Vol. 24, No. 9, pp. 1493-1499, in Great Britain. All rights reserved
~nr. J. Biochem.
Printed
1992 Copyright
0
0020-711X/92 $5.00 + 0.00 1992 Pergamon Press Ltd
SUCCINIC SEMIALDEHYDE DEHYDROGENASE FROM MAMMALIAN BRAIN: SUBUNIT ANALYSIS USING POLYCLONAL ANTISERUM KEN
L. CHAMBLISS and K. MICHAELGIBSON*
Kimberly H. Courtwright and Joseph W. Summers Metabolic Disease Center and Baylor Research Institute, Baylor University Medical Center, Dallas, TX 75226, U.S.A. [Tel. (214)820-2687; Fax (214)820-49521 (Received IO December
1991)
Abstract-l. NAD+-dependent succinic semialdehyde dehydrogenase was purified to apparent homogeneity from rat brain and highly purified from human brain. 2. Molecular exclusion chromatography of the purified enzymes on Sephadex G-150 and G-200 revealed M, values of 203,000 and 191,000 for rat and human, respectively. 3. Electrophoresis on sodium dodecylsulfate polyacrylamide gels revealed a single subunit of M, 54,000 for rat and 58,000 for human. Isoelectric focusing of the purified rat enzyme yielded a pl of 6.1. 4. For both proteins, K,,, values for short-chain aldehydes acetaldehyde and propionaldehyde ranged from 0.33 to 2.5 mM; Km values for succinic semialdehyde were in the 2-4 PM range. 5. The subunit structure of both enzymes was investigated in brain extracts and purified preparations by immunoblotting, using a polyclonal rabbit antiserum against the purified rat brain enzyme. 6. For rat and human extracts, single bands were detected at M, 54,000 and 58,000, comparable to findings in the purified preparations. Immunoblotting analyses in other species (guinea pig, hamster, mouse and rabbit) revealed single subunits of M, 54,000-56,500.
INTRODUCTION Gamma-aminobutyric acid (GABA) is produced from glutamic acid in a reaction catalyzed by glutamic acid decarboxylase (GAD; EC 4.1. I. 15) and further metabolized to succinic acid by the successive action of GABA transaminase (GABA-T; EC 2.6.1.19) and succinic semialdehyde dehydrogenase (SSADH; EC 1.2.1.24). Succinic acid produced from this pathway enters the Krebs’ cycle. GABA metabolism has been well characterized in mammalian central nervous system (CNS) where GABA functions as an inhibitory neurotransmitter. The presence of GABA metabolizing enzymes also has been demonstrated in kidney (Lancaster et al., 1973), platelets (White, 1979), lymphocytes (Gibson et al., 1983), intestine (Miki et al., 1983) and adrenal medulla (Gonzalez et al., 1987). The physiological role of the GABA metabolic pathway in non-CNS tissue is poorly understood. Inherited metabolic diseases caused by a deficiency of a single enzyme have been reported for two of the enzymes in this pathway. While only a single patient with deficiency of GABA-T (Gibson et al., 1985) has been identified, several cases of SSADH deficiency have been reported. SSADH deficient patients have a
characteristic elevation of the reduction product of succinic semialdehyde (gamma-hydroxybutyric acid; GHB) in body fluids indicating a block in the normal oxidative pathway. This is of interest because GHB has been shown to cause a form of absence epilepsy when administered to mammals (Godschalk et al., 1977) as well as increased brain dopamine levels (Gessa ef al., 1966) and decreases in glucose metabolism (Wolfson et al., 1977). SSADH has been partially purified and studied in rat (Kammeraat and Veldstra, 1968), pig (Blaner and Churchich, 1979), monkey (Albers and Koval, 1961) and human brain (Embree and Albers, 1964; Cash et al., 1977; Ryzlak and Pietruszko, 1988). The subunit molecular weights have only been reported for the rat and human brain enzyme. To better characterize SSADH of different species and to develop tools for future molecular analyses to elucidate genetic defects in patients with gammahydroxybutyric aciduria, we have purified both rat and human SSADH, produced antiserum to the rat enzyme, and examined the SSADH protein of different species by immunoblots. MATERIALSAND
METHODS
Materials
*Address correspondence to: Dr K. Michael Gibson, Baylor Research U.S.A. tic
%,%I
Institute,
3812 Elm Street,
Dallas,
TX 75226,
NAD, succinic semialdehyde, Cibacron-blue, Coomassie Brilliant Blue, 2-mercaptoethanol, Tween 20, nitroblue tetrazolium and phenazine methosulfate were purchased 1493
1494
KEN L. CHAMBLISSand K. MICHAEL GIBBON
from Sigma Chemical Company (St Louis, MO.). Acetaldehyde, propionaldehyde, 3-nitrobenzaldehyde, 4nitrobenzaldehyde, butyraldehyde and valeraldehyde were purchased from Aldrich Chemical Company (Milwaukee, Wis.). Acetaldehyde and propionaldehyde were redistilled before use. Sephadex G-l 50, Sephadex G-200, Blue Sepharose and S-AMP-Sepharose 4B were from Pharmacia LKB Biotechnology, Inc. (Piscataway, N.J.). DEAEcellulose DE-52 was purchased from Whatman Lab Sales (Hillsborom. Ore.). Acrylamide, his-acrylamide, ammonium persulfate and N,N,N’,N-tetramethylethylenediamine (Temed) were from Bio-Rad Laboratories (Richmond, Calif.). SDS and protein molecular weight standards were purchased from Bethesda Research Laboratories (Gaithersburg, Md). Nitrocellulose was from Schleicher and Schuell (Keene, N.H.). Blot quality bovine serum albumin, goat anti-rabbit IgG AP conjugate and immunostaining reagents were purchased from Promega. All reagents were of analytical grade quality. Glutaric and adipic semialdehydes were prepared, and aldehyde concentrations were determined as previously described (Forte-McRobbie and Pietruszko, 1986). Intact brains (except for human) were purchased from Pel-Freez Biologicals (Rogers, Ark.). Live Sprague-Dawley rats were purchased from Charles Rivers Laboratories (Wilmington, Mass.). Human brain tissue was acquired from the tissue procurement program of the National Disease Research Interchange (Philadelphia, Pa) and maintained at -7o’C prior to use.
