Báo cáo khoa học: Brain succinic semialdehyde dehydrogenase Reactions of sulfhydryl residues connected with catalytic activity pdf

Brain succinic semialdehyde dehydrogenaseReactions of sulfhydryl residues connected with catalytic activity Byung Ryong Lee1, Dae Won Kim1, Joung-Woo Hong2, Won Sik Eum1, Hee Soon Choi1,

Brain succinic semialdehyde dehydrogenase

Reactions of sulfhydryl residues connected with catalytic activity

Byung Ryong Lee1, Dae Won Kim1, Joung-Woo Hong2, Won Sik Eum1, Hee Soon Choi1, Soo Hyun Choi1,

So Young Kim1, Jae Jin An1, Jee-Yin Ahn1,*, Oh-Shin Kwon3, Tae-Cheon Kang4, Moo Ho Won4,

Sung-Woo Cho5, Kil Soo Lee1, Jinseu Park1and Soo Young Choi1

1

Department of Genetic Engineering and Research Institute for Bioscience and Biotechnology, Hallym University, Chunchon, Korea;

2

Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, OH, USA;3Department of Biochemistry, Kyungpook National University, Taegu, Korea;4Department of Anatomy, College of Medicine, Hallym University, Chunchon, Korea;5Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, Seoul, Korea

Incubation of an NAD+-dependent succinic semialdehyde

dehydrogenase from bovine brain with

4-dimethylamino-azobenzene-4-iodoacetamide (DABIA) resulted in a

time-dependent loss of enzymatic activity This inactivation

followed pseudo first-order kinetics with a second-order rate

constant of 168M )1Æmin)1 The spectrum of DABIA-labeled

enzyme showed a characteristic peak of the DABIA

alkyl-ated sulfhydryl group chromophore at 436 nm, which was

absent from the spectrum of the native enzyme A linear

relationship was observed between DABIA binding and the

loss of enzyme activity, which extrapolates to a

stoichio-metry of 8.0 mol DABIA derivatives per mol enzyme

tetramer This inactivation was prevented by preincubating

the enzyme with substrate, succinic semialdehyde, but not by

preincubating with coenzyme NAD+ After tryptic diges-tion of the enzyme modified with DABIA, two peptides absorbing at 436 nm were isolated by reverse-phase HPLC The amino acid sequences of the DABIA-labeled pep-tides were VCSNQFLVQR and EVGEAICTDPLVSK, respectively These sites are identical to the putative active site sequences of other brain succinic semialdehyde dehy-drogenases These results suggest that the catalytic function

of succinic semialdehyde dehydrogenase is inhibited by the specific binding of DABIA to a cysteine residue at or near its active site

Keywords: brain succinic semialdehyde dehydrogenase; DABIA; GABA shunt; reactive cysteine residues

c-Aminobutyric acid (GABA) is produced from glutamate

in a reaction catalyzed by glutamate decarboxylase (GAD)

and further metabolized to succinate by the successive

action of GABA transaminase (GABA-T) and succinic

semialdehyde dehydrogenase (SSADH) The carbon

skel-etal of GABA therefore enters the tricarboxylic acid in

the form of succinate GABA metabolism has been well

characterized in the mammalian central nervous system

where GABA functions as a major inhibitory

neurotrans-mitter

SSADH, the final enzyme in GABA metabolism, has been purified from rat, human and bovine brain [1–3] This enzyme is also the site of an inborn error of human metabolism [4] In autosomal recessively inherited SSADH deficiency, now identified in more than 45 patients who manifest varying degrees of psychomotor retardation with speech delay, the normal oxidative pathway is blocked, thereby resulting in the accumulation of succinic semialde-hyde (SSA) Metabolic patterns in physiologic fluids derived from patients show large increases in gamma-hydroxybu-tyrate (GHB) [5], the reduction product of SSA by succinic semialdehyde reductase [6] GHB, the biochemical hallmark

of SSADH deficiency, produces central nervous system effects including altered motor activity and behavior disturbances when administered to animals and humans

at pharmacologic levels [7]

Recently, an SSADH cDNA was cloned from rat brain and human liver [8] The mammalian SSADH bears signi-ficant homology to bacterial NADP+-SSADH and con-served regions of aldehyde dehydrogenases, suggesting that it

is a member of the aldehyde dehydrogenase superfamily SSADH cDNA and genomic sequences have been used to identify two point mutations in the SSADH genes derived from four patients [9] Splicing mutations resulted in exon skipping in all four cases In addition, a frameshift and pre-mature termination was observed in one case and an in-frame deletion in the resulting protein was detected in the other case Parents and siblings were shown to be

Correspondence to S Y Choi, Department of Genetic Engineering,

Hallym, University, Chunchon 200-702, Korea.

