Evidence for endogenous ADP-ribosylation of GTP-binding proteins in neuronal cell nucleus. Possible induction by membrane depolarization.

GTP-binding protein(s) recognized by antibodies against the alpha-subunits of Gi- and Go-proteins were detected in crude nuclei isolated from rat brain stem and cortex. Immunohistochemical staining indicated that in the cortex these proteins are perinuclear, or are embedded in the nuclear membrane. Evidence is presented for an endogenous ADP-ribosylation of these proteins, which competes with their PTX-catalyzed ADP-ribosylation. The endogenous reaction has the characteristics of nonenzymatic ADP-ribosylation of cysteine residues, known to involve NAD-glycohydrolase activity. In vitro experiments showed that the alpha-subunit of Go-proteins in the cell membrane also acts as a substrate of this endogenous ADP-ribosylation. The in situ effect of membrane depolarization on the nuclear GTP-binding proteins may be attributable to their depolarization-induced endogenous ADP-ribosylation, suggesting a novel signaling mechanism in neuronal cells in the central nervous system.

[ 3 H]BTX-B binding was determined in the presence of 300 M veratridine (16). The labeled preparations were filtered and washed on GF/C filters and counted for tritium as described previously (10,15).
[ 32 32 P]ADP-ribosylation of proteins (200 g/50 l) was catalyzed by the A-protomer of PTX (2 g/ml) in the presence of 9 -10 pmol/ml [ 32 P]NAD (1 Ci) in hypotonic buffered solution containing 2 mM ATP, 50 mM Tris-HCl, pH 7.4, 0.5 mM MgCl 2 , 0.05 mM EDTA, and 20 mM DTT, according to a previously described procedure (7). Proteins in the crude nuclei were [ 32 P]ADP-labeled, in the absence of the PTX Aprotomer, by incubation of the nuclei for 30 min at 37°C in the above solution (in which the DTT concentration was changed to 0.5 mM), or by incubation in Krebs-Henseleit buffer, pH 7.4, containing 0.5 mM DTT and 10 pmol/ml [ 32 P]NAD. Membranes were [ 32 P]ADP-labeled, in the absence of the PTX A-protomer, by incubation with crude nuclei and [ 32 P]NAD (50 pmol/ml) at 37°C for 30 min in the same hypotonic buffered solution as that used for the PTX-independent reaction in the crude nuclei, but with the addition of the protease inhibitors aprotinin (5 units/ml), pepstatin A (5 g/ml), and phenylmethylsulfonyl fluoride (0.1 mM). The labeled nuclei or membranes were pelleted, resuspended in sample buffer (17), and subjected to SDS-polyacrylamide gel electrophoresis (PAGE) (7.5 or 10% polyacrylamide). The dried gels were autoradiographed (usually for 24 h at Ϫ70°C). Densitometry was performed with a laser densitometer (LKB Broma Ultrascan II).

P]ADP-ribosylation of Proteins in Crude Nuclei and in Membranes-[
Immunoblotting-Gels were electroblotted onto nitrocellulose paper overnight at 10°C and a constant current of 150 mA, as described elsewhere (18). Nitrocellulose strips were exposed to specific antibodies against the N terminus of the ␣-subunit of G o -protein (19) or ␣-common antibodies (20), or antibodies against the C termini of ␣ o -and ␣ iproteins (19). Protein bands with bound antibodies were detected by binding of peroxidase-conjugated second antibody.
Two-dimensional Gel Electrophoresis-Samples containing 200 g of protein were applied to isoelectric focusing gels (first dimension) containing 1.8% Ampholines, pH 5-8, and 0.3% Ampholines, pH 3-10. Isoelectric focusing followed by SDS-PAGE (second dimension) was performed according to Ferro-Luzzi and Nikaido (23). In the second dimension, gels containing the electrofocused proteins were applied on 10% polyacrylamide slab gels and subjected to SDS-PAGE at room temperature. These gels were then dried and autoradiographed.
