(Received for publication, November 16, 1994; and in revised form, May 15, 1995 )
From the
The isoquinoline carboxamide photoaffinity probe PK14105, a ligand with selectivity for mitochondrial benzodiazepine receptors, has been established to photolabel an 18-kDa protein. When this radioactive probe is used to photolabel rat mitochondrial preparations, a protein of 10 kDa, in addition to the 18-kDa protein, is identified following electrophoretic separation and extended autoradiography. These proteins are referred to herein as pk10 and pk18, respectively. Both proteins exhibited the same specificity to a series of ligands used in competition photolabeling studies and are mutually present at apparently similar ratios across multiple tissues. Subcellular fractionation of rat adrenals indicated that pk10 and pk18 comigrated with the mitochondrial marker enzyme cytochrome c oxidase. In numerous paradigms examining specificity, photolabeling of pk18 invariably coincided with photolabeling of pk10. In detergent-solubilized extracts of rat adrenal mitochondria, pk18 and pk10 coimmunoprecipitated when using antisera raised against pk18. Furthermore, purification of the photolabeled proteins using nondenaturing conditions demonstrated that pk18 and pk10 copurify substantiating their intimate association. A set of three antisera, specific to different regions of pk18, did not recognize pk10 on Western blots. Likewise, partial amino acid sequence of peptide fragments indicate that pk10 is not derived from proteolytic cleavage of pk18. These data suggest that pk10 represents another component of mitochondrial benzodiazepine receptors whose identity is not apparent with any known protein.
The principal pharmacological actions of benzodiazepines, a
class of widely prescribed anxiolytic drugs, is believed to be mediated
primarily through binding to a specific allosteric modulatory site
located on ionotropic -aminobutyric acid receptors. In addition to
binding to this class of inhibitory neurotransmitter receptors, there
is another class of high affinity binding sites for benzodiazepines and
other drugs found in virtually all tissues, primarily localized on
mitochondria(1, 2, 3) , hence they are
referred to as peripheral-type or mitochondrial benzodiazepine
receptors (MBR)(
)(4, 5) .
Despite the prevalent therapeutic use of benzodiazepines, the potential secondary drug effects mediated through MBR are not understood, but recent progress has been made to understand the function of this protein complex(5) . In steroidogenic tissues MBR were found to be coupled to the regulation of steroid biosynthesis(6, 7, 8, 9) . This activity appears to involve a specific interaction with a cytosolic protein known as diazepam binding inhibitor (10, 11, 12, 13) to facilitate intramitochondrial cholesterol transport, the rate-limiting step of steroidogenesis(14) . What remains to be elucidated, however, is the precise molecular mechanism of MBR in this process and what role MBR plays in cells which do not synthesize steroids. It seems probable that MBR might play a fundamental role in the activity of the discreet mitochondrial populations that contain these receptors. With relevance to understanding the relationship of MBR to mitochondrial function several proteins which comprise this complex have tentatively been identified(15) .
In attempts to elucidate MBR function a number of other pharmacological agents have been discovered such as aryl indoleacetamides(16) , imidazopyridines(17) , quinoline propanamides(18) , and isoquinoline carboxamides(19) , along with several other more recently reported classes of compounds(20) . Isoquinoline carboxamides include the highly selective fluoro-nitro photoaffinity probe PK14105 (21) which has been widely reported to photolabel an 18-kDa protein almost exclusively. This mitochondrial protein has been purified (22, 23) and the corresponding cDNA cloned(24, 25, 26, 27) . Transfection experiments with the cDNA demonstrate the expression of binding sites for benzodiazepines and isoquinoline carboxamides with characteristic MBR specificity(24, 25, 28) .
To gain further insight into other proteins associated with MBR, we have made further use of PK14105 as a selective photoaffinity probe. The studies reported here present data showing the existence of a second protein specifically photolabeled by PK14105 which is associated with the 18-kDa protein described above.