Fax: +82 33 241 1463, Tel.: +82 33 248 2112,

E-mail: sychoi@hallym.ac.kr or O.-S Kwon, Department of

Biochemistry, Kyungpook National University, Taegu 702-701,

Korea Tel.: +82 53 950 6356, E-mail: oskwon@knu.ac.kr

Abbreviations: DABIA,

4-dimethylaminoazobenzene-4-iodoaceta-mide; Dys, DABIA-Cys; GAD, glutamate decarboxylase; GABA,

c-aminobutyric acid; GABA-T, c-aminobutyric acid transaminase;

GBH, gamma-hydroxybutyrate; SSA, succinic semialdehyde;

SSADH, succinic semialdehyde dehydrogenase.

*Present address: Department of Pathology, School of Medicine,

Emory University, Atlanta, GA, USA.

(Received 30 August 2004, revised 12 October 2004,

accepted 25 October 2004)

heterozygous for the splicing abnormality [10] In addition,

the intensive analysis on novel mutations in human SSADH

locus suggested that the missense mutations caused by point

mutations, small insertions and small deletions in the genomic

level would be causative of SSADH deficiency [11,12]

Despite the importance of SSADH in the metabolism of

GABA, the structural studies of the enzyme have not yet

been well investigated We have purified and characterized

an NAD+-dependent SSADH from bovine brain [2], and

found that the arginine residues are connected with catalytic

activity of the enzyme [13] Recently, we reported that a

specific lysyl residue is located at or near the coenzyme

binding site of the enzyme [14] In the present study, we

identified a regulatory site of the brain SSADH by a

combination of labeling with

4-dimethylaminoazobenzene-4-iodoacetamide (DABIA) and peptide analysis

Materials and methods

Materials

NAD+, succinic semialdehyde, DABIA, EDTA, bovine

serum albumin, trypsin (treated with

tosylphenylalanylchlo-romethane), and 2-mercaptoethanol were purchased from

Sigma (St Louis, MO, USA) CM-Sepharose,

Blue-Seph-arose, and 5¢-AMP-Sepharose were obtained from

Amer-sham Bioscience (Piscataway, NJ, USA) Bovine brains

were obtained from Majang-dong Packing Company

(Seoul, Korea)

Purification of enzyme and enzymatic assays

SSADH from bovine brain was purified according to a

procedure previously described [2] The procedure exploited

four column chromatographic steps: CM-Sepharose,

Blue-Sepharose, hydroxyapatite and 5¢-AMP-Sepharose For

precise measurement of enzymatic activity, the formation of

NADH was measured by the increase in absorbance at

340 nm All enzymatic assays were performed in duplicate

and the initial velocity data was correlated with a standard

assay mixture containing 30 lMsuccinic semialdehyde and

1 mMNAD+in 0.1Msodium pyrophosphate (pH 8.4) at

25C One unit of enzyme was defined as the amount of

enzyme required to reduce 1 lmol of NAD+per min at

25C Protein concentration was estimated by the Bradford

procedure with a bovine serum albumin standard [15]

Spectroscopic studies

The purified enzyme (5 lM) was incubated with 300 lM

DABIA at 25C, in the dark, for 30 min At the end of

incubation, the sample was dialyzed against 0.1M

potas-sium phosphate (pH 7.0) and the absorption spectra were

recorded with a Kontron UVIKON 930 double beam

spectrophotometer (Tegimenta, Rotkreuz, Switzerland) in

the range 325–500 nm

Modification of succinic semialdehyde dehydrogenase

with DABIA

The purified enzyme was dialyzed against 0.1Mpotassium

phosphate (pH 7.0), 1 m EDTA, and then used

immedi-ately A total of 200 mMof DABIA was freshly prepared in dimethylformamide and kept on ice The final concentration

of dimethylformamide in the incubation mixture was no more than 1% (v/v) and was found to have no effect on enzymatic activity The incubation mixture (1 mL) con-tained SSADH (5 lM), DABIA (100–400 lM) and 0.1M potassium phosphate (pH 7.0) The reaction was initiated

by addition of DABIA in the dark at 25C At intervals after the initiation of inactivation, aliquots were withdrawn for the activity assay Whenever possible a small sample volume (2 lL) was used to minimize artifactual blank due

to the transfer of DABIA

Protection experiments were performed in a similar manner except that the enzyme (5 lM) was preincubated with a substrate SSA (3 mM) or coenzyme NAD+(3 mM)

at 25C for 30 min before the modification was initiated by the addition of DABIA The amount of DABIA bound to the enzyme was determined by measuring the increase in absorbance at 436 nm using a molar extinction coefficient

of 29 000M )1Æcm)1[16]

