ANALYTICAL FRACTIONATION OF HOMOGENATES FROM

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1 VOLUME Published Online: 1 Nvember, 1974 Supp Inf: Dwnladed frm jcb.rupress.rg n May 3, 218 ANALYTICAL FRACTIONATION OF HOMOGENATES FROM CULTURED RAT EMBRYO FIBROBLASTS PAUL TULKENS, HENRY BEAUFAY, and ANDRI~ TROUET Frm the Labratire de Chimie Physilgique, Universit~ Cathlique de Luvain, Luvain, Belgium ABSTRACT Hmgenates f cultured rat embry fibrblasts have been assayed fr acid phsphatase, N-acetyl-B-glucsaminidase, cathepsin D, acid dexyribnuclease, cytchrme xidase, NADH cytchrme c reductase, 5'-nucletidase, insine diphsphatase, acid pyrphsphatase, neutral pyrphsphatase, esterase, catalase, chlesterl, and RNA. The validity f the assay cnditins was checked. Neutral pyrphsphatase is a readily sluble enzyme. Acid hydrlases, except acid pyrphsphatase, are particle-bund enzymes, which exhibit a high degree f structural latency. They are activated and slubilized in a parallel fashin by mechanical treatments and tensi-active agents. Catalase is als particle-bund and latent; activating cnditins strnger than thse fr hydrlases are required t activate the enzyme. Acid pyrphsphatase, 5'-nucletidase and insine diphsphatase are firmly particle-bund, but nt latent; they are nt easily slubilized. In differential and ispycnic centrifugatin, the latent hydrlases, cytchrme xidase and catalase dissciate largely frm each ther; this suggests the ccurrence f lyssmes and perxisme-like structures besides mitchndria. The distributin patterns f 5'-nucletidase and chlesterl are largely similar; digitnin influences their equilibrium density t the same extent; these tw cnstituents are thught t be related t the plasma membrane. Insine diphsphatase and acid pyrphsphatase are als partially assciated with the plasma membrane, althugh sme part f these enzymic activities prbably belngs t ther structures. NADH cytchrme c reductase is assciated partly with the endplasmic reticulum, partly with mitchndria. Cultured mammalian cells represent a chice material fr many physipathlgical studies. They are easily submitted t varius treatments under well-cntrlled cnditins; the mdel is a relatively simple ne, mainly because interactins between different cell types are ablished. Characterizatin f the subcellular rganelles f cultured cells by fractinatin techniques has already been initiated in several labratries. Effrts alng this line have been devted mstly t the islatin and subsequent analysis f their plasma membranes (4, 12, 13, 16, 26, 32, 44, 45, 59, 6, 64). Lyssmes were similarly studied but t a lesser extent (31, 38, 39, 43, 51, 61). In n instance, hwever, has a systematic analytical fractinatin been carried ut. The lyssmal functins are presently being investigated n cultured rat embry fibrblasts in ur labratry, and preliminary reprts n these studies have already appeared (53-56). A better knwledge f the bichemical and physical characters f the subcellular cmpnents f these cells THE JOURNAL OF CELL BIOLOGY 63, 1974 pages

2 was a prerequisite in this wrk. This paper presents the results btained by quantitative fractinatin f cultured fibrblasts. An analytical apprach has been fllwed thrughut (19) in rder t survey the prperties f the varius ppulatins f subceliular cmpnents. MATERIALS Cell Culture AND METHODS Primary cultures f fibrblasts were btained by trypsinizatin f eviscerated 17-day ld embrys (.25% trypsin in Ca ++- and Mg++-free Hanks' slutin). The cultures were initiated by l s cells/cm 9 f grwing surface. After 7 days, cells were detached with.1% trypsin in PBS (phsphate-buffered saline, NaCI,.15 M; KCI, 2.7 mm; NagHPO,-KHgPO,, 3 ram; ph 7.4), and subcultures were started at a density f abut 5 1" cells/era z. Cnfluency was reached in 4 7 days; the density was then abut 2.5 l5 cells/cm 9, r apprximately 9/~g f cell prtein/cm 9. Tightly stppered l-liter Rux flasks were used fr mst experiments. They cntained 1 ml f culture medium (ca.5 ml/cm 2 f grwing surface). Eagle's minimum essential medium (25) was used with the fllwing mdificatins: amin-acids were replaced by.5% lactalbumin hydrlysate; glucse and glutamine were, respectively, 25 mm and.2 mm; the initial cncentratin f sdium bicarbnate was 9 mm; small additins f a.6 M slutin were made when necessary t maintain the ph arund 7.2. Medium was supplemented with 1% calf serum. Sterilizatin was perfrmed n Seitz R filter pads (type EKS I, Bad-Kreuznach, Germany) r n Millipre R membrane filters (type GS,.22 nm, Millipre Crp., Bedfrd, Mass.). Penicillin (1 IU/ml) and streptmycin (1 ~ag/ml) were rutinely added t the culture media. Cells at the secnd and third subculture were harvested sn after cnfluency. The appearance f these cells under the electrn micrscpe is illustrated in Fig. I. Nuclei display cnvluted prfiles, with chrmatin present in a peripheral layer and scattered thrughut the nuclear matrix. In the cytplasm, the rugh endplasmic reticulum presents dilated cisternae, limited by membranes heavily laded with ribsmes and plyribsmes. The Glgi apparatus (nt shwn) is well develped and restricted t a perinuclear zne. Dense bdies are numerus and display a plymrphic cntent. Many small vesicles are bserved, especially in the peripheral areas f the cell; sme f them are related t the pericellular membrane. In essence, the appearance f the cells is similar t that described fr type II cells, derived frm human embry lung in culture, by Franks and Cper (29). These fibrblast-like cells were dminant under ur cnditins f culture; n ther cell type, including muscle cells, culd be clearly recgnized. The cell sheet was washed twice with PBS and detached frm glass with.5 mm EDTA in the same medium. The use f trypsin was avided because f pssible deleterius effects n membranes and interference with prtease assays. The cells were gently suspended, spun dwn at 8 rpm fr l min, washed twice in ice-cld.25 M sucrse and resuspended in the same medium, All subsequent peratins were carried ut clse t C. Cell Fractinatin The harvest frm abut eight Rux bttles was hmgenized in 7 ml f.25 M sucrse cntaining 1 mm EDTA at ph 7.4 (sucrse-edta) by six strkes f the tight pestle f a Dunce hmgenizer (Kntes Glass C., Vineland, N. J.). Hmgenizatin was checked by phasecntrast micrscpy. EDTA was fund necessary t prevent agglutinatin f subcellular particles. Fractinatin by differential centrifugatin was carried ut in several ways. In sme experiments, the methd f de Duve et al. (23) was fllwed exactly. In shrt, fur particulate fractins (N, M, L, P) and a final supernate (S) were separated by successive centrifugatins at increasing speeds and times. The angular velcities (rpm) and the time integral f the squared angular velcities W = f t 2dt (rad 9 s "1) were respectively; 1,7 and , 12,5 and 3 18, 25, and 2.5 l b, 4, ad 3 x 11. Centrifugatins were carried ut in the rtr n. 4 (Beckman Instruments, Inc., Spinc Div., Pal Alt, Calif.) except fr the runs at 1,7 rpm, which were perfrmed in an I.E.C. centrifuge (Damn Crp. Needham Heights, Mass., mdel PR-J, head n. 253, meniscus and bttm f the clumn fluid at, respectively, 13.5 and 21.5 cm frm the axis). All pellets were washed twice in sucrse-edta and the supernates were cmbined fr sedimenting the next fractin. In ther experiments, three articulate fractins were btained: N, ML, and P. The pst-n fractin supernate (cytplasmic extract) was then centrifuged directly a 25, rpm (W = rad z s -1) and the ML fractin was equivalent t the sum f the M and L fractins. T study the influence f digitnin n the buyancy f micrsmal cnstituents, the pst-n fractin supernate was divided int M', LP anu S fractins by centrifuging successively at 12,5 rpm fr a lnger time (W = 5.9 l s rad 9 s -1) and at 4, rpm as usual (W = 3 11 rad 2 s-l). Analysis by density equilibratin in a sucrse gradient was perfrmed in a special znal rtr whse principle and advantages have already been described (8, 1 I, 35). We have intrduced successively (a) 1 ml f sample in sucrse-edta; (b) 32 ml f a sucrse gradient, the density f which increased linearly frm 1.1 t 1.25; (c) 6 ml f sucrse slutin f 1.34 density, acting as a cushin. All slutins cntained I mm EDTA. The gradient was centrifuged fr 3 h at 35, rpm (W = rad 9 s- 1); under these cnditins, mst subcellular particles are brught t their equilibrium psitin (1). Finally, the cntent f the rtr was divided int THE JOURNAL OF CELL BIOLOGY. VOLUME 63, 1974

