Biochtmwa et Biophysica Acta, 1073 (1991) 203-208 6) 1991 ElsevierScience PublishersB.V.(Biomedical Division)0304-4165/91./503.50 ADONIS 030,~165910005SZ
203
BBAGEN 23447
D-Aspartate oxidase, a peroxisomal enzyme in liver of rat and man P a u l P. V a n V e l d h o v e n , C h a n t a l Brees a n d G u y P. M a n n a e r t s Kathoheke Unwersitett Leu~,en. Fakultctt geneeshunde. Campt~ Ga~rhutsberg. A[dehng Farrnat ~,logte. Leu~'en (B¢lgtum)
(Received 17 April 1990)
Key words: Peroxisome:Oxidase; Dicarboxylicaminoacid: Zellweg,:rsyndrome;Excitotoxicity: (Humanliver) By means of subcellular fractionation D-aspartate oxidase was shown to be Ioc',dized in peroxisomes in rat and human liver. The oxidase from both sources was most active on D-aspartate and N-methyI-D-aspartate. In different rat tissues, the highest enzyme activity was found in kidney, followed by liver and brain. In these tissues, oxidase activities became detectable 1-4 days after birth, reaching adult values after 4 weeks. Analysis of liver samples from patients with Zellweger syndrome, a generalized peroxisomal dysfunction, demonstrated no significant deficiency of this particular oxidase.
Introduction Through the initial studies of Still and co-workers [1,2] the presence in mammalian renal tissue cf separate oxidases acting on neutral (or basic) and on dicarboxylic D-amino acids was established. These oxidases are generally referred to as 0-amino acid oxidase and 0aspartate oxidase, respectively (see Ref. 3 for a review). The latter one has been found mainly in kidney, liver and brain of mammals and catalyzes the deaminative oxidation of D-aspartic acid, and less effectively of 0-glutamate and D-amino adipate [3]. Whereas 0-amino acid oxidase is since long known to be associated with (micro)pcroxisomes in different tissues (see Ref. 4), the subcellular distribution of 0-aspartate oxidase has only recently been investigated. Thus, Zaar et al. [5] were able to show a pcroxisomal localization of D-aspartate oxidas¢ in rat, bovine and sheep kidney cortex. The presence of this enzyme in purified rat liver peroxisomes was also demonstrated. Until now no data have been published on the localization of 0-aspartate oxidase in human tissues. Such data might be of interest with regard to peroxisomopathies (see Ref. 6 for a review), a newly recognized group Abbreviations: EDTA. ethylencdiaminetetraacetate: D'rl', dithiothreitol; FAD, flavin¢-adenine dinucleotide; NMDA. N-methyl-~ aspartate; Hepes. 4q2-hydroxyethyl)-I-pipcrazinecthanesulfonicacid; FMN. flav/ne mononucleolide. Correspondence: P.P. Van Veldhoven, K.U. Leuven - Campus Gasthuisbcrg Department of Pharmacology, Herestraat, 13-3000 Leuven. Belgium.
of metabolic disorders in which one or more peroxisomal enzymes are missing or malfunctioning. Indeed, 0-aspartat¢, which is present in appreciable quantities in fetal tissues and especially in fetal brain [7], is a potent agonist of the excitatory amino acid receptors, ,,dfich play a role in brain function and development (see Refs. 8-10 for reviews). The generalized peroxisomopathies such as the Zellweger syndrome, in which no recognizable peroxisomes can be found, are characterized by a severe neurological dysfunction and by disturbances in brain development [6]. We, therefore, decided to investigate the subcellular distribution of 0-aspartate oxidase in human and rat liver and we also measured the enzyme in liver tissue from patients with peroxisomopathies. Materials and Methods N-Methyl-0-aspartic acid, 0-glutamic acid and 0amino adipic acid were obtained from Aldrich Europe, Brussels (Belgium); 0-aspartic acid, sodium benzoate. L-tartaric acid. and homovanillic acid from Janssen Chimica, Beerse (Belgium). FAD, Triton X-100, and peroxidase were from Boehringer, Mannheim (F.R.G.) and Percoll from Pharmacia, Uppsala (Sweden), 0-Homocysteic acid, meso-2,3-diaminosuccinic acid, 0-hydroxy glutaric acid, racemic cis-2.3-piperidinedicarboxylic acid, and mesotartaric .~cid were from Sigma, St. Louis, MO (U.S.A.), and lauryldimethylammonium oxide from Serva, Heidelberg (F.R.G.). Flavine mononucleotide was obtained from Fluka, Buchs (Switzerland).