Labeling and tryptic digestion of succinic semialdehyde dehydrogenase

To identify the DABIA binding site, 2.9 mg of enzymes (50 lM) were treated with DABIA as described previously [14,16] Labeling of protein was conducted for 30 min in the dark at 25C The excess reagent was removed by Sephadex G-25 superfine gel filtration The solution was dried, dissolved by first adding 20 lL of 50% (v/v) formic acid and then 600 lL water, transferred to a smaller tube suitable for enzymatic digestion and dried again

DABIA-labeled protein (20 nmol) was suspended in 0.5 mL of 0.1Mammonium bicarbonate buffer (pH 8), and digested with trypsin, previously treated with tosylphenyl-alanylchloromethane, for 20 h at 37C The substrate/ enzyme molar ratio was 50 : 1

Purification of DABIA-labeled cysteine-containing peptides

To a 0.5 mL tryptic digest, 40 lL acetic acid was added and the precipitate was removed by centrifugation (10 000 g, 20 min, 4C) Peptides in the sample solution were lyophilized and separated by reverse-phase chromatography (LKB Instruments, Uppsala, Sweden) using a Vydac C18column (0.46· 25 cm) The separation was performed with a linear gradient from 0 to 70% B in 40 min at a flow rate of 0.8 mLÆmin)1 Eluant A: 10 mM potassium phosphate (pH 7) containing 2% dimethylformamide; eluant B: acetonitrile containing 4% dimethylformamide

Further purification was achieved by rechromatograph-ing the peptides on a Vydac C18 column with a linear gradient of 0–60% B in 60 min Eluant A: 0.1% trifluoro-acetic acid (pH 2.15); eluant B: 0.1% trifluorotrifluoro-acetic acid in acetonitrile/H2O (80 : 20, v/v) Cysteine derivatized with DABIA and the corresponding phenylthiohydantoin deriv-ative were prepared and characterized according to the procedure described by Chang et al [17] The latter was used as standard in the quantitative evaluation of other phenylthiohydantions obtained after automated Edman degradation The absorption properties of the

