Photoaffinity Labeling of Brain Glutamate Dehydrogenase Isoproteins with an Azido-ADP*

Sung-Woo ChoDagger § and Hye-Young YoonDagger

From the Dagger  Department of Biochemistry, University of Ulsan College of Medicine, Seoul 138-736, Korea and the  Department of Medical Technology, College of Health Science, Yonsei University, Wonju 222-701, Korea

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The ADP binding site within two types of bovine brain glutamate dehydrogenase isoproteins (GDH I and GDH II) was identified using photoaffinity labeling with [alpha -32P]8-azidoadenosine 5'-diphosphate (8N3ADP). 8N3ADP, without photolysis, mimicked the activatory properties of ADP on GDH I and GDH II activities, although maximal activity with 8N3ADP was about 75% of maximal ADP-stimulated activity. Saturation of photoinsertion with [alpha -32P]8N3ADP occurred at around 40~50 µM photoprobe with apparent Kd values near 25 and 40 µM for GDH I and GDH II, respectively. Photoinsertion of [alpha -32P]8N3ADP was decreased best by ADP in comparison with other nucleotides. With the combination of immobilized aluminum affinity chromatography and reversed-phase high performance liquid chromatography, photolabel-containing peptides generated by tryptic digestion were isolated. This identified a portion of the adenine ring binding domain of GDH isoproteins as in the region containing the sequence, EMSWIADTYASTIGHYDIN. Photolabeling of the peptide was prevented over 90% by the presence of 1 mM ADP during photolysis, while other nucleotides could not reduce the amount of photoinsertion as effectively as ADP. These results demonstrate selectivity of the photoprobe for the ADP binding site and suggest that the photolabeled peptide with the residues Glu179-Asn197 is within the ADP binding domain of the brain GDH isoproteins.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Glutamate dehydrogenase (GDH1; EC 1.4.1.3) is a family of enzymes that catalyze the reversible deamination of L-glutamate to 2-oxoglutarate using NAD+, NADP+, or both as coenzyme (1). Since the pathology of the disorders associated with GDH defects is restricted to the brain, the enzyme may be of particular importance in the biology of the nervous system. The importance of the pathophysiological nature of the GDH-deficient neurological disorders has attracted considerable interest (2). The enzyme isolated from one of the patients with a variant form of multisystem atrophy displayed a marked reduction of one of the GDH isoproteins (3). Although the origin of the GDH polymorphism is not known, it has been reported that the presence of differently sized mRNAs and multiple gene copies for GDH occur in the human brain (4). A novel cDNA encoded by an X chromosome-linked intronless gene also has been isolated from human retina (5). However, it is not known whether the distinct properties of the GDH isoproteins are essential for the regulation of glutamate metabolism. Therefore, it is essential to have a detailed structural and functional description of the various types of brain GDH to elucidate the pathophysiological nature of the GDH-deficient neurological disorders.

Mammalian GDH is composed of six identical subunits, and the regulation of GDH is very complex (1). It has been a major goal to identify the substrate and regulatory binding sites of GDH. It is the only recent years that the three-dimensional structure of GDH from microorganisms is available (6, 7). Recently, crystallization of bovine liver GDH was reported for the first time from the mammalian sources (8). However, remarkably little is known about the detailed structure of mammalian GDH, especially the brain enzymes. Although regulatory and substrate binding sites of GDH have been reported, the results are quite controversial. Several classical chemical probes have been used to attempt resolution of these binding sites. The studies using classical chemical probes to identify the NADH and GTP binding site within bovine liver GDH, however, gave a wide scatter of modified residues throughout most of the proposed three-dimensional structure of GDH. For instance, the NADH binding site was proposed to be modified by an ATP analogue at Cys319 (9), by a GMP probe at Met169 and Tyr262 (10), and by the adenosine analogue at Lys420 and Tyr190 (11). It seems, therefore, that the base moiety has not been effective at directing the site of modification by classical chemical probes.

Alternatively, identifying nucleotide binding sites of variety of proteins has been advanced by the use of nucleotide photoaffinity analogues that selectively insert into a site upon photoactivation with ultraviolet light. For instance, [32P]2N3NAD+ was shown to be a valid active-site probe for several proteins (12-15). The ATP binding site of adenylate kinase and creatine kinase and the protein unique to cerebrospinal fluids of Alzheimer's patients successfully have been identified using 2N3ATP and 8N3ATP (16). We previously isolated and characterized two soluble forms of glutamate dehydrogenase isoproteins (designated GDH I and GDH II) from bovine brain (17-20) and identified the GTP and NAD+ binding site of the GDH isoproteins using [32P]8N3GTP and [32P]2N3NAD+, respectively (21, 22). Very recently, Stanley et al. (23) have reported that the hyperinsulinism-hyperammonemia syndrome is caused by mutations in GDH gene that affects enzyme sensitivity to GTP-induced inhibition. The mutations identified in the patients with hyperinsulinism and hyperammonemia (23) exactly lie within a sequence of 15 amino acids that we previously suggested to contain GTP binding site of the brain GDH isoproteins (21). On the other hand, the location of the mutations on GDH in those patients is quite distinct from the GTP binding site identified by using the classical chemical probe (24). These results prove selectivity and specificity of the photoaffinity probe as a valid active-site probe.