During purification, the enzyme activity was assayed spectrophotometrically in pooled fractions by following the increase of absorbance at 340 nm, at 25°C in a total volume of 3 ml. In all cases, the initial reaction velocities were proportional to protein concentration. Standard assays contained 0.1 M sodium pyrophosphate buffer, pH 9.0, I mM EDTA, 500 FM NAD+ and 20 PM succinic semialdehyde. Individual column fractions were more conveniently assayed using the same assay mixtures and following the production of NADH fluorometrically in an AMINCO fluoro-calorimeter model 54-7439 (excitation 355 nm, emission 470nm). One unit of enzyme activity was defined as the amount of enzyme producing 1nmol NADH min-’ under the assay conditions, using an extinction coefficient of 6.22mM-’ cm-’ at 340nm. Protein concentrations were determined by the method of Lowry (Lowry et al., 1951) using bovine serum albumin as a standard or the Micro Bio-Rad (Bio-Rad Protein Assay Instruction Manual, Bio-Rad Laboratories) procedure utilizing gamma-globulin as standard. The concentration of affinity-purified enzyme was estimated by comparison to known concentrations of protein molecular weight markers (0.5-4 pg) following SDS-polyacrylamide gel electrophoresis and densitometric scanning of the Coomassiestained bands. Measurements of Km and V,,, values for aldehyde substrates were determined spectrophotometrically at 340 nm and 25°C in 3 ml total volume, in an assay buffer consisting of 0.1 M sodium pyrophosphate, pH 9.0, supplemented with 1 mM EDTA. Data were analyzed using the Lineweaver-Burk plot (Lineweaver and Burk, 1934) and least-square analysis. Purification Extraction. Forty rat brains (60-70 g) were homogenized in a Waring blender for 6min at high speed in 240ml of
ice-cold water supplemented with 0.1 mM EDTA and 0. I % 2-mercaptoethanol. The homogenate was centrifuged at 48,000 g for 30 min at 4°C. The supematant was saved and the pellet was homogenized and recentrifuged. The supernatants were pooled. For human brain, 300 g tissue was similarly homogenized in 6OOml of water/EDTA/Zmercaptoethanol. Centrifugation of extracts for human tissues was at 27,OOOg. Blue-Sepharose CL-6BIDEAE-cellulose DE-52. The pooled supernatants were applied to a Blue-Sepharose CL-6B column (2.5 x 30cm for the rat enzyme and 2.5 x 40cm for the human enzyme) equilibrated with buffer A (2 mM potassium phosphate, pH 7.2, 0.1 mM EDTA, and 0.1% v/v 2-mercaptoethanol) (Cash et al., 1977). The column was washed with buffer A until no further protein eluted from the column as monitored by absorbance at 280nm. The Blue-Sepharose column was eluted directly onto a DEAE-cellulose DE-S2 column (2.5 x 15cm for the rat enzyme, and 2.5 x 4Ocm for the human enzyme, both equilibrated with buffer A) with 0.1 mM Cibacron Blue dissolved in buffer A (2 1). The DEAE-cellulose column was then washed with buffer A as above. SSADH was eluted using a O-45 mM linear gradient of KC1 in buffer A for rat (0.5 1 total volume) and for human (1 liter total volume). S-AMP-Sepharose 48. The active fractions from the DEAE-cellulose column were pooled and applied to a 5’-AMP-Sepharose 4B column (1 x 20 cm) previously equilibrated with buffer B (10mM potassium phosphate. pH 7.2, 1 mM EDTA, 0.1% 2-mercaptoethanol). Unbound material was washed from the column with buffer B until no further protein eluted as monitored by absorbance at 280 nm. SSADH was eluted with a 1 mg/ml solution of NAD+ in buffer B. Active fractions were pooled and stored at 4’C in buffer B, or at -20°C in 50% glycerol. Gel chromatography The native M, of the rat and human brain SSADH was determined using Sephadex G-150 and Sephadex G-200 for equilibrated with buffer A. Protein standards calibration were obtained from Bio-Rad Laboratories and included thyroglobulin (670,000), gamma-globulin (I 58,000), ovalbumin (44,000) myoglobin (17,000) and cyanocobalamin (1350). Polyacrylatnide gel eleclrophoresis SDS-polyacrylamide gel electrophoresis was performed as described by Laemmli (1970). The stacking gel contained 4% w/v acrylamide and the resolving gel contained 10% acrylamide. Samples were boiled in protein sample buffer (50 mM Tris, pH 6.8, 2% w/v SDS, 0.01% v/v 2-mercaptoethanol and 10% glycerol) for 5min prior to loading. Protein bands were visualized by Coomassie staining. For subunit molecular weight determinations, high-range molecular weight markers from Bethesda Research Laboratories were used. Isoelectric focusing Isoelectric focusing was carried out on PhastGel IEF 3-9 and 5-8 gels (PhastSystem, Pharmacia LKB Biotechnology, Inc.) according to manufacturer’s specifications. The protein was visualized by Coomassie staining or activity staining. Activity staining was performed by permeating the gel in 0.1 M Tris-HCl, pH 8.5, with NAD+ (0.3 mg/ml), nitroblue tetrazolium (0.3 mg/ml), phenazine methosulfate (0.03 mg/ml) and 50 FM succinic semialdehyde. For pl
1495
SSADH from mammalian brain
still contained several Coomassie staining bands on SDS-polyacrylamide gels. An additional 24-fold purification, yielding highly purified enzyme, could be achieved with affinity chromatography on S-AMPSepharose 4B. Data on recovery (Table 1) indicate that all purification steps were efficient, with a final yield of about 71% based on total activity in the pooled supematants. Total content of SSADH in rat brain was about 0.48 units/g of tissue, which was comparable to the enzyme from human brain where the level was approx. 0.20 units/g of tissue. The purified enzyme was stable for several weeks in 10 mM potassium phosphate buffer (pH 7.2), with 0.1 mM EDTA and 0.1% v/v 2-mercaptoethanol, and could be stored long term in 50% glycerol at -20°C. As glycerol content was decreased to lo%, the enzyme lost activity. Freezing the protein in buffer without glycerol resulted in complete inactivation.
determination, a broad range (PH 3-10) calibration kit was obtained from Bio-Rad Laboratories. Antibodyproduction The purified rat brain SSADH was electrophoresed on SDS-acrylamide gels, and the 54,000 Da band was excised from the gel after localization of the band by submerging the gel in ice-cold 0.1 M KC1 for 5-10min. Gel slices were emulsified in phosphate buffered saline, pH 7.2, by homogenization. The resulting emulsion, containing l-2 mg of protein, was injected subcutaneously into two New Zealand White female rabbits. Booster immunizations were given at 4 and 6 weeks. Ten days following the second boost, 50-60 ml of blood were taken from the marginal ear vein
and serum prepared. Immunoblotting on SDSProtein samples were electrophoresed polyacrylamide gels and transferred to nitrocellulose sheets by electroblotting (EC 230 Electroblot System) as previously described (Towbin et al., 1979) with minor modifications. The blotting buffer was 12.5 mM Tris, pH 8.6, 96mM glycine, and 0.01% w/v SDS. Electroblotting was carried out at 200 mAmps for 1-2 hr at 4°C. After transfer, the blot was immunostained according to procedures provided by Promega (ProtoBlot Western Blot AP System Technical Manual, 1987) using a l/1000 dilution of the SSADH antisera. The second antibody was goat anti-rabbit IgG alkaline phosphatase conjugate.
Molecular weight The SSADH purified from rat brain was apparently homogenous with only one Coomassie-staining band evident on SDS-polyacrylamide gels (Fig. 1). The subunit A4, was determined to he 54,000 k 3500. The subunit size of rat SSADH that we observed was smaller than that reported by Cash et al. (1977)
(IV, = 68,000). The average M, of the native enzyme was determined by gel filtration using both Sephadex G-150 and G-200 and was found to be 203,000 + 39,000. These data indicate a tetrameric structure consisting of four identical subunits which differs from the previously reported dimeric structure (Cash et al., 1977). A tetrameric structure also has been demonstrated for human SSADH (Ryzlak and Pietruszko, 1988).
Bruin lysates Whole cell lysates were made from frozen brain tissue by homogenization for 4-5 min in ice-cold water. After protein concentrations were determined, 1 volume of 2X protein sample buffer was added. Between 200 and 250 pg of brain protein were loaded into each gel lane for electrophoresis and subsequent
immunoblotting.
