Further purification of mouse liver gap junctions with deoxycholate and protein composition

CellBio/ogylnternat/ona/Repo~s, VOL~No, F~November 1951

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FURTHER PURIFICATION OF t4OUSELIVER GAP JUNCTIONS WITH UEOXYCHOLATE AND PROTEIN COMPOSITION J.C. EHRHART Institut de Recherches Scientifiques sur ]e Cancer g4802 VilleJulf Codex, France ABSTRACT ~ensive purification of ~use liver gap junctions has been achieved with 1.2% deoXyobelate, after clostripain pretreatment of plasma membranes. The presence of a major poIypeptide of 26 go0 daltons is confim~ed. A component of molecular weight 34 O00 which has been mp cared as a bessib e constitutive junctional protein or suspected to represent a combination of the 26 805 dalton be ypept de w th a proteolytic product, does not copurify with gap junctions. This 34 UO0 dalton component is not urate oxidase from detergent~insoluble peroxisomal cores which contaminate crude junotional preparations. iNTRODUCTION Highly-purified fractions of liver gap junctions have been obtained with type A detergents 8elenius and Simons, 1975) such as Sarkosyl or Triton X-tOO, in absence of proteolyt c pretreatment of plasma membranes The so ub lization efficiency of these detergents was enhanced by combination with sonication (Mertzberg and Gilula, 1979), urea (Henderson e t a ] . , 1979) or high 98 (finbow et a l . , 1950 n these conditions and after removal of urate oxidase from detergent-inso ub e peroxisomal core contaminants by high RH treatment (Hertzberg and Gilula, 1579), there is evidence that mammalian flyer gap junctional, preparations are comprised primarily of an intrinsic go ypept de of 26 050 daltons (Hertzberg and Gilula, ~79; Henderson et a l . , 1979; Finbow et a l . , IR50; Mertzberg, 1950 . addition to this major polypeptide, componen:s of molecular weight 34 O00 and 37 000 have also been observed after type A detergent treatment. The 37 K compo~ent has recently been shown not to copur i f y with gap junctions in presence of urea (Nertzberg, 1980 . Although the 34 K component has been suspected to be urate oxidase, the poss hi' ty has been discussed that i t may represent a f o ~ of the junctional protein (Finbow et a l . , 1980; Reve e t a .. Ig80). Detergents of type B such as deoxycholate, the properties of wh ch differ in many respects from bhoseof type A d~tergents, have also been used for the preparation of liver gap junctions (8enedetti and Emme]ot, 1968). Components of molecular weight 34 950 (Ehrhart and Cbeuveau, 1977) and 30 OOO (Zampighi and Unwin, 1979) were observed. Urea and b 9h pH which have been used w th type A detergents are conditions knownto enhance the dissociatio~ of proteinprotein interactions, in addition to their increase of the solubilization efficiency of detergents (Helenius and Simbns. 1975). I t is thu~ usefu to get evidence that the junctional protein composition o~served is not dependent uoon the conditions of isolation of enric~e'd gap juections; In this report me show that mouse liver gap jum~-~ns are cUaracterized by a m~jor p61ypeptide of 26 K after

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Cell E#ologv lnternatlonaf Reports, VME 5, No. l l. Novernb~r1981