3 FIGURE 1 Ultrastructural aspec' cultured rat embry fibrblasts. Cells were cllected and examined as previusly described (56). A. General view f a peripheral part f the cytplasm. Cisternae f the endplasmic reticulum are dilated by an electrn-dense material and are heavily cated with ribsmes. Many small smth vesicles are bserved, sme f which are in cntinuity with the plasma membrane (arrws). Mitchndria, dense bdies, and lipid drplets are present (x 13,). B. Alternating smth and rugh regins f the endplasmic reticulum are clearly visible. Dense bdies (arrws) shw a well-defined membrane surrunding a plemrphic material. The cnvluted aspect f the nucleus prfile is illustrated (x 23,). C. Plysmal arrangement f ribsmes, prbably assciated with a membrane f the endplasmic reticulum seen in an blique sectin (x 3,). Curtesy f Dr. F. Van hf.

4 fractins whse weights and densities were determined (9, 35). The results are expressed as histgrams f the density distributin f the cnstituents (9). In rder t average the data f several experiments, the distributins were standardized by dividing the density interval in 15 virtual fractins f equal increment f density. The percentage amunt f cnstituent crrespnding t each interval was cmputed, fllwing published methds (35), frm primary plts f the cncentratin and f the density in the actual fractins versus their cumulated vlumes. Tw additinal fractins are shwn n each side f the histgram; they represent, respectively, the material recvered belw 1.7 and abve Bichemical Assays Enzymes were assayed by published methds, with minr mdificatins fr increasing their sensitivity. The references and a survey f the methds used are given in Table t. Fr Mg +-dependent enzymes, the catin is in large excess ver the amunt f EDTA added with the sucrse slutins in which the particles are suspended. Activities are expressed in standard miuiunits (nanmles f substrate degraded per minute under assay cnditins), except fr the fllwing enzymes. Cytchrme xidase and catalase activities are expressed in the units defined by Cperstein and Lazarw (17) and by Baudhuin et al. (6), respectively. 1 U f cathepsin has been taken as the enzyme activity releasing per minute an amunt f TCA-sluble ligpeptides equivalent t 1 mg f bvine serum albumin in the Lwry assay (36). Unless therwise stated, the activities given refer t the ttal activity, which is that measured when the structural barriers slwing dwn the reactins catalysed by particle-bund enzymes have been suppressed. In mst cases, the structural latency was released by adding Tritn X-1 in the assay mixture at.1% final cncentratin. Cmplete activatin f catalase was achieved by pretreating the sample with.5% Tritn X-1, the detergent being thereafter diluted five times in the assay mixture. Fr cytchrme xidase, the sample was treated in.2 ml f digitnin at.25% befre starting the reactin by additin f 3 ml f slutin cntaining the substrate and the buffer. In this way, the inhibitry effect f the detergent at the cncentratin which suppresses the latency was avided. In sme cases, activities, referred t then as free activities, were measured under cnditins that preserve the structural-latency f en, zymes: additin f sucrse at.25 M cncentratin t the assay mixture and shrtening f the incubatin time t a maximum f 1 min. The ph was increased t 5 fr determinatin f free cathepsin. The part f the ttal activity which is sedimented by centrifuging 3 min at 4, rpm (rtr 4) will be referred t as the sedimentable activity, Ribnucleic acid was measured accrding t Schneider (46), care being taken t wash ut the sucrse cmpletely (63). Prteins were determined by the methd f Lwry et al. (36). Chlesterl was assayed by gas chrmatgraphy, with stigmasterl as an internal standard (1). Materials All chemicals were f analytical grade. Mst were purchased frm E. Merck A. G. (Darmstad, West Germany), Sigma Chemical C. (St. Luis, M.), and Kch-Light Labratries Ltd. (Clnbrk, Buckinghamshire, England); Tritn X-1 is the prduct f Rhm and Haas C. (Philadelphia, Pa.); lactalbumin hydrlysate was supplied by ICN Nutritinal Bichemicals Div., Internatinal Chemical and Nuclear Crp. (Cleveland, Ohi); vitamins fr Eagle's minimal essential medium were btained as a 1 times cncentrated slutin frm. either Labratires Eurbi, Paris, France, r Flw Labratries, Irvine, Sctland; trypsin was the TC pwder trypsin 1:25 f Difc Labratries, Detrit, Mich.; calf-serum was either btained frm bld cllected at a lcal slaughter-huse and prcessed in ur labratry, r purchased frm Labratires Eurbi r Flw Labratries. Pregnant rats were f the Wistar strain, bred in the Animal Huse f the University f Luvain. RESULTS Enzyme Kinetics A preliminary kinetic study was perfrmed n the varius enzyme reactins, in rder t establish ptimal assay cnditins. Fig. 2 shws the effects f ph at the substrate cncentratins given in Table I. Acid phsphatase, cathepsin, N-acetyl-~glucsaminidase, dexyribnuclease, and pyrphsphatase (in presence f 2 mm EDTA) are mst active at ph 5 r belw. The ther enzymes require a neutral r slightly alkaline ph fr maximum activity. Esterase was assayed nly at ph 7.4, since the substrate readily decmpses at acid r alkaline ph. Except fr cytchrme xidase and catalase, which bey first-rder kinetics, the K,~ values were determined, and the cncentratins f substrate selected prvide the reactin with at least 75% f the maximum velcity. Calibratin experiments accrding t amunt f tissue and t time f incubatin are presented in Fig. 3, which shws the ranges f linearity. Additin f MgC12 enhanced cnsiderably the activities f 5'-nucletidase, insine diphsphatase, and neutral pyrphsphatase. The catin was present in the assay mixtures at cncentratins three t fur times the minimum required t gain full activity. Sucrse was fund t inhibit N-ace- THE JOURNAL OF CELL BIOLOGY VOLUME 63, 1974