204 N-AcetyI-D-aspartate was prepared as follows. DAspartic acid (5 retool) and anhydrous triethylamine (12 retool) were added to 10 ml anhydrous methanol. While stirring. 5 ml acetic anhydride was added dropwise. and the mixture allowed to react overnight. After neutralization of the reaction mixture with concentrated ammonium hydroxide, methanol was removed by rotary evaporation. The residue was dissolved in water, neutralized with ammonium hydroxide and lyophilized. This step was repeated twice. No free amino groups could be detected in the residue by means of trinitrobenzenesulfonic acid. Male Wistar rats, maintained on a standard chow diet, were killed by decapitation. The liver, or other tissues, were removed and homogenized in 0.25 M sucrose/1 mM EDTA (pH 7.2)/1 mM DTT/0.1% (v/v) ethanol (SEDE). Liver homogenates were fractionated as described in Ref. 11 and the light mitochondrial fraction was further subfractionated in self-generating Percoll density gradients [11]. Human liver was obtained at autopsy or as excess tissue after transplantation of livers from adult donors in children. A detailed description of the procedures was submitted to the university hospital Ethics Committee. which granted its approval. A portion of the tissue from a donor liver was cut into small pieces, homogenised in SEDE, containing 5 ~tM FAD, and fractionated as described for rat liver. The remaining tissue and tissues obtained at autopsy were divided into smaller portions, quickly frozen in liquid N 2 and stored at - 8 0 ° C . Marker enzymes were measured as described before [11 ]. 5'-Nucleotidase was measured as follows. 50 .al of the subcellular fractions, appropriately diluted, were mixed with 200 .al of reaction mixture containing 5 mM AMP/12.5 mM MgCI2/12.5 mM L-tartrate/125 mM KCI/62.5 mM Hepes buffer (pH 7.5)/0.25% ( w / v ) lauryldimethylammonium oxide. After 20 rain incubation at 37°C, reactions were stopped with 1 ml of 10% (w/v) trichloroacetic acid. After removal of denatured proteins by centrifugation, 0.5 ml of the supernatants was mixed with 1 ml of 0.45 M H2SO4/0.375% ( w / v ) ammoniumheptamolybdate • 4H20/1.5% (w/v) ascorbic acid and placed at 45"C for 30 rain. After cooling the absorbance of the mixtures was read at 820 nm. Incubations in the absence of subst~ate served as blanks. Oxidases were measured according to Ref. 12, modified as follcws. Samples ,,f 40 .al, diluted in SEDE, were mixed with 10 .al of 100 .aM FAD (or 25 .aM FMN in the case of glycolate oxidase)/25 mM NaN.~ and incubated at 0 ° C for 5 rain in order to inactivate catalase and saturate the oxidases with cofactor. To these samples 0.15 ml of reaction mixture, consisting of 83.3 mM pyrophosphate buffer (pH 8.5)/0.83 mM Nethylmaleimide/16.7 U / m l peroxidase/l mM homo-
vanillic acid/0.083% ( w / v ) Triton X-100. was then added. After another 5 min at 0 ° C , needed to alkylate the DTT and other interfering sulfydryl compounds, reactions were started by adding 50 p.I of 50 mM neutralized substrate, followed by an incl,~ation at 37°C in subdued light. After 30 rain (glycolat¢ oxidase) or 60 min (D-amino acid and D-aspartate oxidase), reactions were stopped with 50 .al of 12c~ ( w / v ) perchloric acid. Denatured proteins were removed by centrifugation and an aliquot (0.2 ml) of the supernatant was diluted 15-fold with 0.5 M carbonate buffer (pH 10.7). containing 10 mM EDTA, before reading the fluorescence of the homovanillic acid dimer [12]. The slow increase in fluorescence, observed after alkalinization of the acidified supernatants, could be almost totally suppressed by adding EDTA to the carbonate buffer. Substrates used were D-aspartate (D-aspartate oxidase), Dproline or D-alanine (D-amino acid oxidase) and glycolate (glycolate oxidase). Fluorescence readings were corrected by means of appropriate blanks and standardized with known amounts of peroxide added to blank assay mixtures. Tissues homogenates were sufficiently diluted to obtain at least 90% recovery of exogenously added peroxide. Results and Discussion Subcellular fractionation of rat liver revealed that D-aspartate oxidase is mainly found in the light mitochondrial and cytosolic fractions and that its activity follows closely that of catalase, a peroxisomal marker (Fig. IA). When a light mitochondrial fraction was further separated on a Percoll gradient, again the distribution patterns of catalase and D-aspartate oxidase coincided (Fig. IB). These data are in agreement with those obtained by Zaar et al. [5] for rat kidney cortex. The enrichment of D-aspartate oxidase (and other peroxisomal enzymes) in the light mitochondrial fraction from human liver was rather low. This is partly due to the gently conditions used to homogenize the liver, which, although satisfactory for rat liver, were not severe enough to disrupt all cells in the human liver. Indeed, a high percentage of all marker enzymes was found in the nuclear fraction (Fig. 2A). The amount of lactate dehydrogenase, a cytosolic marker, present in the nuch:ar fraction indicates that approx, one fifth of the cells remained intact..Secondly, during homogenization peroxisomal matrix enzymes were released to a higher extent in human liver than in rat liver (Fig. 2A). This phenomenon has been observed by others too [13]. in how far this is a consequence of the non-optimum preservation condition of the donor liver is not known. As indicated by the relative specific activity, more catalase was found in the cytosolic fraction than other peroxisomal matrix enzymes. Whether this is due to differential release, as reported before in rat liver [14,15],
205 o r d u e t h e p r e s e n c e o f a c y t o s o l i c f o r m o f c a t a l a s e is n o t clear. D e s p i t e t h e s e d i f f i c u l t i e s , t h e a s s o c i a t i o n o f Daspartate oxidase with peroxisomes became evident
L
2-
r, i1
iLl
I
:I
¼ L
g 2~ 1L
n
I
E
:
R
7
....