In the present work, we report the identification of an ADP binding site in the overall sequence by a combination of peptide analysis and photolabeling with [alpha -32P]8N3ADP. The results obtained using photoaffinity probe place the ADP binding domain within a proposed catalytic cleft defined in the crystal structure (25).

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- NADP+, NAD+, NADH, 2-oxoglutarate, ADP, GTP, L-glutamate, aluminum chloride, and L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin were purchased from Sigma. 8N3ADP was purchased from RPI Corp. [alpha -32P]8N3ADP was synthesized by the method as described previously (26, 27). Epoxy-activated iminodiacetate-Sepharose 6B was obtained from Sigma. All other chemicals and solvents were reagent grade or better.

Enzyme Purification and Assay-- The GDH isoproteins were purified from bovine brains by the method developed in our laboratory (17) and were homogeneous as judged by Coomassie-stained gradient SDS-polyacrylamide gel electrophoresis. Only homogeneously purified GDH isoproteins were used unless otherwise indicated. GDH activity was measured spectrophotometrically in the direction of reductive amination of 2-oxoglutarate by following the decrease in absorbance at 340 nm as described previously (17). Activation of GDH I and GDH II by ADP and 8N3ADP were examined by incubating the enzymes with ADP or 8N3ADP at various concentrations in 50 mM triethanolamine, pH 8.0, at 25 °C. At intervals after the initiation of the activation, aliquots were withdrawn for the assay of enzyme activity.

Photolabeling of GDH Isoproteins-- Photolabeling of GDH isoproteins were performed by the method of Shoemaker and Haley (28) with a slight modification. For saturation studies, GDH I and GDH II (0.1 mg each) in 10 mM Tris acetate, pH 8.0, were separately incubated with various concentrations of [alpha -32P]8N3ADP in Eppendorf tubes for 5 min with a hand-held UV lamp at a distance of 4 cm. For competition studies, 0.1 mg of GDH isoproteins were incubated with various concentrations of ADP for 10 min in the same buffer prior to the addition of 100 µM [alpha -32P]8N3ADP and then allowed to incubate with the photoprobe for 5 min as described above. The samples were then irradiated with a hand-held 254-nm UV lamp for 90 s twice at 4 °C. The reaction was quenched by the addition of ice-cold trichloroacetic acid (final 7%). The reaction mixtures were kept on ice bath for 30 min and then centrifuged at 10,000 × g for 15 min at 4 °C. The pellets were washed and resuspended with 10 mM Tris acetate, pH 8.0. The remaining free photoprobe, if any, was further removed from the protein by exhaustive washing using Centrifree (Amicon), and 32P incorporation into protein was determined by liquid scintillation counting.

Tryptic Digestion of Photolabeled GDH Isoproteins-- To determine the site modified by [alpha -32P]8N3ADP, 2.0-mg samples of each GDH isoprotein in 10 mM Tris acetate, pH 8.0, were separately incubated with 100 µM [alpha -32P]8N3ADP for 5 min at 4 °C. Glutamate (5 mM), NAD+ (1.4 mM), NADP+ (0.1 mM), and GTP (0.1 mM) were also included in the incubation mixture to saturate their respective binding sites. The mixtures were irradiated for 90 s twice. In some experiments, GDH I and GDH II were photolyzed in the presence of additional 1 mM ADP to validate that the isolated peptide(s) was specific for the ADP binding site and could be protected from photomodification. The reaction was quenched by the addition of ice-cold trichloroacetic acid (final 7%) and kept at 4 °C for 30 min. The protein was precipitated by centrifugation at 10,000 × g for 15 min at 4 °C, and the pellet was resuspended in 75 mM NH4HCO3, pH 8.5, containing 2 M urea. GDH isoproteins were proteolyzed by the addition of 15 µg of trypsin and kept at room temperature for 3 h, after which 15 µg of trypsin was added again. After 3 more hours at room temperature, 20 µg of trypsin was added, and the digestion mixture was kept at 25 °C overnight.