The tissue was then washed in phosphate-buffered saline, dehydrated, and embedded in paraffin. Polyclonal antibodies against the C terminus of G␣ i -proteins (kindly donated by Dr. G. Milligan) were localized by a standard immunoperoxidase method (24). Paraffin sections (4 m thick) were mounted on slides that were precleaned in 1 N HCl and coated with poly-L-lysine. After drying overnight at 37°C the sections were dewaxed, treated with 0.3% H 2 O 2 for 30 min, and incubated with 3% goat serum in phosphate-buffered saline for 30 min. Antibody against the C terminus of G␣ i -proteins was diluted (1:200) and incubated with the tissue for 16 h at 4°C or for 1 h at 37°C. The antibody was detected by the use of goat anti-rabbit peroxidase conjugated antibody, or alternatively by the use of biotinylated anti-rabbit IgG and avidin-biotin complex peroxidase (ABC; Vector Labs, Burliname, CA). The peroxidase activity was visualized with diaminobenzidine. Antibody specificity was determined by omitting the antibody against the C terminus of G␣ i -proteins. All tissue sections were counterstained with hematoxylin.

Characteristics of GTP-binding Proteins in Crude
Nuclei of Cells in Brain Stem and Cortex-Crude nuclei prepared from rat brain stem and cortical slices were found to contain proteins that are specifically photolabeled with [azido-32 P]GTP (Fig.   1A). Like G␣ o -and G␣ i -proteins in the cell membrane (20), these nuclear proteins (39 -40 kDa on SDS-PAGE) acted as substrates for PTX-catalyzed ADP-ribosylation (Fig. 1B). The presence of these GTP-binding proteins in the crude nuclei could not be attributed to cytosolic contamination, as only traces of these proteins were found in cytosolic fractions of homogenates prepared from brain stem or cortex (Fig. 1B). In addition, the possibility that they are embedded in membrane residues in the crude nuclear fraction was ruled out by using specific markers (see below).
The GTP-binding nuclear proteins reacted with antibodies against both G␣ i -and G␣ o -proteins in the cell membrane (19) (Fig. 1C). Immunolabeling in crude nuclei revealed, however, that these nuclear proteins were not extractable by treatment with detergents that extract G␣ o -and G␣ i -proteins from the cell membrane (6) (Fig. 1C). Moreover, the PTX-catalyzed ADPribosylation of these nuclear proteins was even enhanced following detergent treatment, as if the proteins had now become FIG. 1. Evidence for the presence of GTP-binding proteins in nuclei of cells in rat brain stem and cortex. A, autoradiograms of proteins in crude nuclei isolated from rat brain stem (lanes 1 and 2) and brain cortex (lanes [3][4][5] were specifically photolabeled with [azido-  4) were pretreated with Triton X-100 (2%) (lanes 2 and 4). Proteins in control and pretreated preparations were immunolabeled with antibodies against the N terminus of G␣ o -proteins (lanes 1-4). Crude nuclei, untreated or pretreated with Triton X-100 (2%) (lanes 5 and 7, respectively), were treated with citric acid (1%) (lanes 6 and 8, respectively). Proteins in these nuclei were immunolabeled with antibodies against the C terminus of G␣ i -proteins more available as substrates for ADP-ribosyltransferase (PTX A-protomer) (Fig. 1D). The possibility that these proteins are embedded in the nuclear membrane was examined by treatment with citric acid (1%), which separates the nuclear membrane from the inner matrix (25). Compared with controls, the treated nuclei exhibited only faint labeling with antibodies against the C terminus of G␣ i -proteins (Fig. 1C). Traces of the [ 32 P]ADP-ribosylated proteins detected in the nuclear extracts were attributed to extraction of these proteins by the citric acid treatment.
The possibility that these proteins are embeded in the nuclear membrane was supported by the results of immunohistochemical staining. Post-embedding labeling of paraffin sections of brain cortex (see "Materials and Methods") with antibodies against the C terminus of G␣ i -proteins and the N terminus of G␣ o -proteins resulted in intense antibody binding to the cell nuclei (which were counterstained by hematoxylin), with a halo of dark staining at the nuclear borders that was clearly distinguishable from the immunolabeling of the cytoplasm (Fig. 2). Some of the cell nuclei in the section were not labeled (Fig. 2, A  and B), and no labeling was observed in control samples in which the anti-G␣ i or anti-G␣ o -antibody was omitted (Fig. 2C). The specific immunolabeling supports the presence of these proteins in the crude nuclei (Figs. 1C and 2).
