Comp Btochem Phvsml

1976 l, ol 54B pp 107 to 110 Pergamon Press Printed In Great Brttaln

STUDIES ON THE HETEROGENEITY OF MAMMALIAN fl-GLUCURONIDASES ROGER T DEAN AND MICHAEL MESSER* Department of Experimental Pathology, Umverslty College Hospital Medical School, London, England

(Recewed 23 September 1974) Abstract--1 Gel electrophoresm showed that the hver of rabbit (Oryctolegus cumculus), and probably of rat (Rattus norveg~cus), contains molecular size varmnts of fl-glucuromdase corresponding to those of mouse (Mus musculus) tissues (described by Swank & Palgen, 1973) 2 Experiments on charge characteristics, xmmunologlcal relationships, mterconvers~on and subcellular dlstnbut~on of the enzyme forms in rabbit hver prowded no ewdence for the inclusion m the larger molecular size forms of an accessory polypeptlde very different from the subumt of the smaller tetramenc forms 3 Extracts of mlcrosomal and lysosomal fractions from rabb~t liver &ffered only quantltatwely m their complements of fl-glucuromdase forms INTRODUCTION

onldase was done as before (Dean, 1974a) except that fast Garnet GBC was included m the incubations, which were extended to a maximum of 12 hr Recoveries of fl-glucuromdase activity from gel electrophoresls and lsoelectnc-focusmg m polyacrylamlde gels, as determined by direct assay of homogemsed polyacrylamlde beads, were 90-95°0 fl-Glucuromdase activity was eluted from gels for re-running by homogenlsmg m dlstdled water with an Ultraturrax homogenlser, followed by sonlcat~on (MSE 150 watt Ultrasomc Disintegrator), at 4°C The polyacrylamlde beads were then shaken m~dlstllled water at 37°C overmght, and the beads removed by centrxfugatlon Only 35°0 of the actwlty was eluted, the remainder being still detectable m the beads For focusing enzyme forms from electrophores~s, gel segments were fragmented by passage through syringe needles, apphed directly to the surface of gels, and overlaid with a small volume of 1°,o ampholm~20% w/v sucrose Electrophoresls m gel slabs containing continuous nonhnear polyacrylamlde gradients (Gradlpore Batch No 197, Umversal Sclenttfic, 231, Plashet Road, London E 13) was also performed with the buffer system of Tommo & Palgen (1970), in the Gra&pore reorculatmg buffer apparatus (Universal Scientific) at operating temperatures of 5-8~C A potentml of 200V was applied for 18 hr Liver homogenates were prepared m 025 M sucrose, 0 02 M lmldazole adjusted to pH 7 4 with HC1, by passage through a Harvard Press, followed by ten passes of a loosefitting Dounce homogenizer, the operatxons were conducted at 0-4°C Subcellular fractions of rabbit liver were obtained by three different procedures (1) The hypotomc METHODS AND MATERIALS shock method of Ganschow & Palgen (1968) In this method, the homogenate ~s centrifuged at 100,000 g to give These were as described by Dean (1974a, b) with the a soluble fractxon consisting of cystosol components and following addmons and exceptions particulate material solubdlsed during the mltml homoCrystalhne trypsin was purchased from Boehrmger Rats genlzauon (including some lysosomal components) The (Wistar-derived) and mice were obtained from laboratory residue is dispersed m a hypotomc medium, and recentnstock Rabbit liver fl-glucuromdase was purified as de- fuged to gwe a fractxon solubdxsed thereby (mainly lysososcribed prewously (Dean, 1974a) and stored m 100mM mal and mltochondrxal components) and a residue which Trls-HCl, pH 7 8 50°o glycerol, at -20°C includes mlcrosomal and other membrane material (2) Gel electrophoresxs was performed with 7~o polyacryla- Rate sedimentation through a hnear sucrose gradxent mide gels in the buffer system descrxbed by Tomlno & (10-60% w/v) at 16,000 0 for l h r m an SW 252 rotor Palgen (1970), using a Shandon &sc-electrophoresls appar- (Swank & Pmgen, 1973) (3) Dlfferentml centrffugatlon by atus run m an LKB-Cold Rac at 4°C Current flow was the method of de Duve et al (1955) but omitting the reholnltmlly 3 mA/tube, at an upper voltage hmlt of 400 V Elec- mogemzat~on and recentrlfugat~on of the nuclear-debris trophoresas was normally for 90 mm Staining for fl-glucur- fracuon fl-Glucuromdase acuwty m the mlcrosomal fraction * Present address Department of Biochemistry, Umver- obtained by each method was solubdlsed wxth 5°0 Triton s~ty of Sydney, New South Wales, Austraha X-100 (Swank & Palgen, 1973) or sometimes with 0 1% 107