RESULTS Purification of rat brain SSADH
Isoelectric focusing
Data representing an average of three purifications of rat brain SSADH are shown in Table 1. Because two of the three chromatographic steps were connected in series (Blue-Sepharose CL-6B and DEAE DE-52), the purification could be accomplished readily in 4-5 days. After the first two chromatographic steps, the enzyme was 250-fold purified but
To further evaluate the purity of the rat SSADH, the enzyme was analyzed by isoelectric focusing. The purified protein was electrophoresed on gels with pH gradient ranges from 3 to 9 and 5 to 8. Protein bands were visualized by Coomassie staining or by “enzyme activity” staining as described in Materials and Methods. Gels of both pH ranges and both staining
Table 1. Purification of succinic semialdehyde dehydrogenase from rat and human brain’
Total activityb (units) step Extraction Pooled-supernatants Blue-Sepharose CLdB/ DEAE cellulose 5’-AMP-Sepharose 48
Protein (mg) 7135 (22,074)e 3893 (3326) ::, 1.1 (3.8)
Specific activity (units/mg)
% Recovery
Enrichment
A
B
A
B
A
B
A
B
60.8
25.3
0.0098
0.0035
-
-
-
-
37.2
32.3
0.0145
0.0081
100
100
13.6
29.5
0.275
0.876
22
91
19
250
10.0
23
2.74
16
71
280
6111
21.39
1.5
2.3
‘Results presented are the mean values for three independent purifications from 65-70 g of rat brain; values for human are the means for three independent purifications from 300 g of tissue. bA, human; B, rat. ‘Values in parentheses are human brain protein contents,
1496
KEN
L. CHAMBLISS andK. MICHAELGIBSON
methods revealed a single band with an average pl of 6.1 (Fig. 2). Activators, inhibitors and pH optimum
Substrates studied for optimum pH with the purified rat brain enzyme included succinic semialdehyde, glutaric semialdehyde and propionaldehyde. For succinic semialdehyde and propionaldehyde, maximum activity was observed at pH 9; for glutaric semiaidehyde, activity appeared maximal between pH 9 and 10. Enzyme activity with 20 PM succinic semialdehyde in Tri-HCI was only 52% of the activity in phosphate or pyrophosphate buffers. 3-Hydroxybenzaldehyde inhibited enzyme activity by 88% at 0.5 mM and 95% at 1 mM; 4-hydroxybenzaldehyde was a more potent inhibitor, decreasing enzyme activity by 97% at 0.5 mM. Addition of increasing amounts of iodoacetamide at concentrations of 0.5, 1 and 2mM resulted in 9, 31 and 46% inactivation, respectively, when iodoacetamide was added to cuvettes at the time of reaction. A variety of metal ions (500 PM) were studied as activators or inhibitors of purified SSADH. Fe+‘, Ca+’ Mgt2 and Mn+’ did not stimulate or inhibit the reaction; conversely, Cut’, Cd+2, Zni2, Hg+’ and Cr+3 inhibited the reaction by 74, 72, 76, 38 and 59%, respectively. The purified enzyme was sensitive to disulfiram inhibition at 5 PM final concentration (Ryzlak and Pietruszko, 1988). Enzyme activity was not restored by addition of 50 FM glutathione or cysteine, but was restored by 50 PM dithiothreitol; in the reaction cuvettes to which cysteine and glutathione had been added, the enzyme was reactivated upon addition of 50mM 2-mercaptoethanol.
where the Km values were in the millimolar range. The carboxylic acid semialdehydes were the best substrates with K,,, values between 2 and 4pM; however, the V,,, was the highest with SSA as substrate at a value about 6-fold higher than the other semialdehydes tested. Immunoblots
Antisera prepared in rabbit against the purified rat brain SSADH were used to probe protein blots of rat and human brain Iysates which had been separated on SDS-polyacrylamide gels. The antisera were specific for only one band in each homogenate (Fig. 3). The M, of the band in the immunoblot of the rat brain homogenate was identical to the M, of the purified rat protein, The IV, of the single immunostaining band in the human brain homogenate blot was 58,000. This contrasted with the report by Ryzlak and Pietruszko (1988) of two subunits for human brain SSADH with &f, of 61,000 and 63,~O. Purification of human SSADH
Because the data we generated by immunoblots pertaining to the human SSADH subunit structure were different than published data, we undertook purification of the human enzyme. Our goal was to clarify the issue of subunit number and molecular weight; moreover, purified protein would allow N-terminal amino acid sequence analysis which would be of value in verification of cDNA clones. The purification of human brain SSADH was carried out by a comparable procedure used for rat brain SSADH. Table 1 shows mean data from three preparations of the human enzyme. When the purified human enzyme was analyzed by Kinetic characterization of aldehyde substrates SDS-PAGE, a single Coomassie staining band was K,,, and V,,, values for several aldehyde substrates observed with a A4, of 58,000. We occasionally were calculated and are listed in Table 2. Suitability observed a second contaminating band (M, 37,000) of each aldehyde as a substrate for the enzyme was in the final preparation of the human enzyme. This assessed by comparison of the V/K, ratios which was readily removed by inclusion of a Sephadex range from as low as 0.002 for a~taldehyde to as high G-150 ~hromatographic step (2.5 cm x 1 m) after as 7.49 for succinic semialdehyde. The lowest V/K+,, the DEAE-DES2 column and prior to the 5’-AMP values were for straight-chain aliphatic aldehydes Sepharose column. Even when the human enzyme Table 2. Kinetic properties
Substrate Acetaldehyde Propjonaldehyde Butyraldehyde Vaieraldehyde Succinic semialdehyde Glutaric semialdehyde Adipic semialdehyde 3-Nitrobenzaldehyde 4-Nitrobenzaldehyde
of rat and human brain succinic semialdehyde Concentration range (IBM) 400--24,300 (100-24.300) 250-16,300 (30-3700) ~lO,~O 4-10,000 0.6-500 (0.6-500) 0.5-1000 0.5-1000 4&10,000 40-10,000
dehydrogenase”
vtK,n 2500 (1670) Iwo (333) 190 27 3.9 (1.7) 2.3 3.3 380 250
3.91 (1.22) 4.50 (0.74) 5.50 4.23 29.2 (3.1) 5.62 4.22 1.52 4.61
0.002 (O.Ooa7) 0.005 (0.002) 0.03 0.16 7.49 (1.82) 2.44 1.28 0.02 0.02
PAll assays were in 0.1 M sodium pyrophosphate buffer, pH 9.0, containing I mM EDTA and 5OOpM NAD. Values in parentheses were obtained using purified human brain SSADH.
SSADH
from mammalian
1497
brain
+ -
200 k Da
5.1 97.4 k Da
6.0 68kDa
6.5
43 k Da
7.1
a.4
29 k Da
Fig. 1. SDS-polyacrylamide gel electrophoresis of purified human (Lane I) and rat (Lane 2) brain SSADH. Lane 3 contains protein molecular weight standards. The gel was electrophoresed as described in Materials and Methods and was stained with Coomassie Brilliant Blue. Protein contents were: Lane 1, 6 pg; Lane 2, 3 pg; Lane 3, 5 pg. M, of the rat and human brain enzymes was 54,000 and 58,000, respectively.
1
2
3
4
5
Fig. 2. Determination of isoelectric point of rat brain SSADH. Protein was analyzed using PhastSystem (Pharmacia LKB Biotechnology, Inc., Piscataway, N.J.) according to manufacturer’s specifications. Purified SSADH was electrofocused, and the gel was developed for protein with Coomassie Brilliant Blue. The relative positions of the isoelectric focusing standards are shown in the right margin.
6
7
-
97.4 k Da
-
68 k Da
-
43 k Da
8
Fig. 3. Immunoblot (Western) analysis of succinic semialdehyde dehydrogenase in brain extracts and purified fractions from different species. Electrophoresis and immunoblotting using rabbit polyclonal antiserum raised against purified rat brain SSADH were as described in Materials and Methods. Lane and protein contents were: 1. rat purified (1 pg); 2, rat extract (0.14mg); 3, human purified (21(g); 4. human extract (0.4 mg); 5, guinea pig extract (0.46 mg); 6. hamster extract (0.39 mg); 7, mouse extract (0.22 mg); 8, rabbit extract (0.55 mg).
