LYSOSOMAL FRACTIONS FROM

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Published Online: 1 February, 1965 Supp nf: http://di.rg/10.1083/jcb.24.2.259 Dwnladed frm jcb.rupress.rg n Octber 6, 2018 LYSOSOMAL FRACTONS FROM TRANSTONAL EPTHELUM NORBERT M. KANCZAK, Ph.D., JOSEPH.

1 Published Online: 1 February, 1965 Supp nf: Dwnladed frm jcb.rupress.rg n Octber 6, 2018 LYSOSOMAL FRACTONS FROM TRANSTONAL EPTHELUM NORBERT M. KANCZAK, Ph.D., JOSEPH. KRALL, E. RUSSELL HAYES, Ph.D., and WLLARD B. ELLOTT, Ph.D. Frm the Departments f Anatmy and Bichemistry, State University f New Yrk at Buffal, Schl f Medicine, Buffal. Dr. Kanczak's present address is The Labratry f Cellular Pathlgy, The Ohi State University, Cllege f Medicine, Clumbus ABSTRACT Histchemical data suggested that the s called lipid granules f transitinal epithelium in sme species are equivalent t lyssmes. Scrapings f bvine and canine transitinal epithelium were subjected t differential centrifugatin t cnfirm this identificatin bichemically. Fractins f rat liver, the classic surce f lyssmes, were als prepared by the same methds t cmpare with the fractins btained frm urinary epithelium. n cntrast t rat liver, urepithelial fractins with a high relative specific activity fr hydrlases were sedimented befre the heavy mitchndria. Micrscpically, these fractins cntained the highest prprtin f lipid granules. The size and sedimentatin characteristics f lyssmes frm transitinal epithelium mre clsely resembled thse f lyssmes derived frm rat kidney than thse islated frm liver. NTRODUCTON The centrifugal separatin and characterizatin f a class f subcellular particles frm rat liver by de Duve et al. (6) has led t the widespread experimental investigatin f these particles which have been called lyssmes. Althugh bichemical, histchemical, and electrn micrscpe studies f lyssmes have been numerus, the number f investigatins in which lyssmes have been separated frm ther cellular cnstituents and characterized enzymatically have been few. The presence f lipid-rich drplets in transitinal epithelium was nted as early as 1911 by Bauch (2). Takahashi (17) cined the term "lipid granules" fr these cytplasmic structures because he felt that they did nt represent neutral lipid inclusins. Since the lipid granules in the transitinal epithelium f several species had been histchemically identified as lyssmes (9), an attempt was made t islate them by differential centrifugatin frm the epithelium f species in which there was a crrespndence between lipid granules and histchemically detectable lyssmes, and t determine their enzymatic characteristics bichemically. MATERALS AND METHODS Histchemistry Lipids were demnstrated in frzen sectins with Sudan black B after fixatin in 10 per cent phsphate-buffered neutral frmalin. Acid phsphatase activity was visualized in frml-calcium-fixed frzen sectins emplying the napthl AS-pararsaniline methd f Barka and Andersn (1) and a mdified Gmri technique (11). Preparatin f Fractins Fr each f the centrifugal fractinatins f bvine transitinal epithelium, the urinary bladclers 259

