE 5 Changes in phsphlipid/prtein ratis f ttal, rugh, and smth liver micrsmes in respnse t treatment f rats with phenbarbital. Ttal, rugh, and smth micrsmes prepared as described in Materials and Methds were suspended in cacdylate buffer, and aliquts were used fr assay f prtein and fr extractin f lipids and assay f phsphlipid as described in Materials and Methds. Phsphlipid/prtein ratis are pltted against time after injectin f phenbarbital. Each pint is the average f determinatins n micrsmes frm at least fur animals. Standard deviatins are indicated by bars. t peak at 3.0-4.5 h after injectin at a level apprximately 150% that f the untreated smth micrsmes (p < 0.02%). The phsphlipid/prtein rati f the rugh micrsmes fell slightly during the 12 h f the experiment, while that f the ttal micrsmes rse, paralleling the smth micrsmes, with values intermediate between that f rugh and that f smth. Micrsmal fractins ptentially may cntain secretry r sluble prteins trapped within the vesicles. The existence f such prteins is f cnsiderable significance in studies f phsph- lipid/prtein ratis f membranes. In cntrl experiments, therefre, bth rugh and smth micrsmes frm livers f nrmal and phenbarbitaltreated rats were subjected t smtic shck as described by Glaumann and Dallner (32). Phsphlipid/prtein ratis in this case were nt significantly different frm thse f micrsmes washed in 0.25 M sucrse shwn in Fig. 5. Since the cmpletin f this wrk, an investigatin has been reprted in which rugh micrsmes were subjected t lw cncentratins f dexychlate t release adsrbed and trapped materials (44). Similar prcedures were applied t rugh and smth micrsmal fractins in ur studies. These caused an elevatin in the phsphlipid/prtein ratis f fractins, but the relative differences between rugh and smth micrsmes frm livers f nrmal rats and rats treated with phenbarbital fr 4.5 h and 18 h were maintained. The rise in phsphlipid/prtein rati f the micrsmal membranes was fllwed by a fall 4.5 h after injectin f phenbarbital, while the rise in specific activity f the acyltransferases peaked 12 h after injectin. Increased phsphlipid synthesis ccurs fr a cnsiderable time after injectin f phenbarbital, as smth membrane prliferatin is nt marked until after tw daily injectins f phenbarbital and cntinues during repeated daily injectins. Hwever, sy~athesis f new smth membrane enzymes ccurs at the same time as the membrane prliferatin, and the incrpratin f these prteins int smth membranes is respnsible fr the fall in the phsphlipid/prtein rati, and als fr the later fall in specific activity f the acyltransferases. Changes in the Specific Activity f NADPH Cytchrme c Reductase and NA DPH Diaphrase f Micrsmes in Respnse t Phenbarbital The enzymes which rise in liver micrsmes in respnse t phenbarbital are thse invlved in the detxificatin f drugs. Orrenius (7) has demnstrated that in the early times after injectin f phenbarbital, the specific activities f a number f these enzymes rise in parallel first in the rugh micrsmes, then in the smth. Of these enzymes, NADPH cytchrme c reductase and NADPH diaphrase were selected as markers f increased, prtein synthesis under ur experimental cnditins. In experiments parallel t thse J. A. HIGGINS Bigenesis f Smth Endplasmic Reticulum Membranes 641

in which the specific activity f the acyltransferases and the phsphlipid/prtein ratis were determined, the specific activities f the NADPH cytchrme c reductase rse rapidly in the rugh micrsmes in respnse t phenbarbital and subsequently rse in the smth micrsmes (Fig. 6). Similar changes, althugh less marked, als ccurred in the case f NADPH diaphrase. The time sequence f these changes is cnsistent with increased enzyme incrpratin int the smth membranes, accunting fr the fall in phsphlipid/prtein rati in these membranes at 4.5 h after injectin f phenbarbital. Effect f lnhibitrs f Prtein Synthesis n the Acyltransferase Activity and the