328
BIOCHEMICAL SOCIETY TRANSACTIONS
The preparation of a cardiac plasma-membrane fraction containing intercalated discs
CAMILO A. L. S. COLACO and W. HOWARD EVANS
National Institute for Medical Research, Mill Hill,
London NW7 IAA, U.K.
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 1min.
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.
C. A. L. S. C. thanks the Medical Research Council for a
studentship.
Culvenor, J. G. & Evans, W. H. (1977)Bimhem. J. 168,475-481
Gilula, N. B., Reeves, 0. R. & Steinbach, A. (1972) Nature (London)
235,262-265
Lowenstein,W. R. (1966)Ann. N.Y. Acad. Sci. 137,441-472
Page, E. & McAllister, L. P. (1973) J. Ultrastruct.Res. 43,388-4 1 1
Table 1. Fractionation of cardiac muscle: distribution of protein and enzymes in isolatedfractions
Values are the means 5 S.D.for five or more determinations.
Specific activitiesof marker enzymes m o l of substrate hydrolysedh per mg of protein)
Fraction
Homogenate
‘Light’plasma membranes
‘Heavy’ plasma membranes
Protein
(mg)
3647
6.3
7.4
A
r
5’-Nucleotidase
4.8 f 1.4
14.3 f 3.4
8.1 f 2.8
Na+/K+-ATPase
6.6 f 1.8
23.0 f 5.8
17.0 f 4.3
\
Succinate
dehydrogenase
10.2 f 2.0
8.1f3.2
6.8 f 3.1
Caz+
ATPase
4.2k 1.6
3.1 k2.6
2.5 k 1.1
1980