5 .=.-~.-~.=,. ~=,..~ e~ tm m m Z "-~ "~ ~ z, 6 -. ~-..~ ~r~ J ' "-"~ "~1~1 ~ ~.~_ ~ ~ ~ -~ ~ z~ -._.O '.." = ~:a.~ ~.~.~ ~ ~ ~ ~ ~ ~ ~ =~ ii.c: ~ +-+ P. TULKENS, H. BEAUFAY, AND A. TROUET Analytical Fractinatin f Hmgenates 387

6 I 1 J' " A N-Acetyl -n~d-as e DexyrJbnul l clease s~ ~ ~- Catat > I-- r.j ~ s x - Cytchrme xidase I I I I u- I I I I 1 ~, NADH cyt. c - reductase I I I I I I I I J l I "~.m i Pyrphspha- D TA ) -2 phsphrtase L I I I1" O Insine L diphsphatase a J a I '- Nucletidase ~- lamp) I I I I ph I FIGURE 2 ph activity curves f enzymes in hmgenates f cultivated fibrblasts. Incubatins were carried ut in the fllwing buffers: acetate (), citrate (O), cacdylate (&), phsphate (O), imidazle-hc1 (El), Tris-HCl (m), glycine-naoh (A), each at 5 mm cncentratin. Other cnditins f the assays are given in Table 1. tyl-/3-glucsaminidase t an extent requiring suitable crrectins when the cncentratin exceeded.1 M. The fibrblast enzymes acting n phsphric mnester bnds r n phsphric anhydride bnds were further characterized by inhibitrs. Tartrate (1 mm D-L-sdium ptassium tartrate) inhibited acid phsphatase as bserved in ther tissues (5, 4). Sdium fluride (1 mm) inhibited acid phsphatase, 5'-nucletidase and neutral pyrphsphatase, lnsine diphsphatase and acid pyrphsphatase were unaffected by either treatment, but the latter was strngly inhibited by ammnium mlybdate, 1 mm (41). Specific activities f fibrblast enzymes are cmpared, in Table II, t values reprted fr their hmlgues in rat liver hmgenates. Insine diphsphatase, esterase, catalase, and the tw pyrphsphatases are much less active in cultivated fibrblasts; the ther enzyme activities are f the same rder f magnitude in the tw materials. We have als fund that glucse-6- phsphate is slwly hydrlyzed by fibrblast hmgenates. Hwever, we are mst likely dealing here with an unspecific phsphatase, nt with the specific glucse-6-phsphatase described in liver and kidney, fr the fibrblast enzyme has an ptimum at ph 5 and is nt inactivated by preincubatin withut substrate at ph 5 and 37 C (22). Structural Latency and Assciatin f Enzymes with Subcellular Cmpnents When hmgenates f fibrblasts are centrifuged fr 3 rain at 4, rpm (Beckman/ Spinc rtr n. 4), the neutral pyrphsphatase, abut ne-third f the esterase activity and nethird f the RNA are recvered in the supernate. The ther cnstituents assayed in this wrk are fund in the pellet t a very large extent. Repeated freezing and thawing r additin f digitnin released 75% f N-acetyl-~-glucsaminidase, acid phsphatase, cathepsin, and acid dexyribnucle- 388 THE JOURNAL OF CELL BIOLOGY VOLUME 63, 1974