Phsphlipid/Prtein Ratis f Micrsme Fractins f Livers f Phenbarbital- Treated Rats Administratin f actinmycin D r purmycin at the same time as phenbarbital resulted in ver 60% inhibitin f prtein synthesis during the 12 h after treatment. When the inhibitrs were administered at hurly intervals and the animals sacrificed at 4.5 h after injectins f phenbarbital, inhibitin f prtein synthesized was greater than 80%. The rise in specific activity f the acyltransferases f smth micrsmes 12 h after injectin f phenbarbital was unaffected by inhibitin f prtein synthesis by either actinmycin D r purmycin (Table III). The specific activity f these enzymes in rugh micrsmes was als unaffected by these inhibitrs, althugh at this time pint the activity was lw cmpared with the untreated cntrl. At 4.5 h after phenbarbital, the specific activity f the acyltransferases f the rugh micrsmes was elevated cmpared with the untreated cntrl (p < 1.0%) (Fig. 1, Table II). Administratin f actinmycin D r purmycin resulted in a fall in this activity (p < 0.2% in each case), indicating that this rise is due t new prtein synthesis and may be blcked at the level f transcriptin r translatin. In cntrast t this, at 4.5 h after phenbarbital, the specific activity f the acyltransferases f the smth micrsmes frm rats treated with actinmycin D r purmycin was elevated cmpared with that f the cntrl treated with phenbarbital alne (p < 0.2% in each case) (Table II). The rise in acyltransferase activity f the smth micrsmes at bth 4.5 and 2.0 ~,. _. 1.2 E E c 0.8 g 0.4 0 ~. 0,5 ~.4 c,i E 0.1 O NADPH cytchrme c reductle ~ ~ 0 m itr ~$%hme'~ O micrime6 I I I I I I I I I I I I I NADPH ~ -~---e" diphrse micrllmell I I I I I I I I I I I I I 0 2 4 6 8 10 12 hurs after injectin f phenbarbital FIGURE 6 Changes in NADPH cytchrme reductase and NADPH diaphrase activity f rugh and smth liver micrsmes in respnse t treatment f rats with phenbarbital. Rugh and smth micrsmes were prepared frm livers f nrmal and phenbarbitaltreated rats, and the specific activities f NADPH cytchrme c reductase and NADPH diaphrase were determined as described in Materials and Methds. The specific activity f these enzymes is pltted against the time after injectin f phenbarbital. Each pint is the average f determinatins n micrsmes f three animals. 12 h after phenbarbital is nt due t new prtein synthesis. At the earlier time pint, inhibitin f ther membrane prtein synthesis results in an apparent increased specific activity f these enzymes. This may be due t an inhibitin f increased synthesis f smth membrane prtein, which nrmally dilutes the acyltransferases, r pssibly t sme cmplex cntrl mechanism, in which synthesis f a cntrlling prtein may be ablished. The rise in phsphlipid/prtein rati f the smth micrsmes at 4.5 h after phenbarbital (Fig. 9) was nt affected by administratin f actinmycin D r purmycin (Table III). There was a slight increase in the phsphlipid/prtein 642 THE JOURNAL OF CELL BIOLOGY - VOLUME 62, 1974

TABLE II Effect f Actinmycin D r Purmycin n the Specific Activity f the A cyltransferases f Micrsmal Fractins f Phenbarbital-Treated Rat Liver Specific activity f acyltransferases Treatment Rugh micrsmes Smth micrsmes Nne 1.07 ±0.310(6) 0.994 ±0.4613(6) 12 h after phenbarbital 0.52 ±0.0176(4) 3.63 ±1.2(5) 12 h after phenbarbital plus purmycin 0.772 ± 0.443 (6) 3.31 ± 0.255 (6) 12 h after phenbarbital plus actinmycin D 0.87 ~ 0.62 (4) 3.26 ± 0.327 (4) 4.5 h after phenbarbital 1.51 ~ 0.456 (5) 2.26 ± 0.63 (5) 4.5 h after phenbarbital plus purmycin 0.207 ~: 0.023 (4) 3.95 ± 1.2 (4) 4.5 h after phenbarbital plus actinmycin D 0.595 ± 0.084 (5) 3.616 ± 0.93 (5) Rats were treated with phenbarbital and actinmycin D r purmycin as described in Materials and Methds, and the rugh and smth micrsmes were islated at the end f the treatment. Acyltransferase activity was determined as nml f [~4C]glycerl-3-phsphate incrprated int lipid per min per mg prtein as