i:-s
•
u
i i
---~
8
il ° N
MLP
I
a: 3r
I
G
H
I i
F I
: i I 2I~
L_2:'LA
S
N~.P
~/~ 0 I: TOTALPROTEIN
S
%
of
t0rAL
8
'
C
•
0
F
-
~
-
H
PROTLIN
•
B
~,o
-
:,
•
~.o.
~i
2o,
:',
-~ ~o:
E
:
!'"
b
~.0.
i
•
-
.
J
~3
•
!' -
;
. •
zo.
•
~o
i
',:
!
.
.
;
. i" 4
. :
:
',...... "8....... ~ ........ :" . . . . . . . :::"~" ..... L~=::.:.: ...... FRACTION
i
7
ii. FRACTION
NUMBER
Fig. I. Subcellular distribution of D-aspartate oxidas¢ in rat liver. (A) Rat liver was fractionated into a nuclear (N). a heavy nfitochondrial (M), a light mitochondrial (L). a microsomal (P) and a soluble fraction (S) and marker ¢nzym~-s were measured in each fraction. A: 5'-nuclcondas¢ for plasma membrane; B: glutamate dehydrogenase for mitochondria: C: glucosc-6-phosphatas¢ for endoplasmic reticuhim; D: catalas¢ for peroxisomal matrix protein: E: acid phosphatase for lysnsomes" F: D-aspanate oxidas¢. Results are expressed as relative specific activities versus cumulative pcrccmage of protein. Recoveries varied between 86-102%. (B) The light mitochondrial fraction. shown in panel A was suhfraclionated by isopycnic centrifugation ~n an iso-osmofic Percoll gradient. The fractions were analyzed for protein (A), acid phnsphatas¢ (B). glutamate dehydrogenas¢ (C). glucose-6-phosphatas¢ (D). catalas¢ (E) and D-aspartate oxidas¢ (F). Results are expressed as percentages of total recovered gradient activity or content, present in each fraction. Recoveries varied between 81-123%. Fraction i and 15 represent the fractions of highest and lowest density, respectively.
NUMB[R
Fig. 2. Subcellular distribution of D-aspartate oxidas¢ in human liver• (A) Human liver was fraetionated as described in the legend to Fig. I. Fractions were analysed for 5'-nuclcotidas¢ (A), glutamate dehydrogenase (B). ~ i a pnusphatas¢ (C), catalas¢ (D). glucosc-6-phosphatase (E). lactate dehydrogenase (F: marker for cytosol), glycolate and D-amino acid oxidase (G and H, respectively markers for peroxisomal matrix proteins) and D-aspartare oxidas¢ (1~ Results are expressed as relative spgch~ic activities versus cumularive percentage of protein• Recoveries varied between 84-107%. (B) The light mitochondrial fraction was further fraetionated on a Percoll gradient as described in the legend to Fig. 1. and analyznd for protein (A). acid pho~phatas¢ (B). glutamate dehydrogenase (C). glacose-6-phosphatas¢ (D), catalas¢ (E). glycolal¢ oxidase (F). D-amino acid oxidase (G) and D-aspartate oxidas¢ (H). Results are expressed as percentages of the total recovered gradient activity or content, present in each fraction. Recoveries varied between 92 -t28%. Fractions I and 16 represent thc fractions of highest and lowest density, respectively.
