(Received for publication, February 16, 1995; and in revised form, July 18, 1995)
From the
Polyacrylamide gel electrophoresis and high performance liquid
chromatography of multicatalytic proteinase complexes (MPC) isolated
from bovine pituitary, lung, and liver showed marked differences in the
pattern of subunits. The concentrations of LMP7 in the lung and liver
were 10 and 5 times, respectively, greater than those in the pituitary,
whereas the chymotrypsin-like activity and the amount of a subunit
(BO2), necessary for its expression, were markedly decreased in the
lung and moderately decreased in the liver. Lower trypsin-like, small
neutral amino acid preferring, and peptidylglutamyl-peptide hydrolyzing
activities were also found in the lung and liver. The activity of the
branched chain amino acid preferring component (BrAAP), predominantly
latent in the pituitary, was highly activated in the lung and liver, as
evidenced by a greatly decreased K and a
20-fold increase of the specificity constant V
/K
, indicating
facilitated substrate access to its active site and increased affinity
toward substrates with branched chain amino acids in the P
position. It is suggested that overexpression of LMP7 in the lung is
related to increased exposure of the airways to foreign antigens. The
possible association between amounts of LMP7 and the activation of the
BrAAP component needs further examination.
All eukaryotic cells contain a high molecular mass (700 kDa, 19
S) multisubunit, multicatalytic proteinase complex (MPC, ()proteasome), constituting up to 0.5-1% of the
soluble protein fraction of tissue
homogenates(1, 2, 3) . The complex contains a
total of 28 subunits with molecular masses of 21-32 kDa, of which
14 are nonidentical. It is organized in four stacked rings each
containing seven subunits. A similarly organized particle containing
only two nonidentical subunits designated as
and
, and
having the general structure
has been found in the archaebacterium Thermoplasma
acidophilum(4) . Unlike the archaebacterial proteasome
which primarily exhibits only chymotrypsin-like (ChT-L)
activity(4, 5, 6) , the mammalian proteasome
exhibits five distinct endopeptidase activities(7) . Initial
work on the specificity of the MPC from bovine pituitaries (8, 9, 10) led to the identification of three
distinct catalytic activities, cleaving bonds on the carboxyl side of
hydrophobic, basic, and acidic amino acids, each associated with a
different component of the complex. The three activities have been
designated as ChT-L, trypsin-like (T-L), and peptidylglutamyl-peptide
hydrolyzing (PGPH), based on the nature of the amino acid residue in
the P
position(10) . Subsequent work led to
identification of two additional catalytic components, one showing a
preference toward cleavage of peptide bonds on the carboxyl side of
branched chain amino acids (BrAAP) in natural and synthetic peptides
and proteins, the other showing preference toward bonds between small
neutral amino acids (SNAAP)(7, 11) . That the BrAAP
activity is an important factor in the protein degrading activity of
the MPC was shown in experiments in which inactivation of the other
four activities by exposure of the MPC to 3,4-dichloroisocoumarin (DCI)
did not decrease, but indeed increased degradation of many natural
peptides and such proteins as casein and proinsulin.
It is now well established that the MPC (19 S) constitutes a major extralysosomal proteolytic system and that it is responsible as the ``catalytic core'' of a larger 26 S complex (12, 13, 14, 15) for ubiquitin-dependent and ubiquitin-independent pathways of intracellular proteolysis. These pathways are involved in diverse cellular functions such as cell growth and mitosis, antigen processing, and degradation of short-lived regulatory proteins such as oncogene products, transcription factors, and cyclins(16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27) .
The possibility that the MPC might be involved in antigen processing
was suggested by observations that two genes, LMP7 and LMP 2, located
in the MHC class II region code for low molecular weight proteins
having amino acid sequences closely similar to some MPC
subunits(17, 22, 25, 26, 28, 29) .