Swank & Palgen (1973) recently separated by gel electrophoresls, six forms of fl-glucuronldase (designated L, X a n d M1 to M4 m order of decreasing anodlc mobility at p H 8 1) present in several mouse tissues Two tetramerlc forms (L & X), which varied slightly m charge but not m size, were shown to predominate respectively m lysosomal and mlcrosomal fractions, in addition, a series of mlcrosomal forms M1 to M4, increasing in a p p a r e n t molecular weight In steps of approximately 50,000 from X, were observed These M forms were absent In the ego m u t a n t which has very low microsomal fl-glucuronidase activity (see Palgen, 1971), a n d form X was significantly decreased Swank & Palgen (1973) therefore proposed that the M forms contain from one to four molecules of a n "accessory protein" of molecular weight 50,000 linking form X to the mlcrosomal membrane The present paper indicates that smallar electrophoretlc forms of fl-glucuromdase are also present m liver of rabbit a n d rat, a n d describes the subcellular distribution a n d relationships of the forms m rabbit hver In addition, their relationship to the multiple forms previously separated by lsoelectric focusing (Dean, 1973a. b, 1974a, b) is investigated

ROGER T DEAN AND MICHAIL MESSLR

108

octyl sodium sulphate (Dean, 1974b) The enzyme in the lysosomal fractions from methods (2) and (3) was Slmllarl~ solubdlsed with Triton X-100 at a final concentration of either 5°o (Swank & Palgen, 1973) or 0 1°o (Dean, 1974b} Total liver homogenate fl-glucuromdase was sometimes solublhsed with 5°o Tmon X-100 All the solubdlsed samples were centrifuged at 100000O for l hr, and the supernatants (containing 85-95°° of the fl-glucuronldase originally present) used for electrophoresls lsoelectrlc focusing or immunological experiments To test possible means of lnterconvertlng rabbit liver enzyme forms, incubations under various conditions ~ere performed and the resultant mixtures lmmedmtelv electrophoresed With the exception of that using percl;tlorate, no loss of actlwty occurred during such incubations, and thus the disappearance of a form indicated its conversion into other forms RESLLTS

Existence and ~ubcellular dtstttbutzon oJ the electmphorett¢ for ms of fl-glucut omdase Gel electrophorests oJ e',tra~ts o] unJracttonated tissue homoyenates The six forms of fl-glucuronldase described by Swank & Paigen (1973), were observed m extracts of both mouse and rabbit hver, although forms M I-M4 were rather faint (Fig 1) The resolution of rat liver fl-glucuromdases was not so clear, but by running duphcate electrophoreses for 30, 60, 90, or 120 ram, and staining one of each pair briefly to demonstrate the active bands, and the other for a more extended period to demonstrate the weak bands, it was again possible to distinguish six bands (Fig 1) O f the M forms, MI was the most intense m the rat and M , in the rabbit, m mouse liver all the M forms were of relatively low activity Since //-glucurontdases of both rat hver (Stahl & Touster, 1971) and rabbit hver (Dean, 1974a) have rather small ranges of lsoelectric points (4 5-5 8), it seems likely that the M forms are retarded because they are larger than the L and X forms, as m the case of the mouse kidney (Swank & Paigen, 1973)