7 ase in sluble frm, whereas insine diphsphatase, 5'-nucletidase, and acid pyrphsphatase remained essentially particle bund; sme release f esterase was nticed (Fig. 4). In fresh hmgenates, acid dexyribnuclease, cathepsin, N-acetyl-B-glucsaminidase, acid phsphatase, and catalase were latent t the extent f at least 75% f their ttal activity. The latency was partially r cmpletely ablished by repeated freezing and thawing and by additin f digitnin r Tritn X-1 (Fig. 5). The activatin curves f acid phsphatase, acid dexyribnuclease, N-acetyl-B-glucsaminidase, and cathepsin are hardly distinguishable frm ne anther, but they dissciate clearly frm the activatin curve f catalase. The latter enzyme is mre resistant t activating means, especially t digitnin. Full activatin f acid phsphatase and N-acetyl-B-glucsaminidase is achieved as sn as digitnin is stichimetrically in excess ver the amunt f chlesterl present in the preparatin; at that digitnin cncentratin the latency f catalase is still unaffected. The activities f 5'-nucletidase and neutral pyrphsphatase were nt mdified by repeated freezing and thawing r by additin f detergents. Insine diphsphatase was nly slightly activated (less than 15%) by these treatments, which were checked t cause cnsiderable activatin f hepatic insine diphsphatase as previusly described (42). Fractinatin by Differential Centrifugatin The distributins btained after fractinatin f fibrblast hmgenates by differential centrifugatin int N, ML, P, and S fractins are presented in Fig. 6. As expected frm the data f Fig. 4, neutral pyrphsphatase is recvered essentially in the S fractin. The lw activity fund in the N fractin demnstrates that very few cells were left unbrken by the hmgenizatin. Anther typical pattern is given by cytchrme xidase, N-acetyi- B-glucsaminidase, acid phsphatase, and dexyribnuclease, which are largely recvered in the large granule fractin (ML), the remainder being 1c..., ---p, i n m n.!..... m m I 75 z 5c abe abe ab ab ~ abc ab c a b FIGURE 3 Kinetics f fibrblast enzymes. Fibrblast hmgenates were incubated under the cnditins given in Table 1. &, times f incubatin were varied with the fllwing cnstant amunts f cell prtein: acid phsphatase, 85 tzg; cathepsin, 166 ~g, N-acetyl-B-glucsaminidase, 7.5 vg; 5'-nucletidase, 12 pg; insine diphsphatase, 275 pg; catalase, 36 ~g. O, tissue cncentratins were varied, with incubatin time kept at 3 min fr acid phsphatase, cathepsin, N-acetyl-B-glucsaminidase, and acid dexyribnuclease; 2 min fr 5'-nueletidase, insine diphsphatase, acid and neutral pyrphsphatases; 1 min fr catalase NADH cytchrme c reductase, cytchrme xidase, and esterase were fllwed spectrphtmetrically fr 1 min. abc a P. TULKENS, H. BEAUFAY, AND A. TROUET Analytical Fractinatin f Hmgenates 389

8 TABLE 1I Cmparisn f the specific cntent in enzymes and chemical cnstituents in hmgenates f fibrblasts and f rat liver Enzyme r cnstituent Fibrblasts Activity r cntent Reference Acid phsphatase 46.1 ± 7.2 (12) N-Acetyl-/3-glucsaminidase 8.7 ± 12.3 (21) Cathepsin 18.6 ± 7.2 (18) DNAse 14.2 ± 6.7 (5) Catalase 2.6 ± 6.3 (12) '-Nucletidase 73.2 ± 7.5 (1) insine diphsphatase 39.3 ± 15.7 (12) Acid pyrphsphatase 16.3 ± 5.8 (9) Neutral pyrphsphatase 23.6 ± 9.1 (6) 17 5 Cytchrme xidase 61.2 ± 18.7 (14) NADH cytchrme c reductase 115 ± 18 (5) Esterase 162 ± 45 (4) RNA 87.1 ± 7.2(6) Chlesterl 38.4 ± 3.1 (8) Results are given as means ±SD in milliunits (enzymes) r in micrgrams (chemical cnstituents) per milligram prtein; figures in parentheses refer t number f experiments. Liver mstly divided between the N and P fractins in a way particular fr each enzyme. The activities in fractin N are nt attributable nly t unbrken cells, fr they largely exceed that f neutral pyrphsphatase. Finally, NADH cytchrme c reductase, acid pyrphsphatase, insine diphsphatase, 5'-nucletidase, and catalase attain the highest specific activity in the micrsmal (P) fractin. Hwever, althugh smewhat variable, the amunts f these enzymes sedimented with the large granules are nearly equal t thse fund in the micrsmes. In the experiment reprted in Fig. 7, the large granule fractin has been reslved int an M and an L fractin. Cytchrme xidase nw shws a distributin pattern distinct frm thse f acid phsphatase, N-acetyl-fl-glucsaminidase, and cathepsin: the three acid hydrlases have a smewhat higher specific activity in L than in M, whereas cytchrme xidase is mre cncentrated in the M fractin. The dissciatin is, hwever, much less marked than it is in liver tissue (23). The difference between the M and L fractins is mre prnunced fr the enzymes that attain their highest specific activity in the micrsmes. In this grup, t which chlesterl may be added, catalase differs clearly frm the ther enzymes by having a sevenfld higher specific activity in the L than in the M fractin. RNA is mainly assciated with fractins S (34%) and P (29%), and shws the highest specific cntent in fractin P. An appreciable amunt f RNA sediments with the N fractin. Esterase exhibits the bradest distributin pattern, with a small peak in the micrsmes and a lw activity in fractin N. Fractinatin by Density Equilibratin The results btained by density equilibratin in linear sucrse gradients are shwn in Table III and Fig. 8. Cytplasmic extracts, free f nuclei and unbrken cells, were used rather than cmplete hmgenates in these experiments, in rder t avid artifacts caused by the presence f nuclei, it can be seen frm the data presented abve that, except fr RNA, n mre than 1% f each cnstituent is discarded with the N fractin. Several patterns f density distributin can be distinguished. (a) Prfiles f N-acetyI-B-glucsaminidase, cathepsin, acid phsphatase, and dexyribnuclease are very similar and extend asymmetrically ver the whle length f abscissa. Their mdes cincide with the! fractin; the median densities are cmprised between and 1.26, except fr acid phsphatase (1.189) which exhibits a secnd small but reprducible mde near the density (b) Catalase presents a sharper prfile, with a mde in the fractin and a median density within these limits. 39 THE JOURNAL OF CELL BIOLOGY - VOLUME 63, 1974

9 (c) The distributin pattern f cytchrme xidase is the sharpest and peaks at a lwer density, in the fractins. NADH cytchrme c reductase shws a peak in the same range f density, but is much mre bradly spread thrugh the gradient, and ccurs in fractins virtually free f cytchrme xidase activity. (d) 5'-nucletidase and insine diphsphatase shw similar, highly skewed prfiles, with a mde in the fractin and extending asymmetrically ver the whle density range. Chlesterl and acid pyrphsphatase behave similarly, except that their ==.9 g ~.6 ~.3 j. Acid phsphatase Q J Cytchrrne xidase = ~ _.2 E I I i I 8 16 mg x rain I I I I.2 rng.4 I I mg x rain._ E -~.3 <.~.1 tr i.u f Cathepsin / ' ~4 -~ 2 i i i i 1 2 mg xmin NADH cyt c reductase / I I I mg ~.6 f Insine / diphspha- // "= I 5 I 1 ll5 mgxmin " ~8 6 z & 4 E 2 N-Acety[- ~- :~. 3 9[ucsaminidase ".5 1. mgxmin ~ ~ E = Esterase ~ mg ~2.2 Acid pyrphsphatase J I I I mgxmin.2.1 g 5 a ~.9.,.2.3 m.g ~.5 ~ 4.3 Catatase Neutral pyrphsphatase g.e ~r'r ~,.4 E min ls rngxmin I I I 5 to 15 mgxmm FIGURE 4 Slubilizatin f enzymes f the hmgenate (1: acid phsphatase; 2: N-acetyl-fl-glucsaminidase; 3: acid dexyribnuclease; 4: cathepsin; 5: 5'-nucletidase; 6: acid pyrphsphatase; 7: insine diphsphatase; 8: esterase; 9: neutral pyrphsphatase) by varius treatments: (a) cntrl hmgenate; (b) hmgenate frzen and thawed five times; (c) hmgenate treated with digitnin at a final cncentratin f.2% (.92 mg f cell prtein ml- ~; mlar digitnin/chlesterl rati: 1.93). After treatment, fractins f hmgenates were centrifuged at 4, rpm fr 3 min (rtr n. 4 Beckman/Spinc). Enzymes were assayed under "ttal" cnditins in resuspended pellets, supernates, and riginal hmgenates; recveries ranged between 92 and 14%. The figure shws the sedimentable (white blcks) and sluble (shaded blcks) activities as percentage f the ttal activity f the untreated hmgenate. P. TULKENS, H. BEAUFAY, AND A. TROUET Analytical Fractinatin f Hmgenates 391