Poly(ADP-ribose)-polymerase (PARP) activity was used as a marker of the nuclear fraction (26) (see Figs. 1, 7, and 8). Significant contamination of the crude nuclei by cell membranes during preparation was excluded by the use of [ 3 H]BTX-B (a labeled derivative of the lipophilic neurotoxin, batrachotoxin), which binds specifically to voltage-dependent Na ϩchannels in the cell membrane (15,27) (Fig. 3). No such binding was detected in crude nuclei isolated from slices of brain stem or brain cortex, while binding of [ 3 H]BTX-B to membranes prepared from these brain regions was similar to that observed previously in membranes prepared from brain synaptoneurosomes (15). Moreover, in agreement with our previous findings, the specific binding of [ 3 H]BTX-B in membranes was stimulated by muscarinic agonists (10, 15), indicating a possible interaction between muscarinic receptors and voltage-dependent Na ϩ -channels in the cell membrane (8,10,15); this effect was not observed in the crude nuclear fraction (Fig. 3). These differences between BTX-B binding in cell membranes and in the crude nuclei were attributed to the lack of voltage-dependent Na ϩ -channels and muscarinic receptors and hence of membrane residues in the crude nuclei.
Evidence for Endogenous ADP-ribosylation of GTP-binding Proteins in Crude Nuclei-GTP-binding proteins (39)(40) in crude nuclei prepared from brain stem or cortical slices, that act as substrates for [ 32 P]ADP-ribosylation catalyzed by the PTX-A protomer, were [ 32 P]ADP-labeled by incubation of the nuclei with [ 32 P]NAD even in the absence of the PTX A-protomer (Fig. 4). These proteins were labeled in nuclei incubated at 37°C for 30 min, either in the hypotonic buffered solution

FIG. 3. Specific binding of [ 3 H]BTX-B to voltage-dependent
Na ؉ -channels as a marker for membrane residues in the crude nuclear preparation. Specific binding of [ 3 H]BTX-B (control (q) and stimulated by 10 M carbamylcholine (E)) to voltage-dependent Na ϩchannels was measured in membranes prepared from brain stem synaptoneurosomes and in crude nuclei prepared from brain stem slices. Nonspecific binding was measured in the presence of veratridine (300 M) (n ϭ 3).

FIG. 2.
Immunohistochemical demonstration of the distribution of G␣ iproteins in sections prepared from rat brain cortex. Specific binding of antibodies to the C terminus of G␣ i -proteins was detected by anti-rabbit IgG conjugated to peroxidase (panel A). In panel B, the avidin-biotin amplification method was used (see "Materials and Methods"). In panel C, exposure of tissue sections to antibodies against G␣ i -proteins was omitted. All sections were counterstained by hematoxylin (panel D) (ϫ 400).
used for their PTX-catalyzed ADP-ribosylation, in which DTT concentration was reduced to 0.5 mM, or in Krebs-Henseleit buffer, pH 7.4, containing 0.5 mM DTT. PTX-catalyzed [ 32 P]ADP-ribosylation of GTP-binding proteins in crude nuclei that had been preincubated under the above experimental conditions in the absence of [ 32 P]NAD and PTX A-protomer was inhibited by 62 Ϯ 5% (n ϭ 10) (Fig. 4, lane 2), suggesting that an endogenous reaction of NAD with these nuclear proteins during preincubation may have interfered with their subsequent PTX-catalyzed ADP-ribosylation. The PTX-independent [ 32 P]ADP-labeling of the nuclear proteins in the presence of [ 32 P]NAD (Fig. 4, lane 3) was temperature-dependent (inactive at 4°C and optimal at 37°C), suggesting the possible involvement of an enzymatic process. In addition, this labeling was inhibited by 40 Ϯ 5% (n ϭ 3) in the presence of GTP␥S (100 M) (Fig. 4, lane 8), reminiscent of the effect of GTP␥S on ADPribosylation of membrane G i -and G o -proteins, which are better substrates for PTX-catalyzed ADP-ribosylation in their nonactivated than in their activated state (28). However, labeling of the nuclear G-proteins in control preparations was not enhanced by GDP␤S (100 M) (n ϭ 3) (Fig. 4, lane 7). Changes in [ 32 P]ADP-labeling of the nuclear proteins were quantified by scanning analysis (see "Materials and Methods").