Isolation of the Photolabeled Peptide and Protein Sequencing-- An immobilized aluminum chromatography procedure (29) modified to isolate photolabeled peptides was used as described elsewhere (16, 28). For preparation of the immobilized aluminum column, 1.5 ml of an epoxy-activated iminodiacetate-Sepharose 6B was chelated to Al3+ by slow passage through 20 ml of 50 mM AlCl3. The resin was then washed successively with H2O and 50 mM ammonium acetate, pH 5.9 (buffer A); 0.5 M NaCl in buffer A (buffer B); buffer A, 2 M urea in buffer A (buffer C); buffer A, 5 mM glutamate in buffer A (buffer D); and buffer A. Buffer A was added to a fraction of the tryptic digestion mixture (1:1), and dithiothreitol was added to a final concentration of 1 mM. The pH was adjusted to 6.0, and the tryptic digested peptides were applied to the column. The column was washed with approximately 15 ml of buffers A, B, A, C, A, D, and A, respectively (0.5 mM/min). The photolabeled peptides were eluted with 5 mM KH2PO4 in 50 mM ammonium acetate, pH 8.0. The absorbance of the fractions was measured at 220 nm and the photoincorporation was determined by liquid scintillation counting.

Fractions from immobilized aluminum column were further purified by reversed-phase HPLC. Fractions containing photolabeled peptides were desalted, freeze-dried, resuspended in 0.1% trifluoroacetic acid, and subjected to reversed-phase HPLC using an Waters C18 column on the same HPLC system. The mobile system consisted of 0.1% trifluoroacetic acid solution and 0.1% trifluoroacetic acid, 80% acetonitrile solvent system. The gradient for HPLC was 0-10 min, 0% acetonitrile; 10-60 min, 0-80% acetonitrile; 60-70 min, 80% acetonitrile at a flow rate 0.5 ml/min. HPLC fractions containing photolabeled peptides were pyridylethylated by the method described elsewhere (30) and sequenced by the Edman degradation method as described previously (17).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Activation of GDH Isoproteins by ADP and 8N3ADP-- The vertebrates GDHs have been well known to be regulated by ADP. To show that 8N3ADP could mimic the regulatory properties of ADP, the photoanalogue should be able to activate GDH as well as ADP. As Fig. 1 shows, both ADP and 8N3ADP activated the activities of the GDH isoproteins, although maximal activity with 8N3ADP was about 75% of maximal ADP-stimulated activity. These results were consistent with an earlier report showing interaction of 8-azido-ADP with bovine liver glutamate dehydrogenase (31). These results show that the azidonucleotide, 8N3ADP, is able to elicit the similar biological effects on GDH isoproteins as the natural nucleotide, ADP.


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Fig. 1.   Activation of GDH isoproteins by ADP and 8N3ADP. The effects of ADP and 8N3ADP on GDH I and GDH II were examined in the direction of reductive amination of 2-oxoglutarate with NADH as a coenzyme at pH 8.0. , GDH I + ADP; open circle , GDH I + 8N3ADP; black-square, GDH II + ADP; , GDH II + 8N3ADP.

Saturation and Competition of Photoinsertion-- To show specificity of the photoprobe-protein interaction, saturation of photoinsertion should be observed. To demonstrate saturation effects with the photoprobe, the enzymes were photolabeled with increasing concentrations of [alpha -32P]8N3ADP in the presence of glutarate. Under the experimental conditions described, saturation of photoinsertion with [alpha -32P]8N3ADP occurred at around 80~100 µM photoprobe with apparent Kd values of 25 µM and 40 µM for GDH I and GDH II, respectively (Fig. 2). The apparent Kd values were also measured from the double-reciprocal plot (data not shown), and the results were almost identical from those obtained in Fig. 2. In all photolabeling experiments, the ionic strength was kept low to enhance binding affinity, as we have observed in general that the lower the ionic strength the tighter the binding of nucleotide photoaffinity probes and the more efficient the photoinsertion. Therefore, the reported apparent Kd values obtained from photoaffinity labeling should be interpreted considering that photolabeling is done under conditions that enhance binding site occupancy. Under saturating conditions, 0.95 mol and 0.90 mol of photolabel/mol of GDH subunit were introduced for GDH I and GDH II, respectively. The results in Fig. 2 indicate the saturability of ADP specific site of GDH isoproteins with this photoprobe and therefore decrease the possibility of nonspecific photoinsertion.


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Fig. 2.   Saturation of [alpha -32P]8N3ADP phosphoincorporation into GDH isoproteins. GDH I and GDH II in the reaction buffer were photolyzed with the indicated concentrations of [alpha -32P]8N3ADP, and 32P incorporation into protein was determined by liquid scintillation counting (see "Experimental Procedures" for details). Relative 32P incorporations were expressed relative to each control. 100% relative 32P incorporation corresponded to 75,000 and 71,000 cpm for GDH I and GDH II, respectively. , GDH I; , GDH II.

To further demonstrate specific labeling of GDH isoproteins, the enzymes were photolabeled with [alpha -32P]8N3ADP in the presence of increasing ADP concentrations. As shown in the results of the competition experiments (Fig. 3), increasing ADP concentration decreased the photolabeling of 100 µM [alpha -32P]8N3ADP. The apparent Kd values of this interaction were 33 µM and 55 µM for GDH I and GDH II, respectively. NAD+ was able to inhibit photoinsertion, but not as effectively as ADP (Table I). ATP and GTP had only a minor effect on the photoinsertion (Table I). When concentrations of the nucleotides were reduced, these effects were less extensive. These results show the specificity of [alpha -32P]8N3ADP and the utility of this probe as a good candidate for determining the ADP binding site.