described in Materials and Methds. The results are expressed as average specific activity ± standard deviatin (bs.). TABLE II1 Effect f Actinmycin D r Purmycin n the Phsphlipid/Prtein Rati f Micrsmal Fractins Prepared frm Phenbarbital-Treated Rat Liver Treatment Phsphlipid/prtein rati Rugh micrsmes Smth micrsmes Nne 4.5 h after phenbarbital 4.5 h after phenbarbital plus purmycin 4.5 h after phenbarbital plus actinmycin 0.686 ± 0.0741 (6) 0.688 ± 0.059 (5) 0.601 ± 0.09667 (4) 0.951 ± 0.0792 (5) 0.736 ±0.088(4) 0.922 ±0.126(4) 0.6204 ±0.08(4) 0.901 ±0.1175(4) Rats were treated with phenbarbital and actinmycin D r purmycin, rugh and smth micrsmes were islated, and the phsphlipid/prtein ratis were determined as described in Materials and Methds. Results are expressed as average ± standard deviatin (bs.) rati f the rugh micrsmes after purmycin treatment, which may be significant (p = 5.0%); but there was n change in the phsphlipid/ prtein rati f the rugh micrsmes after actinmycin D treatment. In the case f purmycintreated rat liver, the yield f rugh micrsmes was cnsistently lw, suggesting that the treatment may mdify this fractin. DISCUSSION Studies f membrane bigenesis in eukarytic cells are cmplicated by the fact that there is a cntinual turnver f all cmpnents f cell membranes in these cells. In additin, in the nrmal hepatcyte a variety f metablic events ccurs, sme f which invlve synthetic prcesses similar t thse invlved in membrane frmatin. Fr example, frmatin f plasma lipprteins invlves synthesis f lipids and prtein and packaging f these t prduce a cmplex structure f regulated cmpsitin which is exprted frm the cell. In the nrmal hepatcyte, therefre, it is difficult if nt impssible t ascertain the events in lipid synthesis related t membrane frmatin. Thus, a basic assumptin f.ur studies is that the changes related t new membrane frmatin are superimpsed n thse ccurring in the nrmal cell, when smth endplasmic reticulum membrane frmatin is induced experimentally by phenbarbital. Fr this reasn, althugh bth rugh and smth micrsmes have the enzymic machinery fr the synthesis f phsphlipid, the changes induced by phenbarbital have been related t smth membrane frmatin. Previus cytchemical experiments reprted frm this labratry indicated that during prliferatin f smth endplasmic reticulum membranes induced by phenbarbital, acyltransferases were active in the smth endplasmic reticulum J. A. HIGGINS Bigenesis f Smth Endplasmic Reticulum Membranes 643

and essentially inactive in the rugh endplasmic reticulum. These results were interpreted as indicating that the phsphlipid f the newly synthesized smth membranes is synthesized in situ, while the prtein which is apparently synthesized by bund ribsmes may be inserted int the smth membranes after synthesis f the phsphlipid cmpnent (11). The bservatins reprted here supprt this hypthesis. First, the specific activity f the acyltransferases f the smth membranes rse rapidly after injectin f phenbarbital. Acyltransferases catalyze the first steps in the synthesis f glycerlipids, and an increase in their specific activity may reflect an increased synthesis either f triglyceride r f phsphlipids in the smth membranes. Incrpratin f [~'C]glycerl int phsphlipids f smth membranes was increased by phenbarbital, while incrpratin int the phsphlipid f rugh micrsmes was elevated nly slightly and this may be accunted fr by exchange ccurring during the islatin prcedure. Incrpratin f [~'C]glycerl int triglyceride was nt increased by phenbarbital. The rise in acyltransferase was therefre directed twards phspblipid synthesis rather than triglyceride. This is cnsistent with the secnd bservatin that the phsphlipid/prtein rati f the smth micrsmes rse after injectin f phenbarbital. There was als a slight but significant rise in the acyltransferase activity f the rugh micrsmes after injectin f phenbarbital. This was nt accmpanied by a rise in the phsphlipid/prtein