DABIA derivative of cysteine (molar extinction coefficient

e¼ 29 000M )1Æcm)1at 436 nm) was used to determine the

cysteine content of the derivatized peptides

Amino acid analysis and peptide sequencing

Peptides (6 nmol) were hydrolyzed for 24 h in 6M HCl,

containing 0.1% thioglycolic acid at 110C in vacuum

Amino acids derivatized with phenylisothiocyanate were

identified and quantified by HPLC (Waters, Milford, MA,

USA) Pico-Tag system, using a Nova-Pak C18column run

at room temperature with a flow rate of 1 mLÆmin)1

For the amino acid sequence analysis, the labeled peptide

was subjected to automated Edman degradation on a

Beckman Model 890M sequenator according to the

manu-facturer’s instructions

Results

Inactivation of succinic semialdehyde dehydrogenase

by DABIA

The relevance of sulfhydryl groups in the catalytic activity

of SSADH, was examined by reacting the enzyme with

DABIA DABIA, a chromophoric reagent, was chosen for

our study for the following reasons: (a) it reacts with the SH

groups of SSADH under native conditions; (b) the labeled

peptides and amino acid residues are easily monitored at

436 nm because of the large extinction coefficient of the

chromophore; (c) in a case to purify the peptides alkylated

by DABIA, the derivatized peptides are more hydrophobic

than unlabeled peptides, allowing their separation by reverse

phase HPLC

Incubation of SSADH with increasing concentrations of

DABIA resulted in a progressive decrease in its enzymatic

activity (Fig 1) This inactivation followed pseudo

first-order kinetics with concentrations of DABIA in the range

100–400 lM The pseudo first-order rate constants, obtained

at each DABIA concentration, were plotted as a function of

DABIA concentration (Fig 1, inset) The second-order rate

constant for the inactivation of DABIA was 168M )1Æmin)1,

as determined from the slope of this plot

In an effort to demonstrate that DABIA is bound to

sulfhydryl groups of SSADH, 5 lM of SSADH was

incubated with or without 400 lMof DABIA at pH 7.0 in

the dark for 30 min and absorption was monitored from 325

to 500 nm The spectrum of DABIA-labeled enzyme showed

a characteristic peak at 436 nm (Fig 2, curve 2), which was

absent from the spectrum of the native enzyme (Fig 2, curve

1) The absorption at 436 nm corresponds to a DABIA

alkylated sulfhydryl group chromophore The value for the

incorporation of DABIA labeled on SSADH was measured

using an extinction coefficient of 29 000M )1Æcm)1 at

436 nm DABIA gave overall incorporation values of about

7.5 mol per enzyme tetramer, indicating that 8 mols of

sulfhydryl groups of SSADH were masked The correlation

between DABIA incorporation and SSADH enzyme

activ-ity is shown in Fig 3 During the inactivation process, a

linear relationship was observed between DABIA and the

loss of enzyme activity, which extrapolates to a

stoichio-metry of 8.0 mol DABIA derivatives per mol enzyme

tetramer, based on increased absorbance at 436 nm

The inactivation studies were carried out in the presence

of substrate or coenzyme to define the site(s) modified by DABIA The reaction between SSADH and DABIA was effectively prevented by incubating SSADH with the substrate, SSA, but not with coenzyme, NAD+(Table 1)

Fig 1 Determination of the rate constant (K obs ) for the inactivation of SSADH at different concentrations of DABIA The enzyme (5 l M ) was incubated with 100 l M (d), 200 l M (s), 300 l M (j) and 400 l M (h)

of DABIA in 0.1 M potassium phosphate (pH 7.0) at 25 C in the dark Aliquots withdrawn from the incubation mixtures were tested for enzymatic activity The inset shows the dependence of the observed rate constant (K obs ) on DABIA concentration.

Fig 2 Absorption spectra of native (curve 1) and DABIA-treated (curve 2) SSADH At the end of incubation, the absorption spectra were determined as described in Materials and methods.

These results suggest that the loss of SSADH enzymatic

activity may be the result of the binding of DABIA to

specific sulfhydryl groups located at or near the substrate

binding site of SSADH

Isolation of modified peptides

To identify the peptides modified by DABIA, SSADH was

treated with DABIA and digested with trypsin as described

above After overnight trypsin digestion, the digested

sample was loaded on to a reverse-phase column (Vydac

C18) Two peptides, designated I and II, were detected by

monitoring the absorption spectrum at 436 nm (data not

shown), indicating that the modification induced by

DABIA was restricted to at least two amino acids in the

SSADH subunits

Each peptide tagged with DABIA was further purified by

a second chromatography through a Hypersil ODS column

using a different solvent system as described previously

[14,16] After the second chromatography, two single pure

peptides derivatized with DABIA were isolated from the originally labeled peptides, respectively, as shown in Fig 4 Amino acid analysis and protein sequencing

The amino acid analysis and sequence of peptides I and II were examined and the observed sequences were found to be

in reasonable agreement with the amino acid compositions determined after the acid hydrolysis of each DABIA-labeled peptide (data not shown)

The stoichiometric study (Fig 3) and the analysis of amino acid sequence (Table 2) showed that one cysteine residue in each peptide (peptides I and II) was labeled with DABIA The amino acid sequence analyses of peptides I and II revealed that the peak fractions contained the

Fig 3 Stoichiometry of DABIA inactivation SSADH (5 l M ) was

incubated with 400 l M of DABIA in 0.1 M potassium phosphate

(pH 7.0) at 25 C in the dark The inactivation of SSADH is plotted as

a function of mol DABIA incorporated per mol enzyme.

Table 1 Inactivation of succinic semialdehyde dehydrogenase by

DABIA Enzyme (5 l M ), DABIA (300 l M ), NAD+(3 m M ) and succinic

semialdehyde (3 m M ) were used The data represent the mean of three

independent experiments with the difference expressed as ± deviation.

