Biomedical Engineering Reference
In-Depth Information
Originally identified as monomeric enzymes, both DP8 and DP9 are now
readily known to form catalytically active dimers.
122-124
It has recently been
shown that DP8 monomers still retain activity.
125
Recombinant human DP8
and DP9 display optimal enzyme activity between pH 7.0 and 8.5 with low
levels of residual activity still observed at acidic pH 5.0.
6,21,124
Recently, a
natural form of DP9 was purified from bovine testes
126
and identified as being
the short form of DP9 by matrix-assisted laser desorption ionization time of
flight mass spectrometry (MALDI-TOF/TOF MS) analysis of the N-terminal
sequence.
35
This natural form of bovine DP9 displays a similar pH profile to
the human DP9 expressed and purified from insect cells.
126
Enzymatic activities
of both recombinant DP8 and DP9 can be inhibited in the presence of
Zn
21
.
6,126
DP9 activity is also reduced in the presence of Ni
1
and almost
completely inhibited by the presence of Cu
21
and Hg
1
. These results suggest a
physiological role for intracellular Zn
21
or other metal ions in the regulation of
cytosolic DP8/9 activity. Recently, in vitro evidence has emerged suggesting
that the activity of DP8/9 might also be regulated by intracellular redox
events.
126,127
Evidence of redox-state regulation of related PEP enzyme activity
has been previously demonstrated.
128,129
Both DP8 and DP9 are ubiquitously expressed at the mRNA level in all
human adult and foetal tissues as assayed by master RNA blot or Northern
blot hybridization.
6,34
High levels of DP8 transcript can be found in the testis,
heart, liver, skeletal muscle, kidney, pancreas,
6
and a number of endocrine-
related tissues.
52
In testis, a smaller alternative transcript of DP8 with
high expression has been detected.
6,51
Displaying a similar pattern of
expression, the highest levels of DP9 transcript can be found in the heart,
liver, skeletal muscle, spleen, prostate, testis, ovary, small intestine, colon,
peripheral blood leukocytes, and also a number of other endocrine related
tissues.
34
In comparison with DP8, a much lower level of DP9 mRNA was
detected in the testis;
52
thus, it was surprising that natural DP9 was purified
from bovine testis and not DP8.
35
The presence of different mRNA tran-
scripts for DP8 and DP9 also suggests their importance in the male repro-
ductive system. Expression of DP8/9 in this system has thus been further
investigated.
Using individual DP specific inhibition profiles, Dubois et al. found that the
majority of detectable DP activity in bovine and rat testis could be attributed to
the activities of DP8/9, whereas in the epipididymis, DP4 was the predominant
contributor.
130
Immunohistochemistry revealed specific, and distinct staining
of DP8 and DP9 in sperm cells from late stages of spermatogenesis; however,
little to no DP8/9 activity was detectable in preparations of bovine sperm,
130
suggesting that DP8/9 play important roles in the late stages of development
but play no role in mature male gametocytes. In support of these findings, a
recent study by Yu et al. using microarray data analysis to determine the levels
of DP8 and DP9 mRNA during the different stages of murine sperm cell
development found a high abundance of DP9 in later stage spermatocytes.
131
In addition, in situ hybridization of baboon testis found DP8/9 distributed
in spermatogonia and spermatides but only weak staining of DP9 in