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Fig 2 Separation by electrophore~ls and lsoelectnc focusing of multiple forms of fl-glucuronldase In subcellular fractions from rabbit hver Electrophoresls was performed as described in the text, wnh the noted modifications Migration was towards the cathode which ~s at the top of the plate Gels were stained for fl-glucuromdase actlva2¢ From left to right, the samples applied ~ere (1) Purified rabbit fl-glucuronldase, treated overnight with 8 M urea at 4 C and electrophoresed in the presence of 8 M urea (2-4) Rabbit hver fractions from the hypotonlc shock procedure [2) Soluble fraction (3) Osmotlcall~ sensltl~e fraction (4) Mlcrosomal residue after solublllSatlon with 5°0 Triton X-100 (5 7) Rabbit hver fractions from the gradient sedimentation procedure (5) Soluble fraction (61 Microsomal fraction (7) Lysosomal fraction (8) Isoelectrlc focusing of the sample used for gel 3 Range pH 3 (towards top) to 6

Gel elecoophorests oJ rabb~t hver fractlon~ obtalrved by the hypotomc ~hock procedure The results of gel electrophoresis of these fractions from rabbit liver are shown in Fig 2 The Triton X-100 extract of the "'mlcrosomal" residue fraction contained all six forms, but their resolution was often masked by enzyme migrating on the surface of the gels An octyl sodium sulphate extract of this fraction contained the same forms In contrast to the results of Swank & Palgen (1973) with mouse kidney, the hypotonically sensitive fraction also contained all six forms The soluble fraction contained large amounts of forms L and X, but only very small amounts of M 1 - M 4

Gel electrophore~l~ oJ ~abbtt hvel Jracttons obtauved h5 density qradtent and differential centl t[ugatton To

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Fig l Polyacrylamlde gel electrophoresls of extracts of mouse, rat and rabbit livers Electrophoresls was performed as described In the text, except that where indicated, times other than 90mm were used Migration was towards the cathode which is at the top of the plate Gels were stained for fl-glucuronldase activity From left to right, the samples apphed were (1) Mouse liver 5°0 Triton X-100 extract (30 rain electrophoreslsl (2) Rat liver 5°o Triton X-100 extract (30 rain electrophoresls 30 mm staining) (3) Rat liver 5°o Triton X-100 extract (30mln electrophoresls, 4hr staining) (4) Rat liver 5°o Triton X-100 extract (120rain electrophoresls, 3 hr staining) (5) Purified rabbit liver fl-glucuronadase (15mln staining) (6) Rabbit hver 5°i, Triton X-t00 extract (120mln electrophoresls, 60 mm staining)

confirm that rabbit hver lysosomes contain larger amounts of forms X and M I - M 4 than those of mouse kidney, the organelles were separated by sucrose density gradient centrlfugation Two overlapping peaks of fl-glucuronldase actwity near the top of the gradient, the upper representing soluble and the lower, mlcrosomal enzyme (cosedtmentlng with glucose-6phosphatase) were observed, m addition to the major lysosomal peak near the bottom Each of the three fractions was electrophoresed (Fig 2) after extraction of the lysosomal and m~crosomal fractions with 5°,, T m o n X-100 All three fractions contained all the forms, but form L predominated in the lysosomal and soluble fractions, while form X predominated in the microsomal fraction The presence of forms X and M 1 - M 4 m the lysosomal fraction again suggests that rabb~t liver lysosomes contain larger amounts of M I - M 4 than do those of mouse kidney Similar results were obtained when the lysosomal and microsomal fractions were prepared by the differentlal centnfugatlon method of de Duve et al (1955)