Furthermore, interferon- (IFN-
) stimulation of human cells
was shown to lead to an increased expression of the LMP7 and LMP2
genes, with the products apparently replacing two highly homologous
subunits X and Y (the latter also referred to as subunit
)(29, 30, 31, 32) ,
respectively. Incorporation of these subunits into the MPC was
suggested to generate so called ``immunoproteasomes'' (33) that could represent a proteasomal subpopulation capable
of more efficiently processing protein antigens into the short 8 to 9
amino acid-containing peptides that are transferred to class I
molecules encoded by the MHC for presentation on the cell surface to
cytotoxic lymphocytes. Since the majority of peptides generated during
antigen processing contain hydrophobic or basic amino acid residues at
the COOH terminus, support for the above hypothesis could have been
anticipated to come from experiments showing that cells stimulated by
IFN-
would show an increase in expression of the ChT-L and T-L
activities of the MPC. Indeed, reports that stimulation of
lymphoblastic and mononuclear cell lines in culture by IFN-
leads
to an increase in the rate of cleavage of bonds after hydrophobic and
basic residues (34) in synthetic peptide substrates, and to
similar activity increases in LMP7 gene transfection
experiments(35) , seemed to support this possibility. However,
a subsequent study of the effect of exposure of the same cell lines to
IFN-
failed to detect any effect on the ChT-L and T-L activities
of the MPC(36) . Moreover, exposure of mouse fibroblast cells
to IFN-
was reported to decrease rather than increase the ChT-L
activity and to have no effect on the T-L activity(32) .
We
report here that the contents of LMP7 and BO2 subunits (the latter
identical with X) differs markedly in MPCs isolated from bovine lung,
pituitary, and liver and that marked changes are also detected in the
catalytic profile of complexes isolated from the three organs. Thus,
the concentration of LMP7 was found to be greatly increased in the lung
compared with that in the liver and pituitary, and this increase was
associated with a decrease in the concentration of subunit BO2. Changes
in the catalytic profile of MPCs isolated from lung and liver involved
a moderate depression of the ChT-L, T-L, PGPH, and SNAAP activities,
and a prominent activation of the BrAAP activity with an increase in
affinity (lowering of K) toward
substrates containing branched chain amino acid residues in the P
position. The possible significance of these changes for antigen
processing is discussed.
SDS-PAGE electrophoresis of equal amounts of protein from MPC
preparations obtained from pituitary, lung, and liver are shown in Fig. 1. About 14 different protein bands are clearly visible
with the overall subunit pattern being similar in all three complexes.
Nevertheless, closer inspection shows marked differences, both
quantitative and qualitative, in the band patterns of the three
complexes. The intensity of some bands is greater in the lung MPC (for
example the slowest moving band), whereas some other bands show higher
density either in the liver or in the pituitary. The structural and
functional significance of these differences, with the exception of
those discussed below, remains to be established. The fastest moving,
somewhat diffuse band in the pituitary preparation was subjected to
NH-terminal amino acid sequencing. The obtained 23 amino
acid sequence RFSPYAFNGGTVLAIAGEDFSIV showed complete identity with the
amino acid sequence of component C5 of the rat hepatoma
proteasome(42) . The second fastest moving band, more intense
in the pituitary than in the lung and appearing in the liver as a
doublet, was shown to correspond to the second peak eluting during HPLC
of the MPC isolated from each of the three organs (Fig. 2).
Because of its mobility in PAGE and HPLC, the component was designated
as BO2 (BO referring to bovine). Identical NH
-terminal
amino acid sequences were found for this component both when BO2 was
isolated by HPLC and subjected to amino acid sequencing, or when the
second fastest moving band in PAGE was blotted to polyvinylidene
difluoride membranes and then subjected to sequencing (see below). A
faint band, clearly visible in the lung but not in the pituitary
complex, was identified by immunoblotting as corresponding to LMP7. Its
identity with LMP7 was also confirmed by NH
-terminal amino
acid sequencing (see below and Table 1). This band was also
visible on direct inspection in PAGE of the liver complex, but its
intensity was fainter than that in the lung and is not visible in the
photographic prints (Fig. 1). The quantitative differences in
the amounts of LMP7 in the three preparations are clearly visible in
immunoblots shown in Fig. 3. The intensity of the band in the
lung is many times greater than that in the pituitary and also greater
than in the liver. Densitometric scanning of the bands in immunoblots
showed that the amount of LMP7 in the lung and liver was respectively
10 and 5 times greater than that in the pituitary MPC (for details see
legend to Fig. 3, and ``Material and Methods'').
Figure 1: Polyacrylamide gel electrophoresis of MPC preparations obtained from bovine pituitaries, lung, and liver. Equal amounts of proteins were subjected to SDS-PAGE as described under ``Materials and Methods.'' The molecular mass markers are derived from the known amino acid sequence of the subunits represented by the respective protein bands.
Figure 2: Subunit patterns obtained by HPLC from MPC preparations isolated from bovine pituitaries (panel A), lung (panel B), and liver (panel C). The conditions of HPLC are given under ``Materials and Methods.'' Arrows indicate the position of the BO2 peak.
