Aging/Tenderization Mechanisms 89Geesink et al. 2005 ). Although the mRNA
levels for p94 are 10 times greater than μ -
and m - calpain mRNA (Kinbara et al. 1998 ),
purifi cation from muscle is diffi cult because
it is highly unstable. The p94 has both a cys-
teine protease domain and a calmodulin - like
Ca +2 binding domain in the same polypeptide
chain that binds to the N2A and M - line
regions of titin (Ojima et al. 2007 ). The
endogenous N - terminal (but not C - terminal)
domain of p94 is localized in the Z - bands and
also directly binds to sarcomeric α - actinin,
suggesting incorporation of proteolytic frag-
ments into the Z - bands. It has been suggested
that p94 protects titin from degradation by
μ - and m - calpains (Ojima et al. 2007 ;
Beckmann and Spencer 2008 ; Hayashi et al.
2008 ). Because p94 is active even in the
absence of Ca 2+ (Sorimachi and Suzuki
2001 ), it is thought to have a role in the regu-
lation of μ - and m - calpain activity or be a
negative regulator of apoptosis (Goll et al.
2003 ).
Less than 10% of total calpain is normally
activated in the skeletal muscle (Goll et al.
2003 ). Research has demonstrated that the
optimal condition for calpain activity is pH
7.5 at 25 ° C (Zeece et al. 1986 ), but activity
is still detected at pH 5. Meat tenderization
begins at about pH 6.3 (approximately 6 h
postmortem in beef), as μ - calpain is activated
at low Ca +2 concentrations. M - calpain, or
calpain 2, is optimally active at pH range of
6.5 to 8.0 and 1 – 2 mM Ca 2+ but shows
minimal activity at pH 5.5 and 5 ° C, condi-
tions achieved in 24 to 48 hours postmortem
in the beef carcass. It is estimated that
approximately 30% of m - calpain remains
inactive and can be detected up to 56 days
postmortem (Geesink and Koohmaraie
1999 ). With this limited postmortem activity
range and reported discrepancies between in
vitro and intracellular Ca 2+ concentrations,
some researchers doubt that calpains alone
are responsible for aging of meat. Furthermore,
purifi cation of calpains and calpastatin is dif-
fi cult to accomplish, and techniques for mea-temperature of activity, autolysis, and inhibi-
tors of calpains have been extensively
reviewed (Allen and Goll 2003 ; Geesink and
Veiseth 2009 ). Calpain 1, or μ - calpain,
requires micromolar (10 – 50 μ M) calcium
concentration for full activity. Calpain 2,
m - calpain, is activated at 0.3 – 1.0 mM Ca 2+ ,
while the Ca 2+ requirement of p94/calpain 3
is reported to be at submicromolar levels
(Branca et al. 1999 ; Ono et al. 2004 ). Chicken
muscle expresses a distinct μ /m - calpain,
intermediate to μ - and m - calpains in Ca 2+
requirements for activation (Sorimachi et al.
1990 ; Sorimachi and Suzuki 2001 ; Lee et al.
2008 ). In postmortem chicken muscle, μ /m -
calpain activity remains steady during aging,
in contrast to μ - calpain activity, which disap-
pears by 6 hours postmortem (Lee et al.
2008 ). From this fi nding it was postulated
that chicken muscle tenderizes more rapidly
than beef due to greater activation of the
calpain system (Lee et al. 2008 ).
Ubiquitous μ - and m - calpains are het-
erodimers that dissociate in the presence of
Ca 2+ into a regulatory 28 kDa subunit that is
identical in both isoforms (Carafoli and
Molinari 1998 ) and into a large catalytic
subunit ( ∼ 80 KDa). It is the catalytic subunit
(Dutt et al. 2002 ) that dictates the Ca 2+ level
for activation. The μ - and m - calpains are
located in the sarcoplasm and are concen-
trated around the Z - disk region bound to
their inhibitor, calpastatin (Allen and Goll
2003 ). The equilibrium binding of calpains
to calpastatin also is Ca 2+ - and pH - dependent,
with binding decreasing as pH decreases
(Dransfi eld 1993 ). Moreover, the Ca +2
required for calpains to bind to calpastatin is
signifi cantly lower than that for activating
μ - calpain proteolysis (Cong et al. 2000,
2002 ).
The p94/calpain 3 isoform, unique to skel-
etal muscle, is not fully inhibited by and can
degrade calpastatin (Ono et al. 2004 );
however, its role in postmortem aging is not
fully understood (Parr et al. 1999 ; Ilian et al.
2000 , 2001a, b , 2004 ; Stevenson et al. 2002 ;