In some experiments, rat adrenals
were subfractionated into four subcellular compartments. A
nuclear-enriched pellet was obtained from the centrifugation at 750
g which was resuspended in HEPES/sucrose buffer and
centrifuged again at 750
g yielding the nuclear
pellet. The mitochondrial-enriched fractions in these experiments were
obtained by centrifugation of the 750
g supernatants
for 10 min at 8,000
g, wherein the pellets were washed
by resuspension and centrifugation. A final centrifugation of the 8,000
g supernatant at 100,000
g for 30 min
produced a pellet consisting of microsomal membranes and a supernatant
representing the cytosolic fraction.
Protein of all subcellular
fractions was quantified by the method of Bradford (29) using
bovine -globulin as a standard. All fractions were stored in
HEPES/sucrose buffer at -70 °C until ready for use.
For immunizations, the peptide conjugates were emulsified with an equal volume of complete Freund's adjuvant and 800 µl, corresponding to about 800 µg of antigen, was injected intradermally into New Zealand White rabbits. For boostings, the conjugates were emulsified in Freund's incomplete adjuvant and injected subcutaneously 2 months and 8 months following the initial injections. The quantities of antigen used for these subsequent injections were 250 and 100 µg, respectively. One week following boosting injections, rabbits were bled from ear veins and antisera were prepared.
For immunoblot analysis the nitrocellulose
membranes were blocked with Dulbecco's phosphate-buffered saline
containing 0.1% Tween 20 and 3% bovine serum albumin for 2 h, incubated
with antisera (1:1000) in blocking buffer for 2 h, then washed three
times (10 min each) with phosphate buffer. The membranes were incubated
for 60 min with a 1:1000 dilution of secondary antibody (alkaline
phosphatase-linked goat anti-rabbit IgG) in blocking buffer, washed
three times for 10 min with phosphate buffer and developed in 0.1 M Tris-HCl (pH 9.5), 0.1 M NaCl, 0.05 M MgCl with nitro blue tetrazolium
chloride/5-bromo-4-chloro-3-indolylphosphate.
Partially purified receptor fractions were next applied to Q-Sepharose columns previously equilibrated with 0.1% digitonin in Buffer A at a ratio of 4 ml of column bed volume per 50 mg of initial mitochondrial protein used in each preparation. All the columns were packed to a diameter:length ratio of 1:50 and a flow rate of 0.2 ml/min was used where 3-ml fractions were collected. Under such conditions the photolabeled protein did not bind. The flow-through fractions were thus pooled, supplemented with additional digitonin to 1%, and applied to another Q-Sepharose column equilibrated in 0.1 M glycine (pH 9.5), 0.1% digitonin. Again, with this second buffer system, the photolabeled protein was found predominantly in the flow-through fractions.
The pooled MBR-containing fractions from the
second column were concentrated to about 2 ml with an Amicon
Centriprep-30 unit and applied to a 20-40% sucrose gradient
prepared with 0.025% digitonin in Buffer A. The gradient was
centrifuged overnight at 40,000 rpm in a Sorvall TH-641 rotor.
Fractions of 1 ml were collected where the radioactive peak was found
near the middle of the gradient. These fractions were pooled, diluted
to 9 ml in Buffer A (without digitonin) and placed in the centrifuge
tube underlaid with 25% sucrose in Buffer A. After another overnight
centrifugation at 40,000 rpm the photolabeled protein was concentrated
at the bottom. The upper portion of the discontinuous gradient was
carefully removed. The sucrose fraction was collected, diluted with an
equal volume of ethanol, and kept on ice. This caused the protein to
precipitate within 10 min where centrifugation at 15,000 g for 10 min produced a protein pellet relatively free of digitonin,
suitable for electrophoretic separation after dissolution in SDS-PAGE
loading buffer.
The autoradiograms in Fig. 1show photolabeling of mitochondrial fractions from 10 different rat organs. Adrenals exhibit specific photolabeling of three proteins having apparent molecular masses of about 36, 15, and 10 kDa. In order to simplify referring to these specifically photolabeled proteins in the context of this report they are signified as pk36, pk18, and pk10, respectively. Because a protein of 10 kDa was identified, all studies described here were performed using a Tris/Tricine/SDS-PAGE system (30) which affords excellent resolution of polypeptides in the range of 100 to 1 kDa. With this buffer system pk18 migrates with an apparent mass of 15 kDa despite the fact that the corresponding cDNA has been cloned and sequenced, from which a mass of about 18 kDa is predicted for this protein(24) . Furthermore, earlier studies using the SDS-PAGE protocol of Laemmli (31) give a relative migration more consistent with 18 kDa (21, 22) .