10 mdes appear in the next denser fractin. In additin, the distributin pattern f acid pyrphsphatase shws a shulder n its dense side, which increases smewhat the median density accrdingly. Other distributins are mre cmplex. Esterase is spread ver the whle gradient; its activity in the lw density regin is largely due t sluble enzyme (25-35%, see Figs. 5 and 7). The same cmment applies t prtein which attains the highest cncentratin at densities arund RNA is rather bradly, but symmetrically distributed. It prbably represents the sum f the density distributin f RNA-cated vesicles and f the sedimentatin pattern f free ribsmes which are still remte frm their equilibrium psitin. Effect f Digitnin n the Density Distributin f Micrsmal Cnstituents It has been reprted that digitnin, at cncentratins t lw t disrupt mst subcellular particles, causes a distinct increase in the equilibrium density f chlesterl-rich membranes frm rat liver, particularly the plasma membrane and the micrsmal cmpnents related t the plasma membrane (3, 52). In view f this finding, the influence f digitnin n the density distributin f micrsmal cnstituents frm cultured fibrblasts was investigated with the hpe that different patterns f behavir might be revealed in this way. Tw enlarged micrsmal fractins (LP fractins, see Materials and Methds) were prepared. One was washed twice with.25 M sucrse; the ther was washed nce with sucrse-edta, resuspended in sucrse-edta supplemented with.3% digitnin, and recentrifuged as usual. The cncentratin f micrsmes was such that the mlar rati digitnin/chlesterl was clse t 1. It ean be seen frm Table IV that the tw preparatins had similar bichemical cmpsitins. The density distributins bserved with the tw micrsmal preparatins are presented and cmpared in Fig. 9. Cnsidering first the untreated preparatin, it appears that, except fr prtein and RNA, the median densities and the prfiles resemble thse btained with cytplasmic extracts. Insine diphsphatase, hwever, and, t a lesser extent, acid pyrphsphatase, exhibit an imprtant tailing twards high densities. The distributin f prtein is largely similar t that f loo > 8 > ~ 6 U- -~ = 2'! I I I t I I I I J I I /,.6.8 n f FREEZING-THAWING [DIGITONIN]% [TRITON X-11% FIGURE 5 Suppressin f structural latency f acid phsphatase (O), N-acetyl-B-glucsaminidase (O), cathepsin (-~), acid dexyribnuclease (V) and catalase (A) by varius treatments. Graphs shw free activities in percent f ttal activities, bth measured after the treatment (A and B). The treatments never mdified the ttal activities significantly. A, hmgenate (2.4 mg f prtein/ml) repeatedly frzen in dry ice-acetne mixture and thawed in water at 1 C. B, hmgenate (1.7 mg f prtein/ml) supplemented with digitnin at varius cncentratins. The pint f stichimetric equivalence between digitnin and chlesterl f hmgenate is indicated by the arrw. C, hmgenates (5 /~g f prtein/ml fr phsphatase; 1 #g/ml fr N-acetyl-fl-glucsaminidase and catalase) were treated with Tritn X-1 at the cncentratins presented n the abscissa. The reactin was started thereafter by additin f 4 vi f buffered substrate. Assays were perfrmed under "free activity" cnditins; activities are expressed in percentage f the activity bserved in the same hmgenate under "ttal activity" cnditins. 392 THE JOURNAL OF CELL BIOLOGY - VOLUME 63, 1974

11 3 Cytchrme (3) xidase NAOH:cyt. c reductase Catalase (3) >- I-.) < O,T.) tu O. O UJ _> i-- < iv, N-Acetyl- [I- Acid phsphatase (3) (31 Acid dexyribnuclease (3) j Acid pyrphsphatase Insine diphsphatase 5'-Nucletidase Neutral pyrphsphatase II1 r l / ~ r//.'[ I I I I I I I I I I too % OF PROTEIN I I I I 5 1 FIGURE 6 Distributin patterns f enzymes after fractinatin by differential centrifugatin. Fibrblast hmgenates were divided int fur fractins: N, ML, P, and S. These are represented by blcks rdered accrding t the same sequence n the abscissa where they span a length prprtinal t their prtein cntent. The rdinate (heights f the blcks) gives the relative specific activities (percentage f activity ver percentage f prtein) f the fractins. Graphs represent the mean result f several experiments; the actual number is given between parentheses fr each enzyme. Percentages relate t the sum f N, ML, P, and S fractins. Averaged recveries frm the hmgenates ranged between 9 and 114%. NADH cytchrme c reductase. The distributin f the micrsmal RNA is symmetrical with a mde in the fractins Digitnin treatment causes marked changes in distributin patterns, characterized by an increased median (Table IV) and mdal density and a smaller dispersin f density fr bth 5'-nucletidase and chlesterl. The shift f these prfiles is such that they dissciate frm the nrmal nes by 7% f their surface area. In cntrast, the distributins f NADH cytchrme c reductase and RNA are nt significantly influenced. Acid pyrphsphatase, insine diphsphatase, and prtein exhibit an intermediate behavir. The tw enzymes shw a mdal shift cmparable t that f 5'-nucletidase and chlesterl, but the prfiles are slightly asymmetrical, as if the shift cncerned nly part f the activities. DISCUSSION Hmgenates f cultured rat embry fibrblasts have been analyzed by quantitative fractinatin techniques. The distributin patterns btained have led t the bichemical characterizatin f several subcellular cmpnents. In the fllwing discussin, these are identified with a given cmpnent f the intact cell. Obviusly sme f these identificatins remain tentative, awaiting a crrelated mrphlgical and bichemical study. Mitchndria Mitchndria, detected by their specific marker cytchrme xidase (24), are recvered largely with the large granules after differential centrifugatin, and after equilibratin in a sucrse gradi- P. TULKENS,. H. BEAUFAY, AND A. TROUET Analytical Fractinatin fltmgenates 393