Inhibition of PTX-catalyzed ADP-ribosylation of the 39 -40-kDa proteins in preincubated brain stem crude nuclei was also detected by two-dimensional gel electrophoresis. Crude nuclear proteins were [ 32 P]ADP-ribosylated by PTX A-protomer after preincubation of the cell nuclei under the experimental conditions employed for their PTX-independent ADP-ribosylation, but in the absence of [ 32 P]NAD (i.e. 37°C, 30 min, Krebs-Henseleit buffer containing 0.5 mM DTT) (Fig. 5A). Subsequent PTX-catalyzed [ 32 P]ADP-ribosylation of the 39 -40-kDa proteins, which reacted with antibodies against the C terminus of G␣ i -proteins, was inhibited in the preincubated nuclei (Fig.  5A). This inhibition may be attributable to an endogenous ADP-ribosylation of these proteins, as indicated by their PTXindependent reaction with [ 32 P]NAD (Fig. 4). Since ADP-ribosylation of the 39 -40-kDa proteins would decrease their isoelectric pH, the possibility that these proteins act as substrates of an endogenous ADP-ribosylation was further examined by  (39)(40) in crude nuclei prepared from brain stem slices were [ 32 P]ADPribosylated by PTX A-protomer and subjected to two-dimensional SDS-PAGE analysis (pH 3-8 in the first dimension, 10% acrylamide in the second dimension). [ 32 P]ADP-ribosylation was performed in crude nuclei exposed to [ 32 P]NAD and PTX A-protomer after preincubation at either 4°C (left) or 37°C (right) for 30 min in Krebs-Henseleit buffer, pH 7.4, containing 0.5 mM DTT (see "Materials and Methods"). Blots of these proteins were autoradiographed (upper frames) and immunostained (lower frames). PTX-catalyzed [ 32 P]ADP-ribosylation of the 39 -40-kDa nuclear proteins did not occur following their preincubation at 37°C (upper right frame). In both preparations these proteins were specifically immunostained with antibodies against the C terminus of G␣ i -proteins. The protein bands (lower left frame) represent 30-, 43-, and 67-kDa protein markers (n ϭ 3). Each sample contained 150 g of protein (41). comparing their isoelectric pH in crude nuclei before and after incubation under conditions enabling their PTX-independent [ 32 P]ADP-labeling on exposure to [ 32 P]NAD. Measurement of the mobility shifts of these nuclear proteins on two-dimensional gel electrophoresis revealed a shift toward acidic pH, detected by immunolabeling of the blotted proteins with antibodies against G␣ i -proteins following either their PTX-catalyzed ADP-ribosylation or their incubation under conditions inducing competition with their PTX-catalyzed ADP-ribosylation. The isoelectric pH of the blotted 39 -40-kDa proteins in untreated nuclei was less acidic by approximately 0.5 pH units (n ϭ 3) (Fig. 5B).
The possibility that cysteine residues in the 39 -40-kDa proteins are acceptors for endogenous ADP-ribosylation in the crude nuclei was examined by the use of cell membrane G␣ iand G␣ o -proteins as substrates (29). Membranes prepared from brain stem or cortical synaptoneurosomes (7) were incubated for 30 min at 37°C with crude nuclei prepared from these brain regions, in the presence of protease inhibitors, in buffered hypotonic solution containing [ 32  . After incubation, the labeled membranes were separated from the mixture by the following procedure. The incubated mixture was centrifuged (30,000 ϫ g, 15 min), and the pellet was resuspended in isotonic sucrose (0.32 M) 1:50 (v/v) and centrifuged (800 ϫ g, 15 min). The pellet, which contained the crude nuclei, was removed. A protein band (100 kDa), which includes poly-(ADP-ribosylated) proteins (see Figs. 1B, 7, and 8), was used as a marker for the crude nuclear fraction (Figs. 6A). The supernatant was centrifuged (800 ϫ g, 15 min), the pellet was removed, six volumes of 20 mM Tris, pH 7.4, were added to the supernatant, and the mixture was centrifuged (30,000 ϫ g, 15 min). The resulting pellet contained cell membranes (12). Proteins in the last pelleted fraction were separated by SDS-PAGE and blotted. Proteins (39 kDa) that were specifically immunolabeled with antibodies against the N terminus of G␣ o -proteins had also been [ 32 P]ADP-labeled during incubation with the crude nuclei and [ 32 P]NAD (Fig. 6). No [ 32 P]ADP-labeling of membrane proteins was observed following their incubation with [ 32 P]NAD in the absence of crude nuclei (Fig. 6A). The [ 32 P]ADP-labeling of the 39-kDa membrane protein was susceptible to proteases (see "Materials and Methods") and to cleavage of thioglycoside bonds by mercuric ions (4), suggesting that an endogenous reaction in the crude nuclei that competes with PTX-catalyzed ADP-ribosylation (Figs. 4 and 5) might have catalyzed ADP-ribosylation of cysteine residues in membrane G␣ o -proteins. For further verification, the products of the PTX-independent reaction, i.e. the [ 32 P]ADP-labeled proteins in membranes prepared from brain cortex, were analyzed by two-dimensional gel electrophoresis, blotted, and subjected to immunolabeling with antibodies against the N terminus of G␣ o -proteins (Fig. 6B). Proteins (39 kDa) in membranes preincubated with crude nuclei and [ 32 P]NAD by the above procedure were found to be both [ 32 P]ADP-labeled and immunolabeled with antibodies against G␣ o -proteins, suggesting that [ 32 P]ADP-labeling of the membrane G␣ o -proteins had occurred during their incubation with the crude nuclei in the presence of [ 32 P]NAD.