Figure 1:
Specific
[H]PK14105 photolabeling in mitochondrial
fractions from various rat tissues. Mitochondrial-enriched membrane
fractions from different rat organs (Ad, adrenal; Ov,
ovary; Te, testis; He, heart; Ki, kidney; Lu, lung; Sp, spleen; SM, diaphragm skeletal
muscle; Pa, pancreas; SC, spinal cord) at
concentrations of 1 mg of protein/ml were photolabeled in the presence
of 10 nM [
H]PK14105 as described under
``Experimental Procedures.'' Nonspecific photolabeling (N), performed with 10 µM PK11195 included in the
incubation buffer, is compared with total photolabeling (T)
where no competitor was introduced. After solubilization in
electrophoresis sample buffer the following amounts of protein were
electrophoresed in their respective lanes: A-SM, 100 µg; P-SC, 150 µg. Following transfer to nitrocellulose, the
membranes were autoradiographed for either 7 days (A-Sp) or 21 days
(SM-SC). Migration of molecular weight markers is indicated and the
three specifically photolabeled protein bands of adrenals are
denoted.
All of the rat
mitochondrial preparations shown in Fig. 1are known to have
MBR, but their levels vary by 2 orders of magnitude. For example,
mitochondrial fractions from rat adrenals contain densities of >100
pmol/mg of protein, whereas those from the skeletal muscle or neuronal
tissue have 1-3 pmol/mg of protein(2) . In all of these
mitochondrial preparations pk18 and pk10 were detected as proteins
specifically photolabeled by [H]PK14105. Note
also that the relative ratio of intensity between these two
photolabeled adducts remained fairly constant despite the fact that MBR
density is highly variable among the different tissue sources.
In contrast to the similar pattern observed for these two proteins, pk36 was detected only in adrenals. Although MBR was shown to be functionally coupled to steroid biosynthesis (6, 7, 8, 9) the presence of pk36 does not seem to be a feature common to all steroidogenic tissues because this protein was not identified in mitochondrial fractions from testes or ovaries (Fig. 1). In several instances, other photolabeled proteins were observed in different tissues. Many of these are nonspecifically labeled as discriminated using 10 µM PK11195 as a competitor, but in certain tissues such as kidney or skeletal muscle other specifically labeled proteins are faintly detected. The remaining experiments were performed with adrenals because this tissue contains the highest MBR levels.
Figure 2:
Specificity of
[H]PK14105 photolabeling in rat and bovine
adrenal mitochondrial fractions. Either rat adrenal (100 µg of
protein/ml) or bovine adrenocortical (300 µg of protein/ml)
mitochondrial fractions were photolabeled with 10 nM [
H]PK14105 in the presence of 1 µM of the following competing ligands: Co, control (no
competitor); PK, PK11195; Q-,(-)-PK14067; Q+, (+)-PK14068; A, alpidem; Z,
zolpidem; Ro, Ro5-4864; FM, flumazenil; FG,
FGIN-2. The quantities of photolabeled mitochondrial protein
electrophoresed on each lane were 20 µg of protein for rat samples
and 60 µg of protein for bovine samples. Autoradiograms of the
corresponding transblots were exposed for 14 days. Beneath each
autoradiogram are listed values in µM of the IC
each competing ligand displayed in competing for specific
[
H]PK14105 binding. These binding experiments
were performed as described previously (33) in an incubation
volume of 0.2 ml containing mitochondrial protein and
[
H]PK14105 at concentrations equal to those used
for photolabeling in this experiment. Five concentrations of each
competing ligand were tested to displace specific
[
H]PK14105 binding for estimation of IC
values by nonlinear regression
analysis.