w h e n t h e light m i t o c h o n d r i a l f r a c t i o n w a s s u b j e c t e d to Percoll d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n . T h e a c t i v i t y followed that of the other pcroxisomal enzymes catalase,
206 D-amino acid oxidase and glycolate oxidase (Fig. 2B). In both species, hepatic D-aspartate oxidase was not sensitive to 10 m M sodium benzoate, a selective inhibitor of D-amino acid oxidase [16]. On the other hand, 20 mM mesotartrate, an inhibitor of rabbit and pig kidney D-aspartate oxidase [17,18] inhibited the enzyme by 85% (data not shown). A comparison of different rat tissues revealed the highest D-aspartate oxidase activity in kidney, followed by liver and nervous tissues (Table I), in agreement with the data reported by Y a m a d a et al. [19]. According to Barker and H o p k i n s o n [20], in man D-aspartate oxidase is present mainly in liver and kidney, whereas traces of activities are found in small intestine and lung, but surprisingly v,ot in brain. On the other hand, o-asparrate oxidase activity has been d e m o n s t r a t e d in mouse I191. hog and cat brain [211. Table iI shows the activity of D-a,~partate oxidase with different dicarboxylic D-amino acids and their analogues for rat kidney, rat liver, and h u m a n liver. The three enzymes show similar substrate specificities, except for N-methyl-D-aspartate ( N M D A ) . While the hum a n liver oxidase is most active on o-aspartate, the rat enzymes show a slight preference for N M D A . A negative charge in the side chain, preferentially carried by a carboxyl group, is an i m p o r t a n t feature of possible substrates recognized by the enzyme. The distance between the two carboxyl groups seems to be critical (about 3 /~) since only D-aspartate and N M D A are oxidized to a significant extent. This agrees well with the conclusion derived from inhibitor studies performed on the octopus enzyme [22]. Similar findings have been reported with the oxidase from other tissues [5,17,18,23]. In contrast to methylation, acetylation of the a m i n o group results in an inactive c o m p o u n d , suggesting the importance of a positive charge on the nitrogen atom. The role of this a m i n o group is further stressed by the TABLE I Tissue distribution of o-aspartate oxidase in rat
n-Aspartate oxidase was measured in tissue homogenates as described in the Materials and Methods section in the presence of 10 mM sodium benzoate. Activities are expressed in mO (nmol substrate oxidized per min) and are the means4-S.E.M, for the number of experiments indicated in parentheses. Tissue Kidney Liver Cerebrum Cerebellum Lung Muscie Heart Spleen Small intestine Testis
mU/g tissue 130.2 ±10.6 (7) 37.6 :t: 1.8 ( 1 1 ) 11.3 4. 1.4 (6) 8.354- 0.74 (4) 6.39± 1.87 (4) 4.97± 0.98 (4) 4.494- 0.97 (4) 3.29± 0.82 (4) 1.81 ± 0.63 (4) 1.78+ 0.85 (4)
mU/mg protein 1.01 +0.11 (7) 0.204+0.010(9) 0.105±0.018(6) 0.080:t:0.003(4) 0.060+0.017(4) 0.0654-0.018(4) 0.044±0.011 (4) 0.0224-0.006(4) 0.020±0.006 (4) 0.024±0.012(4)
TABLE I1 Substrate specificiO' of D-aspartate oxidg~e in rat and human tissues
I.,-Aspartate oxidase activity was measured as described in the Materials and Methods section in the presence of I0 mM sodium benzoate. The substrales were used at a final concentration of 10 raM. Results are expressed as percentage of the activity observed with D-aspartate and are means for two to three experimenls. ( - : not detectable; N.M.: not measured) Substrate D-Aspartate D-Glutamate D-Amino adipate N-Methyl-D-aspartate Meso-2,3-diaminosuccinate D-Homocysteate N-Acetyl-P-aspartate o-Hydroxyglutarate cts-2,3-piperidinedicarboxylate
Rat liver 100 17.4 6.2 134 29.5 2.0 8.3
Rat kidney 100 6 7 1.6 118 4.4 0.2 12.9
Human liver a 100 1.6 0.7 63.6 25.9 0.7 NM.
• The substrate specificity in a liver sample of a Zellweger patient was similar to that observed in control subjects.
inhibitory action of dicarboxylic c o m p o u n d s in which this substituent is replaced by a hydroxy group, like malate, tartrate and h y d r o x y g l u t a r a t e [17,22,23]. In analogy with o - a m i n o acid oxidase which is most active on n-proline [24], we tried a cyclic acidic a m i n o acid, cis-2,3-piperidine dicarboxylic acid as substrate. Like N M D A , this synthetic c o m p o u n d interacts w i t h the N M D A receptor [251. A l t h o u g h the position of the carboxyl groups a n d the charged N - a t o m are c o m p l e t e l y s u p e r i m p o s a b l e in the D-forms of a s p a r t a t e a n d the heterocyclic c o m p o u n d , a racemic m i x t u r e of the latter one was only poorly recognized by the rat enzymes a n d not at all by the h u m a n liver oxidase. A n inhibition by the L-form c a n n o t be excluded, but in general the L-forms are neither oxidized, nor act as inhibitors [17]. In contrast to neutral o - a m i n o acids, which are only rarely found in a n i m a l tissues, appreciable levels of free l > a s p a r t a t e have been d e m o n s t r a t e d in different tissues (brain, pituitary, liver, a n d kidney) a n d blood of rodents, and also in h u m a n b l o o d [7]. Free n - g l u t a m a t e could not be detected, however [7]. Highest D-aspartate levels were found in fetal tissues in which they decrease rapidly after birth [7]. W e therefore studied the ontogenie development of D-aspartate oxidase in rat liver, kidney and cerebrum (Fig. 3). The oxidase b e c a m e detectable early after birth in these tissues a n d the activity rose rapidly to adult values in 6 weeks. This rise in activity fits nicely with the p o s t n a t a l decrease in tissue D-aspartate levels [7]. Apparently, a low D-asparrate oxidase activity was already present in liver d u r i n g the last week of gestation. Generally, peroxisomal enzyme activities are absent or very low in fetal rat liver and start to rise only after birth. Peroxisomal profiles, containing catalase, b e c o m e visible from d a y 14 of
207
I
i 120L ? ' ->:
~ ~0~ :
i
/a ~;"
= I '
i ,
i~
I
,_
'"
,
i ~ '
'
~ . ' , / &
203
BBAGEN 23447
D-Aspartate oxidase, a peroxisomal enzyme in liver of rat and man P a u l P. V a n V e l d h o v e n , C h a n t a l Brees a n d G u y P. M a n n a e r t s Kathoheke Unwersitett Leu~,en. Fakultctt geneeshunde. Campt~ Ga~rhutsberg. A[dehng Farrnat ~,logte. Leu~'en (B¢lgtum)
(Received 17 April 1990)
Key words: Peroxisome:Oxidase; Dicarboxylicaminoacid: Zellweg,:rsyndrome;Excitotoxicity: (Humanliver) By means of subcellular fractionation D-aspartate oxidase was shown to be Ioc',dized in peroxisomes in rat and human liver. The oxidase from both sources was most active on D-aspartate and N-methyI-D-aspartate. In different rat tissues, the highest enzyme activity was found in kidney, followed by liver and brain. In these tissues, oxidase activities became detectable 1-4 days after birth, reaching adult values after 4 weeks. Analysis of liver samples from patients with Zellweger syndrome, a generalized peroxisomal dysfunction, demonstrated no significant deficiency of this particular oxidase.