Studies on the heterogeneity of mammalian fl-glucuronldases

Nature of the heterooenelty of rabbtt liver fl-olucuromgases

109

ceptlble to proteolytlc attack Nor was any evidence obtained for the presence of variable amounts of phosphohpld or related components [as shown Gel electrophoresis studtes That the six forms were recently for monoamme oxldase (Houslay & Tlpton, not artifacts of the procedures was confirmed by re- 1973)] Slight loss of M forms was observed by elecelectrophoresmg single forms extracted from gels trophoresls after overnight treatment of a rabbit liver rates of migration were retained, and no extra bands 5°0 Triton X-100 extract with 8 M urea at 4°C, but were formed Furthermore, the electrophoresls pat- when electrophoresis was done In gels containing 8 M terns of all the fractions which had been prepared urea, only forms with the moblhtles of L and X were without detergent were unchanged by the inclusion found [gels were stained following reduction of the of Triton X-100 (5% w/v) in samples applied to the urea concentration by washing (Dean, 1974a)] Thus gels and the resolution of forms was not prevented in the case of the rabbit enzyme, rapid reassoclatlon by electrophoreslng in gels containing 5°o Triton of accessory chains presumably occurs at the start X-100 throughout, although much longer electro- of electrophoresls m the absence of urea phoresis times were required (3 hr) Electrophoresls, in gradlpore gels equilibrated with Swank & Paigen (1973) used the method of Hed- 8 M urea, of rabbit liver or mouse kidney 5~o Triton rick & Smith (1968) of electrophoresis an gels of X-100 extracts pretreated overnight at 4°C with 8 M vaned acrylamlde concentration, to show that the urea, produced only two narrowly separated forms molecular weights of mouse forms X and M1-M4 in- These migrated further than ferrltln monomer, to poscreased from 260,000 to 470,000 with regular incre- itions sLmilar to those of bands L and X in gels run ments of approx 50,000 The variation in size of the in the absence of urea, presumably, therefore, any dismouse and rabbit liver enzyme forms was confirmed sociated accessory chain was Inactive In addition, It by us, using electrophoresis In gels containing poly- would seem that forms L and X differ slightly In size acrylamide gradients ("gradlpore electrophoresls") in the presence of 8 M urea which allows a &rect demonstration of size &fferIsoelecow focusing Previous work using rabbit ences (Rodbard et al, 1971. Andersson et al, 1972) liver mlcrosomal and lysosomal fractions prepared by Five forms were observed in Triton X-100 liver differential centrlfugatlon (Dean, 1974b) had shown extracts from both species, three migrated further that both fractions contain a complex array of lsoelecthan the monomer of horse spleen ferrltln [mol wt, tric forms of fl-glucuronldase (pI's 4 5-5 5) whose pat440,000 (Crichton. 1972)], and thus were smaller, terns were not clearly distinguishable (Dean, 1974a, while the other two did not penetrate as far as the b) In the present work the focusing patterns of the marker ferritin Using serum albumin and ferrltln extracts prepared by the hypotonlc shock or density monomer and dimer standards, a linear plot of mig- gradient methods were again very similar, despite the ration distance w log (mol wt) was obtained (Anders- fact that the lysosomal and microsomal fractions conson et al 1972) and values for the rabbit liver fl-glu- tamed different proportions of forms M1-M4 The curonldase forms crudely estimated by interpolation mouse enzyme showed a narrow range of forms, with They were as follows--280,000, 330.000, 370.000, a higher pI (cf Coutelle, 1971) 420 000,500,000 Whereas previously, resolution with narrow range The most intense band corresponded to the form ampholytes was rather unsatisfactory (Dean. 1974a), of smallest molecular size Gradlpore electrophoresls we found that ff the focusing time was reduced from of the 100,000 0 supernatant of rabbit liver homo- the normal 5 hr at 250V. or overnight at 75 V, to genate (soluble fraction), which contained negligible the time required for focusing of horse spleen ferritln amounts of forms M i - M 4 (see above) gave only the (visible as brown bands) plus 30-60 rain (giving a total band corresponding to the smallest form This band, of .~-..'~l ~i2hr), good resolution of fl-glucuronidase on therefore, represented a mixture of L and X, and so pH 3-6 gels was obtained (Fig 2) As mentioned prethe four larger forms correspond to M i - M 4 viously (Dean, 1974a) focusing patterns were not lnterconverston of molecularforms Swank & Palgen altered by the presence of 8 M urea in the gels (1973) showed that mouse kidney fl-glucuronidase Swank & Palgen (1973) found that Ferguson plots forms M I-M4 were almost completely converted into for mouse forms X-M4 intersect at a single point, form X without loss of activity, during incubation an and thus concluded that these forms have identical 6 M urea for 3 5 hr at 4°C, or by prolonged incuba- charges However, when the bands L-M4 from a tion at 37°C, or by addition of trypsin This was attri- group of electrophoresls gels were rerun in gel focusbuted to dissociation of the 50,000 mol wt accessory lng using Amphohnes of pH 3-10, each gave a single chain We observed no significant loss of forms main band of similar lsoelectrlc point, and a group M i - M 4 in a 5% Triton X-100 extract of rabbit liver of surrounding bands after incubation in 100 mM Trls-HC1, pH 7 8, at 37°C Heterogenezty of purified rabbit fl-glucuromdase All for up to 5 days, or up to 24 hr with 6 M urea Simi- the experiments so far described, including those of larly, incubation with trypsin (I #g/ml) for 4 h r at Swank & Paigen (1973), were done with crude fl-glu37°C in 50mM Tris-HC1, pH 7 8, 50#M CaCI/, curonidases It was important to show that similar caused no loss of M forms or total activity Brief results could be obtained In the absence of other treatment with the chaotroplc perchlorate ion (pre- cellular materials All six electrophoretlc forms of the cisely as described by Houslay & Tlpton, 1973) enzyme were found m purified rabbit fl-glucuronidase, caused a 40% loss of activity, but had little effect on and thus the forms had not been destroyed by the the electrophoretlc profile Thus these experiments autolysis step of the purification, and none were lost gave no evidence for lnterconverslon of rabbit fl-glu- during gel filtration on Sepharose 6 B (Dean, 1974a) curonidase forms, or of any accessory material sus- The gel filtration was repeated with a 5°~ Triton