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Fig. 3.   The effect of ADP on [alpha -32P]8N3ADP phosphoincorporation into GDH isoproteins. GDH I and GDH II in the reaction buffer were photolyzed with 100 µM [alpha -32P]8N3ADP in the presence of the indicated concentrations of ADP. Relative 32P incorporation into protein was determined and expressed as described in the legend to Fig. 2. , GDH I; , GDH II.

                              
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Table I
Effects of nucleotides on photolabeling of brain GDH isoproteins with [alpha -32P]8N3ADP
GDH isoproteins were incubated with the indicated concentrations of various nucleotides for 1 min in 50 mM triethanolamine, pH 8.0, at 25 °C prior to the addition of 100 µM [alpha 32P]8N3ADP. Relative 32P incorporation into protein was determined as described in the legend to Fig. 3.

Tryptic Digestion of Photolabeled Proteins and Isolation of the Photolabeled Peptide-- To identify the peptides modified by [alpha -32P]8N3ADP, GDH isoproteins were photolabeled twice in the absence and presence of 1 mM ADP and digested with trypsin. To reduce any possible nonspecific labeling and at the same time to optimize the specific labeling of the enzymes, 100 µM [alpha -32P]8N3ADP was used, which is the concentration at which photoinsertion approaches saturation. In addition, 5 mM glutamate, 0.1 mM NADP+, 1.4 mM NAD+, and 0.1 mM GTP were included in the reaction mixture to reduce the nonspecific binding of [alpha -32P]8N3ADP to those sites, although they decreased the level of photoinsertion of [alpha -32P]8N3ADP (Table I). The photolabeled GDH isoproteins were separated from most of the noncovalently bound nucleotide by acid precipitation and proteolyzed by trypsin. The digested samples were applied to an immobilized aluminum column. The immobilized aluminum column retained over 90% of the radioactivity (Fig. 4), and the photolabeled peptide was eluted with 5 mM KH2PO4, pH 8.0. One major radioactive peak was recovered from the immobilized aluminum column. Over 85% of the loaded radioactivity from the sample was associated with the eluates from the immobilized aluminum column (fractions 20-30 in Fig. 4). ADP was able to reduce [alpha -32P]8N3ADP photoinsertion into this peak. When 1 mM ADP was originally present in the incubation mixture, more than 90% of the radioactivity of the peak was eliminated as shown in Fig. 4, and the UV absorption peak eluted in fractions 20-26 was dramatically decreased (omitted in Fig. 4 for clarity purposes). This result indicates that the radioactive peak represent a peptide in the ADP binding domain of the GDH isoproteins.


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Fig. 4.   Purification of tryptic peptides from GDH I photolabeled with [alpha -32P]8N3ADP on an aluminum chelate column. GDH I was photolabeled with [alpha -32P]8N3ADP as described under "Experimental Procedures" in the absence and presence of 1 mM ADP, and the mixture was subjected to an immobilized aluminum chromatography. Each 1-ml fraction was monitored for absorbance at 220 nm by UV spectrophotometer and for radioactivity by liquid scintillation. The plain solid line represents the absorbance at 220 nm of the sample photolabeled in the absence of 1 mM ADP. The plot also represents the radioactivity profiles from the aluminum chelate column of the sample photolabeled in absence () and the presence (open circle ) of 1 mM ADP. Fractions 1-5 and 6-20 represent the fractions collected while loading and washing, respectively. Fractions 20-30 represent fractions eluted with 5 mM K2HPO4.

When the flow-through fractions from aluminum column were subjected to separation on a reversed-phase HPLC column, multiple peaks were observed as determined with UV-absorption spectrophotometer at 220 nm, indicating that many of the peptides were not retained on the resin (data not shown). When the radioactive eluates from the immobilized aluminum column were subjected to reversed-phase HPLC, one major radioactive peak and one minor nonradioactive peak were clearly observed (Fig. 5). Although some radioactivity was found in the HPLC flow-through and wash fractions, over 90% of the total radioactivity coeluted with the major peak. The radioactivity associated with the HPLC flow-through fractions represents unbound probe including any decomposition products of photoadduct produced as peptide binds to the HPLC column matrix. These flow-through fractions were subjected to analysis and no significant amounts of amino acids were detected. The radioactive fractions were collected and identified by sequence analysis. GDH II gave almost identical chromatographic profiles to GDH I on both immobilized aluminum chromatography and reversed-phase HPLC, even though the intensities of the radioactivity was slightly lower than those of GDH I (data not shown). These results demonstrates that the microenvironmental structures of the two GDH isoproteins are very similar to each other. The photolabeled peptides of GDH II were, therefore, treated and sequenced by the same method as described above.


