A structural characterization of gap junctions isolated from mouse liver

Cell Biology

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Vol. 7, No. 7 1, November

1983

897

A STRUCTURAL CHARACTERIZATION OF GAP JUNCTIONS ISOLATED FROM MOUSE LIVER S. S. Sikerwar and S. K. Mdhotra

Biological SciencesElectron Microscopy Laboratory. Department of Zoology, University of Alberta, Edmonton, Alberta, Canada T6G 2E9 Abstract Mouse liver gap junctions have been isolated by using an anionic detergent, n-dodecanoyl sarcosine.in combination with non-ionic polyoxyethylene ethers (Brij 35 and Brij 58) and (W-l) detergents. Purified gap junctions are obtained in a sucrose step gradient containing l-o-n- octyl-/3- D- glucopyranoside. This procedure is aimed at minimizing proteolysis. The junctions thus isolated have a hexagonal lattice of connexons with a lattice constant of 7.6-8.4 nm. Presence of a major Mr 26,000 gap junctional protein has been confirmed by SDS-PAGE. Introduction The polypeptide composition of the gap junctions is as yet uncertain because investigations of the polypeptide are complicated by endogenousand exogenousproteolysis and a marked propensity of gap junctional polypeptides to aggregate in the presence of SDS. This has resulted in the apparent heterogeneity in the reported polypeptide composition (Culvenor and Evans, 1977; Fallon and Goodenough, 1981; Finbow et al.. 1980; Henderson and Weber, 1979; Hertzberg and Gilula, 1979). Furthermore, gap junctions isolated by conventional methods usually display a high degree of mosaicity (Unwin and Zampighi, 1980) and/or short-range disorder (Caspar et al., 1977) which has restricted the resolutions attained in the structural studies thus far to 1.8 nm. We wish to report here on a new method for isolating gap junctions from mouse liver, modified from Fallon and Goodenough (1981), that aims at minimizing proteolysis and eliminates the exposure to l-6M urea. Materials and Methods (a) Isolation of Gap Junctions: The plasma membraneswere isolated from 40 mouse livers by a method due to Fallon and Goodenough (1981). The isolation medium employed consisted of 1 mM EGTA, 1 mM NaHCO:, 1 mM PMSF (phenylmethylsulphonylfluoride), 1 mM PCMB (parachloromercuribenzoate)and 1 mM DTT (dithiothreitol), pH 8.0. The resulting plasma membranes were suspended in approximately 40 ml of solubilization medium at room temperature (Fig. 1). To this suspension, approximately 40 ml of n-dodecanoyl sarcosinewas added slowly drop by drop with constant stirring provided by a magnetic stirrer at room temperature. After 5 min., the suspensionwas centrifuged at 4-5 ‘C in a SS-34 rotor (Sorvall), at 3100 rpm for 10 min. Supernatant was kept at room temperature, and the small pellets (which were unevenly distributed at the bottom and had mostly collagen) were discarded. 10 ml of Brij 35 was slowly added to the supernatant, followed by an incubation at room temperature for 5 min. and cenuifupation in a SS-34 rotor at 17,000 rpm for 15 min. at 4-5 ‘C. The supernatant was aspirated out and discarded. The pellets were suspended in 2 ml of the solubilization medium. The 0309-I

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MEMBRANES 1) SOLUBILIZATION MEDIUM (1mM EGTA, 2mM NaHC03,lmM DTT and 1mM PMSF, pH 9 ) at room temperature 2) l%(w/v) n-dodecanoyl sarcosine in solubization medium, room temp., 5 MIN 3) 3100 rpm, SS-34 rotor, 10 MIN

N-DODECANOYL

SARCOSINE

SUPERNATANT

1) 0.5% (w/v) BRIJ 35 in solubilization medium, rooz, 5 MIN 15 MIN 2) 17,000 rpm, SS-34 rotor,

w

BRIJ-35 AND N-DODECANOYL I

POLYOXYETHYLENE

SARCOSINE

PELLET

1) solubilization medium at room temp. 2) D.z%(w/v) BRIJ 58 and polyoxyethylene ether W-1( 0.25% w/w) in solubilization medium at room temperature

ETHER W-l

AND BRIJ-58

TREATMENT

0 CENTRIFUGATION sample sucrose 34% w/v sucrose 550/, ,

1

L

WI3z

interface in gap junctions W/V

THE ISOLATION

OF GAP JUNCTIONS

SUCROSE GRADIENTS IN O.lmM l-O-n-octlylY B-D-glucopyranoside in the soubilization medium at

4-E’C.