KENL. CHAMBLISS and K. MICHAEL GIBSON
1498
was underioaded and el~trophoresed over relatively long distances (10 cm), only one band was visible. The native IV, of the human brain enzyme was determined to be approx. 191,000 by Sephadex G-200 molecular exclusion chromatography. This result is comparable to previous reports (Ryzlak and Pietruszko, 1988). Comparison of rat and human enzymes with different aldehyde substrates is shown in Table 2. Like the rat enzyme, the human enzyme had high I(m values for propionaldehyde and acetaldehyde, with low I’,,,,,. The human enzyme, like the rat enzyme, was inactive with pyrroline-5carboxylic acid as substrate. The K,,, values for both species for succinic semiaidehyde were in close agreement (Table 2). Analysis of the purified human brain enzyme by IEF was unsuccessful. Repetitive analyses on gels with pH ranges of 3-9 or 5-8 revealed a broad staining region (activity or Coomassie stained) of approx. 6.1-7.1. Suruey of subunit M, of other mammalian species To determine the subunit h4, of brain SSADH from other mammalian species, lysates were made from guinea pig, hamster, mouse, rabbit, bovine calf, sheep, pig, dog and chicken brain. The lysates were el~trophoresed on SDS-~~yacrylamide gels, blotted to nitrocellulose sheets, and immunostained with rabbit anti-rat SSADH. Only faint bands could be detected in brain lysates of calf. sheep, pig, dog and chicken (data not shown), whereas with the more closely related guinea pig, hamster and mouse brain lysates, a distinct band was observed for each species (Fig. 3). Estimated M, values were 56,500 for guinea pig, 54,000 for hamster, 54,000 for mouse and 54,000 for rabbit. DISCtiSSlON
AND SUMMARY
The purification of NAD+-dependent of SSADH has been reported in two mammalian species, rat and human (Cash et al., 1977; Ryzlak and Pietruszko, 1988). Cash and coworkers described the purification of both rat and human brain SSADH. These workers reported a subunit M, of 70,000 and a native M, value of about 140,000 for both. These data are consistent with molecular weight determinations and Km values reported by Forte-McRobbie and Pietruszko (1986) for human liver and by Farres et al. (1988) for human placenta I-pyrroline-5-carboxylic acid dehydrogenase. Further, 1-pyrroline-5carboxylic acid dehydrogenase was identified in the mitochondrial fraction during the purification of human brain SSADH (Ryzlak and Pietruszko, 1988), suggesting that the protein isolated by Cash er af. (1977) might be I-pyrroline-5-carboxylic acid dehydrogenase. A recent report on the purification of pyrroline-5-carboxylate dehydrogenase from rat liver mitochondrial matrix indicated a dimeric structure with weight identical subunits of M, 59,000 (Small and Jones, 1990).
Ryzlak and Pietruszko (1988) reported the purification of human brain SSADH to homogeneity. These workers presented evidence that the human brain enzyme was composed of weight non-identical subunits of &f, 61,000 and 63,000. We were intrigued by this divergence of subunit composition and undertook the purification of human brain SSADH in addition to the rat brain enzyme. Our preparation revealed only a single subunit of I%&58,000. To rule out the possibility of proteotytic degradation during enzyme purification, we prepared polyclonal antisera in rabbit using purified rat brain SSADH as antigen. Analysis of crude rat and human brain extracts revealed the same results as the purified preparations (Fig. 3) i.e. single subunits of !4, 54,000 and 58,000. This suggested that proteolysis during enzyme preparation was not artefactually producing our 58,000 M, subunit. For studies with human brain, we were unsuccessful in attempts to obtain fresh post mortem tissue from local or outlying hospitals. Human brains were obtained from the NDRI Tissue Procurement Program. Although tissue was obtained as rapidly as possible post mortem, intervals ranged from g-20 hr between death and storage of brain tissue at - 70 C. This may have contributed to the low specific activity (-3 ~mol/min/mg protein) for the final purified human brain SSADH, which was a value about 5% of that reported by Ryzlak and Pietruszko (1988). Similarly, the antiserum revealed single subunit bands in other rodent species, including guinea pig, hamster, mouse and rabbit. Immunoblot analyses revealed strong reactive bands for rat, guinea pig, hamster, mouse, rabbit and human; fainter bands were observed in brain extracts of bovine calf. sheep, pig, dog and chicken (data not shown). This may have resulted from using rodent antigen for a generation of antiserum. We demonstrated that the human brain enzyme had high I& values for short-chain aliphatic aidehydes such as acetaldehyde and propionaldehyde (1.67 and 0.33, respectively). Our K, value for succinic semialdehyde of 1.7 FM was in good agreement with the value of 1 PM reported by Ryzlak and Pietruszko (1988). We showed that rat brain SSADH had a pH optimum of about 9 and was sensitive to inactivation by hydroxybenzaldehyde derivatives and sul~ydryl reagents, features consistent with the results of Ryzlak and Pietruszko (1988) for the human brain enzyme. We also demonstrated the sensitivity of the rat brain enzyme to the drug disuifiram, as was the case for the preparation of Ryzlak and Pietruszko (1988). We were unable to resolve distinct bands by isoelectric focusing of the human brain enzyme. A broad staining region from ~16.1-7.1 was observed; conversely, Ryzlak and Pietruszko (1988) reported distinct bands of pi6.3, 6.6, 6.8, 6.95 and 7.15. However, data obtained during purification of human brain SSADH in the present study did support the possibility of isozymes. At least three distinct bands of activity, as detected
SSADH from mammalian brain
with succinic semialdehyde as substrate, were routinely observed during chromatography on DEAE DE-52 (data not shown). Although we only detected the single subunit of M, 58,000, it remains possible that different subunits exist with only subtle molecular weight differences which are not resolved by conventional SDS-polyacrylamide gel electrophoresis. Conversely, charge differences may exist in subunits with identical or nearly identical molecular weights. Further analyses will be required to determine the existence of multiple isozyme forms of human brain SSADH. A number of patients suffering from an acquired deficiency of SSADH have been reported in recent years. In response to this defect, presumably succinic semialdehyde in brain accumulates and is reduced to the neuropharmacologically-active 4-hydroxybutyric acid, a compound of unique activity in nervous tissue. The molecular genetics of this disease, an inherited defect in the GABA degradative pathway, have not been reported. In rat, it is likely that one gene is responsible for the single subunit comprising active SSADH. However, recent data presented for the human brain proteins may be more complex (Ryzlak and Pietruszko, 1988). The results of the present report suggest that human brain SSADH is a tetramer of weight-identical subunits. The data of Ryzlak and Pietruszko (1988) suggest a tetrameric structure of az& subunits. Human brain SSADH may be composed of non-identical subunits whose subunit M, is identical or so similar that resolution on normal SDS-polyacrylamide gels is impossible. Resolution of the number of genes encoding human brain SSADH should be revealed by molecular analyses. These studies are in progress. Acknowledgements-The
authors are indebted to Dr Christopher D. Cash for advice regarding protein purifications and Carol F. Lee for technical assistance. REFERENCES
Albers R. W. and Koval G. J. (1961) Succinic semialdehyde dehydrogenase: purification and properties of the enzyme from monkey brain. Biochim. biophys. Acta 52, 29-35. Bio-Rad Protein Assay Instruction Manual (1989) Bio-Rad Laboratories, Richmond, California. Blaner W. S. and Churchich J. (1979) Succinic semialdehyde dehydrogenase: reactivity of lysyl residues. J. biol. Chem. 254, 179441798. Cash C., Ciesielski L., Maitre M. and Mandel P. (1977) Purification and properties of rat brain succinic semialdehyde dehydrogenase. Biochimie. 59, 257-268. Embree L. J. and Albers R. W. (1964) Succinic semialdehyde dehydrogenase from human brain. Biochem. Pharmac. 13, 1209-1217.
Farres J., Julia P. and Pares S. (1988) Aldehyde oxidation in human placenta: purification and properties of lpyrroline-5carboxylate dehydrogenase. Biochem. J. 256, 461-467.
Forte-McRobbie C. M. and Pietruszko R. (1986) Purification and characterization of human liver “high K,”
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aldehyde dehydrogenase and its identification as glutamic-y-semialdehyde dehydrogenase. J. biol. Chem. 261, 2154-2163. Gessa G. L., Vargiu L., Crabai F., Boero G. C., Caboni F. and Camba R. (1966) Selective increase of brain dopamine induced by gamma-hydroxybutyrate. Life Sci. 5, 1921-1930. Gibson K. M., Sweetman L., Nyhan W. L., Jansen I. and Jaeken J. (1985) Demonstration of 4-aminobutyric acid aminotransferase deficiency in lymphocytes and lymphoblasts. J. Inherit. Metab. Dis. 8, 204-208. Gibson K. M., Sweetman L., Nyhan W. L., Jakobs C., Rating D., Siemes H. and Hanefeld F. (1983) Succinic semialdehyde dehydrogenase deficiency: an inborn error of gamma-aminobutyric acid metabolism. C/in. chim. Acta 133, 33-42.