Introduction Through the initial studies of Still and co-workers [1,2] the presence in mammalian renal tissue cf separate oxidases acting on neutral (or basic) and on dicarboxylic D-amino acids was established. These oxidases are generally referred to as 0-amino acid oxidase and 0aspartate oxidase, respectively (see Ref. 3 for a review). The latter one has been found mainly in kidney, liver and brain of mammals and catalyzes the deaminative oxidation of D-aspartic acid, and less effectively of 0-glutamate and D-amino adipate [3]. Whereas 0-amino acid oxidase is since long known to be associated with (micro)pcroxisomes in different tissues (see Ref. 4), the subcellular distribution of 0-aspartate oxidase has only recently been investigated. Thus, Zaar et al. [5] were able to show a pcroxisomal localization of D-aspartate oxidas¢ in rat, bovine and sheep kidney cortex. The presence of this enzyme in purified rat liver peroxisomes was also demonstrated. Until now no data have been published on the localization of 0-aspartate oxidase in human tissues. Such data might be of interest with regard to peroxisomopathies (see Ref. 6 for a review), a newly recognized group Abbreviations: EDTA. ethylencdiaminetetraacetate: D'rl', dithiothreitol; FAD, flavin¢-adenine dinucleotide; NMDA. N-methyl-~ aspartate; Hepes. 4q2-hydroxyethyl)-I-pipcrazinecthanesulfonicacid; FMN. flav/ne mononucleolide. Correspondence: P.P. Van Veldhoven, K.U. Leuven - Campus Gasthuisbcrg Department of Pharmacology, Herestraat, 13-3000 Leuven. Belgium.
of metabolic disorders in which one or more peroxisomal enzymes are missing or malfunctioning. Indeed, 0-aspartat¢, which is present in appreciable quantities in fetal tissues and especially in fetal brain [7], is a potent agonist of the excitatory amino acid receptors, ,,dfich play a role in brain function and development (see Refs. 8-10 for reviews). The generalized peroxisomopathies such as the Zellweger syndrome, in which no recognizable peroxisomes can be found, are characterized by a severe neurological dysfunction and by disturbances in brain development [6]. We, therefore, decided to investigate the subcellular distribution of 0-aspartate oxidase in human and rat liver and we also measured the enzyme in liver tissue from patients with peroxisomopathies. Materials and Methods N-Methyl-0-aspartic acid, 0-glutamic acid and 0amino adipic acid were obtained from Aldrich Europe, Brussels (Belgium); 0-aspartic acid, sodium benzoate. L-tartaric acid. and homovanillic acid from Janssen Chimica, Beerse (Belgium). FAD, Triton X-100, and peroxidase were from Boehringer, Mannheim (F.R.G.) and Percoll from Pharmacia, Uppsala (Sweden), 0-Homocysteic acid, meso-2,3-diaminosuccinic acid, 0-hydroxy glutaric acid, racemic cis-2.3-piperidinedicarboxylic acid, and mesotartaric .~cid were from Sigma, St. Louis, MO (U.S.A.), and lauryldimethylammonium oxide from Serva, Heidelberg (F.R.G.). Flavine mononucleotide was obtained from Fluka, Buchs (Switzerland).