2 SLURRY 1, Q-rain. REHOMOGEN'ZE "g 1 SEDMENT SUPERNATANT A... ~.#COMBNED SUPERNATANT 6,000 g-rain..-"./ 0,000 ~-min. l ~.'". 1 // '1 S D.MENT SU.ERNA ANT.".-" RA,ON' [RE.O.O EN. E / 6,000 g" rain. // / / 1 // FRACTON ]~Z" SUPERNATANT SEDMENT SUPERNATANT C/" 155,000 9.rain. 3,130 g_-min. ; FRACTON SO NAT"T FRACTON "r FRACTON ~. SUPERNATANT ] 250,000 g-min.. 16' ] SO ERNATANT 1 FRACTON SUPERNATANT (DSCARDED) ]5,000,OOO g-rain. FRACTON ~ FRACTON FGURE 1 Schematic representatin f the prcedure utilized in the differential centrifugatin f transitinal epithelium and rat liver. The term "g-min." is that advcated and utilized by de Duve and Berthet (5) t enhance the reprducibility f results. t is defined as tile time integral f the field prevailing in the middle f the centrifugal fluid. f 4 freshly slaughtered steers were pened and pinned ut with the urinary surface upward. The transitinal epithelium was scraped with a razr blade and cllected in tared beakers chilled in ice. Each beaker cntained 2.0 ml f an islatin medium which was cmpsed f 0.25 M sucrse and M ethylenediaminetctraacetate (EDTA) and whse ph was adjusted t 7.4. The pled scrapings prvided 4 t 7 gm f tissue. The tissue was further diluted t 5 times the riginal weight f the tissue in grams. n the cld rm the tissue was hmgenized in a glass tube with a mtr-drlven Tefln pestle by 10 passes f the pestle. The resultant slurry was centrifuged in a refrigerated Serval preparative centrifuge (Mdel RC ) maintained at --2 C using rtr N. SS 34 (Rm,x = 10.8 cm, Rmln = 4.5 era). n initial experiments, the fractinatin scheme f de Duve et al. (6) fr rat liver was fllwed. With this scheme hydrlase-rich fractins frm bvine transitinal epithelium were sedimented befre thse with cytchrme xidase activity. Because f this and the fact that the lipid granules, which were histchemically identified as lyssmes, ranged up t 3 ~ in diameter, a mdified fractinatin scheme was develped which finally resulted in 8 fractins. This scheme is shwn schematically in Fig. 1. The sediment resulting frm the riginal hm- genizatin was rehmgenized 2 times by passing the pestle thrugh the tissue 5 times after diluting with the same amunt f medium in rder t insure cmplete hmgenizatin f remaining intact cells. The "cmbined supernatant" was diluted with the islatin medium t a final vlume in ml equal t 10 times the riginal wet weight f the tissue in grams. Fractin was washed nce by resuspending the sediment with a single pass f the pestle after diluting with 10 ml f islatin medium and then resedimenting by the same field-time as used fr its initial islatin. T remve nuclear cntaminatin, Fractins t V were resuspended as abve and centrifuged by 3,130 g-min. 1 with the sediment being discarded. The supernatants were then centrifuged by the same field-time used t btain the given fractin. The given fractin was then washed nce, again emplying the same field-time fr resedimentatin and discarding the wash-supernatant. Fractin V was washed nce. Each f the fractins was examined micrscpically with reduced illuminatin. The unstained specimen was prepared by mixing a small quantity f sediment with 2 r 3 drps f islatin medium. n the study f canine transitinal epithelium, the same separatin scheme was fllwed as utlined 1 See legend t Fig THE JOURNAL OF CELL BOLOGY VOLUME ~, 1965

3 abve. The urinary bladders were remved frm 4 dgs under Nembutal anesthesia. The pled scraping prvided 0.8 gm f epithelium. Rat liver was als fractinated by the revised scheme used fr the transitinal epithelium. Bichemical Enzyme Assays ACD PHOSPHATASE : Determinatins were perfrmed accrding t the methd described in Hawk, Oser, and Summersn (8). ~-GLUCURONDASE" The enzyme was assayed by a slight mdificatin f the methd detailed in Sigma Technical Bulletin N. 105 using M phenlphthalein glucurnide (Sigma) in the incubatin mixture. CYTOCHROME OXDASE : This enzyme was measured plargraphically in a G.X.E. Oxygraph. The incubatin mixture cnsisted f 2.0 ml f 0.1 ~t ptassium phsphate buffer (ph 7.3.), 0.2 ml f 6 X 10-4 M cytchrme c, and 0.3 ml f 0.25 M as ACD PHOSPHATASE 0... CYTOCHROME OXDASE 8' g i v,: 6 i <[ ~ (n 4 g~ t ~.!/" ~ 'm TO" 'v "VT ~ FRACTONS J FmUaE ~ Rat liver. Sedimentatin patterns frmed by the simultaneus pltting f the relative specific activities (Rel. Sp. Act.) fr cytchrme xidase and acid phsphatase in fractins btained by differential centrifugatin. TABLE A Cmparisn between the Relative Specific Enzyme Activities in Fractins Obtained by the Differential Centrifugatin f Canine Transitinal Epithelium and Thse Obtained frm Rat Liver under the Same Cnditins Fractin V V V V r~ Canine transi- Cytchrme tinal epithelium xidase Acid phs phatase Rat liver Cytchrme xidase Acid phs phatase KANCZAK, KRALL, HAYES, AND ELLOTT Lyssmalfractins 261