Godschalk M., Dzoljic M. R. and Bonta I. L. (1977) Slow wave sleep and a state resembling absence epilepsy induced in the rat by y-hydroxybutyrate. Eur. J. Pharmac. 44, 1055111. Gonzalez M. P., Oset-Gasque M. J., Gimenez Solves A. and Canadas S. (1987) Succinic semialdehyde dehydrogenase activity in bovine adrenal medulla and blood platelets: a comparative study with the brain enzyme. Comp. Biochem. Physiol. 86B, 489-492. Kammeraat C. and Veldstra H. (1968) Characterization of succinic semialdehyde dehydrogenase from rat brain. Biochim. biophys. Acta 151, I-10.
Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Lancaster G., Mohyuddin, F., Striver C. R. and Whelan D. T. (1973) A y-aminobutyrate pathway in mammalian kidney cortex. Biochim. biophys. Acta 297, 229-240. Lineweaver H. and Burk D. (1934) The determination of enzyme dissociation constants. J. Am. them. Sot. 56, 658-666.
Lowry 0. J., Rosebrough J. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Miki Y., Taniyama K., Tanaka C. and Tobe T. (1983) GABA, glutamic acid decarboxylase, and GABA transaminase levels in the myenteric plexus in the intestine of humans and other mammals. J. Neurochem. 40, 861-865.
ProtoBlot Western Blot AP System Technical Manual (1987) Promega, Madison, Wisconsin. Ryzlak M. T. and Pietruszko R. (1988) Human brain “high K,,,” aldehyde dehydrogenase: purification, characterization, and identification as NAD+-dependent succinic semialdehyde dehydrogenase. Archs Biochem. Biophys. 266, 386-396.
Small W. C. and Jones M. E. (1990) Pyrroline 5-carboxylate dehydrogenase of the mitochondrial matrix of rat liver: purification, physical and kinetic characteristics. J. biol. Chem. 265, 18.668%18,672.
Towbin H., Staehelin T. and Gordon J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. natn. Acad. Sci. 76, 4350-4354.
White H. L. (1979) CAminobutyrate: aminotransferase in blood platelets.
2-oxoglutarate Science
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696-698.
Wolfson L. I., Sakurada 0. and Sokoloff L. (1977) Effects of y-butyrolactone on local cerebral glucose utilization in the rat. J. Neurochem. 29, 777-783.
~nr. J. Biochem.
Printed
1992 Copyright
0
0020-711X/92 $5.00 + 0.00 1992 Pergamon Press Ltd
SUCCINIC SEMIALDEHYDE DEHYDROGENASE FROM MAMMALIAN BRAIN: SUBUNIT ANALYSIS USING POLYCLONAL ANTISERUM KEN
L. CHAMBLISS and K. MICHAELGIBSON*
Kimberly H. Courtwright and Joseph W. Summers Metabolic Disease Center and Baylor Research Institute, Baylor University Medical Center, Dallas, TX 75226, U.S.A. [Tel. (214)820-2687; Fax (214)820-49521 (Received IO December
1991)
Abstract-l. NAD+-dependent succinic semialdehyde dehydrogenase was purified to apparent homogeneity from rat brain and highly purified from human brain. 2. Molecular exclusion chromatography of the purified enzymes on Sephadex G-150 and G-200 revealed M, values of 203,000 and 191,000 for rat and human, respectively. 3. Electrophoresis on sodium dodecylsulfate polyacrylamide gels revealed a single subunit of M, 54,000 for rat and 58,000 for human. Isoelectric focusing of the purified rat enzyme yielded a pl of 6.1. 4. For both proteins, K,,, values for short-chain aldehydes acetaldehyde and propionaldehyde ranged from 0.33 to 2.5 mM; Km values for succinic semialdehyde were in the 2-4 PM range. 5. The subunit structure of both enzymes was investigated in brain extracts and purified preparations by immunoblotting, using a polyclonal rabbit antiserum against the purified rat brain enzyme. 6. For rat and human extracts, single bands were detected at M, 54,000 and 58,000, comparable to findings in the purified preparations. Immunoblotting analyses in other species (guinea pig, hamster, mouse and rabbit) revealed single subunits of M, 54,000-56,500.
INTRODUCTION Gamma-aminobutyric acid (GABA) is produced from glutamic acid in a reaction catalyzed by glutamic acid decarboxylase (GAD; EC 4.1. I. 15) and further metabolized to succinic acid by the successive action of GABA transaminase (GABA-T; EC 2.6.1.19) and succinic semialdehyde dehydrogenase (SSADH; EC 1.2.1.24). Succinic acid produced from this pathway enters the Krebs’ cycle. GABA metabolism has been well characterized in mammalian central nervous system (CNS) where GABA functions as an inhibitory neurotransmitter. The presence of GABA metabolizing enzymes also has been demonstrated in kidney (Lancaster et al., 1973), platelets (White, 1979), lymphocytes (Gibson et al., 1983), intestine (Miki et al., 1983) and adrenal medulla (Gonzalez et al., 1987). The physiological role of the GABA metabolic pathway in non-CNS tissue is poorly understood. Inherited metabolic diseases caused by a deficiency of a single enzyme have been reported for two of the enzymes in this pathway. While only a single patient with deficiency of GABA-T (Gibson et al., 1985) has been identified, several cases of SSADH deficiency have been reported. SSADH deficient patients have a
characteristic elevation of the reduction product of succinic semialdehyde (gamma-hydroxybutyric acid; GHB) in body fluids indicating a block in the normal oxidative pathway. This is of interest because GHB has been shown to cause a form of absence epilepsy when administered to mammals (Godschalk et al., 1977) as well as increased brain dopamine levels (Gessa ef al., 1966) and decreases in glucose metabolism (Wolfson et al., 1977). SSADH has been partially purified and studied in rat (Kammeraat and Veldstra, 1968), pig (Blaner and Churchich, 1979), monkey (Albers and Koval, 1961) and human brain (Embree and Albers, 1964; Cash et al., 1977; Ryzlak and Pietruszko, 1988). The subunit molecular weights have only been reported for the rat and human brain enzyme. To better characterize SSADH of different species and to develop tools for future molecular analyses to elucidate genetic defects in patients with gammahydroxybutyric aciduria, we have purified both rat and human SSADH, produced antiserum to the rat enzyme, and examined the SSADH protein of different species by immunoblots. MATERIALSAND
METHODS
Materials
*Address correspondence to: Dr K. Michael Gibson, Baylor Research U.S.A. tic
%,%I
Institute,
3812 Elm Street,
Dallas,
TX 75226,
NAD, succinic semialdehyde, Cibacron-blue, Coomassie Brilliant Blue, 2-mercaptoethanol, Tween 20, nitroblue tetrazolium and phenazine methosulfate were purchased 1493
1494
KEN L. CHAMBLISSand K. MICHAEL GIBBON
from Sigma Chemical Company (St Louis, MO.). Acetaldehyde, propionaldehyde, 3-nitrobenzaldehyde, 4nitrobenzaldehyde, butyraldehyde and valeraldehyde were purchased from Aldrich Chemical Company (Milwaukee, Wis.). Acetaldehyde and propionaldehyde were redistilled before use. Sephadex G-l 50, Sephadex G-200, Blue Sepharose and S-AMP-Sepharose 4B were from Pharmacia LKB Biotechnology, Inc. (Piscataway, N.J.). DEAEcellulose DE-52 was purchased from Whatman Lab Sales (Hillsborom. Ore.). Acrylamide, his-acrylamide, ammonium persulfate and N,N,N’,N-tetramethylethylenediamine (Temed) were from Bio-Rad Laboratories (Richmond, Calif.). SDS and protein molecular weight standards were purchased from Bethesda Research Laboratories (Gaithersburg, Md). Nitrocellulose was from Schleicher and Schuell (Keene, N.H.). Blot quality bovine serum albumin, goat anti-rabbit IgG AP conjugate and immunostaining reagents were purchased from Promega. All reagents were of analytical grade quality. Glutaric and adipic semialdehydes were prepared, and aldehyde concentrations were determined as previously described (Forte-McRobbie and Pietruszko, 1986). Intact brains (except for human) were purchased from Pel-Freez Biologicals (Rogers, Ark.). Live Sprague-Dawley rats were purchased from Charles Rivers Laboratories (Wilmington, Mass.). Human brain tissue was acquired from the tissue procurement program of the National Disease Research Interchange (Philadelphia, Pa) and maintained at -7o’C prior to use.