Reaction mixture

Remaining activity (%)

Enzyme + NAD + + DABIA 23 ± 3

Enzyme + succinic semialdehyde + DABIA 91 ± 4

Fig 4 Second chromatography of the tryptic peptides labeled with DABIA Peptides I and II were purified by HPLC using a Vydac

C 18 column and a linear gradient of acetonitrile (0–60%) containing

5 m M sodium phosphate (pH 6.4) for 120 min at a flow rate of 0.5 mLÆmin)1 Elution was monitored at 436 nm and the DABIA-labeled peptides (I and II) were sequenced by Edman degradation.

Table 2 Sequences of the cysteine-containing tryptic peptides from succinic semialdehyde dehydrogenase DABIA-Cys (Dys) was deter-mined as the DABIA phenylthiohydantoin (residues indicated in bold).

Peptide Sequence

I Val-Dys-Ser-Asn-Gln-Phe-Leu-Val-Gln-Arg

II

Glu-Val-Gly-Glu-Ala-Ile-Dys-Thr-Asp-Pro-Leu-Val-Ser-Lys

amino acid sequences

Val-Xaa-Ser-Asn-Gln-Phe-Leu-Val-Gln-Arg and

Glu-Val-Gly-Glu-Ala-Ileu-Xaa-Thr-Asp-Pro-Leu-Val-Ser-Lys, respectively, where Xaa represents an

assayable phenylthiohydantoin amino acid This residue can

be designated DABIA-Cys (Dys), based on amino acid

analysis Of interest, the amino acid sequences of peptides I

and II were found to be identical to regions of human

SSADH, i.e amino acids 341–350 and 266–279,

respect-ively

Discussion

Little is known about the chemistry of the active site of

SSADH, partly because the crystal structure of this enzyme

is not available Therefore, it is essential that a detailed

structural description of SSADH is elucidated Previously,

we purified the homotetramer SSADH from bovine brain

homogenate [2] Recently, an investigation on the catalytic

role of specific amino acid residues in the enzyme indicated

the involvement of a lysyl residue in its enzymatic activity

[14]; this conclusion was reached based on evidence

obtained by chemically modifying SSADH with

pyrid-oxal-5¢-phosphate, a specific lysine residue modifying

rea-gent In the present study, we identified at least one

substrate binding domain in brain SSADH by combining

DABIA labeling and peptide analysis

DABIA has been widely used in structural and functional

studies to selectively label reactive cysteine residues in

particular, which are often directly involved in the catalytic

mechanisms of active sites [16–18] Haloacetamide

deriva-tives such as DABIA react with cysteine via a SN2 reaction

mechanism to give the corresponding carboxamidomethyl

derivatives The reaction of haloacetamide derivatives with

cysteine is 20–100 times as rapid as with other

cysteine-modifying reagents In addition, the two benzene rings

provide DABIA with a large extinction coefficient, which

allows DABIA-labeled cysteine to be measured efficiently

and rapidly [19]

The incubation of SSADH with increasing

concentra-tions of DABIA resulted in a progressive reduction in

enzyme activity (Fig 1) The evidence for the specific

modification of cysteine residues by DABIA was provided

by monitoring the absorption of DABIA-alkylated

cyste-ines at 436 nm (Fig 2) The nature of the inhibitory effect

exerted by DABIA was studied in detail The possibility that

DABIA inhibition is the result of the reaction of cysteine

residues critically connected with catalysis was investigated

by performing inhibition studies in the presence and absence

of the substrate SSA or in the presence or absence of

coenzyme NAD+ At pH 7.0, the inhibitory effect of

DABIA was influenced by SSA at 3.0 mM(Table 1) The

near complete protection afforded by SSA, strongly

suggests that the inactivation occurred because of an

interaction between DABIA and cysteine residues located

at or near the substrate binding site of SSADH In marked

contrast to SSA, the coenzyme NAD+, did not afford any

protection against DABIA inactivation

Although differences in the absorption spectra of native

and DABIA-labeled SSADH were observed by absorption

spectroscopy (Fig 2), we have evidence that the

conform-ational changes of SSADH did not occur when it reacts with

DABIA We investigated these conformational changes

indirectly by fluorometric anisotropy, but no differences in the anisotropies (A) of native (A¼ 0.174) and modified enzyme (A¼ 0.179) were observed This observation demonstrates that the inactivation of SSADH occurs due

to the interaction between DABIA and cysteine residues on SSADH, and that it is not due to conformational changes of SSADH