FGURE 8 Frzen sectin f bvine transitinal epithelium stained with Sudan black B. Mderate t intensely sudanphilic lipid granules are visible arund the nuclei. X 1100.

Enzymatic activity is present in paranuclear drplets which crrespnd in size, number, and distributin t the lipid granules f Fig. 3. )< 1~00. crbic acid. Added t this was 0. l ml f 0.

4 FGURE 8 Frzen sectin f bvine transitinal epithelium stained with Sudan black B. Mderate t intensely sudanphilic lipid granules are visible arund the nuclei. X Fret:rE 4 Bvine transitinal epithelium incubated fr acid phsphatase activity (1). Enzymatic activity is present in paranuclear drplets which crrespnd in size, number, and distributin t the lipid granules f Fig. 3. )< 1~00. crbic acid. Added t this was 0. l ml f 0.04 M EDTA t minimize autxidatin. The reactin was started by the additin f 0.1 ml f sample. Prtein was determined by the methd f Lwry et al. (12). All enzyme assays were perfrmed n fractins which were stred vernight at 2 C. RESULTS Rat Liver The sedimentatin patterns btained fr the activity f acid phsphatase and cytchrme xidase resembled thse btained by de Duve et al. (6) wh emplyed 5 fractins. Fractin cnsisted f islated nuclei with cntaminatin by red cells and smc mitchndria. Fractins t V all cntained slightly refractile particles f apprximately the same size within a given fractin. The particle size, hwever, did decrease gradually frm Fractin t Fractin V. Frac- tin V shwed n visible particulates under the micrscpe, and grssly the sediment had the typical red jelly-like appearance f the micrsmal fractin. n Fig. 2 and Table it can be seen that the highest relative specific activities fr cytchrme xidase were present in fractins sedimenting befre fractins with the highest relative specific activity fr acid phsphatase. The relative specific activity fr each fractin was btained by dividing the per cent f the ttal enzyme activity by the per cent f the ttal prtein in that fractin. The ttal enzyme activity and the ttal prtein were determined by the summatin f the respective values fr each f the fractins. The highest relative specific activities fr acid phsphatase were present in Fractins V and V. Fractin V was sedimented by a field-time equivalent t that used by de Duve et al. (6) in the preparatin f the light mitchndrial r lyssmal fractin f rat liver. 262 T~E JurNxL OF CELL BOLOGY VOLVME ~4, 1965