rati f this fractin. Hwever, increased phsphlipid synthesis in these membranes may be accmpanied by increased prtein syntheses. The specific activity f the NADPH cytchrme c reductase and NADPH diaphrase f the rugh micrsmes rse immediately after injectin f phenbarbital, indicating that a synchrnized synthesis f phsphlipid and prtein may ccur. It has been demnstrated by mrphmetric analysis that there is an increased surface area f rugh endplasmic reticulum the 1st day after injectin f phenbarbital (5). Althugh this is small cmpared with the later prliferatin f smth membranes, an increase in rugh membrane phsphlipid and prtein must be invlved in this prliferatin. It is als pssible that the increased acyitransferase activity f the rugh micrsmes is due t increased synthesis f these enzymes fr transfer t the smth membranes fr grwth ver several days f phenbarbital treatment. The rise in acyltransferase activity f the rugh micrsmes is ablished by inhibitin f prtein synthesis, in cntrast t the acyltransferase activity f the smth micrsmes which is nt altered by inhibitin f prtein synthesis. Hltzmann and Gillette (31)have reprted that in male rats there is an increased synthesis f phsphlipid in respnse t phenbarbital, but that the predminant effect f this drug appears t be t decrease the breakdwn f phsphlipid in micrsmes, resulting in its accumulatin. Direct cmparisn between ur results and thse f Hltzmann and Gillette is nt pssible because f the difference in experimental prtcl; hwever, the increase in incrpratin f [14C]glycerl int phsphlipids fund here is f the same rder as that reprted by these investigatrs. If the predminant effect f phenbarbital is t decrease breakdwn f phsphlipid, hwever, synthesis f phsphlipids must cntinue at either the same r an elevated rate. Our results indicate that the site f this synthesis shifts n treatment with phenbarbital t take place predminantly in the prliferating smth membranes. This is especially marked in the livers f rats treated fr 2 r mre days with phenbarbital, when the acyltransferase activity f the rugh micrsmes is very lw (11). It is well established that ne site f synthesis f new smth membrane enzymes is the rugh endplasmic reticulum. This has been demnstrated in the nrmal rat liver (12, 14, 33), the phenbarbital-treated rat liver (2, 3, 7, 12-14), and the develping rat liver, in which prliferatin f smth membrane ccurs (34 37). As the enzymes investigated remain elevated in bth the rugh and smth micrsmes, it is nt clear frm these investigatins whether the prteins f the rugh and smth membranes are a single pl, appearing first in the rugh membranes, r whether rugh and smth membrane prteins are individual pls and incrprated int their specific membrane structure separately. The data presented here indicate that the smth membrane phsph- lipid is synthesized in situ. The new prtein synthesized at the site f the rugh membranes either may be released, pssibly int the cisternae, and incrprated int the smth membrane after phsphlipid synthesis, r alternatively, may be incrprated'int the structure f the rugh membrane and mve by lateral diffusin t the smth membranes. The pssibility f a rapid lateral mvement f plasma membrane prteins has been demnstrated by Frye and Edidin (38) and is an imprtant cncept in the fluid msaic mdel f 644 THE JOURNAL OF CELL BIOLOGY VOLUME 62, 1974