204 N-AcetyI-D-aspartate was prepared as follows. DAspartic acid (5 retool) and anhydrous triethylamine (12 retool) were added to 10 ml anhydrous methanol. While stirring. 5 ml acetic anhydride was added dropwise. and the mixture allowed to react overnight. After neutralization of the reaction mixture with concentrated ammonium hydroxide, methanol was removed by rotary evaporation. The residue was dissolved in water, neutralized with ammonium hydroxide and lyophilized. This step was repeated twice. No free amino groups could be detected in the residue by means of trinitrobenzenesulfonic acid. Male Wistar rats, maintained on a standard chow diet, were killed by decapitation. The liver, or other tissues, were removed and homogenized in 0.25 M sucrose/1 mM EDTA (pH 7.2)/1 mM DTT/0.1% (v/v) ethanol (SEDE). Liver homogenates were fractionated as described in Ref. 11 and the light mitochondrial fraction was further subfractionated in self-generating Percoll density gradients [11]. Human liver was obtained at autopsy or as excess tissue after transplantation of livers from adult donors in children. A detailed description of the procedures was submitted to the university hospital Ethics Committee. which granted its approval. A portion of the tissue from a donor liver was cut into small pieces, homogenised in SEDE, containing 5 ~tM FAD, and fractionated as described for rat liver. The remaining tissue and tissues obtained at autopsy were divided into smaller portions, quickly frozen in liquid N 2 and stored at - 8 0 ° C . Marker enzymes were measured as described before [11 ]. 5'-Nucleotidase was measured as follows. 50 .al of the subcellular fractions, appropriately diluted, were mixed with 200 .al of reaction mixture containing 5 mM AMP/12.5 mM MgCI2/12.5 mM L-tartrate/125 mM KCI/62.5 mM Hepes buffer (pH 7.5)/0.25% ( w / v ) lauryldimethylammonium oxide. After 20 rain incubation at 37°C, reactions were stopped with 1 ml of 10% (w/v) trichloroacetic acid. After removal of denatured proteins by centrifugation, 0.5 ml of the supernatants was mixed with 1 ml of 0.45 M H2SO4/0.375% ( w / v ) ammoniumheptamolybdate • 4H20/1.5% (w/v) ascorbic acid and placed at 45"C for 30 rain. After cooling the absorbance of the mixtures was read at 820 nm. Incubations in the absence of subst~ate served as blanks. Oxidases were measured according to Ref. 12, modified as follcws. Samples ,,f 40 .al, diluted in SEDE, were mixed with 10 .al of 100 .aM FAD (or 25 .aM FMN in the case of glycolate oxidase)/25 mM NaN.~ and incubated at 0 ° C for 5 rain in order to inactivate catalase and saturate the oxidases with cofactor. To these samples 0.15 ml of reaction mixture, consisting of 83.3 mM pyrophosphate buffer (pH 8.5)/0.83 mM Nethylmaleimide/16.7 U / m l peroxidase/l mM homo-
vanillic acid/0.083% ( w / v ) Triton X-100. was then added. After another 5 min at 0 ° C , needed to alkylate the DTT and other interfering sulfydryl compounds, reactions were started by adding 50 p.I of 50 mM neutralized substrate, followed by an incl,~ation at 37°C in subdued light. After 30 rain (glycolat¢ oxidase) or 60 min (D-amino acid and D-aspartate oxidase), reactions were stopped with 50 .al of 12c~ ( w / v ) perchloric acid. Denatured proteins were removed by centrifugation and an aliquot (0.2 ml) of the supernatant was diluted 15-fold with 0.5 M carbonate buffer (pH 10.7). containing 10 mM EDTA, before reading the fluorescence of the homovanillic acid dimer [12]. The slow increase in fluorescence, observed after alkalinization of the acidified supernatants, could be almost totally suppressed by adding EDTA to the carbonate buffer. Substrates used were D-aspartate (D-aspartate oxidase), Dproline or D-alanine (D-amino acid oxidase) and glycolate (glycolate oxidase). Fluorescence readings were corrected by means of appropriate blanks and standardized with known amounts of peroxide added to blank assay mixtures. Tissues homogenates were sufficiently diluted to obtain at least 90% recovery of exogenously added peroxide. Results and Discussion Subcellular fractionation of rat liver revealed that D-aspartate oxidase is mainly found in the light mitochondrial and cytosolic fractions and that its activity follows closely that of catalase, a peroxisomal marker (Fig. IA). When a light mitochondrial fraction was further separated on a Percoll gradient, again the distribution patterns of catalase and D-aspartate oxidase coincided (Fig. IB). These data are in agreement with those obtained by Zaar et al. [5] for rat kidney cortex. The enrichment of D-aspartate oxidase (and other peroxisomal enzymes) in the light mitochondrial fraction from human liver was rather low. This is partly due to the gently conditions used to homogenize the liver, which, although satisfactory for rat liver, were not severe enough to disrupt all cells in the human liver. Indeed, a high percentage of all marker enzymes was found in the nuclear fraction (Fig. 2A). The amount of lactate dehydrogenase, a cytosolic marker, present in the nuch:ar fraction indicates that approx, one fifth of the cells remained intact..Secondly, during homogenization peroxisomal matrix enzymes were released to a higher extent in human liver than in rat liver (Fig. 2A). This phenomenon has been observed by others too [13]. in how far this is a consequence of the non-optimum preservation condition of the donor liver is not known. As indicated by the relative specific activity, more catalase was found in the cytosolic fraction than other peroxisomal matrix enzymes. Whether this is due to differential release, as reported before in rat liver [14,15],
205 o r d u e t h e p r e s e n c e o f a c y t o s o l i c f o r m o f c a t a l a s e is n o t clear. D e s p i t e t h e s e d i f f i c u l t i e s , t h e a s s o c i a t i o n o f Daspartate oxidase with peroxisomes became evident
L
2-
r, i1
iLl
I
:I
¼ L
g 2~ 1L
n
I
E
:
R
7
....