Figure 3: Visualization of LMP7 in preparations of the MPC from bovine pituitaries (1), lung (2), and liver (3) by immunoblotting with a specific antibody (for details see ``Materials and Methods''). Densitometric scanning of immunoblots was carried out in order to determine the relative amounts of LMP7 in the three MPC preparations. The photographic negative obtained during the chemiluminescence procedure was transilluminated with a light box and photographed with a Cohu video camera. The video image was captured on a MacIntosh computer as a digitized black and white image. Intensity of the bands on immunoblots was quantitated using the NIH Image program (version 1.53b), according to the recommendations of the manufacturer. This procedure showed that the amounts of LMP7 in the lung and liver were, respectively, 10 and 5 times greater than in the pituitary. The molecular mass of the LMP7 band is about 23 kDa as derived from the amino acid sequence of this subunit and its position in relation to the other MPC bands.
HPLC of MPC preparations results in dissociation of subunits of the
complex and a reproducible separation of 13 peaks. When equal amounts
of protein (25 µg) from each of the pituitary, lung, and liver MPCs
were subjected to HPLC, the subunit patterns shown in Fig. 2were obtained. Several quantitative differences in the
patterns are clearly visible in the three preparations. The second
well-separated peak (designated as BO2) was markedly higher in the
pituitary than in either the lung or the liver. Integration of the
areas under the peak in several preparations showed that the BO2
subunit in lung (panel B, in Fig. 2) amounted on
average to only 60% of that present in the pituitary (panel A, Fig. 2), and that the corresponding amount in the liver (panel C, Fig. 2) constituted about 80% of that in the
pituitary. When 0.5 mg of protein of pituitary MPC was separated by
HPLC and the effluent containing the BO2 component was concentrated and
subjected to amino-terminal sequencing, the sequence shown in Table 1was obtained. An identical NH-terminal
sequence was obtained from the second fastest moving band in PAGE of
the bovine lung MPC (Fig. 1). The NH
-terminal amino
acid sequences of the first 10 amino acids in both bands of the doublet
visible in PAGE of the liver MPC were also identical with those of the
lung, and the separation of the two bands could have resulted from
small differences arising from post-translational modifications in the
part of the protein, COOH-terminal to the sequences shown in Fig. 1.
The NH-terminal sequence of BO2 (Table 1) shows complete identity with sequences reported
previously for human subunit X(43) , bovine pituitary 21-kDa
subunit(44) , bovine lens L2(45) , the
subunit of
human reticulocytes(46) , and also close similarity to the
NH
-terminal sequence of LMP7 from bovine lung, the sequence
of a rat liver proteasome subunit(47) , the internal stretch of
amino acid sequence encoded by the LMP7 gene from a human T cell
line(22) , and the subunit PRE2 from yeast(48) .
Examination of the complete amino acid sequence of BO2 and LMP7 showed
that within a stretch of 204 amino acids 135 (66%) showed identity, and
a considerable number of the remaining amino acids represented
conservative replacements.
Several lines of evidence indicate that BO2 is necessary for expression of the ChT-L activity of the MPC. Thus, the ChT-L activity is the only catalytic activity of the complex that was reported to be sensitive to inactivation by diisopropylfluorophosphate, and indeed two separate studies have reported incorporation of labeled DFP into this subunit (49, 50) . Furthermore, the PRE2 gene encoding a subunit showing high homology to the BO2 and LMP7 sequences was shown to be necessary for expression of the ChT-L activity, since yeast pre2 mutants show very low ChT-L activity and accumulation in cells of ubiquitin-protein conjugates(48) .