Characteristics of the PTX-independent Reaction of the 39 -40-kDa Proteins with NAD in the Crude Nuclei-In order to determine whether the PTX-independent reaction of the proteins with NAD is their ADP-ribosylation, we examined the characteristics of the PTX-independent [ 32 P]ADP-labeling of the 39 -40-kDa crude nuclear GTP-binding proteins that also act as substrates for PTX-catalyzed ADP-ribosylation. First we examined the possibility that their PTX-independent [ 32 P]ADP-labeling might be a result of reactions other than ADP-ribosylation. A nitric oxide-induced reaction of nicotinamide with cysteine residues of the GTP-binding proteins was excluded by the negative results obtained following the protocol of McDonald and Moss (4) for NO-induced binding of nicotinamide-labeled NAD to cysteine residues in the cytosolic enzyme glyceraldehyde-3-phosphate dehydrogenase.
Both the PTX-independent [ 32 P]ADP-labeling and the PTXcatalyzed ADP-ribosylation of the 39 -40-kDa crude nuclear proteins were eliminated as a result of the cleavage of thioglycoside bonds by mercuric ions (4) (Fig. 7D). Thioglycoside bonds are reportedly formed as a result of PTX-catalyzed ADP-ribosylation of cysteine residues in membrane G␣ i -and G␣ o -proteins (29). These observations may therefore indicate the formation of thioglycoside bonds in the PTX-independent [ 32 P]ADP-labeling of the 39 -40-kDa nuclear proteins, suggesting an endogenous ADP-ribosylation of cysteine residues in these proteins in the crude nuclei.
Effect of Membrane Depolarization on the ADP-ribosylation of Nuclear GTP-binding Protein(s)-A possible role for the endogenous ADP-ribosylation of GTP-binding proteins in the crude nuclei may be inferred from the effect of membrane depolarization on their ADP-ribosylation (Figs. 9 and 10). Membrane depolarization of cells in rat brain stem and cortical slices was induced either by their exposure to high-[K ϩ ] Krebs-Henseleit buffer (7) or by prolonged activation of voltage-dependent Na ϩ -channels induced by BTX (14,35).
The in situ effect of membrane depolarization on the 39 -40-kDa nuclear GTP-binding proteins could not be directly detected in this preparation by the technique used in synaptoneurosomes to examine its in situ effect on membrane G-proteins (7,8). We therefore used the subsequent PTX-catalyzed [ 32 P]ADP-ribosylation and [azido-32 P]GTP labeling of the GTP-binding nuclear proteins as probes for detecting the in situ effect of membrane depolarization on these proteins. This procedure has been found useful in the study of PTX-sensitive G-proteins in the cell membrane (7)(8)(9)(10). PTX-catalyzed [ 32 P]ADP-ribosylation of the 39 -40-kDa proteins in crude nuclei prepared from depolarized cells in brain stem or cortical slices was 60 Ϯ 7% less efficient (n ϭ 10) than that occurring in crude nuclei prepared from brain stem or cortical slices at resting potential (Fig. 9, A and C). Similar inhibition (78 Ϯ 5%, n ϭ 5) of their subsequent PTX-independent [ 32 P]ADP-ribosylation was observed in crude nuclei prepared from depolarized cells, while no significant effect was observed either in their subsequent labeling with [azido-32 P]GTP (Fig. 9B) or in their immunolabeling with ␣-common antibodies (36). On the basis of previous results (8,9) these findings are not consistent with a depolarization-induced activation of the nuclear GTP-binding proteins (see also Fig. 4).