The competitor ligands tested are derived
from five classes of aromatic compounds, each having a well
characterized specificity for MBR. In understanding the results
obtained with rat adrenal mitochondria it is best to consider each
class of compounds individually. 1) The isoquinoline carboxamide
PK11195, a high-affinity congener of PK14105 (19) , effectively
competed against photolabeling of all three proteins. 2) A pair of
quinoline propanamide stereoisomers,(-)-PK14067 and
(+)-PK14068, display stereoselective specificity of MBR. The
(-)-isomer exhibits a low nanomolar affinity being over 2 orders
of magnitude more potent than the (+)-isomer(18) . This
stereoselective specificity was observed in the competition
photolabeling of all three proteins. 3) Alpidem and zolpidem are
imidazopyridines showing subnanomolar and micromolar affinities for
MBR, respectively(17) , a pattern which is again reflected in
the photolabeling paradigm. 4) The benzodiazepines Ro5-4864 and
flumazenil show reciprocal selectivity for MBR and type A
-aminobutyric acid receptors(20) . Consistent with the
specificity of mitochondrial benzodiazepine recognition sites, Ro5-4864
competed against the photolabeling of all three proteins whereas
flumazenil had no apparent effect. 5) FGIN-2, an aryl indoleacetamide
displaying high affinity for MBR(16) , also proved to be an
effective competitor. In summary, with rat adrenal mitochondria,
photolabeling of pk36 and pk10 exhibited the same sensitivity to all
competitors as the established pk18 MBR protein.
An equivalent scenario can be drawn with the photolabeling of bovine adrenocortical mitochondria (Fig. 2, lower panel). There are several important differences, however, with respect to the results from rat adrenals. Several other proteins with masses of 30-60 kDa are found to be photolabeled by PK14105, but these represent nonspecifically labeled adducts as neither PK11195 nor any of the other compounds competed against their photolabeling. Therefore, a bovine counterpart of pk36 is not observed, but it may be masked by the nonspecifically labeled proteins evident here. The photolabeling of pk18 and pk10 is easily detected in bovine samples and these show the same sensitivity to the different ligands as was found in the rat except for one major difference; Ro5-4864 did not appreciably compete against their photolabeling. This is an important point because it is well documented that Ro5-4864, while having nanomolar affinity for rodent MBR, exhibits micromolar affinity in bovine tissues(25) . As this result was anticipated with pk18, a conspicuous relationship is evident in that photolabeling of pk10 not only shows the chemical and stereoselective specificities of MBR, but it also displays this characteristic species-specific difference. These results alone provide strong evidence that pk10 may be another component of MBR in close association with pk18.
Figure 3:
Subcellular distribution of photolabeled
proteins. Rat adrenal homogenates were subfractionated to yield pellets
at 750, 8,000, and 100,000 g, indicated as P-750,
P-8K, and P-100K, respectively. These membrane pellets
and the supernatant remaining following the 100,000
g centrifugation (Supe) were photolabeled at a
concentration of 50 µg of protein/ml. Membranes were pelleted
whereas the supernatant samples were concentrated to 0.1 ml on
Centricon-30 units and 10 µg of protein was processed for Western
transfer and autoradiography. The lower panel of the figure
shows quantitation of radioactivity migrating with each protein
species. The open bars represent specific activity of
cytochrome c oxidase measured as described in earlier
studies(2) . Quantitation of enzyme activity and radioactivity
associated with each of the three protein species are normalized
relative to the values determined in P-8K which were: cytochrome c oxidase (open bars), 381 nmol/min/mg of protein; pk36 (hatched bars), 5.2
10
dpm; pk18 (solid bars), 2.0
10
dpm; pk10 (cross-hatched bars), 1.2
10
dpm. Means
± S.D. of at least three photolabeling experiments are
shown.
Figure 4:
Comparison of photolabeling properties for
pk18 and pk10. All photolabeling experiments included rat adrenal
mitochondrial fractions at a concentration of 100 µg of protein/ml.
In Panel A the concentration of
[H]PK14105 was varied and radioactivity migrating
with pk18 (
) and pk10 (
) was quantified following
electrophoresis and transfer to nitrocellulose. Means ± S.E. of
duplicate experiments are shown. Note that both axes are plotted on log
scales. In Panel B 10 nM [
H]PK14105 was used in the presence of
different concentrations of either nonradioactive PK14105
or(-)-PK14067 and radioactivity was determined for both proteins. Symbols indicate mean values of duplicate determinations; the
S.E. was <5% in competition photolabeling experiments with
(-)-PK14067. Data using nonradioactive PK14105 are virtually
superimposable with the data plotted for(-)-PK14067. The
competition curve was fitted by nonlinear regression analysis (assuming
a Hill coefficient of 1) from the data for pk18 using nonradioactive
PK14105 as a displacer.