12 Cytchrme xidase NADH cyt. c reductase Catalase Acid phsphatase Acid pyrphsphatase Esterase N-Acetyl- {3-5LNucletidase RNA Cathepsin Insine diphsphatase Chlesterl I I I I I I I I I I I I I I % OF PROIEIN FIGURE 7 Distributin patterns f enzymes and cnstituents after fractinatin by differential centrifugatin. Fibrblast hmgenate was divided int five fractins: N, M, L, P, and S. Same mde f representatin as in Fig. 6. Recveries frm the hmgenate ranged between 84 and 118%. TABLE III Median Densities and Recveries f Cnstituents after Equilibratin f Cytplasmic Extracts in Sucrse-H2 Gradient Median density f equili- N. f experi- Cnstituent bratin Recvery in the gradient ments Insine diphsphatase I. 137 ± ± 5. I 4 Chlesterl I. 144 ± ± '-Nucletidase ± ± Acid pyrphsphatase ± ± Esterase ± RNA Cytchrme xidase ± ± NADH cytchrme c reductase ± ± Catalase ± ± Acid phsphatase ± ± Cathepsin ± ± Acid dexyribnuclease N-Acetyl-/3-glucsaminidase 1.26 ± ± Prtein , ± Values listed are means ± SD. % 394 THE JOURNAL OF CELL BIOLOGY VOLUME 63, 1974

13 Tlnsine DNASE ~ Cytchrme xidase j ~j TTr N-Acetyl- IE}- rhl 5LNucleti - I glucsamin- -, NADH cyt. c Treductase 15 >- 1 1 U,,=,s,, ~" IL Acid i _ L.J- -- I--T,=IT~ J.I.kTT.-Z 5 I RNA I Esterase Prtein i l I I I I I I DENSITY FIGURE 8 Distributin patterns f enzymes and chemical cnstituents after fractinatin by density equilibratin in linear sucrse gradient. Cytplasmic extracts were equilibrated as described under Materials and Methds. Results are pltted in the frm f nrmalized histgrams (11, 35). The abscissa is the density scale divided in 15 equal sectins f density increment Ap =.13, ver the span The frequency given in rdinate (± standard deviatin) is AQ/(ZQ.Ap), where AQ is the amunt f cnstituent present within the sectin, and ~Q the sum f the amunts fund n all the subfractins. The surface area f each sectin f the diagram gives the fractinal amunt f cnstituent present within the sectin. Slid blcks n each side f the distributin prfile represent material recvered belw 1.7 and abve 1.27; they are arbitrarily cnstructed ver the density spans and The ttal area f each histgram is then equal t 1. Cmplementary data are presented in Table I11. ent, shw a sharp peak at a density f abut 1.17, a value smewhat lwer than that bserved fr liver mitchndria (9, 35) but cmparable t that reprted. fr the mitchndria f several ther tissues, including spleen (15), skeletal muscle (48), Ehrlich ascites tumur cells (34), and Chinese hamster vary fibrblasts (38). The mitchndria seem t cntain sme NADH cytchrme c reductase activity but nt all f it, since after fractinatin by differential centrifugatin r den- P. TULKENS, H. BEAUFAY, AND A. TROUET Analytical Fractinatin f Hmgenates 395

14 TABLE IV Effect f Digitnin n Prperties f Fibrblast Micrsmes Cntent f hmgenate (LP fractin) Median density f equilibratin Digitnin- Digitnin- Untreated treated Untreated treated fractin fractin fractin fractin Dissciatin f patterns % % % Prtein '-Nucletidase I Chlesterl i Acid pyrphsphatase I nsine diphsphatase Ribnucleic acid NADH cytchrme c reductase I. 162 I N-Acetyl-~-glucsaminidase '-Nucletidase Acid pyrphsphatase Prtein 2 1 ~ 3 2 Chlesterl I: Insine diphsphatase AC RNA I.L i DENSITY i 1,1 1.2 FIGURE 9 Influence f digitnin n the density distributin f micrsmal cnstituents in a sucrse gradient. Enlarged micrsmal fractins (LP) were equilibrated in a linear sucrse gradient. Results are presented as described in Fig. 8. Density distributins were btained frm untreated micrsmes (thick line) and frm micrsmes treated with digitnin as described in the text (shading). Cmplementary data are presented in Table IV. 3~6 THE JOURNAL OF CELL BIOLOGY VOLUME 63, 1974