To gain
additional evidence that BO2 is indeed involved in expressing ChT-L
activity, we took advantage of the previously described high
susceptibility of this activity to DCI
inactivation(7, 37, 51) . Treatment of the
MPC with DCI is associated with some proteolytic degradation of BO2, as
evidenced by the disappearance of the peak in HPLC, and a decrease in
the intensity of the BO2 bands in PAGE (Fig. 4). In addition
chemical modification of this subunit by covalent attachment of one or
more DCI molecules could be likely the cause of the appearance of a
slower migrating BO2-derived band having the same
NH-terminal sequence as native BO2. All the other
components remain unchanged under these conditions, a conclusion
confirmed by scanning densitometry carried out by the procedure
described in the legend to Fig. 3. The possibility was therefore
considered that the inactivation of the ChT-L activity and changes in
the BO2 component observed by HPLC and SDS-PAGE are an expression of
the same event. We proceeded therefore to determine the
pseudo-first-order rate constant of inactivation of the ChT-L activity
and to compare it with the rate constant of disappearance of the BO2
peak in HPLC. A summary of the data obtained for pituitary MPC is given
in Table 2. Almost identical rate constants were obtained for the
two processes, further adding to the evidence that BO2 is necessary for
expression of the ChT-L activity, and that changes in BO2 after DCI
treatment and inactivation of the ChT-L activity represent two linked
processes. It is notable that the rate constants of inactivation (k
/I) of the other components of the pituitary
MPC greatly differ from those of the ChT-L activity. Previous studies
of the kinetics of inactivation by DCI of the activities of the bovine
pituitary MPC showed that inactivation rate constants for the PGPH,
T-L, and SNAAP activities are, respectively, 3, 7.5, and 9 times lower
than for the ChT-L activity, and that the BrAAP activity is resistant
to inactivation by DCI(7) . Additional confirming evidence for
the conclusion that BO2 is necessary for expression of the ChT-L
activity was obtained by showing that treatment of the MPC with DCI in
the presence of the substrate of the ChT-L activity Cbz-Gly-Gly-Phe-pAB
(5 mM), prevented disappearance of the BO2 peak in HPLC and
that this substrate also prevented incorporation of
C-labeled DCI into this peak (data not shown). The
association of a decreased ChT-L activity and a decreased amount of the
BO2 subunit in MPC preparations from bovine lung compared with those in
bovine pituitary also supports the same conclusion. Collectively, all
these findings firmly establish the conclusion that the BO2 component
is necessary for the expression of the ChT-L activity.
Figure 4: Effect of exposure of the lung MPC to 3,4-dichloroisocoumarin. The lung enzyme was exposed to 20 µM DCI as described under ``Materials and Methods.'' A, SDS-PAGE of the native lung enzyme. B, SDS-PAGE of lung DCI-treated enzyme. Molecular markers are derived from the molecular mass of subunits subjected to amino acid sequencing.
The possibility that incorporation of the LMP7 subunit into the MPC is related to the antigen-processing function, and the finding that antigen processing of cytoplasmic proteins for presentation by class I molecules involves in the majority of cases the generation of peptide fragments having a hydrophobic residue at the carboxyl terminus, a reaction consistent with the involvement of an activity with ChT-L specificity, prompted examination of whether changes in the contents of BO2 and LMP7 subunits of the three preparations are reflected in changes of the ChT-L activity. Furthermore, the finding in both PAGE and HPLC of quantitative differences in the pattern of subunits of the three preparations beyond those related to LMP7 and BO2, induced us to extend such study to examination of differences between the activities of all five known catalytic components. A summary of the catalytic activities measured at a single substrate concentration is given in Table 3. The ChT-L activities measured with two different substrates (Cbz-GGF-pAB and Suc-LLVY-MCA) were moderately but statistically significantly decreased both in the lung and the liver when compared with those in the pituitary. Although the ChT-L activities appeared to be higher in the liver MPC than those in the lung, the differences were not significant. The lung and liver MPCs showed also significantly lower T-L and PGPH activities compared with those in the pituitary. No differences were found between the T-L activity in the lung and liver, but the PGPH activity of the liver MPC was significantly higher than that in the lung. The SNAAP activities were somewhat higher in the pituitary MPC than those in the lung and liver complexes.
The most conspicuous differences between the three MPC preparations were found in the BrAAP activity. The highest activity of this component was found in the lung, whereas the pituitary showed the lowest activity. These differences were found in three consecutive preparations. The activity in the liver, although lower than in the lung, was still significantly higher than in the pituitary. Thus, when measured with ZGPALA-pAB as the substrate the activities in the lung and liver were more than six and three times higher, respectively, than in the pituitary.
To gain insight into the
kinetic basis of the differences between the activities of the five
catalytic components, we determined the maximal velocity (V) as a measure of catalytic efficiency, the
affinity toward substrates (K
), and the
specificity constants (V
/K
). A summary of these
kinetic parameters is given in Table 4. Neither the ChT-L nor the
PGPH component exhibited Michaelis-Menten kinetics when Suc-LLVY-MCA
and Z-LFE-2NA, respectively, were used as substrates for
activity measurements. Plots of velocity versus substrate
concentration yielded sigmoidal curves for both substrates, and as a
result both the K
and V
values were dependent on the range of substrate concentrations
used, which in turn were limited by the solubility of the substrates.