The depolarization-induced effect on the subsequent PTXcatalyzed ADP-ribosylation of these proteins could be reversed by membrane repolarization (Fig. 9C). In addition, the depolarization induced effect did not occur following in situ PTXcatalyzed ADP-ribosylation (7) (Fig. 9A), suggesting the involvement of PTX-sensitive G-proteins in the effect of depolarization on the nuclear GTP-binding proteins.
Similarly, BTX-induced membrane depolarization evoked marked inhibition (65 Ϯ 5%, n ϭ 3) of the subsequent PTXcatalyzed [ 32 P]ADP-ribosylation of the 39 -40-kDa crude nuclear proteins in brain stem and cortex (Fig. 9D). The effect of BTX has been attributed to membrane depolarization induced by the delayed inactivation of inward Na ϩ current (10,35), and was indeed antagonized by blocking of the Na ϩ current with tetradotoxin, a specific blocker of the voltage-dependent Na ϩchannel (10) (Fig. 9D). Exposure of cells in brain stem and cortical slices to BTX or high [K ϩ ] apparently did not change the amount of 39 -40-kDa GTP-binding nuclear proteins, as estimated from immunolabeling with ␣-common antibodies (36) (Fig. 9D). This may exclude a possible transfer of these proteins from the cell nucleus in response to membrane depolarization or inward Na ϩ -current.
Since neither the exposure of crude nuclei (rather than cells) to high [K ϩ ], nor their treatment with BTX affected the ADPribosylation of these nuclear proteins, the effect of these agents, when applied on cells, was attributed to their known induction of membrane depolarization (7,8,15). Fig. 10 presents the effect of membrane depolarization induced by high extracellular [K ϩ ] on the isoelectric pH (pI) of the 39 -40-kDa proteins in crude nuclei prepared from depolarized cells in cortical slices. Slices were exposed for 10 min to Krebs-Henseleit buffer containing either 4.7 or 50 mM [K ϩ ] at 37°C and 95% O 2 , 5% CO 2 . Crude nuclei prepared from cortical slices, either depolarized or at resting potential, were subjected to two-dimensional SDS-PAGE analysis and blotting (Western blots). The blotted 39 -40-kDa proteins were immunolabeled with antibodies against G␣ i -proteins (Figs. 1C and 2). The results indicated a shift of approximately 0.4 -0.5 pH units (n ϭ 3) toward a lower pH in the pI of these proteins in nuclei of depolarized cells.
The possibility that the depolarization-induced shift in the isoelectric pH of these proteins reflected their phosphorylation was also examined. Brain cortical slices were exposed to 10 Ci of [ 32 P]phosphorus (400 mCi/ml) for 30 min prior to their depolarization, in order to achieve labeling of phosphorylated proteins during membrane depolarization. High [K ϩ ]-induced membrane depolarization did not enhance the 32 P-labeling of the 39 -40-kDa nuclear proteins, excluding the possibility of their depolarization-induced phosphorylation. Under the same experimental conditions, a depolarization-induced 32 P-phosphorylation of G-proteins was observed in the cells membrane.
In view of the presented evidence for an endogenous ADPribosylation of the 39 -40-kDa proteins in the crude nuclei (Figs. 4 -8), the depolarization-induced shift in their isoelectric pH may support the notion that membrane depolarization induces ADP-ribosylation of these proteins, resulting in inhibition of their subsequent PTX-catalyzed ADP-ribosylation (Fig.  9).

DISCUSSION
This study provides evidence for the presence of GTP-binding proteins (39)(40) in cell nuclei in the rat brain stem and cortex (apparently perinuclear proteins) that are recognized by antibodies against the ␣-subunits of membrane G i -and G oproteins ( Figs. 1 and 2). Proteins immunoblotted with antibodies against these membrane proteins have previously been detected in perinuclear structures of PC12 cells (37). Nuclear proteins (40 kDa) that act as substrates for PTX-catalyzed ADP-ribosylation and react with antibodies against membrane G␣ i -proteins have been detected in cell nuclei isolated from rat liver (38).
