587th MEETING, DUNDEE
327
Investigations as to the nature of the junction between transverse tubules and sarcoplasmic
reticulum of skeletal muscle
JOHN J. S. CADWELL,* ANTHONY H. CASWELL*
and N. MICHAEL GREEN
National Institute for Medical Research, Mill Hill, London
NW7 IAA, U.K.
could be increased by increasing either enzyme concentration or
time of incubation. Trypsin and chymotrypsin at O.Olmg/ml
and 37OC caused almost complete breakage of the triad junction
in lOmin, and Pronase required 0.05mg/ml. In contrast,
neuraminidase at 0.1 mg/ml was without detectable effect.
Membrane-to-membrane communication occurs between cells
Polyacrylamide-gel electrophoresis of terminal cisternae/
and within cells. Examples of the former include the gap junction triads digested for various lengths of time indicated that
and the desmosome, and an example of the latter is the triad or relatively few of the protein bands were affected by the enzyme
diad junction which joins the transverse tubule to the sarco- treatment. Ca2+-stimulated ATPase was digested into 45 000plasmic reticulum. Electron-micrographic evidence has shown and 55 000-dalton fragments, whereas calsequestrin and a
that interposed between the transverse tubules and sarco- number of minor proteins were not affected. In addition to the
plasmic reticulum are electron-dense particles termed sarco- CaZ+ATPase, a band of approx. 80000 daltons was hydroplasmic-reticulum feet. These feet form tetragonal arrays and lysed by trypsin or chromotrypsin.
are embedded in the terminal cisternae, as shown by the
The next series of experiments was designed to test whether
organization of intercalated particles into arrays (Franzini- the enzymes were selective for the organelle which they
Armstrong, 1975). After nerve stimulation, an action potential is attacked. Terminal cisternaehriads were passed through a
transmitted throughout the fibre by the plasma membrane. French press and free terminal cisternae and transverse tubules
Depolarization spreads to the interior of the fibre via the were isolated by density gradient centrifugation, The free
transverse tubules and subsequently induces the release of Ca2+ transverse tubules were separately treated for 5 min with trypsin
from the sarcoplasmic reticulum. Schneider 8c Chandler (1973) (O.Olmg/ml). The reaction was terminated by addition of
have proposed that the movement of charge which accompanies trypsin inhibitor (0.02mg/ml). The trypsin-treated transverse
depolarization of the muscle occurs through the junctional feet tubules were mixed with undigested terminal cisternae in the
to induce Ca2+ release from the sarcoplasmic reticulum. presence of 0.3 M-potassium cacodylate (pH 7.4). DensityTherefore the triad junction may manifest a physiological role as gradient centrifugation was carried out to determine the degree
well as a structural one.
to which junctions had been re-formed. The control experiment
This study was aimed at observing and describing in with undigested transverse tubules demonstrated full rejoining.
biochemical terms the junctional feet through fractionation of In contrast, the trypsin-treated transverse tubules showed no
the muscle fibre. Transverse tubules and associated terminal detectable rejoining to terminal cisternae. This indicated that a
cisternae were isolated by the method of Lau et al. (1977). This protein was required in transverse tubules for recombination into
technique utilizes [ )Hlouabain entrapment by the transverse a triad junction.
tubules as a specific marker. Microsomal preparations separate
The analogous experiment was performed in which isolated
on density-gradient centrifugation into two bands identified as terminal cisternae were treated with trypsin and trypsin
longitudinal reticulum and terminal cisternae with transverse inhibitor, incubated with undigested transverse tubules in the
tubules attached in the form of triad junctions. The isolated triad presence of 0.3 M-potassium cacodylate and then centrifuged on
junctions consist of one transverse tubule attached to two heavy a sucrose density gradient. Three bands were observed. These
terminal cisternae. The intactness of the triad junction was were free transverse tubules, free terminal cisternae and a band
assayed in sucrose density gradients by examination of the of intermediate isopycnic point (32% sucrose). The interisopycnic density at which the I )HIouabain radioactivity mediate band contained transverse tubules, as indicated by
appears (22-28% sucrose for free transverse tubules and [)H]ouabain radioactivity. It appears that this band consists of
3 8 4 2 % sucrose for intact triad junctions). Triadic junctions transverse tubules associated with a fragment of terminal
can be broken, and free transverse tubules produced, by passage cisternae which had been released by proteolytic digestion.
of the suspension through a French press (Lau er al., 1977). The
From these results we conclude that the junction between
two populations of vesicles produced in this manner could be terminal cisternae and transverse tubules is at least partly
induced to re-form the junction in the presence of 0.6~-protein in nature. Its relatively high susceptibility to proteolytic
potassium cacodylate (Caswell el al., 1979). Rejoining was enzymes indicated that this protein should be accessible to other
accompanied by a light-scattering change, which occurred in a protein-modifying agents (such as covalent labels). The data
period of a few seconds, indicating the rapidity of the process. also suggests that some junction-forming activity might remain
The selectivity of the rejoining reaction suggested that a protein in a proteolytic fragment of the junction. These properties could
(or proteins) might be involved in formation of the junction. To be exploited in order to achieve biochemical characterization of
investigate this possibility, several proteolytic enzymes were the junction.
tested for their ability to break the junction. Intact triad
junctions were incubated with digestive enzymes for various
This work was supported by N.I.H.grant I ROI AM-21601 and
times and concentrations and centrifuged on sucrose density 1 T32 HL07188 student research training grant.
gradients in order to determine whether the junction had been
broken and the two organelles separated. The results showed Caswell, A. H.,Lau, Y. H., Garcia, M. & Brunschwig, J.-P.(1979)J.
that several proteolytic enzymes removed nearly all of the
Biol. Chem. 254,202-208
I)Hlouabain from the zone of the terminal cisternaeitriads to the Franzini-Armstrong, C. (1975)Fed. Proc. Fed. Am. Soc. Exp. Biol. 34,
zone of free transverse tubules. Mild treatment with these
1382-1389
enzymes resulted in partial separation of the organelles, which Lau, Y. H., Caswell, A. H. & Brunschwig, J.-P. (1977)J. B i d . Chem.
Present address: Department of Pharmacology, University of
Miami School of Medicine, Miami, FL 33152,U.S.A.
Vol. 8
252,5565-5574
Schneider, M. F. & Chandler, W. K. (1973) Nature (London) 242,
244-246