deoxycholate treatment, the solubilization efficiency of which was enhanced by controlled clostripain digestion of plasma membranes, In addition we present evidence that the 34 K component, which is not urate oxidase, cannot be considered as a junctional protein. I~TBRIAL AND METHODS PREPARATION OF PURIFIED GAP JUNCTIONS : I. Clostripain pretreatment of plasma membranes : Contiguous plasma membrane subfractiens from mouse liver were prepared as described previously (Ehrhart and Chauveau. 1977), Membranes (8 mg of protein) ~ere t~suspended in 20 ml of 0.030% clostripain (Sigma). 0.5 mMdithiobhreitol, i mMNaHEO 3, pH 7.5. Incubation was done at room temperature for 1 h and ended by the addition of 0.6 ml of 0.1% N-itosyl-L-lysyl chloromethyl ketone (TECK) for 15 min. 2. Deoxycholate treatment : The resulting pellet was processed as described previously (Ehrhart and Chauveau. 1977) except that the final concentration of deoxyeholate was O.8. 1.0 or 1,2%. 3. First isopycnic centrifugation : The resulting 2EEOOxgav pellet was resuspended in B.B ml of ice-cold 1 mMNaHCU3, layered on top of two 4 ml HaHCOg-buffered 20-60% (w/v) linear sucrose gradients and centrifuged at 4~ for 15 h in a 5W 50.I rotor at 30 eBB rev/ min. 4. Carbonate treatment : The resulting combined pellet was resuspended n 2 B m of 50 n~4 sodium carbonate and incubated on ice for 15 min (Hertzber9 and Gilula. 1979). The carbonate-treated junct ons were centrifuged at 26 SOOxgav for 20 min at 4~ Whenmentioned. carbenete treatment was also performed immediately after the deoxychelate solubilization step and followed by one isopycnic centrifuBation as described above. S. Second isopycnic centrifugation : The pellet was resuspended in ice-cold 1 ~ NBHCO 3, layered on top of a 4 ml NaHCOg-buffered 3248% linear sucrose gradient and centrifuged at 4~C for 15 h in a SW 50.I rotor at 30 BOO rev/min. The gap junctions were collected. diluted with ice-cold water and pelleted. PREPARAT ON OF MOUSELIVER URATEOXIDASE : Crude urate oxidase was prepared from deoxycholate-inso]uble peroxisomal cores (Goldmanand Blobel. 1978). Advantage was then taken of theresistance of urate oxidase to clostripain digestion. 0.8 ml of suspension (190 mg of liver} in 50mMTris-HEl. pH 8 was incubated at 37~ for I h with 0.2 ml of 0.030% clostripain buffered solution containing 0,5 ~M dithiothreitol. Clostripain digestion was terminated bythe addition of eL1 ml of 0.014% buffered TLCK for 15 min. The suspension was then mixed with 3.8 ml of ice-cold buffer and pelleted at 06 50Oxgav for 20 min at g~ Whenmentioned, urate oxidase was submitted to carbonatetreatment as described above. THIN SECTIONING ELECTRON MICROSCOPY : Pellets were processed as described previous y(Ehrhart and Chauveau 1977). SDS-POLYACRYLAMIDE SLAB GEL ELECTROPMORESIS : Electrophores s was carried Out in 13% acry]amide, 0.1% bisecrylamide slab ge]s in 9.1% SOS, using the discontinuous buffersystem of Laemmli and Favre (1973). DissO red samp es were nhuhatedat room temperature for 1 h prio? to elddbrophoresis (Nehderson et al, 1979). Molecular weight Standards Used for calibratidn of the gels were : phosphorylase a

