23% increase in the protein accretion rate,
whereas a 10% increase in the fractional
synthesis rate will result in an 11%
increase in the protein accretion rate. One
could theorize even greater increases in
growth if synthesis and degradation were
both affected.
Protein synthesis and breakdownIt is evident that both synthesis and break-
down of proteins are necessary to evaluate
the regulation of protein turnover. A better
understanding of these processes is needed
before we proceed. A brief overview of the
mechanisms involved is presented.
Protein synthesis
Protein synthesis (translation) requires the
coordination of >100 macromolecules
working together. They include DNA,
mRNA, tRNA, rRNA, activating enzymes
and protein factors. Information encoded
in DNA is transcribed into the RNA
molecules, which are responsible for the
synthesis of the individual protein. The
formation of single-stranded mRNA
occurs in the nucleus and is called tran-
scription. The mRNA is transported to the
cytosol, where it associates with ribo-
somes, and the translation of the mRNA
sequence into an amino acid sequence
occurs. There are three phases of pro-
tein synthesis: initiation, elongation and
termination.
Initiation occurs when the mRNA
and ribosome bind. The elongation cycle
proceeds with aminoacyl-tRNA (tRNA
molecules bound to specific amino acids)
assembling on a specific codon on the
mRNA. Many ribosomes can attach to a
single mRNA and translate a protein.
Synthesis is terminated when a stop codon
is encountered. A newly synthesized
protein may undergo post-translational
modifications before it can become a func-
tional protein. The protein synthetic
process is probably regulated in two ways
which can affect the rate of protein
synthesis measured: the amount of RNA
and the rate of translation to form protein.
Protein breakdown
Once a protein is synthesized, it is sub-
ject to breakdown. The mechanism of
protein breakdown involves the hydroly-
sis of an intact protein to amino acids.
Protein breakdown is selective, and spe-
cific proteins degrade within the cell at
widely different rates. There are two gen-
eral mechanisms involved in breakdown,
lysosomal and non-lysosomal systems.
The lysosomal system is characterized by
the following: (i) it is located in lyso-
somes at pH 3–5 and includes the cathep-
tic peptidases (cathepsins B, D, H and L);
(ii) it is involved in degradation of endo-
cytosed proteins; and (iii) it is involved
in bulk degradation of some endogenous
proteins. It is unclear how such a degra-
dation system can produce different half-
lives for different proteins.
A second system is the error-eliminat-
ing system which includes peptidases
located in the cell cytoplasm. This system
is specific for proteins containing errors
of translation (abnormal), short-lived pro-
teins, long-lived proteins and membrane
proteins, and it requires ATP. This system
is the ubiquitin–proteasome pathway
reviewed by Mitch and Goldberg (1996).
Proteins are degraded by this pathway
when ubiquitin binds to the protein. It is
accomplished by three enzymes: (i) the E1
enzyme activates ubiquitin in an ATP-
requiring reaction; (ii) activated ubiquitin
is transferred to E2 carrier protein; and (iii)
this is transferred to the protein, catalysed
by the E3 enzyme. This process is repeated
to form a ubiquitin chain. The ubiquitin-
conjugated proteins are recognized by the
proteasome and degraded within the
proteasome by multiple proteolytic sites.
The peptides are released and degraded in
the cytoplasm.
Another cytoplasmic system is the
calpain system consisting of two iso-
enzymes, μ- and m-calpain. This system is
regulated by Ca2+-binding, autoproteolytic
modification, and its inhibitor, calpastatin
(Emori et al., 1987). It has been hypothe-
sized that the calpain system is involved in
the rate-limiting step of myofibrillar protein
breakdown (Reeds, 1989). The calpains areMeasurement and Significance of Protein Turnover 27