15 sity equilibratin a substantial reductase activity is fund in fractins pssessing little r n cytchrme xidase activity. L yssmes Fibrblast hmgenates were fund t cntain fur hydrlases that are: active at acid ph, largely latent in fresh preparatins, unmasked tgether by repeated freezing and thawing and by additin f detergents, assciated with particles f fairly high sedimentatin cefficient and f high equilibrium density in sucrse gradient, and released t a large extent in sluble frm by treatments that suppress latency. These prperties crrespnd t thse described previusly fr lyssmal enzymes frm rat liver (9, 23) and t the bichemical cncept f lyssmes as it has been prpsed by de Duve (18). It may be taken that they characterize als the lyssmes f cultured fibrblasts. High sensitivity t disruptin by digitnin represents anther similarity between fibrblast and liver (47) lyssmes and suggests that chlesterl is a cnstituent f their membranes. As in many ther tissues (5, 4), the lyssmal acid phsphatase f fibrblasts is inhibited by fluride and tartrate. The lyssmes dissciate clearly frm catalase in differential centrifugatin, frm mitchndria in density equilibratin, and frm ther cmpnents in bth methd. Our data, hwever, are nt incmpatible with a lcalizatin f sme esterase activity in lyssmes. Whatever the separatin methd applied, the distributin prfiles f the lyssmal enzymes are never quite identical. The small differences between cathepsin, N-acetyl-fl-glucsaminidase, and acid dexyribnuclease reflect prbably sme bichemical hetergeneity within the lyssmal ppulatin f cultured fibrblasts. Anther pssible surce f partial dissciatin is the interference by enzymes assciated with ther particles in assays f lyssmal enzymes. In this respect, t much significance shuld nt be attributed t the excess f acid phsphatase at the density f 1.15, where ther enzymes capable f bydrlyzing fl-glycerphsphate may equilibrate. Catalase-Bearing Particles (Perxismes?) Catalase is essentially assciated with subcellular particles in fibrblast hmgenates. The high degree f latency exhibited by fresh preparatins (7-8%) and the manner in which it can be suppressed indicate that this assciatin is nt merely the cnsequence f an adsrptin at the surface f subceuular particles. The greater resistante f the catalase-bearing particles t activating agents, particularly digitnin, differentiates them clearly frm lyssmes. The results f differential centrifugatin shw cnvincingly that we are nt dealing with mitchndria, and the density distributins btained by equilibratin in sucrse gradient distinguish the catalase-bearing particles frm ther micrsmal cnstituents. In liver and kidney, catalase has been shwn t be a typical cnstituent f perxismes (21). Even thugh the structural latency f fibrblast catalase and its relative resistance t activatin by digitnin are reminiscent f prperties reprted fr rat liver perxismes, identificatin f the catalase-bearing rganelles f fibrblasts with perxismes wuld be premature. Indeed, liver (9) and kidney (7) perxismes sediment mre rapidly and equilibrate at a higher density. Furthermre, they have been fund t cntain several hydrgen perxideprducing xidases which culd nt be detected in the present case. Plasma Membrane and Related Structures Chlesterl, 5'-nucletidase, insine diphsphatase, and acid pyrphsphatase share several prperties. All are assciated with sedimentable cmpnents f the hmgenate; they are purified in the micrsmal fractin but differ frm typical micrsmal cmpnents in cntributing cnsiderable amunts t the heavier N, M, and L fractins. They shw similar patterns f distributin in the sucrse gradient, althugh slight differences are bserved, especially after digitnin treatment. The linkage f the enzymes t subcellular structures is nt suppressed by treatments that release lyssmal enzymes int the suspensin medium. Fragments f plasma membrane r related structures are likely t be the cytlgical entity bearing the bulk f 5'-nucletidase in cullured rat fibrblasts. This enzyme exhibits mst prperties f the hepatic plasma membrane-bund 5'- nucletidase: activatin by Mg ++, inhibitin by F- ins and resistance t tartrate (27), absence f structure-linked latency, assciatin with subcellular cmpnents f lw density, density shift fllwing additin f digitnin (3, 2, 52). The characteristic "nucle-micrsmal" distributin f the liver enzyme has nt been btained with fibrblast P. TULKENS, H. BEAUFAY, AND A. TROUET Analytical Fractinatin f Hmgenates 397

16 hmgenates. But this is easily explainable, since the result f mechanical disruptin f the cell membrane by hmgenizatin may differ, depending n whether we are dealing with an rganized tissue r with a suspensin f cells. The clse parallel between the distributins f 5'-nucletidase and chlesterl indicates that, as in the liver, mst f the latter cnstituent is assciated with the pericellular membrane and related structures f cultured fibrblasts. Judging frm the digitnin shift, these cmpnents represent a sizeable fractin f the micrsmal prtein. They are likely t bear als the larger part f the acid pyrphsphatase activity. Sme f this activity, hwever, may be linked t anther subcellular cmpnent equilibrating at a higher density (Fig. 8), less sensitive t the treatment with digitnin (Fig. 9) and still t be identified. The distributin f insine diphsphatase fllws clsely that f 5'-nucletidase in differential and ispycnic centrifugatin. Only partial dissciatin is achieved by digitnin treatment. It is therefre likely that insine diphsphatase belngs largely t plasma membrane. The presence f an enzyme splitting the pyrphsphate bnd f insine diphsphate in the plasma membrane f cultured fibrblasts may be related t that f an unspecific nucleside diphsphatase, assayed with ADP, in the plasma membrane f rat liver (62), chick embry fibrblasts (44), and Ehrlich ascites cells (59). In liver, nucleside diphsphatase activity is mainly due t an enzyme acting preferentially n IDP, UDP, and GDP and assciated with micrsmal elements derived frm the endplasmic reticulum (11, 28) which, in cntrast t the ADPase f plasma membrane, is strngly activated and released in sluble frm by sdium dexychlate (42). The insine diphsphatase f fibrblast hmgenates lacks these prperties, thereby resembling the enzyme lcalized in rat liver plasma membranes. The absence f a specific nucleside diphsphatase in the endplasmic reticulum wuld accunt fr the lwer specific activity f insine diphsphatase fund in fibrblast hmgenates (Table li). The partial dissciatin achieved between 5'-nucletidase and insine diphsphatase by digitnin may reflect a bichemical hetergeneity f the plasma membrane, r the assciatin f sme f the insine diphsphatase activity with anther type f subcellular cmpnent. Endplasmic Reticulum and Other Cmpnents The mrphlgy f the cells, illustrated in Fig. 1, leaves little dubt that in the cytplasmic fractins f cultured fibrblasts the bulk f the RNA is cntributed by the RNA f ribsmes. In the case f the S fractin, this has been cnfirmed by the bservatin that mst f the RNA sedimented by a centrifugatin f W = rad ~ s- 1 (4, rpm fr 2 h, rtr 4, Beckman/Spinc Centrifuge). In the M, L, and P fractins, RNA must be, t a large extent, ribsmal RNA assciated with the vesicles derived frm the rugh endplasmic reticulum which is very expanded in these cells. After density equilibratin f LP fractins, the distributin f RNA thus reflects, in a smewhat crude fashin, the density distributin f ribsme cated vesicles. Since the density distributin f RNA is nt shifted after the treatment f LP fractins with digitnin, the endplasmic reticulum distinguishes itself clearly frm the plasma membrane in fibrblast hmgenates, as it des in the case f liver (3, 52). The enzyme respnsible fr the nnmitchndrial activity f NADH cytchrme c reductase is the nly ne which may be tentatively attributed t elements derived frm the endplasmic reticulum. It is assciated with micrsmal entities that shw n digitnin shift, and are therefre largely different frm thse bearing the 5'-nucletidase and the bulk f chlesterl. Hwever, the dual lcalizatin f NADH cytchrme c reductase makes this activity f little use as an enzymic marker f the endplasmic reticulum membranes. Esterase des nt seem t belng t a single subcellular cmpnent. In liver, the micrsmal activity prevails (2, 57) and belngs t elements derived frm the endplasmic reticulum (11). This lcalizatin, hwever, is disputed by cytchemical data which display reactin prducts f esterase activity in cytplasm, in the endplasmic reticulum, and in lyssmal bdies (33, 37). In spleen, the enzyme assayed with -nitrphenyl acetate has been attributed partially t lyssmes (14). In fibrblast hmgenates, esterase activity ccurs in the final supernate and in subcellular particles which cver a wide range f size and density. This may reflect the adsrptin f a sluble enzyme n varius subcellular cmpnents. 398 THE JOURNAL OF CELL BIOLOGY VOLUME 63, 1974