5 /~ -- ACD PHOSPHATASE... CYTOCHROME OXDASE [',, [', 10- "5 $ t-- >" 6-! -4 ~. _ ~t FGURE 5 Bvine transitinal epithelium. Altered sedimentatin patterns f cytchrme xidase and acid phsphatase btained with epithelial scrapings using the same fractinatin scheme emplyed fr rat liver (Fig. ~). FRACTONS Bvine Transitinal Epithelium The lipid granules f the bvine epithelium range up t 3 # in diameter and are distributed as paranuclear rings ~" (Fig. 3). Their number and average size increase as ne prceeds frm basal t surface cells. n unstained sectins, a yellwbrwn pigment can be demnstrated in them. Sectins incubated fr acid phsphatase activity (Fig. 4) reveal that there is a crrespndence in size, number, and distributin between lipid granules and histchemically demnstrable lyssmes. When fractins which were prepared by differential centrifugatin were examined micrscpically, the larger size, pigment, and higher refracfility f the lipid granules permitted their ready differentiatin frm mitchndria which were nly slightly refractile. Fractin was fund t cnsist mainly f islated nuclei and rare intact cells. Fractins t V cntained lipid granules whse average size decreased frm Fractin t V. n additin, these fractins cntained mitchndria, the prprtin f these increasing frm Frac- 2 Data n further histchemical prperties f lipid granules in transitinal epithelium will be published elsewhere. tin t V. Fractin V shwed hmgeneus fields f slightly refractile particles. Fig. 5 shws that in cntrast t rat liver the highest relative specific activity fr acid phsphatase was present in Fractin V. Fractins t V always displayed higher relative specific activities fr acid phsphatase than fr cytchrme xidase, and it was these fractins in which the highest prprtin f lipid granules was fund. Fractin V, sedimented under the same cnditins as Fractin V f rat liver, shwed relatively high cytchrme xidase activity and lw acid phsphatase activity. Studies cmparing ~-glucurnidase with cytchrme xidase activity in bvine transitinal epithelium were made prir t the develpment f the final fracfinatin scheme cnsisting f 8 fractins. Fractin A in Fig. 6, hwever, was sedimented by 6000 g-min, and can be expected t cntain the particulates sedimented in Fractins and f the final fractinatin scheme. Fractin B, likewise, can be cnsidered equivalent t Fractins and V f the 8 fractin scheme since it was sedimented by g-min. Fractins C, D, E, and F were b~ined under cnditins that made them equivalent t Fractins V, V, V and V, respectively. Fig. 6 demnstrates KANeZAK, Y~ALL, HAYF.S, AND ELMOTT Lgssmalfractins 263

6 /~- GLUCURONDASE,\ ~\... CYTOCHROME OXDASE!\ ~c :!,, i 1 t / i A B C 0 E F (z+'rr) ('rrrqyj ('0 3 (~Z) (WT) (vm) 2.O l~a~e 6 Bvine transitinal epithelium. Sedimentatin patterns f eytchrme xidase and/~-glucurnidase. FRACTONS that high relative specific activities fr fl-glucurnidase and lw specific activities fr cytchrme xidase were present in Fractins A ( and ) and B ( and V). Fractin C (V) cntained high relative specific activities fr bth enzymes. t will be recalled that micrscpically this fractin cntained the smallest lipid granules and a high prprtin f mitchndria. Fractin D (V) which micrscpically cnsisted f hmgeneus fields f slightly refractile mitchndria pssessed a realtively high cytchrme xidase activity and a relatively lw ~-glucurnidase activity. Canine Transitinal Epithelium n the dg, there again is a crrespndence in size, number, and distributin between lipid granules (Fig. 7) and histchemically detectable yssmes (Fig. 8). The granules are nn-pigmented in this species and reach a maximum diameter f 6 g. Larger nes demnstrate evidence f internal suaacture discernible even at the light micrscpic level. n Table, a cmparisn is made between the data btained with canine transitinal epithelium and rat liver. n the transitinal epithelium f the dg the highest relative specific activities fr acid phsphatase were fund in Fractins and V which were sedimented befre Fractin V. t was the latter which pssessed the highest relative specific activity fr cytchrme xidase. DSCUSSON Lyssmes were first described and defined as a distinct class f subcellular particles in rat liver (6). n the parenchymal cells f this rgan they have an average diameter f 0.4 p and sediment between mitchndria and micrsmcs. Later, the presence f lyssme-like particles in rat brain was first suggested in anther bichemical study bccause hydrlytic enzymes were bund t particulate cmpncnts within which they were nt fully active unless a lysing agent (Tritn X-100) was added (3). Hwever, they culd nt be separated frm mitchndria. A cncentratin f acid phsphatasc in a "light mitchndrial" fractin f rat brain at nearly three times its cncentratin in the ttal hmgenatc has als been reprted (10). Watfiaux (19) has studied the scdimentatin patterns f several hydrlases and cytchrmc xidasc after differential centrifugatin and density-gradient centrifugatin f hmgenized HcLa 264 T~ JOURNAL OF CELL BOLOGY " VOLUME ~4, 1965

FGURE 7 Canine transitinal epithelium stained with Sudan black B. n this species the lipid granules are als paranuclear, but reach a maximum size f 6/z. X 1~. cells.