i:-s
•
u
i i
---~
8
il ° N
MLP
I
a: 3r
I
G
H
I i
F I
: i I 2I~
L_2:'LA
S
N~.P
~/~ 0 I: TOTALPROTEIN
S
%
of
t0rAL
8
'
C
•
0
F
-
~
-
H
PROTLIN
•
B
~,o
-
:,
•
~.o.
~i
2o,
:',
-~ ~o:
E
:
!'"
b
~.0.
i
•
-
.
J
~3
•
!' -
;
. •
zo.
•
~o
i
',:
!
.
.
;
. i" 4
. :
:
',...... "8....... ~ ........ :" . . . . . . . :::"~" ..... L~=::.:.: ...... FRACTION
i
7
ii. FRACTION
NUMBER
Fig. I. Subcellular distribution of D-aspartate oxidas¢ in rat liver. (A) Rat liver was fractionated into a nuclear (N). a heavy nfitochondrial (M), a light mitochondrial (L). a microsomal (P) and a soluble fraction (S) and marker ¢nzym~-s were measured in each fraction. A: 5'-nuclcondas¢ for plasma membrane; B: glutamate dehydrogenase for mitochondria: C: glucosc-6-phosphatas¢ for endoplasmic reticuhim; D: catalas¢ for peroxisomal matrix protein: E: acid phosphatase for lysnsomes" F: D-aspanate oxidas¢. Results are expressed as relative specific activities versus cumulative pcrccmage of protein. Recoveries varied between 86-102%. (B) The light mitochondrial fraction. shown in panel A was suhfraclionated by isopycnic centrifugation ~n an iso-osmofic Percoll gradient. The fractions were analyzed for protein (A), acid phnsphatas¢ (B). glutamate dehydrogenas¢ (C). glucose-6-phosphatas¢ (D). catalas¢ (E) and D-aspartate oxidas¢ (F). Results are expressed as percentages of total recovered gradient activity or content, present in each fraction. Recoveries varied between 81-123%. Fraction i and 15 represent the fractions of highest and lowest density, respectively.
NUMB[R
Fig. 2. Subcellular distribution of D-aspartate oxidas¢ in human liver• (A) Human liver was fraetionated as described in the legend to Fig. I. Fractions were analysed for 5'-nuclcotidas¢ (A), glutamate dehydrogenase (B). ~ i a pnusphatas¢ (C), catalas¢ (D). glucosc-6-phosphatase (E). lactate dehydrogenase (F: marker for cytosol), glycolate and D-amino acid oxidase (G and H, respectively markers for peroxisomal matrix proteins) and D-aspartare oxidas¢ (1~ Results are expressed as relative spgch~ic activities versus cumularive percentage of protein• Recoveries varied between 84-107%. (B) The light mitochondrial fraction was further fraetionated on a Percoll gradient as described in the legend to Fig. 1. and analyznd for protein (A). acid pho~phatas¢ (B). glutamate dehydrogenase (C). glacose-6-phosphatas¢ (D), catalas¢ (E). glycolal¢ oxidase (F). D-amino acid oxidase (G) and D-aspartate oxidas¢ (H). Results are expressed as percentages of the total recovered gradient activity or content, present in each fraction. Recoveries varied between 92 -t28%. Fractions I and 16 represent thc fractions of highest and lowest density, respectively.