During the inactivation process, a linear relationship was observed between DABIA and the loss of enzyme activity, which extrapolates to a stoichiometry of 8.0 mol DABIA derivatives per mol enzyme tetramer, based on increased absorbance at 436 nm (Fig 3) There has been major controversy concerning SSADH protein structure In the early 1970s, Cash et al reported that SSADH was a dimeric protein of mass identical subunits [20], however, in the 1980s, Ryzlak & Pietruszko reported that SSADH was a tetrameric protein of mass nonidentical subunits [3], a finding never repeated in the aldehyde dehydrogenase literature Our results support the cloning data of Chambliss

et al [8], that SSADH is a protein of homotetrameric structure with mass identical subunits Our previous puri-fication of SSADH from bovine brain showed that the SSADH is a tetramer composed of mass identical subunits, although there is minor variation in the molecular mass of a single subunit [2] This result is in keeping with the notion that mammalian SSADH is a homotetramer, and also supports our conclusion that two cysteine residues per single subunit are labeled by DABIA

To identify the site of inactivation, tryptic peptides containing DABIA-labeled cysteine were prepared The results of sequence analysis (Table 2) showed that the modified residues correspond to the cysteine residues already identified in SSADH from human liver and rat brain [8] Even though the amino acid composition and sequence of bovine brain SSADH have not been identified, the labeled cysteine residues in peptides I and II correspon-ded to Cys342 and Cys272, respectively, of mammalian brain SSADH Cys342 is conserved in all members of the aldehyde dehydrogenase superfamily from bacteria to human, and is presumed to be located at an active site based on the sequence homology with bovine aldehyde dehydrogenase, the three-dimensional structure of which has been solved [21] This finding is consistent with our observation that the substrate, SSA, nearly completely blocked the DABIA-mediated inhibition of SSADH, but coenzyme NAD+ did not On the other hand, Cys272, found in peptide II, appears to be located near the coenzyme binding site Sequence similarity between Cys272 and the aldehyde dehydrogenase superfamily shows that Cys272 is located 12 amino acids away from Gly284, which is widely accepted to be a coenzyme binding site In addition, coenzyme binding sites in bovine aldehyde dehydrogenase have been shown to lie in an a-helical structure near the surface of the enzyme [21] If the three-dimensional structure

of SSADH is not much different from the structure of aldehyde dehydrogenase, the DABIA molecule bound to Cys272 seems to be located too far away from the coenzyme binding site to interfere the NAD+–protein interaction

In summary, the study presented here establishes that brain SSADH is inhibited by the binding of DABIA to specific cysteine residues at or near the active site of the protein Knowledge of the interaction between DABIA and

SSADH may provide insights into approaches for the

design of a new class of regulators, which do not resemble

SSADH substrates

Acknowledgements

This work was supported by the 21st Century Brain Frontier Research

Grant (M103KV010019-03K2201-01910), and National Research

Laboratory (NRL) Grant (M1-9911-00-0025) from the Ministry of

Science and Technology, and in part by the Research Grant from

Hallym University.

References

1 Chambliss, K.L & Gibson, K.M (1992) Succinic semialdehyde

dehydrogenase from mammalian brain: subunit analysis using

polyclonal antiserum Int J Biochem 24, 1493–1499.

2 Lee, B.R., Hong, J.W., Yoo, B.K., Lee, S.J., Cho, S.W & Choi,

S.Y (1995) Bovine brain succinic semialdehyde dehydrogenase:

purification, kinetics and reactivity of lysyl residues connected with

catalytic activity Mol Cells 5, 611–617.

3 Ryzlak, M.T & Pietruszko, R (1988) Human brain
high Km

aldehyde dehydrogenase: purification, characterization, and

identification as NAD + -dependent succinic semialdehyde

dehy-drogenase Arch Biochem Biophys 266, 386–396.

4 Jakobs, C., Jaeken, J & Gibson, K.M (1993) Inherited disorders

of GABA metabolism J Inherit Metab Dis 16, 704–715.

5 Hogema, B.M., Akaboshi, S., Taylor, M., Solomons, G.S.,

Jakobs, C., Schutgens, R.B., Wilcken, B., Worthington, S.,

Maropoulos, G., Grompe, M & Gibson, K.M (2001) Prenatal

diagnosis of succinic semialdehyde dehydrogenase deficiency:

Increased accuracy employing DNA, enzyme, and metabolic

analyses Mol Genet Metab 72, 218–222.

6 Cho, S.W., Song, M.S., Kim, Y.G., Kang, W.D., Choi, E.Y &

Choi, S.Y (1993) Kinetics and mechanism of an

NADPH-dependent succinic semialdehyde reductase from bovine brain.