$(18) in an analysis f skeletal muscle have nted that hydrlase activity sedimented in the mitchndrial fractin, in fractins sedimenting between mitchndria and micrsmes, and with the micr0smal fractin.$ $On the ther hand, Rahman (15) has prepared fractins f rat thymus by density-gradient centrifugatin and has examined these bichemically and electrn micrscpically.$

7 FGURE 7 Canine transitinal epithelium stained with Sudan black B. n this species the lipid granules are als paranuclear, but reach a maximum size f 6/z. X 1~. cells. t was cncluded that the lyssmes f these cells were similar t thse f rat liver. Tappel et al. (18) in an analysis f skeletal muscle have nted that hydrlase activity sedimented in the mitchndrial fractin, in fractins sedimenting between mitchndria and micrsmes, and with the micr0smal fractin. N histchemical analysis f the tissue was made and the mrphlgical cmpsitin f the fractins was nt examined. On the ther hand, Rahman (15) has prepared fractins f rat thymus by density-gradient centrifugatin and has examined these bichemically and electrn micrscpically. Electrn-paque bdies with a mean diameter f 0.25 ~ were fund in highest cncentratin in fractins high in acid phsphatase, while fractins rich in cytchrme xidase cnsisted mstly f mitchndria. Thus, in the studies cited abve, the lyssmes f several tissues have exhibited size and sedimentatin characteristics similar t thse f rat liver. n the present study, the separatin f fractins frm bvine and canine transitinal epithelium FGURE 8 Canine transitinal epiflmlium incubated fr acid phsphatase activity (11). The lipid granules f Fig. 7 all appear t cntain enzymatic activity. X cntaining high relative specific activity fr acid phsphatase and fl-glucurnidase, lw cytchrme xidase, and large numbers f refractile granules f the same size range as the lipid granules f these species substantiates the histchemical identificatin f these granules as lyssmes. The presence f lyssmes in transitinal epithelium has, in passing, been previusly suggested frm histchemical data (14). The lyssmal fractins f transitinal epithelium in the species studied here have been shwn t differ frm thse f rat liver in the same study by a direct cmparisn emply= ing identical methds f preparatin. The nly ther islated lyssmal fractins which have been shwn t have size and sedimentatin characteristics different frm rat liver are thse btained frm rat kidney (16). Drplets up t 6 # in diameter have been separated frm rat kidney which have high cncentratins f acid phsphatase, ribnuclease, dexyribnuclease, fl-glucurnidase and cathepsin, and which sediment between nuclei and mitchndria. KANCZAK, KRALL, HAYES, AND E,MOTT Lyssmalfractins 265

$Mrphlgical analysis f the fractins btained frm the bvine epithelium was relatively simple because the lyssmes range up t 3/~ in diameter and cntain pigment.$