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Fig. 5.   Reversed-phase HPLC purification of tryptic peptides eluting from an aluminum chelate column. The radioactive eluates from the aluminum chelate column were loaded onto a C18 reversed-phase column, eluted with an acetonitrile gradient (dashed line) at a flow rate of 0.5 ml/min, and monitored at 220 nm (plain solid line). One-minute fractions were collected. The solid line with closed circles represents radioactivity. Levels of 32P were determined by liquid scintillation counting in aqueous phase.

Sequence Analysis and Amino Acid Composition of the Photolabeled Peptide-- As shown in Table II, the amino acid sequence analysis revealed that the photolabeled peptide of the GDH isoproteins contained the amino acid sequence, EMSWIADTYASTIGHYD and EMSWIADTYASTIGHYDIN for GDH I and GDH II, respectively. The sequences obtained were also compared with those of various GDHs (Table III). Since these are tryptic digests, it was expected to produce a sequence ending with Arg or Lys. The amino acid composition of the photolabeled peptide revealed that the peptide had a composition that was compatible with that of the tryptic peptide spanning residue Glu179-Lys205 of the amino acid sequence of the mature human GDHs (Table IV). Photolabeling of the peptide was prevented over 90% by the presence of 1 mM ADP during photolysis. These results demonstrate selectivity of the photoprobe for the ADP binding site and suggest that the peptide identified using the photoprobe is located in the ADP binding domain of the brain GDH isoproteins.

                              
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Table II
Amino acid sequence of the [alpha 32P]8N3ADP-modified peptide
The amino acids are denoted by the single-letter code.

                              
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Table III
Alignment of [alpha 32P]8N3ADP-modified peptide with homologous sequence from various GDHs

                              
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Table IV
Amino acid composition of the [alpha 32P]8N3-modified peptide


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present work, we identified an adenine binding domain peptide of the ADP binding site of two GDH isoproteins from bovine brain using photoaffinity probe [alpha -32P]8N3ADP and peptide analysis. The specificity of 8N3ADP and the utility of this probe as a good candidate for determining the ADP site were demonstrated by the following. First, in the absence of activating light, 8N3ADP was able to activate GDH similarly to ADP. The ability to mimic a native compound before photolysis has an advantage over determination of the enzyme function after modification. A previous study showed that 8N3ADP and ADP induced similar effects on the structure of GDH (31). This is consistent with the results in Fig. 1. Second, the data showing decreased photoinsertion by addition of ADP demonstrates that photoinsertion occurs only by the bound form of [alpha -32P]8N3ADP. Also, reduction by other nucleotides was not as effective as that observed with ADP. This indicates that proximity controls photoinsertion and that the residues modified are within the adenine binding domain. Third, saturation of photoinsertion at concentrations corresponding to that expected from the reversible binding affinities also strongly supports the site being labeled is within the binding domain. Finally, their selectivity and specificity of 8N3ADP have been successfully utilized to locate the specific base binding domains of nucleotide binding site peptides of many proteins (12-15, 21, 22).

To identify the site of photoinsertion, the photolabeled GDH isoproteins were digested with trypsin and the peptides were purified by aluminum chelate chromatography. The immobilized aluminum chromatography was able to successfully retain the radiolabel of photolabeled peptides and greatly reduces the possibility of any nonphotolabeled peptide coeluting on HPLC with the photolabeled peptide, which could give misleading results. Also, excess protease might be added without a problem with this technique since we have observed no detectable peptide fragments of the protease being retained. Once photolabeled peptides are bound to the metal chelate resin, they can be washed with 0.1 M ammonium acetate solutions containing 0.5 M NaCl or 2 M urea to elute any peptides that may be bound to the resin other than through phosphate-metal interactions. The flow-through fractions from aluminum chelate affinity column had a multiple of peptides (data not shown), while the phosphate eluate contained only two UV peaks from HPLC, one of which was radioactive (Fig. 5). The use of metal chelate chromatography enhances the recovery of photolabeled peptides, because it reduces the number of separation steps. These results indicate that aluminum chelate chromatography could prove to be an effective way of isolating azidonucleotide-photomodified peptides.