This kinetic behavior apparently results from the presence of more than
one binding site for these substrates, and/or from allosteric
interactions between subunits expressing the activities, as reported
previously from several
laboratories(49, 50, 51) . However, when the
ChT-L activity was measured with Cbz-GGF-pAB, the reaction appeared
nevertheless to follow Michaelis-Menten kinetics. These measurements
have shown that depression of the ChT-L activity in the lung was
accompanied by a decrease of both the V
and K
with the V
/K
ratios remaining the
same. V
decreases were also mainly responsible
for the decrease of the T-L and SNAAP activities in the lung with small
changes in the specificity constants.
Of particular interest were
the findings for the BrAAP component. Thus, whereas the V for this component was only moderately
increased in the lung and decreased in the liver, the biggest changes
were observed for the K
values in preparations
from both organs. Indeed, the K
values were as
much as 20 times lower in the lung and liver MPC than those for the
pituitary enzyme. Thus, the high activities observed for the BrAAP
component in the liver and lung MPC preparations shown in Table 2are accounted for by the fact that the 1 mM substrate concentrations used in the assays were more than 2-fold
higher than the K
for the lung and liver
preparations, but several times lower than K
for
the pituitary MPC. Because of the low K
values,
the V
/K
ratios expressing
the specificity constants for substrates containing a branched chain
amino acid in the P
position were about 20 times higher for
the lung and liver MPC than those for the pituitary. By contrast, only
minor changes of the V
/K
ratios were observed for the other activities of the MPC,
indicating that unlike the specificity of the BrAAP component, the
specificities of the other catalytic components are similar in all
three preparations.
Polyacrylamide gel electrophoresis under dissociating conditions of MPCs isolated from bovine pituitaries, lung, and liver clearly demonstrate qualitative as well as quantitative differences between the subunit composition of the complexes from the three organs. These differences were manifested by virtually complete absence of single subunits in some of the preparations, as well as by the different intensities of protein bands representing different subunit concentrations. The functional significance of these differences is not clear, since the identity of the subunits expressing the known catalytic components of the complex, with the exception of the ChT-L activity, is not known. The identification therefore of BO2 as the component that is necessary for the expression of the ChT-L activity, and the finding that its amount is decreased in the lung and liver compared with that in the pituitary, provided an opportunity to examine the relationship between the expression of ChT-L activity and the quantitative contents of the BO2 component in the three organs. The high ChT-L activities in the pituitary and the decreased activities in lung and liver are therefore consistent with the finding of lower amounts of the BO2 subunit in preparations from the latter two organs.
Recent reports have indicated that stimulation of cells with
IFN- is associated with increased expression of LMP7 and LMP2
genes and down-regulation of expression of the X and Y subunits of the
MPC and their partial replacement by products of the LMP7 and LMP2
genes, respectively. This replacement was suggested to change the
specificity of the MPC in a way that favors the generation of the eight
to nine amino acid residue antigenic peptides that are transported to
the cell surface for presentation to cytotoxic T
lymphocytes(29, 30) . The finding in the lung and the
liver of decreased amounts of subunit BO2 (homologous with the X
subunit), provided an opportunity to examine the possibility that this
decrease is associated with an increase in LMP7, and also to study the
effects of these changes on the specificity of the MPC, under
conditions that do not involve experimental stimulation of cells in
culture with IFN-
. The finding of increased concentrations of LMP7
in association with decreased concentrations of BO2 in the lung and
liver, two organs that are considerably more exposed to foreign
antigens than the pituitary, supports reports that increased expression
of the LMP7 gene leads to partial replacement of the X subunit with the
LMP7 subunit(29, 30, 32) . This finding also
extends the validity of this observation from cells in cultures to
intact organs and suggests the presence of an interrelationship between
expression of these two subunits.