Cell Biology Intenna#onaf Reports, VOl. 5, NO, 11, November 1981

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(94 000 , bovine serum albumin (68 go0), ovalhumin (43 OOg}, carbonic anhydrase (go ODD , soybean tryps n inh b tar 20 100 and lysozyme (14 gO0), Gels were processed through fixing, staining, destaining and drying as described previously (Ehrhart and Chauveau, 1977). RESULTS AgO DISCUSSION The isolation of--gap junctions from mouse liver plasma membranes has been achieved by a modified procedure which is described in Material and Methods. The solubilization efficiency of 0.8 to 1.2% deoxycholate on nonjunctional membranes was enhanced by c]ostripain pretreatment of plasma membranes. C]ostr~pain is one of the proCease species which are present in partially purified collagenase (f~l~h~hell and Harrington, 1968) used in most earXy preparative m~thods. This bhio] proteinase (EC 3.4.22,8) shows a narrow specific i t y range which is confined primarily to arginyl bonds (Mitchell, 1977). The use of clostripain was rationalized on the basis of amino acid analyses from HendersoR et al. (1975). The arginine content of gap junctions isolated after collagenase treatment of plasma $~embranes was decreased by only 3 to 4% as compared to collage~aseuntreateg junctions. High pH treatment of junctional membranes has been shown to solubilize urate oxidase from detergent-insoluble peroxisomal cores which contaminate partially purified gap junctions (Hertzherg and Gilula, 1979; Finbow et a l . , 1go0; gertzberg, i580). Addition of a 50mM carbonate step between the deoxycholate treat~nt,and purification of gap junctions by one isopyenic centrifugation on a 20-60% linear sucrose gradient was followed by the presence of components of mo]ecular weight 26 O00 and 34 OOO,whatever the deoxycholate concentration used between 0.8 and 1.2% (Pig. I, lanes a to c). Some degradation of the 26 ODD dalton polypeptide occurred, as revealed by the presence of a series of proteolytic products in the 15 OOOto Zi go0 molecular weight range (Henderson et a l . , ig7g; Finhow et a l , , 1980}. The 18 OO0 dalton-breakdown product consti~ tutes the majority species (Fig. i , lanes a-f). Optimal preservation of the 26 K polypeptide was achieved at a clostripain to membrane protein weight ratio of 0.75. In Sombexperiments performed in absence of high pM treatment of junctional membranes, a major component was observed i~ the 35 OO0 dalton region of the gels, whatever the concentrations of cgostripain and deoxycholate used for the solubilization of nonjunctional membrane components (Fig~ E, lanes d and f ) . This component comigrated with urate oxidase (Fig. 2, lanes d and e) purified from mouse liver by clostripain treatment of deoxycholateinsoluble pe~oxisomal cores (fig. 2i lanes a and b). Addition of a 50 net carbonate step betweent h e deoxycholat~ treatment and purification of gap junctions hyone isopycnic centrifugation on a2oegO%sucrose gradient was followed by the presence of a distinct 34 KmajPrcompohent (FigL gi lane g; Fig. 1, lanes a to c). THis polypeptide did not comigratewithcarbonatetreated urate oxidase, themigration ofwhich is notincreased after high pHtreetment (Fig. 2~ lanes b and c).

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Cell Biology International Reports, Vol. 5, No, 11, November 1981

Fig. I :'SDS-polyacrylamide gel analysis of 50 mMcarbonatetreated junctional fractions purified by one (a,b,o) or two (d,e, f) isopycnic centrifugations (see Material and Methods). Contents of the lanes are : a,d : 0.8% deoxycholate-resistant material; b,e : ].0% deoxycholate-~sistant material; c,f : 1.2~ deoxycholate~resist~nt material. Polypeptides under study are seen as bands of Mr Cx10" j ) 26 and 34.

Fig. 2 : Comparative analysis of the migrations of urate oxidase and putative junctional Components in the 35 000 ~,!r range. Contents of the lanes are i a : crude urate oxidase; b and e : clostripain§ purified Grate o• c ; 5Ohm carbonate-treated urate oxidase; d and f : I% deoxycholate-resistant and carbonate-untreated junctional fractions; g : 50mM carbonate~treated ~unctionai fraction isolated after one isopycnic centrifugation.

Cell Biology lnternadonal Reports, VOL 5, IVo, t 1, November 1981

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I t has been s~ggested that a 34 K component could r e s u l t from aggregation between the Z6 gOO dalton polypeptide and a low molecular weight breakdown product (Revel et a l . , 1980). No change was observed in the relative intensity of the 34K band whether the sample had been boiled before electrophoresis or solubilized at roo~l temperature to minimize aggregation phenomena (Henderson et e l . , 1979). Relative enrichment in the 34 K polypeptide was observed with increasing concentrations of deoxycholate after one isopycnic centrifugation Fig. i , lanes a to c). Mild detergents are often unable to dissociate the protein-protein interactions which keep a number of complexes together (Helenius and Simons, 1975). However, such a view is d i f f i c u l t to reconcgle with a high pH condition knownto enhance the dissociation of proteins. In addition, the relative purity of junctional fractions was not consistently increased with deoxycholate concentration as observed on a morphological basis (not shown) and corroborated by electroghoresis (Fig. 1, lanes a to c). Further purification of gag junctions was therefore undertaken by using two successive isopycnic centrifugations. The deexycholateresistant material was f i r s t fractionated on a 20-60% sucrose gradient. Junctional membranes were subsequently treated with 50 mM carbonate then again fractionated on a 32-49% linear sucrose gradient. AS shown in Fig. i , lanes d to f , the 26 K po]ypeptide was consistently increased relatively to the 34 g component, especially at the deoxycholate concentration of 1.2%. Fig. 3 shows that 1.2% deoxycholate-resistant gap junctions are highly enriched and do not present the multiple junctional disruptions observed at higher concentrations of detergent. The corresponding sgS-gel profile contains the 26 g polygeptide with i t s closbripain-breakdown 18 K product, other bands being much less visible (Fig. k, lane f ) . Though we have no indication about the very nature of the 3d K component, the data taken together demonstrate the absence of copurification of this component with gap junctions. By contrast, they demonstrate copurification of the 26 9OO dalton polypeptide with gap junctions. These observations with a type B detergent thus support the conclusion established with'type A detergents that liver gap junctions are comprised primarily of a polypegtidic camponent of molecular weight 26 OOO. We have also obtained evidence that clostripain, as a component of partially purified preparations of collagenase, is implicated in the varying amounts of proteolysis observed with early isolation procedures of liver gag junctions, Most presumably, clastripain cleaves arginy] bonds which must be close to the surface of junctional membranes, including the channel interiors. As such, this highly specific protease may prove a useful tool ~ primary structure determinations on the 26 Kpolypeptide and in 5%ructurefunctions studies.