The possibility that contamination of the nuclear fraction by membranes was responsible for our observations was excluded by immunohistochemical staining of nuclei (Fig. 2) and by the lack of cell membrane markers in the crude nuclear preparation (Fig. 3). Unlike GTP-binding proteins in the cell membrane (3), the nuclear proteins apparently act as substrates for an endogenous ADP-ribosylation (Figs. 4 -8). They also differ from membrane G-proteins in their response to membrane depolarization: ADP-ribosylation of G␣ i -and G␣ o -proteins in membranes prepared from depolarized brain synaptoneurosomes was similar to that in membranes prepared from synaptoneurosomes at resting potential, unless the synaptoneurosomes were subjected to PTX-catalyzed ADP-ribosylation during depolarization ( Fig. 11) (8,9). This may be attributable to the lack of endogenous ADP-ribosylation of membrane G␣ o -and G␣ i -proteins in these preparations (3). When brain stem synaptoneurosomes were treated with PTX during membrane depolarization, PTX-catalyzed ADP-ribosylation of these proteins was inhibited (Fig. 11) (8,9), apparently as a result of uncoupling of the ␣-subunit from the ␤␥-subunits in PTX-sensitive G-proteins (28) in response to their depolarization induced activation (8,9). As a consequence, their subsequent PTX-catalyzed [ 32 P]ADP-ribosylation was enhanced (by 60 Ϯ 10%, n ϭ 10) (8,9). In contrast, according to the present results the subsequent PTX-catalyzed [ 32 P]ADP-ribosylation of the 39 -40-kDa nuclear GTP-binding proteins in depolarized cells was inhibited by 60 -80% (n ϭ 10) (Figs. 9 and 11). This effect may be attributable to a depolarization-induced endogenous ADP-ribosylation of cysteine residues in these proteins, which would prevent these sites from undergoing subsequent PTX-catalyzed [ 32 P]ADP-ribosylation ( Figs. 9 and 10). This suggestion is supported by evidence indicating an endogenous ADP-ribosylation of the 39 -40-kDa GTP-binding proteins in crude nuclei .
This activity apparently catalyzes the in vivo formation of thioglycoside bonds (29) in these nuclear proteins in the presence of NAD (Figs. 7 and 8), as well as the in vitro ADPribosylation of G␣ o -proteins in membranes incubated with the FIG . 10. Effect of high [K ؉ ]-induced membrane-depolarization on the isoelectric pH of the 39 -40-kDa nuclear proteins in brain cortex. Crude nuclei were prepared from slices of rat brain cortex exposed for 10 min to either 4.7 mM or 50 mM K ϩ in Krebs-Henseleit buffer at 37°C and 95% O 2 , 5% CO 2 . Nuclear proteins were separated by two-dimensional gel electrophoresis, blotted (Western blot) and stained in Ponceau S solution. The 39 -40-kDa nuclear proteins were immunolabeled with antibodies against the C terminus of G␣ i -proteins. In nuclei prepared from depolarized cells the isoelectric pH of the 39 -40-kDa proteins was shifted from approximately 6.5 (at resting potential) to approximately 6.0 (after depolarization) (n ϭ 3). Each sample contained 300 g of protein (41) .   FIG. 11. Comparison of the effects of high [K ؉ ]-induced membrane depolarization on PTX-catalyzed [ 32 P]ADP-ribosylation of GTP-binding proteins in brain stem crude nuclei and membranes. Lanes 1 and 2, Autoradiograms of PTX-catalyzed [ 32 P]ADPribosylated proteins in crude nuclei prepared from brain stem slices preincubated for 10 min at 37°C in Krebs-Henseleit buffer containing 4.7 and 50 mM K ϩ , i.e. at resting potential and during depolarization, respectively. Lanes 3-6, PTX-catalyzed [ 32 P]ADP-ribosylated proteins in membranes prepared from brain stem synaptoneurosomes that were preincubated at resting potential (4.7 mM K ϩ ) (lanes 3 and 5) and during depolarization (50 mM K ϩ ) (lanes 4 and 6) in the presence (lanes 3 and 4) and in the absence (lanes 5 and 6) of PTX (200 ng/ml, 37°C, 2 h, 95% O 2 , 5% CO 2 ) (n ϭ 10 in each preparation). Each lane contained 200 g of protein (41).