The crystal structure of Clostridium symbiosum GDH has been aligned with the primary sequence of the bovine liver GDH (25). The structures of C. symbiosum and mammalian GDHs were suggested to be similar due to considerable identity and the conservation of 13 glycine residues, which probably conserves the structure among species, and to consist of two domains. The first domain has been proposed to contain the catalytically important residues, while the second domain contains a beta -alpha -beta motif that is responsible for coenzyme binding (32, 33). The primary sequence of bovine liver GDH also suggests the presence of a beta -alpha -beta motif corresponding to the motif within the bacterial subunit. Previously, we identified GTP binding site of bovine brain GDH isoproteins as the peptide containing residues Ile440-Arg459 using [alpha -32P]8N3GTP, an effective biomimic of GTP in inhibiting GDH activity (21). Since only the vertebrate GDHs contain the sequence corresponding to Ile440-Arg459, and only GDHs from these sources are regulated by GTP, it seems likely that the regulatory GTP binding domains are located in this region. Our results were clearly consistent with those reported by Shoemaker and Haley (14), who identified the GTP binding site of bovine liver GDH by photoaffinity labeling. Very recently, Stanley et al. (23) have reported that the hyperinsulinism-hyperammonemia syndrome is caused by mutations in GDH gene that affects enzyme sensitivity to GTP-induced inhibition. The sensitivity of GDH to inhibition by GTP was a quarter of the normal level in the patients with sporadic hyperinsulinism-hyperammonemia syndrome. The mutations identified in the patients with hyperinsulinism and hyperammonemia (23) exactly lie within a sequence of 19 amino acids (Ile440-Arg459) that we previously suggested to contain GTP binding site of the brain GDH isoproteins (21). The mutations in the patients with sporadic cases impaired enzyme sensitivity to GTP-induced inhibition but did not affect basal or ADP-stimulated enzyme activity (23). The ADP and GTP binding sites have been thought to be distinct but proximal and possibly overlapping (34). Both nucleotides also elicit different conformational changes within the enzyme (35).

The similarities between the structures of NAD+ and ADP could contribute to the difficulties in identifying the specific ADP binding site. In the present work, the photoprobe [alpha -32P]8N3ADP is shown to mimic ADP kinetically (Fig. 1) and specifically identify the ADP binding site of GDH I and GDH II (Table II). The results also indicate that there are high levels of sequence identity between GDHs compared and the photolabeled peptides corresponds to the tryptic peptide spanning residue Glu179-Lys205 of the amino acid sequence of the mature human GDHs (Table III and Table IV). The peptide within this region were specifically protected from photoinsertion by competing amounts of ADP. Comparisons of this sequence with the bacterial enzyme suggest that these peptides lie within the first globular domain. It was reported that the affinity of ADP and 8N3ADP to GDH is not as tight as that of NAD+, GTP, and their corresponding photoaffinity probes (28). This is most likely due to fewer points of protein-nucleotide contact, which would account for loose binding. While certain residues of the photolabeled peptides showed decreases compared with other residues, the exact residues photolabeled within the adenine ring domain within the ADP binding site remain uncertain. Our results are clearly consistent with those reported by Shoemaker and Haley (28), who identified the ADP binding site of bovine liver GDH using [alpha -32P]8N3ADP and [beta -32P]2N3ADP. Data obtained using photoaffinity probes (13, 14, 21, 22) place all of the base binding domains of NAD+, GTP, and ADP at different locations within a proposed catalytic cleft defined in the structure by Teller et al. (25).

In contrast to our approach, several classical chemical probes have been used to attempt resolution of the binding sites and have shown quite discrepant results. For instance, the ADP binding site was proposed to be modified by an two different AMP analogues at His82 (36) and Arg459 (37). These two residues are outside the catalytic cleft. Similar results with discrepancy using classical chemical probes were also reported by the same research group to identify other regulatory sites within bovine liver GDH. The NADH binding site was proposed to be modified by an ATP analogue at Cys319 (9), by a GMP probe at Met169 and Tyr262 (10), and by the adenosine analogue at Lys420 and Tyr190 (11). The GTP binding site was also proposed to be modified by 5'-p-(fluorosulfonyl)benzoyl-1,N6-ethenoadenosine at Tyr262 (24). It is not clear why a hydrophobic adenosine-containing probe 5'-p-(fluorosulfonyl)benzoyl-1,N6-ethenoadenosine preferentially bind and react at a hydrophilic GTP binding site and not react at the other adenosine binding sites. Above all, the GTP binding site identified by the classical chemical probes are quite distinct from the GTP binding site (Ile440-Arg459) observed in the patients with hyperinsulinism and hyperammonemia (23). It seems, therefore, that the base moiety has not been effective at directing the site of modification by classical chemical probes. Chemically reactive probes, due to their long-lived reactive state, have an increased opportunity to react with the most nucleophilic residues within an enzyme and may not necessarily react with a less reactive or nonreactive residue that may be located within the binding domain. This is especially likely if they display low affinity for the binding site being studies. Their lack of specificity may be the reason for the wide three-dimensional distribution of the residues identified using classical chemical probes as being in the NADH inhibitory site of GDH (9, 10). An analysis of the three-dimensional structure of the mammalian enzyme should supplement the understanding of the nature and location of these regulatory sites.

