90 Chapter 4
Ladrat et al. 2006 ). The proteasome, fi rst
isolated in 1980 (Wilk and Orlowski 1980 ),
is a barrel - shaped polypeptide structure with
active sites in its interior core (Mykles and
Harie 1995 ; Attaix et al. 1998, 2001 ;
Glickman and Cienchanover 2002 ). A 20S
latent form is part of the 26S proteasome
(Attaix et al. 1998, 2001 ) and can be acti-
vated by mild denaturing treatments
(Yamamoto et al. 2009 ) such as heat, chemi-
cals, or high pressure. At least fi ve multipep-
tidase activities (Mykles and Harie 1995 )
have been described: trypsin - like, chymo-
trypsin - like, branched - chain amino acid - pre-
ferring, small neutral amino acid - preferring,
and peptidylglutamyl peptide hydrolase
(PGPH). The MCP proteasome enzymes
have optimal pH activity at pH 7.0 – 8.0, and
the proteasome is found in the sarcoplasm
(Foucrier et al. 2001 ) of skeletal muscle.
Control of indiscriminate proteolysis appears
to be regulated by two methods. The MCP
preferentially degrades polypeptides that
have been ubiquitinated and secondly by
physical size limitation; that is, only poly-
peptides that can pass through the narrow
10 – 13 Å opening to the central core of the
barrel are easily degraded into 6 – 12 amino
acid fragments in a single pass (Attaix et al.
1998, 2001 ; Glickman and Cienchanover
2002 ). It is likely that another protease, cal-
pains for example, acts in concert or synergy
to release proteins from the myofi brillar
assembly in order to make large proteins
available for degradation into amino acids by
the MCP (Hasselgren 1999 ; Allen and Goll
2003 ). The MCP plays a major role in degrad-
ing sarcoplasmic proteins and myofi brillar
fragments; however, there is insuffi cient evi-
dence that MCP breaks down the same pro-
teins in postmortem muscle as in in vitro tests
(Huang et al. 2007 ). Proteasomes remained
relatively stable throughout 7 days of aging
in beef and rabbit muscle (Yamamoto et al.
2009 ), supporting their potential role in meat
tenderization.suring calpain under different pH/temperature
and ionic strength combinations may not
provide an accurate estimation of activity
because of precipitation of calpain or the
alteration of the interaction with substrates.
These issues have been the topic of much
debate in the literature (Prates 2002 ; Goll et
al. 2003 ; Geesink and Veiseth 2009 ), but the
prevailing belief is that μ - calpain is the
essential and predominant enzyme responsi-
ble for postmortem proteolysis and that com-
bined m - and μ - calpain activity may be
responsible for up to 85% of postmortem
meat tenderization (Geesink et al. 2000 ;
Geesink et al. 2006 )
Proteasomes
Evidence has been accumulating that cal-
pains are necessary to initiate the degradation
of myofi brillar proteins by releasing them
from the surface of the myofi bril and making
them available for subsequent degradation.
Given that calpains cleave proteins at a
limited number of sites and produce large
polypeptide fragments rather than small pep-
tides or amino acids, it is clear that other
proteases may be involved in the bulk degra-
dation of sarcomeric structures (Goll et al.
2003 ). For the subsequent breakdown of
myofi brillar proteins, once calpains have
released them from the sarcomere, the main
candidate is the proteasome (Attaix et al.
1998, 2001 ; Delbarre - Ladrat et al. 2006 ;
Yamamoto et al. 2009 ; Geesink and Veiseth
2009 ).
The proteasome, or multicatalytic protein-
ase complex (MCP), is a multisubunit prote-
ase complex with an apparent sedimentation
coeffi cient of 20S. Two types of regulatory
complexes bind to both ends of the cylindri-
cal 20S. One complex, the 26S proteasome,
is a eukaryotic ATP - dependent protease
(Tanaka 1998 ) and hydrolyzes ubiquitin -
conjugated proteins (Tanaka 1998 ; Delbarre -