30,000 rpm,SW-40 at 4oC

rotor,120

MIN

is enriched

FROM PLASMA MEMBRANES

Figure 1. A flow-chart describing the solubilization membranes and the isolation of gap junctions.

of non-junctional

plasma

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Figure 2. Electron micrograph of a negatively stained preparation of isolated pap junctions. The gap junctions often lie fiat on the grid in the form of plaques of various shapes and sizes, often reaching approximately 1 pm on the edge. The hexagonally packed connexons are evident. The encircled area has been subjected to optical diffraction (Fig. 3a). suspension was accomplished by empioying a hand driven Dounce homogenizer with a type ‘B’ pestle (8- 10 strokes). To this 1 ml of Brij 58 and 1 ml of polyoxyethylene ether (W-l) in the solubilization buffer were added at 4 ‘C and mixed. This suspension was loaded on the top of two step gradients prepared in four SW-40 (Beckman) rotor tubes with approximately 5 ml of 50% or 54% (w/v) and approximately 7 ml of 33.81% (w/v) buffer. Incorporation of 0.1 mM l-o-n-octyl-/3-Dsucrose in solubilization glucopyranoside facilitated better separation of gap junctions in the gradient These gradients were centrifuged in SW-40 rotor at 30,000 rpm for 2 hours at 8°C. The gap junctions usually gathered at 33.81-50s (w/v) sucrose interface could be observed as a band and harvested with the help of a syringe. Washings were performed with the isolation medium (SW-40 rotor, 30,000 r-pm, 45 min.). Finally, these gap junctions were suspended in 100-200 ml of the isolation medium. Protein estimation was carried out according to Lowry et al (1951).

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Figure 3. The optical diffraction pattern corresponding to the area enclosed by the circular mask displayed in Fig. 2. The diffraction pattern can be indexed on a hexagonal lattice of a lattice constant of approximately 8.2 nm, and extends to the 2,0 reflection. In a few cases (Fig. 3b) the diffraction pattern extends to the 3.0 reflection. (b) Electron Microscopy: A small aliquot of a gap junctional fraction (1 mg/ml) was deposited on freshly prepared carbon-coated grids and negatively stained with 1% (w/v) aqueous uranyl acetate. The grids were scanned in a Phillips EM400 (operated with a liquid N, cooled anticontamination device) at low magnification to locate the well stained areas. Electron micrographs of stained gap junctions were taken at a magnification of x36000-46000. (c) Optical Diffraction: Optical diffraction experiments were performed on a Poiaron optical-diffractometer (model M802). This instrument employs a 2 milliwat helium-neon

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I’HOSPHORYLASE

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b

CATALAS E OVALSUMIN

aX:HYMOTRYPSINOGEN

-A

Figure 4. A typical SDS-polyacrylamide gel electrophoreLopram of isoIaLed gap junctions. The gels were sLained in the ammonical silver nitrate solution; track A, standard proteins. the number shows mol. wt = xl000 dahons; BI gap junctional proteins dispIal;ing a major component of epproximatel! 26,000 dalrons (black circle). end a minor diffuse componenr in approximateI\, 43,000 dalton region (white arrotl: the diffused component could not b: reproduced photographicall~f; C, displays a band of a-chymotrypsinoger! A of 26,000 mol. wt. that comigrates with the gap junctional polypeptide. Iaser as the source of coherent light (632.8 nm). Electron Mlcrographs of negauvely stained pap junctions, suitably masked to expose onI!- the particular areas of inreresb were used as objects in the optical diffraction experiments. Optical diffracroprams were recorded on Y’ A 4” Polaroid (black and white) positive-negative plates. (d) SDS-PAGE: SDS-PAGE was carried out using the discontinuous buffer system of LaemmIi (1970) employing 5% (U./Y) and 10% {w/v) acrylamide (30 : 0.8 crosslinking ratio) as the stacking and separating gels respectively. The samples for SDS-PAGE were prepared b!. solubilizing fresh11 isolated pap junctions with vigorous pipetting in SDS-sample buffer (10% W/V glycerol, 5% w/v 2-mercaptoethanol, 2% w/v SDS and 0.0625 M Tris-HCI, pH 6.8) at room temperature for 0.5 hr. Gels were stained in the ammonical siiver nitrate solution (Wray et al., 1981). photographed b\; employing transmitted light, on a Kodak Panchromatic-X (ASA 32) film. Results and Discussion A procedure, modified from Fallon and Goodenough (1981) has been developed for isolation of highly enriched gap junction fraction from mouse liver plasma membranes.