Partial deficiency of GDH isoproteins has been reported in some patients with cerebellar degeneration, suggesting that the enzymes are important in brain function (3, 4). The existence of the hyperinsulinism-hyperammonemia syndrome also highlights the importance of GDH in the regulation of insulin secretion (23, 38) and indicates that GDH has an important role in regulating hepatic ureagenesis (39). Further studies of GDH and its isotypes may provide explanations for the absence of central nervous system symptoms due to hyperammonemia (40). As many proteins have functions distinct from those for which they were originally identified, the other roles of GDH isoproteins have been reported. For instance, a membrane-bound form of GDH possesses a microtubule binding activity (41), and GDH reacts as a RNA-binding protein and shows a possible role in regulation of transcription (42, 43). Recently, Cavallaro et al. (44) have identified GDH as one of the late memory-related genes in the hippocampus. It would appear that we have just begun to unravel the mystery of GDH and their role in biological system. To our knowledge, comparison of the detailed structure of active sites and regulatory sites of any GDH isoproteins rarely has been reported. In conclusion, the work presented here clearly identifies the ADP binding site of the brain GDH isoproteins in the overall sequence.

    FOOTNOTES

* This work was supported by the Nondirected Research Fund 1997 from the Korea Research Foundation (to S.-W. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. of Biochemistry, University of Ulsan College of Medicine, 388-1 Poongnap-Dong, Songpa-Ku, Seoul 138-736, Korea. Fax: 82-2-2224-4278; E-mail: swcho{at}www.amc.seoul.kr.