Another criterion that was exploited in this comparison was the fact that MBR affinity for Ro5-4864 at 4 °C is about 1 order of magnitude higher than it is at 37 °C(32) . This property seems particular to benzodiazepines as binding of isoquinoline carboxamides is not appreciably temperature dependent. Given these parameters it would be expected that Ro5-4864 should be a potent inhibitor of photolabeling at 4 °C and not as potent at 37 °C, whereas competition by PK11195 should be independent of temperature. Competition photolabeling experiments with these two ligands at 100 nM demonstrate this expected behavior for both pk18 and pk10 (Fig. 5). This criterion again reveals the cognate features that pk18 and pk10 have in their photolabeling properties. It is worthy to note that photolabeling of pk36 does not show a similar temperature dependence in photolabeling (Fig. 5, upper panel). This finding places further skepticism on the possibility that pk36 is associated with MBR.
Figure 5:
Temperature dependence of photolabeling
competition by Ro5-4864. Rat adrenal mitochondrial fractions at 100
µg of protein/ml were incubated and photolabeled with 10 nM [H]PK14105 at either 4 or 37 °C in the
absence (Co) or presence of 100 nM PK11195 (PK) or Ro5-4864 (Ro). Autoradiography (upper
panel) and quantitation of radioactivity (lower panel) in
the pk18 (solid bars) and pk10 (hatched bars) bands
are shown.
A photolabeled preparation of rat adrenal mitochondria was determined to have 17-fold higher level of radioactivity incorporated into pk18 relative to pk10. This preparation was subjected to immunoblot analysis wherein a 17-fold difference in the amounts of protein were loaded into pairs of neighboring lanes during SDS-PAGE. With this experimental design, if the antisera detect pk18 in the lanes having 17-fold less protein, then the neighboring lane would have an equivalent level of pk10 (based on radioactivity) which should be detected if it is a fragment of pk18. All three antisera vividly revealed pk18 in the lanes loaded with less protein, however, pk10 was not recognized by any of the three antisera (Fig. 6). The largest peptide segment of pk18 spanning a region not recognized by any of the three antisera is about 6.5 kDa, hence, it is unlikely that a 10-kDa fragment of pk18 could escape detection by the battery of antisera. This immunochemical evidence suggests that pk10 is a protein immunologically distinct from pk18. Additional studies to rule against proteolytic cleavage of pk18 are that extended incubations (1-2 h) at 37 °C either before and during (Fig. 5) or after photolabeling (data not shown) do not change the proportion of photolabeled pk10 relative to pk18. Furthermore, the presence of a diversity of protease inhibitors (see ``Experimental Procedures'') did not affect the photolabeling pattern when compared with experiments omitting these inhibitors.
Figure 6:
Immunoblot analysis with antisera against
different regions of pk18. A preparation of photolabeled rat adrenal
mitochondria containing 17-fold higher radioactivity associated with
pk18 compared to pk10 was electrophoresed in four sets of double lanes,
one lane (17 ) having 17 times more protein than its
neighboring lane (1
). The amount of protein signified
as 1
corresponds to 9 µg except for the lanes under aC where this level is 20 µg. After transfer to nitrocellulose
one pair of lanes was autoradiographed ([
H]PK)
whereas the other lanes where processed for immunoblot analysis using
three antisera generated to different segments of pk18. Antiserum from
a rabbit immunized with keyhole limpet hemocyanin alone (NS)
is used as an indicator of nonspecificity arising from immunizations
using this carrier protein conjugated with peptides corresponding to
amino-terminal/internal (aNI), internal (aI), or
carboxyl-terminal (aC) sequences of pk18 as described in the
text.
