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A Biochemical Dissection of the Cardiac Intercalated Disk: Isolation of Subcellular Fractions Containing Fascia Adherentes and Gap Junctions

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A Biochemical Dissection of the Cardiac Intercalated Disk: Isolation of Subcellular Fractions Containing Fascia Adherentes and Gap Junctions J. Cell Sci. 5a, 313-325 (1981) 313 Printed in Great Britain © Company of Biologists Limited 1981 A BIOCHEMICAL DISSECTION OF THE CARDIAC INTERCALATED DISK: ISOLATION OF SUBCELLULAR FRACTIONS CONTAINING FASCIA ADHERENTES AND GAP JUNCTIONS C. A. L. S. COLACO AND W. HOWARD EVANS* National Institute for Medical Research, Mill Hill, London NW-j \AA SUMMARY In view of our limited knowledge of the biochemical composition of intercellular junctions, a method was developed for the preparation from rats and mice of plasma membranes containing cardiac intercalated disks. When these membranes were extracted with deter- gents, e.g. AMauryl sarcosinate or deoxycholate, the detergent-insoluble material contained structures derived mainly from fascia adherentes junctions, but a few gap junctions and maculae adherentes were also present. When the detergent extraction was carried out at an alkaline pH, the maculae adherentes junctions were dissolved. Fractionation of the detergent- insoluble extract on a sucrose gradient yielded a fraction containing fascia adherentes junction of density 1-20-1-26 g/cm'. Gap junctions banded at a lower density, 1-16-1-20 g/cm3. Poly- acrylamide gel electrophoresis showed that the major polypeptide bands in the fascia adherentes-enriched fraction were of molecular weights 134000, 108000, 62-64000, 58000, 47000 and 43000. Although fractions with the gap junctions were contaminated by fascia adherentes junctions, the major polypeptides were calculated by subtraction to be of mol. wt 37000, 26000 and 19000. Two glycoproteins corresponding to minor polypeptides visualized by Coomassie Blue staining were present in the fascia adherentes fraction. Comparison of the fasci aadherentes-ennched fraction with a Z-disc fraction prepared from rabbit hearts indicated a different morphology and polypeptide composition. INTRODUCTION A distinctive feature of cells organized into tissues and organs is the presence on the plasma membrane of various types of junctional specializations on the mediating cell-cell adhesion and communication. Although well-characterized morphologically, little is known of their composition and molecular organization. Methods for the isolation and biochemical characterization of intercellular junctions have been described for cow nose epidermis maculae adherentes (Skerrow & Matolsty, 1974; Drochmans et al. 1978; Skerrow, 1979), synaptic junctions (Matus, 1978) and gap junctions (Evans & Gurd, 1972; Goodenough & Stoeckenius, 1972). The present paper extends our knowledge of the composition of mammalian intercellular junctions by describing procedures for dissecting the junctional components of the cardiac plasma membrane. The cardiac plasma membrane incorporates a specialized region termed the • Address correspondence to this author, u CKL52 314 C. A. L. S. Colaco and W, H. Evans intercalated disk, which facilitates intercardiocytic adhesion and communication. The intercellular junctions present are the macula adherens, a junction mediating cell-cell adhesion, the fascia adherens junction at which the myofibrils attach to the sarco- lemma; and the nexus or gap junction, which provides the intercellular channels mediating intercardiocytic communication (McNutt & Fawcett, 1969; Simpson, Rayns & Ledingham, 1974; McNutt, 1975; De Haan, Williams, Ypey & Clapham, 1981; Page, 1978). Using a cardiac plasma membrane fraction containing intact and fragmented intercalated disks, we describe procedures utilizing detergents that remove the non-junctional membranes and amorphous material, and leave a residue highly enriched in junctional components. The most numerous junctions present j n the preparations were fascia adherens junctions, and these were shown to have a different composition from isolated Z disks. A fraction containing gap junctions was also isolated. MATERIALS AND METHODS Preparation of intercellular junctions Intercalated disks were isolated from mouse or rat hearts as described previously (Colaco & Evans, 1981) with the following modifications. Hearts (15 g) were disrupted using an Ultra- turrax tissue homogenizer (3x53, at setting 2-5) followed by homogenization in a loose- fitting Dounce homogenizer. The homogenate was filtered through 2 layers of muslin and centrifuged at 500 g for 5 min. The pelleted material was extracted overnight at 4 °C with o-6 M-KC1 dissolved in 8% (w/v) sucrose and was then centrifuged at 500 g for 5 min. The pelleted material was resuspended in 25 % (w/v) sucrose, layered onto 37 %, 45 %, 50% and 54% (w/v) sucrose solutions (6 ml of each) and centrifuged at 98000 £ for 2 h. The intercalated