    ABBREVIATIONS

The abbreviations used are: GDH, glutamate dehydrogenase; 8N3ADP, 8-azidoadenosine 5'-diphosphate; HPLC, high performance liquid chromatography.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
  1. Fisher, H. F. (1985) Methods Enzymol. 113, 16-27[Medline] [Order article via Infotrieve]
  2. Plaitakis, A., Berl, S., and Yahr, M. D. (1982) Science 216, 193-196[Medline] [Order article via Infotrieve]
  3. Hussain, M. H., Zannis, V. I., and Plaitakis, A. (1989) J. Biol. Chem. 264, 20730-20735[Abstract/Free Full Text]
  4. Plaitakis, A., Flessas, P., Natsiou, A. B., and Shashidharan, P. (1993) Can. J. Neurol. Sci. 20 Suppl. 3, S109-S116[Medline] [Order article via Infotrieve]
  5. Shashidharan, P., Michaelidis, T. M., Robakis, N. K., Kresovali, A., Papamatheakis, J., and Plaitakis, A. (1994) J. Biol. Chem. 269, 16971-16976[Abstract/Free Full Text]
  6. Baker, P. J., Britton, K. L., Engel, P. C., Farrants, G. W., Lilley, K. S., Rice, D. W., and Stillman, T. J. (1992) Proteins Struct. Funct. Genet. 12, 75-86[Medline] [Order article via Infotrieve]
  7. Yip, K. S. P., Stillman, T. J., Britton, K. L., Artymiuk, P. J., Baker, P. J., Sedelnikova, S. E., Engel, P. C., Pasquo, A., Chiaraluce, R., Consalvi, V., Scandurra, R., and Rice, D. W. (1995) Structure (Lond.) 3, 1147-1158[Medline] [Order article via Infotrieve]
  8. Peterson, P. E., Pierce, J., and Smith, T. J. (1997) J. Struct. Biol. 120, 73-77[CrossRef][Medline] [Order article via Infotrieve]
  9. Ozturk, D. H., and Colman, R. F. (1991) Biochemistry 30, 7126-7134[Medline] [Order article via Infotrieve]
  10. Ozturk, D. H., Park, I., and Colman, R. F. (1992) Biochemistry 31, 10544-10555[Medline] [Order article via Infotrieve]
  11. Schmidt, J. A., and Colman, R. F. (1984) J. Biol. Chem. 259, 14515-14519[Abstract/Free Full Text]
  12. Campbell, S., Kim, H., Doukas, M., and Haley, B. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1243-1246[Abstract]
  13. Kim, H., and Haley, B. (1991) Bioconjugate Chem. 2, 142-147[Medline] [Order article via Infotrieve]
  14. Shoemaker, M. T., and Haley, B. E. (1993) Biochemistry 32, 1883-1890[Medline] [Order article via Infotrieve]
  15. Vaillancourt, R. P., Dhanasekaran, N., and Ruoho, A. E. (1995) Biochem. J. 311, 987-993[Medline] [Order article via Infotrieve]
  16. Olcott, M., Bradly, M., and Haley, B. E. (1994) Biochemistry 33, 11835-11941
  17. Cho, S.-W., Lee, J., and Choi, S. Y. (1995) Eur. J. Biochem. 233, 340-346[Abstract]
  18. Cho, S.-W., and Lee, J. E. (1996) Biochimie (Paris) 78, 817-821[CrossRef][Medline] [Order article via Infotrieve]
  19. Kim, S. W., Lee, J., Song, M.-S., Choi, S. Y., and Cho, S.-W. (1997) J. Neurochem. 69, 418-422[Medline] [Order article via Infotrieve]
  20. Cho, S.-W., Cho, E. H., and Choi, S. Y. (1998) FEBS Lett. 426, 196-200[CrossRef][Medline] [Order article via Infotrieve]
  21. Cho, S.-W., Ahn, J.-Y., Lee, J., and Choi, S. Y. (1996) Biochemistry 35, 13907-13913[CrossRef][Medline] [Order article via Infotrieve]
  22. Cho, S.-W., Yoon, H.-Y., Ahn, J.-Y., Choi, S. Y., and Kim, T. U. (1998) J. Biol. Chem. 273, 31125-31130[Abstract/Free Full Text]
  23. Stanley, C. A., Lieu, Y. K., Hsu, B. Y. L., Burlina, A. B., Greenberg, C. R., Hopwood, N. J., Perlman, K., Rich, B. H., Zammarchi, E., and Poncz, M. (1998) N. Engl. J. Med. 338, 1353-1357
  24. Jacobson, M. A., and Colman, R. F. (1982) Biochemistry 21, 2177-2186[Medline] [Order article via Infotrieve]
  25. Teller, J., Smith, R., McPherson, M., Engel, P., and Guest, J. (1992) Eur. J. Biochem. 206, 151-159[Abstract]
  26. Potter, R. L., and Haley, B. E. (1982) Methods Enzymol. 91, 613-633
  27. Salvucci, M. E., Chavan, A. J., and Haley, B. E. (1992) Biochemistry 31, 4479-4487[Medline] [Order article via Infotrieve]
  28. Shoemaker, M. T., and Haley, B. E. (1996) Bioconjugate Chem. 7, 302-310[CrossRef][Medline] [Order article via Infotrieve]
  29. Anderson, L. (1991) J. Chromatogr. 539, 327-334[CrossRef]
  30. Mak, A. S., and Jones, B. L. (1978) Anal. Biochem. 84, 432-440[Medline] [Order article via Infotrieve]
  31. Koberstein, R., Cobianchi, L., and Sund, H. (1976) FEBS Lett. 64, 176-180[CrossRef][Medline] [Order article via Infotrieve]
  32. Wierenga, R., Terpstra, P., and Hol, W. G. J. (1986) J. Mol. Biol. 187, 101-107[Medline] [Order article via Infotrieve]
  33. Stillman, T., Baker, P., Britton, K., Rice, D., and Rodgers, H. (1992) J. Mol. Biol. 224, 1181-1184[Medline] [Order article via Infotrieve]
  34. Cross, D., and Fisher, H. (1970) J. Biol. Chem. 245, 2612-2621[Abstract/Free Full Text]
  35. Cioni, P., and Strambini, G. (1989) J. Mol. Biol. 207, 237-247[Medline] [Order article via Infotrieve]
  36. Batra, S., and Colman, R. (1986) J. Biol. Chem. 261, 15565-15571[Abstract/Free Full Text]
  37. Wrzeszczynski, K. O., and Colman, R. F. (1994) Biochemistry 33, 11544-11553[Medline] [Order article via Infotrieve]
  38. Fahien, L. A., MacDonald, M. J., Kmiotek, E. H., Mertz, R. J., and Fahien, C. M. (1988) J. Biol. Chem. 263, 13610-13614[Abstract/Free Full Text]
  39. Brusilow, S. W., and Horwich, A. L. (1995) in The Metabolic and Molecular Bases of Inherited Disease (Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D., eds), Vol. 1, pp. 1187-1232, McGraw-Hill, New York
  40. Zammarchi, E., Filippi, L., Novembre, E., and Donati, M. A. (1996) Metabolism 45, 957-960[Medline] [Order article via Infotrieve]
  41. Rajas, F., Gire, V., and Rouset, B. (1996) J. Biol. Chem. 271, 29882-29890[Abstract/Free Full Text]
  42. Preiss, T., Hall, A. G., and Lightowlers, R. N. (1993) J. Biol. Chem. 268, 24523-24526[Abstract/Free Full Text]
  43. Bringaud, F., Stripecke, R., French, G. C., Freedland, S., Turck, C., Byrne, E. M., and Simpson, L. (1997) Mol. Cell. Biol. 17, 3915-3923[Abstract]
  44. Cavallaro, S., Meiri, N., Yi, C. L., Musco, S., Ma, W., Goldberg, J., and Alkon, D. L. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9669-9673[Abstract/Free Full Text]
  45. Nakatani, Y., Banner, C., Herrath, M., Schneider, M. E., Smith, H. H., and Freese, E. (1987) Biochem. Biophys. Res. Commun. 149, 405-410[Medline] [Order article via Infotrieve]
  46. Tzimagiorgis, G., and Moschonas, N. K. (1991) Biochim. Biophys. Acta 1089, 250-253[Medline] [Order article via Infotrieve]
  47. Julliard, J., and Smith, E. L. (1979) J. Biol. Chem. 254, 3427-3438[Abstract]


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