Identification, Characterization and Partial Purification of a Thiol-protease Which
Cleaves Specifically the Skeletal Muscle Ryanodine Receptor/Ca
, W. Feng
, M. Varsanyi
, V. Shoshan-Barmatz
Department of Life Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel
Institute fuer Physiologische Chemie, Ruhr Universitaet, D-44780 Bochum, Germany
Received: 10 December 1996/Revised: 11 August 1997
Abstract. A 94 kDa large subunit thiol-protease, as iden-
tified by anti-calpain antibodies, has been isolated from
skeletal muscle junctional sarcoplasmic reticulum (SR).
This protease cleaves specifically the skeletal muscle
ryanodine receptor (RyR)/Ca
release channel at one
site resulting in the 375 kDa and 150 kDa fragments.
The 94 kDa thiol-protease degrades neither other SR
proteins nor the ryanodine receptor of cardiac nor brain
membranes. The partially purified 94 kDa protease, like
the SR associated protease, had an optimal pH of about
7.0, was absolutely dependent on the presence of thiol
reducing reagents, and was completely inhibited by
, leupeptin and the specific calpain I inhibitor.
However, while the SR membrane-associated protease
at a submicromolar concentration, the iso-
lated thiol-protease has lost the Ca
The 94 kDa thiol-protease had no effect on ryano-
dine binding but modified the channel activity of RyR
reconstituted into planar lipid bilayer: in a time-
dependent manner, the channel activity decreases and
within several minutes the channel is converted into a
subconducting state. The protease-modified channel ac-
tivity is still Ca
-dependent and ryanodine sensitive.
This 94 kDa thiol-protease cross react with anti-
calpain antibodies thus, may represent the novel large
subunit of the skeletal muscle specific calpain p94.
Key words: SR — Ryanodine receptor — Calpains —
Calcium-activated neutral proteases (CANP; EC 3.4.22.
17), also known as calpain (calcium-dependent papain-
like), are a group of cysteine endopeptidases that have
neutral pH optima and are absolutely dependent on Ca
for catalytic activity [29, 38, 46]. There are two homolo-
gous isoenzymes with different Ca
calpain and m-calpain, with low- (
) and high- (m
concentration requirements, respectively [14, 29].
Calpains (m and ) have been isolated from many tis-
sues, including brain  skeletal , cardiac , and
smooth  muscles, and were found to have similar
structural and biochemical characteristics. While m- and
-calpain are expressed ubiquitously, the tissue-specific
novel calpains were recently discovered [38, 44–48].
These calpains are expressed specifically in skeletal
muscle [43, 47, 48] and stomach  and named n-
calpain (p94 or nCL-1). Based on their primary amino
acid sequence deduced from cDNA the large subunit of
n-calpains is expected to have a molecular weight of 94
kDa and to be activated by nano-molar concentrations of
[38, 46, 48]. Muscle-specific calpain, p94, was
identified during fractionation of connectin using spe-
cific antibodies and was found to be associated with
connectin through a p94-specific sequence . The
p94 has a unique feature of rapid autolysis .
Limited tissue distribution of n-calpain suggests a
restricted range of substrate specificity and may be very
Correspondence to: V. Shoshan-Barmatz
Abbreviations: EGTA, ethylene glycol bis (␤-aminoethyl ether)-N,
N,NЈ,NЈ-tetraacetic acid; Tricine, N-[2-hydroxy-1,1-bis (hydroxy-
methyl)-ethyl]-glycine, MOPS, 3-(N-morpholino) propanesulfonic
acid; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electro-
phoresis; SR, sarcoplasmic reticulum; HSR, heavy SR; CHAPS; 3-[(3-
cholamidopropyl) dimethyl-ammonio]-1-propane-sulfonate; PMSF;
phenylmethylsulfonyl fluoride; RyR, ryanodine receptor, ATPase,
-ATPase; CS, Calsquestrin.
J. Membrane Biol. 161, 33–43 (1998)
The Journal of
© Springer-Verlag New York Inc. 1998