Eur J Biochem 211, 757–762.

7 Snead, O.C (1978) Gamma hydroxybutyrate in the monkey I.

Electroencephalographic, behavioral, and pharmacokinetic

stud-ies Neurology 28, 636–642.

8 Chambliss, K.L., Caudle, D.L., Hinson, D.D., Moomaw, C.R.,

Slaughter, C.A., Jakobs, C & Gibson, K.M (1995) Molecular

cloning of the mature NAD(+)-dependent succinic

semialde-hyde dehydrogenase from rat and human cDNA isolation,

evo-lutionary homology, and tissue expression J Biol Chem 270,

461–467.

9 Blasi, P., Boyl, P.P., Ledda, M., Novelleto, A., Gibson, K.M.,

Jakobs, C., Homega, B., Akaboshi, S., Loreni, F & Malaspina, P.

(2002) Structure of human succinic semialdehyde dehydrogenase

gene: Identification of promoter region and alternatively processed

isoforms Mol Genet Metab 76, 348–362.

10 Chambliss, K.L., Hinson, D.D., Trettel, F., Malaspina, P., Novelletto, A., Jakobs, C & Gibson, K.M (1998) Two exon-skipping mutations as the molecular basis of succinic semialdehyde dehydrogenase deficiency (4-hydroxybutyric acid-uria) Am J Hum Genet 63, 399–408.

11 Aoshima, T., Kajita, M., Sekido, Y., Ishiguro, Y., Tsuge, I., Kimura, M., Yamaguchi, S., Watanabe, K., Shimokata, K & Toshimitsu, N (2002) Mutation analysis in a patient with succinic semialdehyde dehydrogenase deficiency: a compound hetero-zygote with 103–121del and 1460T>A of the ALDH5A1 gene Human Heredity 53, 42–44.

12 Akaboshi, S., Hogema, B.M., Novelletto, A., Malaspina, P., Salomons, G.S., Maropoulos, G.D., Jakobs, C., Grompe, M & Gibson, K.M (2003) Mutational spectrum of the succinic semi-aldehyde dehydrogenase (ALDH5A1) gene and functional ana-lysis of 27 novel disease-causing mutations in patients with SSADH deficiency Human Mutation 22, 442–450.

13 Bahn, J.H., Lee, B.R., Jeon, S.G., Jang, J.S., Kim, C.K., Jin, L.H., Park, J., Cho, Y.J., Cho, S.W., Kwon, O.S & Choi, S.Y (2000) Brain succinic semialdehyde dehydrogenase: reaction of arginine residues connected with catalytic activities J Biochem Mol Biol.

33, 317–320.

14 Choi, S.Y., Bahn, J.H., Lee, B.Y., Jeon, S.G., Jang, J.S., Kim, C.K., Jin, L.H., Kim, K.H., Park, J.S., Park, J & Cho, S.W (2001) Brain succinic semialdehyde dehydrogenase: identification

of reactive lysyl residues labeled with pyridoxal-5¢-phosphate.

J Neurochem 76, 919–925.

15 Bradford, M.M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Anal Biochem 72, 248–254.

16 Kim, Y.T & Churchich, J.E (1989) Sequence of the cysteinyl-containing peptides of 4-aminobutyrate aminotransferase Iden-tification of sulfhydryl residues involved in intersubunit linkage Eur J Biochem 181, 397–401.

17 Chang, J.Y., Knecht, R & Braun, D.G (1983) A new method for the selective isolation of cysteine-containing peptides Specific labeling of the thiol group with a hydrophobic chromophore Biochem J 211, 163–171.

18 Cardamone, M., Aslunan, K., Brandon, M.R & Puri, N.K (1993) Identification of cys-containing peptides during peptide mapping of recombinant proteins Pept Res 6, 242–248.

19 Lundblad, R.L (1995) The modification of cysteine In Techniques

in Protein Modification pp 63–83 CRC Press, Boca Raton, FL.

20 Cash, C.D., Maitre, M & Mandel, P (1979) Purification from human brain and some properties of two NADPH-linked akde-hyde reductases which reduce succinic semialdeakde-hyde to 4-hydro-xybutyrate J Neurochem 33, 1169–1175.

21 Steinmetz, C.G., Xie, P., Weiner, H & Hurley, T.D (1997) Structure of mitochondrial aldehyde dehydrogenase: the genetic component of ethanol aversion Structure 5, 701–711.