membrane structure suggested by Singer and Niclsn (39). The rle f free ribsmes in the bigenesis f membrane prteins has nt been clarified. If these ribsmes are invlved in additin t bund ribsmes, it is als necessary t p~.tulate mvement f prtein frm the cytplasm t the area f new phsphlipid synthesis in the smth endplasmic reticulum. Our bservatins that the phsphlipid/prtein rati and the specific activities f the acyltransferases f the smth micrsmes may change withut similar changes in the rugh micrsmes raise questins cncerning the existence f free lateral flw f phsphlipid and acyltransferase mlecules between the rugh and smth endplasmic reticulum membranes. It has been clearly established that the rugh and smth membranes are cntinuus in hepatcytes f nrmal and phenbarbital-treated rat liver (2, 3, 40). It has als been demnstrated, using bth mdel systems and sarcplasmic reticulum vesicles, that there is a rapid lateral diffusin f spin-labeled phsphlipid mlecules within the plane f membranes (41, 42). The present results suggest that such a rapid flw f phsphlipid mlecules and acyltransferase mlecules, sufficient t cause immediate randmizatin f membrane cmpnents, des nt ccur between rugh and smth membranes. This raises the pssibility f barriers t free diffusin f cmpnents between mrphlgically cntinuus membranes r f cntrlling frces invlved in maintaining the individual cmpsitin f the rugh and smth membranes with regard t bth lipid and prtein. The theries cncerning the structure f cellular membranes are at present in a state f flux, and there is increasing evidence that membranes are fluid structures (39). The results described here serve t emphasize this cncept. The phsphlipid/prtein rati f the smth micrsmes rse in respnse t phenbarbital, and this rise ccurred when ver 80% f new prtein synthesis was inhibited. Transient differences in the cmpsitin f smth endplasmic reticulum membranes may ccur, therefre, and a rigid cmpsitin is nt bligatry under all physilgical cnditins. In additin, phsphlipids f smth endplasmic reticulum may be synthesized withut prteins. Thus, synthesis f cmplete lipprtein subunits f these membranes des nt take place. Similar bservatins were made by Mindich (43) in Bacillus subtilis mutants incapable f lipid synthesis. These rganisms synthesized membranes having a high prtein-t-lipid rati cmpared with the wild strain. These bservatins are cnsistent with a membrane structure such as that suggested by Singer and Niclsn (39) in which the prtein cmpnents are embedded t varying depths in a phsphlipid bilayer, and in which the tw cmpnents may change independently f each ther. This research was supprted by Grant AM 03688 frm the Natinal Institute fr Arthritis and Metablic Diseases, TICA 05055 frm the Natinal Cancer Institute, and GM 20001 frm the Natinal Institute f General Medical Sciences f the Natinal Institutes f Health, United States Department f Health, Educatin and Welfare. Received fr publicatin 28 Nvember 1973, and in revised frm 8 April 1974. REFERENCES 1. REMMER, H., and H. J. MERKEL. 1963. Science (Wash. D. C.). 142:1657. 2. ORRENJUS, S., J. L. E. ERICSSON, and L. ERNSTER. 1965. J. Cell Bil. 25:627. 3. ORRENIUS, S., and J. L. E. ERICSSON. 1966. J. Cell Bil. 28:181. 4. ERNSTER, L., and S. ORRENtUS. 1965. Fed. Prc. 25:1190. 5. ST.~UBLI, W., R. HESS, and E. R. WEIBEL. 1969. J. Cell Bil. 42:92. 6. ORRENIUS, S. 1965. J. Cell Bil. 26:713. 7. ORRENIUS, S. 1965. J. Cell Bil. 26:725. 8. CONNEY, A. H., C. DAVISON, R. GASTEL, and J. J. BURNS. 1960. J. Pharmacl. Exp. Ther. 130:1. 9. BRAZDA, F. G., and B. W. BAUCUTI. 1961. J. Pharmacl. Exp. Ther. 132:295. 10. YOUNG, D. L., G. POWEEL, and W. O. MCMILLAN. 1971. J. Lipid Res. 12:1. I 1. HIGGINS, J. A., and R. J. BARRNETT. 1972. J. Cell Bil. 55:282. 12. SARGENT, J. R., and B. P. VALAMUDI. 1968. Bichem. J. 107:839. 13. KURIYAMA, Y., T. OMURA, P. SIEKEVITZ, and G. E. PALAOE. 1969. J. Bil. Chem. 244:2017. 14. ARIAS, I. M., D. DOYLE, and R. T. SCHXMKE. 1969. J. Bil. Chem. 244:3303. 15. CHEDID, A., and V. NAIR. 1972. Science (Wash. D. C.). 175:176. 16. FOLCI-I, J., M. LEES, and G. H. SLOANE STANLEY. 1956. J. Bil. Chem. 226:497. 17. ERNSTER, L., D. SIEKEVITZ, and G. E. PALADE. 1962. J. Cell Bil. 15:541. 18. LOWRY, O. H., N. J. ROSEBROUGH, A. L. FARR, and R. J. RANDALL. 1951. J. Bil. Chem. 193:265. 19. FISKE, C. H., and Y. SUBaAROW. 1925. J. Bil. Chem. 66:375. J. A. HIGGINS Bigenesis f Smth Endplasmic Retieulum Membranes 645

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