During purification, the enzyme activity was assayed spectrophotometrically in pooled fractions by following the increase of absorbance at 340 nm, at 25°C in a total volume of 3 ml. In all cases, the initial reaction velocities were proportional to protein concentration. Standard assays contained 0.1 M sodium pyrophosphate buffer, pH 9.0, I mM EDTA, 500 FM NAD+ and 20 PM succinic semialdehyde. Individual column fractions were more conveniently assayed using the same assay mixtures and following the production of NADH fluorometrically in an AMINCO fluoro-calorimeter model 54-7439 (excitation 355 nm, emission 470nm). One unit of enzyme activity was defined as the amount of enzyme producing 1nmol NADH min-’ under the assay conditions, using an extinction coefficient of 6.22mM-’ cm-’ at 340nm. Protein concentrations were determined by the method of Lowry (Lowry et al., 1951) using bovine serum albumin as a standard or the Micro Bio-Rad (Bio-Rad Protein Assay Instruction Manual, Bio-Rad Laboratories) procedure utilizing gamma-globulin as standard. The concentration of affinity-purified enzyme was estimated by comparison to known concentrations of protein molecular weight markers (0.5-4 pg) following SDS-polyacrylamide gel electrophoresis and densitometric scanning of the Coomassiestained bands. Measurements of Km and V,,, values for aldehyde substrates were determined spectrophotometrically at 340 nm and 25°C in 3 ml total volume, in an assay buffer consisting of 0.1 M sodium pyrophosphate, pH 9.0, supplemented with 1 mM EDTA. Data were analyzed using the Lineweaver-Burk plot (Lineweaver and Burk, 1934) and least-square analysis. Purification Extraction. Forty rat brains (60-70 g) were homogenized in a Waring blender for 6min at high speed in 240ml of
ice-cold water supplemented with 0.1 mM EDTA and 0. I % 2-mercaptoethanol. The homogenate was centrifuged at 48,000 g for 30 min at 4°C. The supematant was saved and the pellet was homogenized and recentrifuged. The supernatants were pooled. For human brain, 300 g tissue was similarly homogenized in 6OOml of water/EDTA/Zmercaptoethanol. Centrifugation of extracts for human tissues was at 27,OOOg. Blue-Sepharose CL-6BIDEAE-cellulose DE-52. The pooled supernatants were applied to a Blue-Sepharose CL-6B column (2.5 x 30cm for the rat enzyme and 2.5 x 40cm for the human enzyme) equilibrated with buffer A (2 mM potassium phosphate, pH 7.2, 0.1 mM EDTA, and 0.1% v/v 2-mercaptoethanol) (Cash et al., 1977). The column was washed with buffer A until no further protein eluted from the column as monitored by absorbance at 280nm. The Blue-Sepharose column was eluted directly onto a DEAE-cellulose DE-S2 column (2.5 x 15cm for the rat enzyme, and 2.5 x 4Ocm for the human enzyme, both equilibrated with buffer A) with 0.1 mM Cibacron Blue dissolved in buffer A (2 1). The DEAE-cellulose column was then washed with buffer A as above. SSADH was eluted using a O-45 mM linear gradient of KC1 in buffer A for rat (0.5 1 total volume) and for human (1 liter total volume). S-AMP-Sepharose 48. The active fractions from the DEAE-cellulose column were pooled and applied to a 5’-AMP-Sepharose 4B column (1 x 20 cm) previously equilibrated with buffer B (10mM potassium phosphate. pH 7.2, 1 mM EDTA, 0.1% 2-mercaptoethanol). Unbound material was washed from the column with buffer B until no further protein eluted as monitored by absorbance at 280 nm. SSADH was eluted with a 1 mg/ml solution of NAD+ in buffer B. Active fractions were pooled and stored at 4’C in buffer B, or at -20°C in 50% glycerol. Gel chromatography The native M, of the rat and human brain SSADH was determined using Sephadex G-150 and Sephadex G-200 for equilibrated with buffer A. Protein standards calibration were obtained from Bio-Rad Laboratories and included thyroglobulin (670,000), gamma-globulin (I 58,000), ovalbumin (44,000) myoglobin (17,000) and cyanocobalamin (1350). Polyacrylatnide gel eleclrophoresis SDS-polyacrylamide gel electrophoresis was performed as described by Laemmli (1970). The stacking gel contained 4% w/v acrylamide and the resolving gel contained 10% acrylamide. Samples were boiled in protein sample buffer (50 mM Tris, pH 6.8, 2% w/v SDS, 0.01% v/v 2-mercaptoethanol and 10% glycerol) for 5min prior to loading. Protein bands were visualized by Coomassie staining. For subunit molecular weight determinations, high-range molecular weight markers from Bethesda Research Laboratories were used. Isoelectric focusing Isoelectric focusing was carried out on PhastGel IEF 3-9 and 5-8 gels (PhastSystem, Pharmacia LKB Biotechnology, Inc.) according to manufacturer’s specifications. The protein was visualized by Coomassie staining or activity staining. Activity staining was performed by permeating the gel in 0.1 M Tris-HCl, pH 8.5, with NAD+ (0.3 mg/ml), nitroblue tetrazolium (0.3 mg/ml), phenazine methosulfate (0.03 mg/ml) and 50 FM succinic semialdehyde. For pl
1495
SSADH from mammalian brain
still contained several Coomassie staining bands on SDS-polyacrylamide gels. An additional 24-fold purification, yielding highly purified enzyme, could be achieved with affinity chromatography on S-AMPSepharose 4B. Data on recovery (Table 1) indicate that all purification steps were efficient, with a final yield of about 71% based on total activity in the pooled supematants. Total content of SSADH in rat brain was about 0.48 units/g of tissue, which was comparable to the enzyme from human brain where the level was approx. 0.20 units/g of tissue. The purified enzyme was stable for several weeks in 10 mM potassium phosphate buffer (pH 7.2), with 0.1 mM EDTA and 0.1% v/v 2-mercaptoethanol, and could be stored long term in 50% glycerol at -20°C. As glycerol content was decreased to lo%, the enzyme lost activity. Freezing the protein in buffer without glycerol resulted in complete inactivation.
determination, a broad range (PH 3-10) calibration kit was obtained from Bio-Rad Laboratories. Antibodyproduction The purified rat brain SSADH was electrophoresed on SDS-acrylamide gels, and the 54,000 Da band was excised from the gel after localization of the band by submerging the gel in ice-cold 0.1 M KC1 for 5-10min. Gel slices were emulsified in phosphate buffered saline, pH 7.2, by homogenization. The resulting emulsion, containing l-2 mg of protein, was injected subcutaneously into two New Zealand White female rabbits. Booster immunizations were given at 4 and 6 weeks. Ten days following the second boost, 50-60 ml of blood were taken from the marginal ear vein
and serum prepared. Immunoblotting on SDSProtein samples were electrophoresed polyacrylamide gels and transferred to nitrocellulose sheets by electroblotting (EC 230 Electroblot System) as previously described (Towbin et al., 1979) with minor modifications. The blotting buffer was 12.5 mM Tris, pH 8.6, 96mM glycine, and 0.01% w/v SDS. Electroblotting was carried out at 200 mAmps for 1-2 hr at 4°C. After transfer, the blot was immunostained according to procedures provided by Promega (ProtoBlot Western Blot AP System Technical Manual, 1987) using a l/1000 dilution of the SSADH antisera. The second antibody was goat anti-rabbit IgG alkaline phosphatase conjugate.
