The preparation of a cardiac plasma-membrane fraction containing intercalated discs [proceedings].

In tissues and organs, mechanisms have evolved that allow the constituent cells to function in an integrative manner. Direct communication between the cells is mediated by a surface region, termed the gap junction, where the plasma membranes of contiguous cells are in contact. In cardiac muscle the spread of excitation is thought to proceed primarily through a series of gap junctions that interconnect cardiocytes. This spread of excitation leads to an orderly recruitment of contraction in different parts of the heart. Various studies show the gap junction to consist of a battery of ionic channels, sealed from the extracellular space, connecting the coupled cells (Lowenstein, 1966; Gilula et al., 1972). The biochemical identification and study of the molecular nature of these channels requires the isolation of the junction and the polypeptides comprising the channels. Using cardiac tissue, we describe a fractionation procedure that yields a plasma-membrane fraction containing the region in which these gap junctions are located. In cardiac muscle, gap junctions are found at the intercalated-disc region of the plasma membrane (Page & McA1lister, 1973). The intercalated disc also contains zones of adhesion between cardiocytes (manrla adherens), as well as regions for the attachment of the thin filaments to the sarcolemma ybscia adherens). Our strategy for the isolation of gap junctions was to isolate a subcellular fraction enriched in intercalated discs. A variety of tissue-disruption procedures and media were first examined (e.g. tissue presses, Ultra-Turrax and Dounce homogenizers) and optimal conditions for efficient disruption of rat and mouse hearts were selected. Rat or mouse hearts were homogenized by using an Ultra-Turrax tissue homogenizer (three bursts of 1 0 s at setting 1) in lOmM-Tris/histidine buffer (pH7.8) containing 0.1 mMEDTA, 2 0 m ~ sodium pyrophosphate and 0.1% phenylmethanesulphonyl fluoride. the homogenate was filtered through a mesh-40 gauze and the filtrate centrifuged at 500g for 1 min. The pellet was washed in the same buffer by repeated low-speed centrifugations until the supernatant was free of protein. The pellet was then resuspended in a hypo-osmotic ‘lysis’ buffer (composition: deionized water, 0.1 mM-EDTA, 20 mM sodiumpyrophosphate, 0.1% phenylmethanesulphonyl fluoride) by using a loose-fitting Dounce homogenizer, left on ice for 30min. rehomogenized and then centrifuged at 5OOg for 5min. This hypo-osmotic lysis step was repeated twice. The final pellet was resuspended in 8% (w/v) sucrose by using a loose-fitting Dounce homogenizer and layered on a discontinuous gradient constructed of 37%, 45% and 54% (w/v) sucrose solutions. After centrifugation at 98000g,,, for 120min, fractions were collected at the 37%/45%-sucrose interface (‘light’ membranes) and the 45%/54% sucrose interface (‘heavy’ membranes). When a tight-fitting Dounce homogenizer was used to resuspend the final pellet, a third fraction also appeared at the 8%/37% sucrose interface. The ‘light’ and ‘heavy’ membrane fractions showed an increase in the specific activities of two plasmamembrane marker enzymes, 5‘-nucleotidase and Na+/K+stimulated ATPase relative to the homogenate (Table 1). Measurement of the activities of succinate dehydrogenase (mitochondrial marker) and Ca*+-stimulated ATPase (sarcoplasmic-reticulum marker) showed that most of these subcellular components were removed by the low-speed-centrifugation steps. Morphological examination showed the ‘light’ plasma-membrane fraction to consist mainly of membrane vesicles. The ‘heavy’ plasma-membrane fraction contained mainly larger membrane vesicles and intercalated discs. Both fractions also contained some fragments of undisrupted muscle fibre. Extraction of the ‘heavy’ plasma-membrane fraction containing the intercalated discs with 1% N-laurylsarcosinate dissolved over 90% of the protein, and centrifugation of the residue on sucrose gradients yielded subfractions containing gap junctions (at 37%/45% sucrose interface) and desmosomes (at 45%/54% sucrose interface). Morphological examination ofthe isolated gap junctions, by using negative staining and freezefracture, indicated a similar particle size (7-8 mm diameter) and centre-to-centre spacing (8-9 mm) to the gap junctions isolated from rodent liver (Culvenor & Evans, 1977). Three major groups of polypeptides have been tentatively identified as candidates for the channel-forming proteins, and further biochemical characterization is required.