8 Mrphlgical analysis f the fractins btained frm the bvine epithelium was relatively simple because the lyssmes range up t 3/~ in diameter and cntain pigment. Nuclei were bvius because f their large size and lack f pigment. Mitchndria were nly slighdy refractile and nn-pigmented. Only the studies f Strans n rat kidney (16), f Nvikff et al. n the liver (13), and f Rahman n the thymus (15) have invlved a mrphlgical analysis f lyssmal fractins frm ther tissues. n additin t mrphlgical analysis, the present study emplyed the mitchndrial enzyme, cytchrme xidase, t determine the degree f mitchndrial cntaminafin f the lyssmal fractins. Althugh Kenig and Jibril (10) speak f btaining a BBLOGRAPHY 1. BARKA, T., and ANDERSON, P. j., Histchemical methds fr acid phsphatase using hexaznium pararsaniline as cupler, J. Histchem. and Cytchem., 1962, 10, BAUCH, M., Vergleichende anatmische und histlgische Untersuchungen fiber die Hamblase der Haustiere, Dissertatin, University f Leipzig, BEAUFAY, H., BERLEUR, A. M., and DOYEN, A., The ccurrence f lyssme-like particles in rat brain tissue, Bichem. J., 1957, 66, 32p. 4. CONCHE, J., HAY, A. J., and LEWY, G. A., Mammalian glycsidases. 3. The intracellular lcalizatin f ~-glucurnidase in different mammalian tissues, Bichem. J., 1961, 79, DvE, C. DE, and BERTHET, J., Reprducibility f differential centrifugatin experiments in tissue fractinatin, Nature, 1953, 1"12, DUVE, C. DE, Pressman, B. G., GANETTO, R., WATTAUX, R., and APPELMANS, F., Tissue fractinatin studies. 6. ntracellular distributin patterns f enzymes in rat-liver tissue, Bichem. J., 1955, 60, GREENBAUM, A. L., SLATER, T. F., and WANG, D. Y., Lyssme-like particles in the rat mammary- gland, Nature, 1960, 188, HAWK, P. B., OSER, B. L., and SU~MERSON, W. H., Practical Physilgical Chemistry, New Yrk and Trnt, Blakistn C., KANCZAK, N. M., Cmparative histlgical, histchemical, electrn micrscpic, and bichemical studies n transitinal epithelium, Ph.D. thesis, State University f New Yrk at Buffal, KOENm, H., and JBRL, J., Acidic glyclipids and the rle f inic bnds in the structure- "light mitchndrial" fractin frm rat brain with a hydrlase cncentratin f three times that in the ttal hmgenate, they make n mentin f mrphlgical cnfirmatin f the cntent f the fractins r determinatin f cytchrme xidase activity. Other bichemical studies (4, 7) have nly established that lyssmes are present in varius rgans by inference, since the nly criteria f identificatin have been the presence f sedimentable hydrlase activity and/r the presence f latent hydrlase activity. This investigatin was supprted in part by a United States Public Health Service Training Grant 5T- GEM-407 and a research career award (5-K3-GM ) t Dr. Ellitt. Received fr publicatin, March 10, linked latency f lyssmal hydrlases, Bichim. et Biphysica Acta, 1962, 65, LmLE, R. D., Histpathlgic Technique and Practical Histchemistry, New Yrk, Trnt and Lndn, Blakistn Divisin, McGraw Hill Bk C., 1954, LOWRY, O. H., ROSEBRAUGH, N. J., FARR, A. L., and RANDALL, R. J., Prtein measurement with the Flin phenl reagent, J. Bil. Chem., 1951, 193, NvmFF, A. B., BEAUFAY, H., and DuvE, C. DE, Electrn micrscpy f lyssme-rich fractins f rat liver, J. Biphysic. and Bichem. Cytl., 1956, 2, N. 4, suppl., Nvmvv, A. B., Lyssmes and related particles, in The Cell, (J. Brachet and A. E. Mirsky, editrs), New Yrk, Academic Press, nc., 1961, 2, RAHMAN, Y. E., Electrn micrscpy f lyssme-rich fractins frm rat thymus islated by density-gradient centrifugatin befre and after whle-bdy irradiatin, J. Cell Bil., 1962, 13, STRAUS, W., Cncentratin f acid phsphatase, ribnuclease, dexyribnuclease, /~-glucurnidane, and cathepsin in drplets islated frm kidney cells f nrmal rats, J. Biphysic. and Bichem. Cytl., 1956, 2, TAKAHASH, T., Zur Zytlgie der Epithelzellen der Harnblasen des V[enschen, Okajimas Flia Anat. Japn., 1938, 16, TAPPEL, A. L., ZALKN, H., CALDWELL, K. A., DESA,. D., and SHBKO, S., ncreased lyssmal enzymes in genetic muscular dystrphy, Arch. Bichem., 1962, 96, WATTAUX, R., Lcalisatin des hydrlases aeides dans les cellules HeLa, Arch. nternat. Physil. et Bichim., 1962, 70, TH JOURNAL OF CELL BOLOGY VOLUME ~4, 1965

The demonstration of lysosomes by the controlled temperature freezing-sectioning method By LUCILLE BITENSKY

The demonstration of lysosomes by the controlled temperature freezing-sectioning method By LUCILLE BITENSKY 205 The demnstratin f lyssmes by the cntrlled temperature freezing-sectining methd By LUCILLE BITESKY (Frm the Department f Pathlgy, Ryal Cllege f Surgens f England, Lincln's Inn Fields, Lndn, W.C. 2)