Molecular weight The SSADH purified from rat brain was apparently homogenous with only one Coomassie-staining band evident on SDS-polyacrylamide gels (Fig. 1). The subunit A4, was determined to he 54,000 k 3500. The subunit size of rat SSADH that we observed was smaller than that reported by Cash et al. (1977)
(IV, = 68,000). The average M, of the native enzyme was determined by gel filtration using both Sephadex G-150 and G-200 and was found to be 203,000 + 39,000. These data indicate a tetrameric structure consisting of four identical subunits which differs from the previously reported dimeric structure (Cash et al., 1977). A tetrameric structure also has been demonstrated for human SSADH (Ryzlak and Pietruszko, 1988).
Bruin lysates Whole cell lysates were made from frozen brain tissue by homogenization for 4-5 min in ice-cold water. After protein concentrations were determined, 1 volume of 2X protein sample buffer was added. Between 200 and 250 pg of brain protein were loaded into each gel lane for electrophoresis and subsequent
immunoblotting.
RESULTS Purification of rat brain SSADH
Isoelectric focusing
Data representing an average of three purifications of rat brain SSADH are shown in Table 1. Because two of the three chromatographic steps were connected in series (Blue-Sepharose CL-6B and DEAE DE-52), the purification could be accomplished readily in 4-5 days. After the first two chromatographic steps, the enzyme was 250-fold purified but
To further evaluate the purity of the rat SSADH, the enzyme was analyzed by isoelectric focusing. The purified protein was electrophoresed on gels with pH gradient ranges from 3 to 9 and 5 to 8. Protein bands were visualized by Coomassie staining or by “enzyme activity” staining as described in Materials and Methods. Gels of both pH ranges and both staining
Table 1. Purification of succinic semialdehyde dehydrogenase from rat and human brain’
Total activityb (units) step Extraction Pooled-supernatants Blue-Sepharose CLdB/ DEAE cellulose 5’-AMP-Sepharose 48
Protein (mg) 7135 (22,074)e 3893 (3326) ::, 1.1 (3.8)
Specific activity (units/mg)
% Recovery
Enrichment
A
B
A
B
A
B
A
B
60.8
25.3
0.0098
0.0035
-
-
-
-
37.2
32.3
0.0145
0.0081
100
100
13.6
29.5
0.275
0.876
22
91
19
250
10.0
23
2.74
16
71
280
6111
21.39
1.5
2.3
‘Results presented are the mean values for three independent purifications from 65-70 g of rat brain; values for human are the means for three independent purifications from 300 g of tissue. bA, human; B, rat. ‘Values in parentheses are human brain protein contents,
1496
KEN
L. CHAMBLISS andK. MICHAELGIBSON
methods revealed a single band with an average pl of 6.1 (Fig. 2). Activators, inhibitors and pH optimum
Substrates studied for optimum pH with the purified rat brain enzyme included succinic semialdehyde, glutaric semialdehyde and propionaldehyde. For succinic semialdehyde and propionaldehyde, maximum activity was observed at pH 9; for glutaric semiaidehyde, activity appeared maximal between pH 9 and 10. Enzyme activity with 20 PM succinic semialdehyde in Tri-HCI was only 52% of the activity in phosphate or pyrophosphate buffers. 3-Hydroxybenzaldehyde inhibited enzyme activity by 88% at 0.5 mM and 95% at 1 mM; 4-hydroxybenzaldehyde was a more potent inhibitor, decreasing enzyme activity by 97% at 0.5 mM. Addition of increasing amounts of iodoacetamide at concentrations of 0.5, 1 and 2mM resulted in 9, 31 and 46% inactivation, respectively, when iodoacetamide was added to cuvettes at the time of reaction. A variety of metal ions (500 PM) were studied as activators or inhibitors of purified SSADH. Fe+‘, Ca+’ Mgt2 and Mn+’ did not stimulate or inhibit the reaction; conversely, Cut’, Cd+2, Zni2, Hg+’ and Cr+3 inhibited the reaction by 74, 72, 76, 38 and 59%, respectively. The purified enzyme was sensitive to disulfiram inhibition at 5 PM final concentration (Ryzlak and Pietruszko, 1988). Enzyme activity was not restored by addition of 50 FM glutathione or cysteine, but was restored by 50 PM dithiothreitol; in the reaction cuvettes to which cysteine and glutathione had been added, the enzyme was reactivated upon addition of 50mM 2-mercaptoethanol.
where the Km values were in the millimolar range. The carboxylic acid semialdehydes were the best substrates with K,,, values between 2 and 4pM; however, the V,,, was the highest with SSA as substrate at a value about 6-fold higher than the other semialdehydes tested. Immunoblots
Antisera prepared in rabbit against the purified rat brain SSADH were used to probe protein blots of rat and human brain Iysates which had been separated on SDS-polyacrylamide gels. The antisera were specific for only one band in each homogenate (Fig. 3). The M, of the band in the immunoblot of the rat brain homogenate was identical to the M, of the purified rat protein, The IV, of the single immunostaining band in the human brain homogenate blot was 58,000. This contrasted with the report by Ryzlak and Pietruszko (1988) of two subunits for human brain SSADH with &f, of 61,000 and 63,~O. Purification of human SSADH
Because the data we generated by immunoblots pertaining to the human SSADH subunit structure were different than published data, we undertook purification of the human enzyme. Our goal was to clarify the issue of subunit number and molecular weight; moreover, purified protein would allow N-terminal amino acid sequence analysis which would be of value in verification of cDNA clones. The purification of human brain SSADH was carried out by a comparable procedure used for rat brain SSADH. Table 1 shows mean data from three preparations of the human enzyme. When the purified human enzyme was analyzed by Kinetic characterization of aldehyde substrates SDS-PAGE, a single Coomassie staining band was K,,, and V,,, values for several aldehyde substrates observed with a A4, of 58,000. We occasionally were calculated and are listed in Table 2. Suitability observed a second contaminating band (M, 37,000) of each aldehyde as a substrate for the enzyme was in the final preparation of the human enzyme. This assessed by comparison of the V/K, ratios which was readily removed by inclusion of a Sephadex range from as low as 0.002 for a~taldehyde to as high G-150 ~hromatographic step (2.5 cm x 1 m) after as 7.49 for succinic semialdehyde. The lowest V/K+,, the DEAE-DES2 column and prior to the 5’-AMP values were for straight-chain aliphatic aldehydes Sepharose column. Even when the human enzyme Table 2. Kinetic properties
Substrate Acetaldehyde Propjonaldehyde Butyraldehyde Vaieraldehyde Succinic semialdehyde Glutaric semialdehyde Adipic semialdehyde 3-Nitrobenzaldehyde 4-Nitrobenzaldehyde
of rat and human brain succinic semialdehyde Concentration range (IBM) 400--24,300 (100-24.300) 250-16,300 (30-3700) ~lO,~O 4-10,000 0.6-500 (0.6-500) 0.5-1000 0.5-1000 4&10,000 40-10,000
dehydrogenase”
vtK,n 2500 (1670) Iwo (333) 190 27 3.9 (1.7) 2.3 3.3 380 250
3.91 (1.22) 4.50 (0.74) 5.50 4.23 29.2 (3.1) 5.62 4.22 1.52 4.61
0.002 (O.Ooa7) 0.005 (0.002) 0.03 0.16 7.49 (1.82) 2.44 1.28 0.02 0.02
PAll assays were in 0.1 M sodium pyrophosphate buffer, pH 9.0, containing I mM EDTA and 5OOpM NAD. Values in parentheses were obtained using purified human brain SSADH.
SSADH
from mammalian
1497
brain
+ -
200 k Da
5.1 97.4 k Da
6.0 68kDa
6.5
43 k Da
7.1
a.4
29 k Da
Fig. 1. SDS-polyacrylamide gel electrophoresis of purified human (Lane I) and rat (Lane 2) brain SSADH. Lane 3 contains protein molecular weight standards. The gel was electrophoresed as described in Materials and Methods and was stained with Coomassie Brilliant Blue. Protein contents were: Lane 1, 6 pg; Lane 2, 3 pg; Lane 3, 5 pg. M, of the rat and human brain enzymes was 54,000 and 58,000, respectively.