Figure 7:
Immunoprecipitation with antiserum against
pk18. Rat adrenal mitochondria were photolabeled at a concentration of
500 µg of protein/ml in the presence of 10 nM [H]PK14105 and processed through an
immunoprecipitation scheme as described under ``Experimental
Procedures.'' The protein recovered from this procedure was
lyophilized, dissolved in 20 µl of water, and prepared for
SDS-PAGE, transfer to nitrocellulose and autoradiography. Shown are
immunoprecipitates obtained using antiserum aI (see Fig. 6)
against pk18, rabbit antiserum against keyhole limpet hemocyanin as a
nonspecific control (Co) for antiserum aI, and
preimmune serum (Pre).
Further support for this possibility was obtained by developing a purification scheme to isolate the photolabeled proteins under nondenaturing conditions. The protocol devised for this was adapted from our previous studies (22, 33) using a detergent different than those used for immunoprecipitation. In all purification methods we tested, pk18 and pk10 were always found to cofractionate as exemplified by the purified preparation shown in Fig. 8. These purified fractions typically show several protein species of which pk18 and pk10 are major components. Several proteins with a mass of 30-34 kDa were verified to be the voltage-dependent anion channel and ADP/ATP carrier using specific antibodies. These proteins were reported earlier to copurify with pk18 in a similar purification scheme with rat kidney mitochondria(15) . The relative abundance of pk18 is much greater, however, using the methods described here, probably because adrenal mitochondria were used which contain much higher MBR levels. The presence of additional proteins at 80, 38, and 5 kDa is under investigation concerning their possible relationship with MBR. Of particular concern with these findings is that the voltage-dependent anion channel and ADP/ATP carrier are known to be among the most abundant mitochondrial proteins (apparently with the 5-kDa protein as well, as seen in Fig. 8) and their cofractionation with pk18 may simply be an unavoidable consequence of their contamination in detergent micelles where pk18 was solubilized. Nevertheless, the photolabeling experiments provide strong support that pk10 is closely tied with MBR and therefore its copurification with pk18 most likely reflects this intrinsic association. The staining intensities seen in Fig. 8suggest that the molar ratios of pk18 and pk10 are more comparable than the relative intensities by which both proteins are photolabeled.
Figure 8:
Purification of pk18 and pk10. Rat adrenal
mitochondria photolabeled with [H]PK14105 were
processed through the purification scheme detailed under
``Experimental Procedures.'' The left panel shows
the electrophoretic pattern of proteins stained with silver according
to the procedure of Morrissey(34) . From a preparation starting
with 50 mg of mitochondrial protein the lanes represent specific
proportions of different fractions as follows: A, 0.5% of
total mitochondrial protein; B, 0.5% of the urea-washed
membrane pellet; C, 3% of the final purified preparation. The right panel shows an autoradiogram of fraction
C.
From preparations as shown in Fig. 8, pk10 is easily resolved from other proteins by SDS-PAGE. This has enabled us to purify pk10 for generation of various peptide fragments. Among these, a tryptic digest of pk10 gave a peptide bearing the sequence XLADK demonstrating that pk10 is not a fragment of pk18. Additional sequence not reported here indicates that pk10 is not currently represented in all sequence data bases of the National Center for Biotechnology Information. Further studies are thus necessary to clone a cDNA for pk10 and reveal the chemical nature of this newly discovered MBR protein.
The development of PK14105 has proven to be a crucial step in unveiling proteins associated with MBR. The protein referred to as pk18 in this report has been unequivocally shown to be an essential component for expressing the specific drug recognition sites of this complex(24, 25, 26, 27, 28) . Other proteins have been reported to be photolabeled specifically by PK14105 (15, 23, 25, 35) but a salient association of these proteins with MBR has not been demonstrated. Of concern in some of these reports is that very long periods of photolabeling are necessary to observe the additional proteins, in which case a broader photolabeling selectivity and damage through extensive ultraviolet irradiation may be occurring. By comparison, all photolabeling studies described here involved only 15 min of irradiation and were scrutinized for specificity with a series of competitor ligands.