Increased expression of LMP7 and
LMP2 was expected to result in a change in the specificity of the MPC
that would increase the formation of peptides that contain the
hydrophobic and also basic amino acid residues that are commonly seen
at the COOH terminus of antigenic peptides presented at the cell
surface by class I molecules (for reviews see Refs. 55, 56). There is
no evidence, however, that LMP7 or LMP2 express either the ChT-L or T-L
activities or that any of the activities of the MPC are expressed by
these subunits. Nevertheless, an increase of the ChT-L and T-L
activities was reported in lymphoblastoid cells stimulated by
IFN-, or transfected with the LMP7 gene(34, 35) ,
with the increased activities being primarily the result of increased
maximal velocities toward substrates with hydrophobic and basic
residues in the P
position. However, in a similar study (36) exposure of lymphoblastoid cells to IFN-
failed to
produce an increase in the ChT-L or T-L activity of MPC preparations
purified from these cells. Furthermore, stimulation with IFN-
of
mouse fibroblast cells was reported to decrease rather than increase
the ChT-L activity and to have no effect on the T-L
activity(32) . It is of interest that in the latter study
IFN-
induced a change in the specificity of the MPC toward a
25-amino acid residue peptide whose internal sequence contained a known
nine amino acid antigenic peptide. It remains to be determined whether
the increased cleavage frequency in this study of a bond on the
carboxyl side of a leucine residue is solely incidental or an
expression of an altered ChT-L activity induced by IFN-
stimulation. It should be noted that these groups have used for
determination of the ChT-L activity Suc-Leu-Leu-Val-Tyr-7-MCA, a
substrate that has limited solubility, and yields with bovine and human
red blood cell MPC preparations sigmoidal velocity versus substrate concentration plots, and therefore different V
and K
values, depending
on the range of substrate concentrations used. It would be therefore
important to reexamine the kinetics toward this substrate of MPC
preparations from cells used in some of the above studies, in order to
determine whether the kinetic behavior of the ChT-L activity is the
same in the different cell lines.
The results reported here indicate
that the increased amounts of LMP7 in MPC preparations isolated from
the lung and liver, while being associated with a decrease in the
concentrations of the BO2 subunit, are not associated with increases in
the ChT-L or T-L activities. Indeed, small but significantly decreased
activities were found not only for these two activities, but also for
those of the PGPH and SNAAP components. It is notable that these
activity changes were associated only with minor changes of the V/K
ratios, parameters that
are generally considered as reliable indicators of specificity. In view
of these results it would seem important to extend studies on the
effect of IFN-
on a wider range of cells in culture, since the
possibility must be considered that differences might exist in response
of different cell strains to IFN-
stimulation.
The most
remarkable change accompanying the increases of LMP7 in the lung and
liver and the commensurate decrease in the contents of the BO2 subunit
was the great decrease of the K value of the BrAAP
component toward the substrate with a branched chain amino acid in the
P
position. This was associated with a 20-fold increase in
the specificity constant V
/K
. The BrAAP component
was shown to be characterized by selectivity toward peptide bonds in
which the carbonyl group is provided by any one of the branched chain
amino acids(7, 51) , and therefore the increase of the
specificity constant in the lung enzyme indicates an increased
selectivity toward this type of bond. Whereas both the ChT-L and BrAAP
components are capable of cleaving bonds after either branched chain or
aromatic amino acid residues, the latter component shows a definite
preference toward bonds after branched chain amino acids in proteins
and natural peptides (7, 51) . In view of our finding
that differences in subunit composition of MPCs isolated from the
pituitary, lung, and liver are not limited to the BO2 and LMP7
subunits, more evidence would be needed to establish that changes in
the specificity of MPC preparations from these organs are solely due to
changes in expression of these two subunits. More evidence would also
be needed to establish whether the high prevalence of branched chain
amino acids at the COOH terminus of class I antigenic peptides (55, 56) is merely coincidental or a true expression
of the involvement of the BrAAP component in reactions that lead to
excision of these peptides from larger molecules.
The data presented
here also pose the question whether LMP7 is indeed expressing ChT-L
activity as measured with such substrates as Suc-LLVY-MCA or Z-GGF-pAB, in spite of the marked amino acid sequence
similarity to the X (BO2) subunit. LMP7 and X subunits have quite
different pI values, the latter being a more basic
protein(30) . It is therefore possible that replacement of X by
LMP7 is not associated with introduction of this subunit to the same
site previously occupied by X, and that this introduction could induce
changes in the organization of the subunits within the four-ring
structure of the MPC. Such changes could impair the ability of the LMP7
subunit to express the ChT-L activity, if its association with a
defined neighboring subunit is necessary for its catalytic function.
Together with a decrease in the contents of the X(BO2) subunit, changes
in the organization and interaction between subunits induced by
introduction of LMP7 and also LMP2 into the MPC could lead to
facilitation of substrate access to the catalytic site of the component
that expresses the BrAAP activity, a change that would explain the
rather remarkable changes in the V/K
ratios observed in the
lung and liver MPC preparations. It is notable that exposure of the MPC
to DCI, an inhibitor that also leads to inactivation of the ChT-L
activity and proteolytic degradation and chemical modification of BO2 (7) , leads to changes in the V
/K
ratio, indicating
facilitated substrate access to the BrAAP component that are similar to
those found in lung and liver MPC preparations.