1
2
3
4
5
Fig. 2. Determination of isoelectric point of rat brain SSADH. Protein was analyzed using PhastSystem (Pharmacia LKB Biotechnology, Inc., Piscataway, N.J.) according to manufacturer’s specifications. Purified SSADH was electrofocused, and the gel was developed for protein with Coomassie Brilliant Blue. The relative positions of the isoelectric focusing standards are shown in the right margin.
6
7
-
97.4 k Da
-
68 k Da
-
43 k Da
8
Fig. 3. Immunoblot (Western) analysis of succinic semialdehyde dehydrogenase in brain extracts and purified fractions from different species. Electrophoresis and immunoblotting using rabbit polyclonal antiserum raised against purified rat brain SSADH were as described in Materials and Methods. Lane and protein contents were: 1. rat purified (1 pg); 2, rat extract (0.14mg); 3, human purified (21(g); 4. human extract (0.4 mg); 5, guinea pig extract (0.46 mg); 6. hamster extract (0.39 mg); 7, mouse extract (0.22 mg); 8, rabbit extract (0.55 mg).
KENL. CHAMBLISS and K. MICHAEL GIBSON
1498
was underioaded and el~trophoresed over relatively long distances (10 cm), only one band was visible. The native IV, of the human brain enzyme was determined to be approx. 191,000 by Sephadex G-200 molecular exclusion chromatography. This result is comparable to previous reports (Ryzlak and Pietruszko, 1988). Comparison of rat and human enzymes with different aldehyde substrates is shown in Table 2. Like the rat enzyme, the human enzyme had high I(m values for propionaldehyde and acetaldehyde, with low I’,,,,,. The human enzyme, like the rat enzyme, was inactive with pyrroline-5carboxylic acid as substrate. The K,,, values for both species for succinic semiaidehyde were in close agreement (Table 2). Analysis of the purified human brain enzyme by IEF was unsuccessful. Repetitive analyses on gels with pH ranges of 3-9 or 5-8 revealed a broad staining region (activity or Coomassie stained) of approx. 6.1-7.1. Suruey of subunit M, of other mammalian species To determine the subunit h4, of brain SSADH from other mammalian species, lysates were made from guinea pig, hamster, mouse, rabbit, bovine calf, sheep, pig, dog and chicken brain. The lysates were el~trophoresed on SDS-~~yacrylamide gels, blotted to nitrocellulose sheets, and immunostained with rabbit anti-rat SSADH. Only faint bands could be detected in brain lysates of calf. sheep, pig, dog and chicken (data not shown), whereas with the more closely related guinea pig, hamster and mouse brain lysates, a distinct band was observed for each species (Fig. 3). Estimated M, values were 56,500 for guinea pig, 54,000 for hamster, 54,000 for mouse and 54,000 for rabbit. DISCtiSSlON
AND SUMMARY
The purification of NAD+-dependent of SSADH has been reported in two mammalian species, rat and human (Cash et al., 1977; Ryzlak and Pietruszko, 1988). Cash and coworkers described the purification of both rat and human brain SSADH. These workers reported a subunit M, of 70,000 and a native M, value of about 140,000 for both. These data are consistent with molecular weight determinations and Km values reported by Forte-McRobbie and Pietruszko (1986) for human liver and by Farres et al. (1988) for human placenta I-pyrroline-5-carboxylic acid dehydrogenase. Further, 1-pyrroline-5carboxylic acid dehydrogenase was identified in the mitochondrial fraction during the purification of human brain SSADH (Ryzlak and Pietruszko, 1988), suggesting that the protein isolated by Cash er af. (1977) might be I-pyrroline-5-carboxylic acid dehydrogenase. A recent report on the purification of pyrroline-5-carboxylate dehydrogenase from rat liver mitochondrial matrix indicated a dimeric structure with weight identical subunits of M, 59,000 (Small and Jones, 1990).
Ryzlak and Pietruszko (1988) reported the purification of human brain SSADH to homogeneity. These workers presented evidence that the human brain enzyme was composed of weight non-identical subunits of &f, 61,000 and 63,000. We were intrigued by this divergence of subunit composition and undertook the purification of human brain SSADH in addition to the rat brain enzyme. Our preparation revealed only a single subunit of I%&58,000. To rule out the possibility of proteotytic degradation during enzyme purification, we prepared polyclonal antisera in rabbit using purified rat brain SSADH as antigen. Analysis of crude rat and human brain extracts revealed the same results as the purified preparations (Fig. 3) i.e. single subunits of !4, 54,000 and 58,000. This suggested that proteolysis during enzyme preparation was not artefactually producing our 58,000 M, subunit. For studies with human brain, we were unsuccessful in attempts to obtain fresh post mortem tissue from local or outlying hospitals. Human brains were obtained from the NDRI Tissue Procurement Program. Although tissue was obtained as rapidly as possible post mortem, intervals ranged from g-20 hr between death and storage of brain tissue at - 70 C. This may have contributed to the low specific activity (-3 ~mol/min/mg protein) for the final purified human brain SSADH, which was a value about 5% of that reported by Ryzlak and Pietruszko (1988). Similarly, the antiserum revealed single subunit bands in other rodent species, including guinea pig, hamster, mouse and rabbit. Immunoblot analyses revealed strong reactive bands for rat, guinea pig, hamster, mouse, rabbit and human; fainter bands were observed in brain extracts of bovine calf. sheep, pig, dog and chicken (data not shown). This may have resulted from using rodent antigen for a generation of antiserum. We demonstrated that the human brain enzyme had high I& values for short-chain aliphatic aidehydes such as acetaldehyde and propionaldehyde (1.67 and 0.33, respectively). Our K, value for succinic semialdehyde of 1.7 FM was in good agreement with the value of 1 PM reported by Ryzlak and Pietruszko (1988). We showed that rat brain SSADH had a pH optimum of about 9 and was sensitive to inactivation by hydroxybenzaldehyde derivatives and sul~ydryl reagents, features consistent with the results of Ryzlak and Pietruszko (1988) for the human brain enzyme. We also demonstrated the sensitivity of the rat brain enzyme to the drug disuifiram, as was the case for the preparation of Ryzlak and Pietruszko (1988). We were unable to resolve distinct bands by isoelectric focusing of the human brain enzyme. A broad staining region from ~16.1-7.1 was observed; conversely, Ryzlak and Pietruszko (1988) reported distinct bands of pi6.3, 6.6, 6.8, 6.95 and 7.15. However, data obtained during purification of human brain SSADH in the present study did support the possibility of isozymes. At least three distinct bands of activity, as detected
SSADH from mammalian brain
with succinic semialdehyde as substrate, were routinely observed during chromatography on DEAE DE-52 (data not shown). Although we only detected the single subunit of M, 58,000, it remains possible that different subunits exist with only subtle molecular weight differences which are not resolved by conventional SDS-polyacrylamide gel electrophoresis. Conversely, charge differences may exist in subunits with identical or nearly identical molecular weights. Further analyses will be required to determine the existence of multiple isozyme forms of human brain SSADH. A number of patients suffering from an acquired deficiency of SSADH have been reported in recent years. In response to this defect, presumably succinic semialdehyde in brain accumulates and is reduced to the neuropharmacologically-active 4-hydroxybutyric acid, a compound of unique activity in nervous tissue. The molecular genetics of this disease, an inherited defect in the GABA degradative pathway, have not been reported. In rat, it is likely that one gene is responsible for the single subunit comprising active SSADH. However, recent data presented for the human brain proteins may be more complex (Ryzlak and Pietruszko, 1988). The results of the present report suggest that human brain SSADH is a tetramer of weight-identical subunits. The data of Ryzlak and Pietruszko (1988) suggest a tetrameric structure of az& subunits. Human brain SSADH may be composed of non-identical subunits whose subunit M, is identical or so similar that resolution on normal SDS-polyacrylamide gels is impossible. Resolution of the number of genes encoding human brain SSADH should be revealed by molecular analyses. These studies are in progress. Acknowledgements-The
authors are indebted to Dr Christopher D. Cash for advice regarding protein purifications and Carol F. Lee for technical assistance. REFERENCES
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