The identification of pk36 and pk10 is a subject which had gone unnoticed in many previous studies. The primary reason for this is that the level of photolabeling of these two proteins is much lower than that of pk18. Much longer times of autoradiography are required to detect these less apparent adducts and thus are normally undetected in the time it takes to clearly visualize pk18. An earlier study reported observing pk36(35) , where it was also indicated that in mitochondrial fractions from several rat tissues, this adduct was only found in adrenals as is confirmed here. The disclosure of pk10 is a novel finding and, for the case of MBR structure, is the most relevant discovery in these studies. This photolabeled species had been observed by us previously (see (2) and (13) ), however, it was inconsistently detected and initially believed to result from a breakdown of pk18. SDS-PAGE performed by the procedure of Laemmli (31) gives poor resolution and reproducibility in this molecular weight range accounting primarily for the inconsistency we initially observed in detecting pk10. Changing to a different SDS-PAGE system which permitted superb resolution as low as 1 kDa proved to greatly facilitate the analysis of pk10. As a result of this improvement in methodology, pk10 is invariably detected when pk18 is present.
Because several tests
showed discrepancies in the photolabeling of pk36 compared to pk18, it
is difficult to validate an association of pk36 with MBR. In the
attempt to correlate other proteins with MBR, the revelation of pk10
fulfills many stringent criteria. 1) pk10 and pk18 are mutually present
in all tissues examined containing MBR. The apparent ratio of PK14105
photoincorporation into these two proteins also seems invariable
despite the fact that MBR levels vary by 2 orders of magnitude among
different tissues. 2) Photolabeling of pk10 and pk18 exhibit identical
responses when testing chemical specificity, stereoselectivity, and
species-dependent variation in benzodiazepine recognition. 3)
Equivalent patterns in photolabeling of both proteins are observed at
PK14105 concentrations between 1 and 100 nM and the same
IC values are obtained in competition photolabeling
studies with nonradioactive PK14105 or(-)-PK14067. 4)
Photolabeling of pk10 displays the characteristic MBR behavior that
affinity for Ro5-4864 is markedly temperature dependent, whereas the
binding of PK11195 is insensitive to temperature. 5) pk10
cofractionates with mitochondrial markers plus, with two different
detergent solubilization schemes, it immunoprecipitates and copurifies
with pk18 suggesting a tight physical association between the two
proteins. Furthermore, partial amino sequence and the use of three
separate antisera recognizing defined regions of pk18 argues against
the possibility that pk10 is a proteolytic fragment of, or is
antigenically related to pk18.
The most plausible explanation to account for this long list of coincident photolabeling patterns between pk18 and pk10 is that these two proteins are associated together in a heteromeric complex. A number of laboratories have verified that pk18 is essential for expression of MBR drug binding sites(15, 24, 25, 26, 27, 28) . The fact that PK14105 intensely labels pk18 upholds the proposal that this protein constitutes the binding domain for isoquinoline carboxamides. It is feasible that pk10 lies in close proximity to this domain such that in only 5% of instances pk10 is covalently modified by PK14105, whereas pk18 is labeled in the remaining 95% of successful photolabeling events. This ratio can account for the 15-20-fold difference in photolabeling efficiency consistently observed between these two proteins. Such a possibility can account for the observation that the molar ratios of pk10 and pk18 are not as disparate as judged by silver staining following SDS-PAGE of subfractionated receptor preparations. pk10 may actually comprise a small facet of the binding domain, or be labeled by virtue of being a near neighbor, where photoactivated ligand may dissociate from its binding site and diffuse a short distance in this excited state to encounter pk10. In studies not shown here, the use of a scavenger such as diethylamine(21) , does not inhibit the photolabeling of pk18 or pk10, implying that PK14105 may not need to leave its binding domain in order to label pk10.
The partial amino acid sequence derived from purified pk10 establishes that this protein is not a fragment of pk18, nor is it the 10-kDa cytosolic protein diazepam binding inhibitor which is suspected of interacting with MBR(10, 11, 12, 13) . An identical protein containing this and other sequence obtained is not represented in any of the current sequence data bases. Because pk10 appears to show a distinct association with pk18 it seems credible that this protein has not been identified in another context outside of MBR.
Evidence is mounting that MBR exists as a hetero-oligomeric complex (15, 33, 36, 37) and identification of all proteins in this entity are essential to understand its biochemical role. Strong data have been reported for the function of MBR in steroidogenesis (5) and mitochondrial respiratory control (38) but the molecular mechanisms underlying these findings remains a mystery. Current research goals include revealing the identity of pk10 to hopefully obtain novel insight into MBR function and the pharmacological consequences of drugs which bind to these sites.