110

ROGER T DEAN AND MICHAELMESSER

X-100 extract of rabbit liver, but only a single enzyme peak was obtained, with only partial separation of forms within the peak (as assessed by gel electrophoresls of successive fractlonst All the experiments on lnterconverslon of forms were repeated with the purified enzyme, in each case results identical to those with the crude extracts were obtained lmmunologzcal investigation of the multiple forms Since the polyspecLfic and monospeclfiC antlsera to fl-glucuronldase produced previously (Dean, 1974b) were raised against preparations which contained all six electrophoretlc forms, they can be expected to contain antibodies against any accessory polypeptade chains The immunological relationships between the enzyme molecules in the various subcellular fractions were assessed by Ouchterlony gel diffusion as described previously, (Dean, 1974b), but with the plates equilibrated with 5°,o Triton X-100 to maintain the solublhty of m~crosomal extracts Each fraction from the hypotonlc shock procedure was tested against the other two, with both monospeclfiC and polyspecLfiC antlsera, and similarly for the sedimentation fractions Every combination of subcellular fractions gave a reaction of complete identity between their fl-glucuronldase components, in agreement with the previous results on fractions obtained by differential centrlfugatlon (Dean, 1974b) A more direct test of the relationship between the electrophoretlc fractions was made by electrophoreslng them d~rectly from previously run polyacrylamlde gels into agarose "rocket" plates (Dean, 1974b) contalnlng 0 5°0 antiserum to rabbit fl-glucuronIdase A single preclpltln hne. showing a major peak (at the most anodic end of the polyacrylamlde gel) and several minor peaks, was obtained, and again, no spur was observed Thus the electrophoretlc forms of rabbit fl-glucuronldase are immunologlcally identical However, evidence for the presence of antibodies to the accessory material has still to be obtained DISCUSSION The rabbit M forms of fl-glucuronldase were far more resistant to conversion into L and X, than were those of mouse kidney (Swank & Palgen, 1973) conversion was only demonstrated when 8 M urea was present throughout incubation and electrophoresls As ferrltln is known not to be dissociated by 8 M urea (Hofmann & Harrison, 1963), the results of gradlpore electrophoresls in 8 M urea imply that the widely reported inactivation of 3-glucuronldase by urea may not be due to subunit dissociation, as is usually assumed but rather to local unfolding Previous rough estimates of the molecular weight of fl-glucuronldases in 8 M urea by gel filtration (Stahl & Touster, 1971, Dean 1974a) may well be in error, because the degree of unfolding and dissociation of proteins in 8 M urea is variable, and so, few reliable molecular weight standards are readily available for urea gel filtration The immunological identity of the separated enzyme forms together with the similar ~pan~ of lSOelectric points of all the forms, suggest that the accessory material may be the normal enzyme polypeptide modified shghtly However, no second subunlt has yet been detected in SDS-gels of purified rabbit fl-glucur-