disk-containing sarcolemmal fraction was collected at the 45% to 50% (w/v) sucrose interface. The intercalated disks were collected and resuspended in JV-lauryl sarco- sinate at a concentration of 1 g detergent per 4 g protein. The iV-lauryl sarcosinate was prepared 83 a 5% (w/v) solution dissolved in C225 M-Tris-HCl and the final pH was 78. In some experiments, the JV-lauryl sarcosinate was adjusted to pH 10. The fractions were dispersed using a Dounce homogenizer fitted with a tight-fitting pestle, left on ice with occasional stirring for 30 min and then centrifuged at 98000 £ for 20 min. The pelleted detergent- insoluble material was resuspended in 25% (w/v) sucrose containing 0-5% Triton Xioo sonicated (Branson Sonicator, 3x53 bursts, setting 4) and introduced onto a discontinuous, density gradient containing 6 ml of 37%, 45%, 54% and 72% (w/v) sucrose dissolved in o-i% Triton Xioo. After centrifugation at 98000 j* for 2 h, the interfaces were collected, diluted with 10 mM-Tris-HCl (pH 10) and centrifuged at ioooo# for 15 min in plastic tubes (Sterilin). The pellets were analysed by sodium dodecyl sulphate/polyacrylamide gel electro- phoresis and electron microscopy. All density-gradient centrifugations were carried out in a Beckman SW27 rotor and all the solutions used in the fractionation procedure contained 1 mM phenylmethylsulphonyl fluoride. Preparation of cardiac Z discs Rabbit hearts (40 g) were homogenized using an Ultraturrax (3x53, at setting 2^5) followed by homogenization using a loose-fitting Dounce homogenizer in 10 mM-Tris-histidine (pH 7'8), o-i mM-EDTA and 20 mM-sodium pyrophosphate. The homogenate was filtered through 2 layers of muslin and centrifuged at 2000 g for 5 min. The pellet was resuspended in 8 % (w/v) sucrose and loaded onto an MSE A XII zonal rotor containing linear gradients of 8 % to 36 % (300 ml) and 36% to 60% (600 ml) (w/v) sucrose. The 2 linear gradients were separated by 36% (w/v) sucrose (150 ml) and underlayered by a cushion of 60% (w/v) sucrose. After centrifugation at 3000 rev./min for 30 min, the rotor was unloaded and material banding Composition of intercellular junctions 315 at 36% (w/v) sucrose was collected, extracted with Sarcosyl (see above) and the composition of the pellet was examined by electron microscopy and shown to contain material resembling Z-disk remnants. Electrophoresis SDS/polyacrylamide gel electrophoresis was carried out using the method of Laemmli (1971). The molecular weight markers used were: /?-galactosidase (130000), phosphorylase a (94000), catalase (60000), aldolase (40000), carbonic anhydrase (29000) and myoglobin (17500). Glycoprotein bands in the gels wer edetected by exploiting their property of binding specifically to iodinated concanavalin A (Gurd & Evans, 1976). Polypeptide patterns in poly- acrylamide gels were traced by using a Joyce-Lobel densitometer connected to a CALCOMP printer. Electron microscopy Fractions were routinely examined by negative staining with 1% sodium silicotungstate. Fractions for conventional transmission electron microscopy were processed as a pellet. The pelleted material was fixed overnight in 4% glutaraldehyde buffered in 50 mM-sodium cacodylate (pH 7-5) and post-fixed in 1% buffered osmium tetroxide. Samples were stained en bloc with buffered 1 % uranyl acetate, dehydrated using graded ethanol and embedded in Araldite using propylene oxide as a penetrating agent. Sections were cut using an LKB microtome, stained with lead citrate and uranyl acetate and examined in a Phillips 300 electron microscope. RESULTS Fractionation of cardiac muscle homogenates yielded a sarcolemmal fraction containing large numbers of intercalated disks (Fig. 1). This fraction was used to prepare the constituent intercellular junctions of the intercalated disk by solubilization of the non-junctional membranes in various detergents followed by a sucrose-density centrifugation step. Detergent extraction and subfractionation of the intercalated disk-containing sarcolemmal fraction Extraction of the intercalated disk-containing sarcolemmal fraction with 1-5% N- lauryl sarcosinate at pH 10 solubilized approximately 95% of the protein. Morpho- logical examination of the detergent-insoluble fraction showed paired filamentous matrices, some gap junctions, structures resembling fascia adherentes junctions and amorphous material (Fig. 2). After extraction with detergent, the structures resembling fascia adherentes junctions showed no unit membrane profile but the cytoplasmic filamentous mats always remained paired (Fig. 2). When the detergent extraction of the parent fraction was carried out at pH 7-9, some maculae adherentes junctions were also observed in the detergent-insoluble fraction (Fig. 2). Analysis by SDS/ polyacrylamide gel electrophoresis of the detergent-insoluble fraction prepared from rat and mouse hearts showed 2 major doublets with average molecular weights of 62-64000 and 58000 as well
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