17 We thank Dr. C. de Duve fr helpful criticism and cntinuus encuragement alng this wrk. The cntributin f Drs. A. Amar-Cstesec, P. Baudhuin, and D. Thin~s-Sempux is gratefully acknwledged. This wrk has been supprted by the Belgian Fnds de la Recherche Fndamentale cllective (grants n. 78 and 1137) and the Belgian Service de la Prgrammatin et de la Plitique Sc&ntifique. Dr. P. Tulkens is Aspirant and Dr. A. Truet, Chercheur Qualifi~ f the Belgian Fnds Natinal de la Recherche Scientifique. Received fr publicatin 19 Nvember 1973, and in revised frm 28 May REFERENCES 1. AMAR-COSTESEC, A., H. BEAUFAY, E., FEYTMANS, D. THINES-SEMPOUX, and J. BERTHET Subfractinatin f rat liver micrsmes In Micrsmes and Drug xidatins. J. R. Gillette, A. M. Cnney, G. J. Csmides, R. W. Estabrk, J. R. Futs, and G. J. Mannering, editrs. Academic Press Inc., New Yrk AMAR-COSTESEC, A., H. BEAUFAY, M. WIBO, D. THINES-SEMPOUX, E. FEYTMANS, M. ROBBI and J. BERTHET Analytical study f micrsmes and islated subcellular membranes frm rat liver. I1. Preparatin and cmpsitin f the micrsmal fractin. J. Cell Bil. 61: AMAR-COSTESEC, A., M. WIBO, D. THINES- SEMPOUX, H. BEAUFAY, and J. BERTHET Analytical fractinatin f micrsmes and islated membranes frm rat liver. IV. Bichemical, physical, and mrphlgical mdificatins f micrsmal cmpnents induced by digitnin, EDTA, and pyrphsphate. J. Cell Bil. 63: BARLAND, P., and E. A. SCHROEDER A new rapid methd fr the islatin f surface membranes frm tissue culture ceils. J. Cell Bil. 45: BARRETT, A. J Prperties f lyssmal enzymes. In Lyssmes in Bilgy and Pathlgy. J. T. Dingle and H. B. Fell, editrs. Nrth-Hlland, Amsterdam. Tme II, BAUDHUIN, P., H. BEAUFAY, Y. RAHMAN-LI, O. Z. SELLINGER, R. WATTIAUX, P. JACQUES, and C. DE DUVE Tissue fractinatin studies. 17. lntracellular distributin f mnamine xidase, aspartate amintransferase, alanine amintransferase, D-aminacid xidase and catalase in rat liver tissue. Bichem. J. 92: BAUDHUIN, P., M. M/2LLER, B. POOLE, and C. DE DUVE Nn mitchndrial xidizing particles (micrbdies) in rat liver and kidney and in Tetrahymena pyrifrmis. Bichem. Biphys. Res. Cmmun. 2: BEAUFAY, H La centrifugatin en gradient de densit6. Applicatins h l'6tude des rganites subcellulaires. Ceuterick Luvain (Belgium). 9. BEAUFAY, H., P. JACQUES, P. BAUDHUIN, O. Z. SELLINGER, J. BERTHET, and C. DE DUVE Tissue fractinatin studies. 18. Reslutin f mitchndrial fractins frm rat liver int three distinct ppulatins f cytplasmic particles by means f density equilibratin in varius gradients. Bichem. J. 92: BEAUFAY, H., A. AMAR-COSTESEC, E. FEYTMANS, D. THINES-SEMPOUX, M. WIBO, M. ROBBI, and J. BERTHET Analytical study f micrsmes and islated subcellular membranes frm rat liver. I. Bichemical methds. J. Cell Bil. 61: BEAUFAY, H., A. AMAR-COSTESEC, D. THINES- SEMPOUX, M. Wm, M. ROBBI, and J. BERTHET Analytical study f micrsmes and islated subcellular membranes frm rat liver. II!. Subfractinatin f the micrsmal fractin by ispycnic and differential centrifugatin in density gradients. J. Cell Bil. 61: BOONE, C. W., L. E. FORD, H. E. BOND, D. C. STUART, and D. LORENZ Islatin f plasma membrane fragments frm HeLa cells. J. Cell Bil. 41: BOSMANN, H. B., A. HAGOPIAN, and E. H. EYLAR Cellular membranes. The islatin and characterizatin f the plasma and smth membranes f HeLa cells. Arch. Bichem. Biphys. 128: BOWERS, W. E., J. T. FINKENSTAEDT, and C. DE DUVE Lyssmes in lymphid tissues. I. The measurement f hydrlytic activities in whle hmgenates. J. Cell Bil. 32: BOWERS, W. E., and C. DE DUVE Lyssmes. in lymphid tissues. 11. lntracellular distributin f acid hydrlases. J. Cell Bil. 32: BUCK, C. A., M. C. GLICK, and L. WARREN A cmparative study f glycprteins frm the surface f cntrl and Rus Sarcma virus transfrmed hamster cells. Bichemistry. 9: COOPERSTEIN, S. J., and A. LAZAROW A micrspectrphtmetric methd fr the determinatin f cytchrme xidase. J. Bil. Chem. 189: DE DUVE, C The lyssme cncept. Ciba Sympsium, Lyssmes. A. V. S. De Reuck, and M. P. Camern, editrs. J. and A. Churchill Ltd., Lndn DE DUVE, C Principles f tissue fractinatin. J. Ther. Bil. 6: DE DUVE, C Tissue fractinatin past and present. J. Cell Bil. 5:2D-55D. 21. DE OUVE, C., and P. BAUDHUIN Perxismes. Micrbdies and related particles. Physil. Rev. 46: DE DUVE, C., J. BERTHET, H. G. HERS, and L. P. TULKENS, H. BEAUFAY, AND A. TROUET Analytical Fractinatin f Hmgenates 399

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