onldase (Dean, 1974b) or lmmunopreclpltated mouse enzyme (Ganschow, 1973) To date, therefore, there is no direct lnformanon on the nature of the accessory material in any system As evidence has been obtained In biosynthetic studies that rabbit hver fl-glucuronldase In transit to the lysosomes passes through the Golgl apparatus (Dean, 1973b), where glycosylatlon is known to occur, it will be of interest to determine whether glycosylatlon of the M-forms differs from that of L and X, and whether the Golgl apparatus has a role in the translocatlon of M forms 4cknowledoements--RTD thanks the MRC for support REFERENCES

ANDERSSONL O, BORG H & MIKAELSONM (1972) Molecular weight estimation of proteins b) electrophoresls on polyacrylamide gels of graded porosity FEBS Lett 20. 199-204 COUTELLER (1971) Resolution of glucuromdase from Ehrhch ascltes carcinoma cells and mouse brain b) lsoelectric focusing m polyacrylamlde 4¢ta hlol reed qerm 27, 681-691 CRICHTON R R (1972) The subunlt structure of apoferritin and other elcosamers Blochem J 126, 761-764 DE DUVE D, PRESSMAN B C, GIANErTO R, WATTIAUX R & APPELMANSF (1955) Tissue frachonation studies 6 Intracellular distribution patterns of enzymes in rat liver tissue Blochem J 60, 604-617 DEAN R T, (1973a) The purification and properties of rabbit liver fl-Glucuronidase Btochem Soc Trans 1, 384 DEAN R T (1973b) The synthesis and translocation of lysosomal enzymes Ph D Thesis, University of Cambridge DEAN R T (1974a) Rabbit B-glucuromdase Purification and properties and the existence of multiple forms Blochem J 138, 395-405 DEAN R T (1974b) Rabbit fl-glucuronidase Subcellular distribution and lmmunochemlcal properties B~o~hem J 138, 407-413 GANSCHOWR E (1973) The Genetic Control of Acid Hydrolases In Metabolic Conjugatmn and Metahohc Hydrolysis Vol 3 (Edited by FISHMANW H ). pp 189-207 Academic Press. New York GANSCHOW R E & PAIGEN K (1968) Glucuronldase phenotypes of inbred mouse strains Genetzcs 59, 33%349 HEDRICK J L & SMITHA J (1968) Size and charge isomer separation and estimation of molecular weight of protelns b) disc gel electrophoresls Archs Bmchem Bzoph~ s 126, 155-164 HOFMANN T & HARRISON P M (1963) The structure of apoferritin degradation into and molecular weight of subunits J molec Btol 6. 256-267 HOUSLA~ M D & TIPTON K F (1973) The nature of the electrophoretically separable multiple forms of rat lever monoamine oxidase Btochem J 135. 173-186 PAIGEN K (1971) The Genetics of Enzyme Realisahon In En=yme Synthesis and Degradatton m Mammahan Systems (Edited by REICHtGL M). pp 1-46 Karger. Basel RODBARD D. KAPADIAG ~: CHRAMBACHA (1971} Pore gradient electrophoresls Analyt Btochem 40. 135 157 STAHL P D & TOUS~R O (1971) fl-Glucuronldase of rat liver lysosomes Purification. properties, subunits J htol Chem 246, 5398-5406 SWANK R T & PAIGEN K (1973) Biochemical and genetic ewdence for a macromolecular fl-glucuronidase complex in microsomal membranes J molec Btol 77, 371-389 TOMINO S & PAIGEN K (1970) In The Lactose Operon (Edited by BECKWITH J R & ZIPSER D) p 233 Cold Spring Harbor. New York