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This procedure avoids the use of exogenousproteasesand urea that have been commonly included in protocols of isolation of gap junctions. The plasma membraneswere first treated with anionic detergent, n-dodecanoyl sarcosine,in combination with a non-ionic polyoxyethylene ether detergent, Brij 35. The insoluble fraction was further solubilized with polyoxyethylene ethers (Brij 58) and (W-l). Subsequentcentrifugation in a sucrose step gradient in the presenceof 0.1 mM l-o-n-octyl-/3-II-glucopyranoside (a non-ionic detergent)yielded a highly enriched gap junction fraction. It is emphasizedhere that some of the detergents (n- dodecanoyl sarcosine, W- 1, 1-o-n-oc;yl-/?-D-glucopyranoside) employed in this isolation protocol have not been previously used for this purpose. The incorporation of 1 mM EGTA throughout the isolation protocol provides exceptionally high yields of gap junctions (200-400 kg protein/40 mice) as previously documentedby Fallon and Goodenough(1981). This correspondsto a 12-26s recovery basedupon the estimate that the gap junctions occupy about 1.5%of the surfacearea of hepatocvtemembranes(Yee and Revel, 1980)and on the assumptionthat the protein is uniformly distributed within the membrane. The gap junctions thus obtained often have their iateral dimensionsas large as 1 pm (Fig. 2). They display a hexagonallattice of connexonsof lattice constant7.6-8.4 nm (Fig 3a) as is characteristicof isolated gap junctions (Unwin and Zampighi, 1980). The optical diffraction patterns of the electron micrographsof negatively stained gap junctions sometimesextend to the 3.0 reflection (Fig 3b). An analysisof these isolated iunctions b) SDS-PAGE has revealed a predominant component of Mr 26.000 tiat coGgrates with a-chymotrypsinogen A. In addition, there is a rather inconspicuouscomponent of Mr 43,000 that is representedby a diffuse weakly stained band comigratmgwith ovalbumin. An important characteristic of the gap junctional polypeptides has been described b\ Hendersonet al. (1979) namely, a strong propensity to form homodimersin SDS and with prolonged incubation even multimers. Heterodimer formation has not been reported. Basedupon this observation,the diffuse band of Mr 43,000may representa dimer of a Mr 21,000polypeptide, possibly a degradationproduct of the major (Mr 26.000)gap junctional protein. Acknowleeements We would like to thank Sanjay PimpIikar for his help In carrying out the SDS-PAGE. One of us (S.S.S.)is grateful to the Alberta Heritage Foundation for Medical Research for award of a studentship. The researchwork has been supported by grants awardedby the Natural Scienceand EngineeringResearchCouncil of Canada.

Caspar,D.L.D., Goodenough,D.A., Makowski, L. and Phillips, W.C. (1977). Gap junction structures,I. correlated electron microscopyand X-ray diffraction. J. Cell Biol. 74, 6C5-626. Culvenor, J.C. and Evans, W.H. (1977). Preparation of hepatic gap (communicating) junctions. Biochem. J. 168, 475-481. Fallon, R.F. and Goodenough, D.A. (1981). Five-hour half-life of a mouse liver gap junction protein. J. Cell Biol. 90, 521-526. Finbow, M.. Yancey. LB., Johnson, R. and Revel, J.P. (1980). Independent lines of evidencesuggestinga major gap junctional protein with a molecular weight of 26,000. Pm. Nan. Acad. Sci. USA. 77, 970-974.

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Henderson, II., Eibl, H., and Weber, K. (1979). Structure and biochemistry of mouse hepatocytegap junctions. .I. Mol. Biol. 132, 193-218. Hertzberg, E.L. and Giiula, N.B. (1979). Isolation and characterization of gap junctions from rat liver. J. Biol. Chem 254, 2138-2147. Laemmli, U.K. (1970). Cleavage of structural proteins during the assemblyof the head of the bacteriophageT4, Nature, 227, 680-685 Lowenstein, W.R. (1981). Junctional intercellular Communication: The Cell- to-Cell Membrane Channel. Physiological Reviews,61, 829-913. Lowr!-. O.H.. Resenbrough. NJ., Fax. AL. and Randall, R..I. (1951). Protein measurementwith the folin phenol reagent. J. Gen. Phpsiol. 193, 265-275. Unwm, P.N.T. and Zampighi, G. (1980). Structure of the junction between communicatingcells. Nature, 283, 54% 549. Wray, W., Boulikas, T., Wra]. V. and Hancock, R. (1981). Silver snining of proteins in polyacrylamide gels. Anal. Biochem.,118, 197-203. Yee, A.G. and Revel, J.P. (1973). Loss and reappearanceof pap Junctions in regenerating iiver. J. Cell Bio!., 78. 554-564.

Received

:

16th

June

1983.

Accepted : 15th August

1983.