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Cell Biology mternationat Repe~s, Va~ 5, No9 11, November 1881

Fig. 3 : Thi~ section~ of 1.2 % deo~ycholate-enrgched ~ap ~une~ion ~ractlons, (a), tb~ pellet cantatas junctional plaques that e~ist as sheets or posslb]8 vesicles x 60gg (bar = 2,0 um); (b), the enriched fraction contains ~me amorphous material and n~njunc~1on~I membranes, x 1~g Dog (bar = O [ ~m). ACKNOWLEDGEMENTS We 9ratef~u~F1y thank Dr J. LQeb for his interest in and support of this work. REFERENCES ~ e g e t t l , E,L. and E~elot, P. (196B) Hexagonal array of subunits in tight junctions separated from isolated rat liver plasma membranes. Journal o~ Cell Biology, 38, 15-g4. s d.C. and Chauveau, 3 (1977) The protein component of m~use hepatomyte gap junctions. FEBS Letters, 78, 29S-29g. F i n ~ , M., ~aneey, S.~., Johnson, R~ and Revel, J;P. (19~0) Independent lines of evidence s~gges~ing a mB~o)- gap jgnctiosal proteinwith a molecular weight of 26 OOO. Proceedings of the ~Btional Academy of Sciences (USA), 77,970-974. Goldman aiM, and globel, G. (1978 Biogenesis of peroxisomms intraceIlular site of s2nthesis of catalase and ur case. Proceedings of Phe National Academy of Sciences (USA), 75, 5066~5070. Helenius, A: and Simons, K. (1975) Solubilization of membranes by detergents9 Bio~himica et Biophysica Acba, 415, 29-79. Henderson, P-, Eibl, H. and ~eber, k, (1979). Struc[ure an~ biochemistry of mouse hepatic gap junctions. Journal o~ Molecular Bi~logy, 132, 193-21B. Hertzber9, E.L. and Gilula, N:9, (1979 Isolation and characterization of gap juncti~n~ from rat liver. Journal of Big ~gical Cb~mis%r~, 25~; 2)3~-2147.

Cell Hiology lnternational Reports, Vol. 5, No 17, November 1987

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Hertzberg E.L. (Iggg) Biochemical and immunological approaches to the study of gap Junctional communication, in V fro, 16, 1057I067, Laemmli, U.K, and Favre, M. 1973 Maturation of the head of bacteriophage T4. Journal of Molecular g o ogy, 80, 575-599. Mitchell, W.M. and Harrington, B,P, (1968) Purif{cation and properties of clostridiopegtidase B (clostripain). Journal of Biological Chemistry, 213, 4683-4692. Mitchell, W.M. (1977) Cleavage at arginine residues by olostripain. Methods in Enzymology, 47, 165~170. Revel J . P . Yancey 5.B., Meyer, O.J. and Nicholson, B. (1980) Cell junctions and intercellular communication, n V tro, 16, 1010-1017. Zampighi, G, and Unwin, P. 1979} Two forms of isolated gap junctions. Journal of Molecular Biology, 135, 451-464.

Received: 6th July 1981

Accepted: 27th July 1981