w h e n t h e light m i t o c h o n d r i a l f r a c t i o n w a s s u b j e c t e d to Percoll d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n . T h e a c t i v i t y followed that of the other pcroxisomal enzymes catalase,
206 D-amino acid oxidase and glycolate oxidase (Fig. 2B). In both species, hepatic D-aspartate oxidase was not sensitive to 10 m M sodium benzoate, a selective inhibitor of D-amino acid oxidase [16]. On the other hand, 20 mM mesotartrate, an inhibitor of rabbit and pig kidney D-aspartate oxidase [17,18] inhibited the enzyme by 85% (data not shown). A comparison of different rat tissues revealed the highest D-aspartate oxidase activity in kidney, followed by liver and nervous tissues (Table I), in agreement with the data reported by Y a m a d a et al. [19]. According to Barker and H o p k i n s o n [20], in man D-aspartate oxidase is present mainly in liver and kidney, whereas traces of activities are found in small intestine and lung, but surprisingly v,ot in brain. On the other hand, o-asparrate oxidase activity has been d e m o n s t r a t e d in mouse I191. hog and cat brain [211. Table iI shows the activity of D-a,~partate oxidase with different dicarboxylic D-amino acids and their analogues for rat kidney, rat liver, and h u m a n liver. The three enzymes show similar substrate specificities, except for N-methyl-D-aspartate ( N M D A ) . While the hum a n liver oxidase is most active on o-aspartate, the rat enzymes show a slight preference for N M D A . A negative charge in the side chain, preferentially carried by a carboxyl group, is an i m p o r t a n t feature of possible substrates recognized by the enzyme. The distance between the two carboxyl groups seems to be critical (about 3 /~) since only D-aspartate and N M D A are oxidized to a significant extent. This agrees well with the conclusion derived from inhibitor studies performed on the octopus enzyme [22]. Similar findings have been reported with the oxidase from other tissues [5,17,18,23]. In contrast to methylation, acetylation of the a m i n o group results in an inactive c o m p o u n d , suggesting the importance of a positive charge on the nitrogen atom. The role of this a m i n o group is further stressed by the TABLE I Tissue distribution of o-aspartate oxidase in rat
n-Aspartate oxidase was measured in tissue homogenates as described in the Materials and Methods section in the presence of 10 mM sodium benzoate. Activities are expressed in mO (nmol substrate oxidized per min) and are the means4-S.E.M, for the number of experiments indicated in parentheses. Tissue Kidney Liver Cerebrum Cerebellum Lung Muscie Heart Spleen Small intestine Testis
mU/g tissue 130.2 ±10.6 (7) 37.6 :t: 1.8 ( 1 1 ) 11.3 4. 1.4 (6) 8.354- 0.74 (4) 6.39± 1.87 (4) 4.97± 0.98 (4) 4.494- 0.97 (4) 3.29± 0.82 (4) 1.81 ± 0.63 (4) 1.78+ 0.85 (4)
mU/mg protein 1.01 +0.11 (7) 0.204+0.010(9) 0.105±0.018(6) 0.080:t:0.003(4) 0.060+0.017(4) 0.0654-0.018(4) 0.044±0.011 (4) 0.0224-0.006(4) 0.020±0.006 (4) 0.024±0.012(4)
TABLE I1 Substrate specificiO' of D-aspartate oxidg~e in rat and human tissues
I.,-Aspartate oxidase activity was measured as described in the Materials and Methods section in the presence of I0 mM sodium benzoate. The substrales were used at a final concentration of 10 raM. Results are expressed as percentage of the activity observed with D-aspartate and are means for two to three experimenls. ( - : not detectable; N.M.: not measured) Substrate D-Aspartate D-Glutamate D-Amino adipate N-Methyl-D-aspartate Meso-2,3-diaminosuccinate D-Homocysteate N-Acetyl-P-aspartate o-Hydroxyglutarate cts-2,3-piperidinedicarboxylate
Rat liver 100 17.4 6.2 134 29.5 2.0 8.3
Rat kidney 100 6 7 1.6 118 4.4 0.2 12.9
Human liver a 100 1.6 0.7 63.6 25.9 0.7 NM.
• The substrate specificity in a liver sample of a Zellweger patient was similar to that observed in control subjects.
inhibitory action of dicarboxylic c o m p o u n d s in which this substituent is replaced by a hydroxy group, like malate, tartrate and h y d r o x y g l u t a r a t e [17,22,23]. In analogy with o - a m i n o acid oxidase which is most active on n-proline [24], we tried a cyclic acidic a m i n o acid, cis-2,3-piperidine dicarboxylic acid as substrate. Like N M D A , this synthetic c o m p o u n d interacts w i t h the N M D A receptor [251. A l t h o u g h the position of the carboxyl groups a n d the charged N - a t o m are c o m p l e t e l y s u p e r i m p o s a b l e in the D-forms of a s p a r t a t e a n d the heterocyclic c o m p o u n d , a racemic m i x t u r e of the latter one was only poorly recognized by the rat enzymes a n d not at all by the h u m a n liver oxidase. A n inhibition by the L-form c a n n o t be excluded, but in general the L-forms are neither oxidized, nor act as inhibitors [17]. In contrast to neutral o - a m i n o acids, which are only rarely found in a n i m a l tissues, appreciable levels of free l > a s p a r t a t e have been d e m o n s t r a t e d in different tissues (brain, pituitary, liver, a n d kidney) a n d blood of rodents, and also in h u m a n b l o o d [7]. Free n - g l u t a m a t e could not be detected, however [7]. Highest D-aspartate levels were found in fetal tissues in which they decrease rapidly after birth [7]. W e therefore studied the ontogenie development of D-aspartate oxidase in rat liver, kidney and cerebrum (Fig. 3). The oxidase b e c a m e detectable early after birth in these tissues a n d the activity rose rapidly to adult values in 6 weeks. This rise in activity fits nicely with the p o s t n a t a l decrease in tissue D-aspartate levels [7]. Apparently, a low D-asparrate oxidase activity was already present in liver d u r i n g the last week of gestation. Generally, peroxisomal enzyme activities are absent or very low in fetal rat liver and start to rise only after birth. Peroxisomal profiles, containing catalase, b e c o m e visible from d a y 14 of
207
I
i 120L ? ' ->:
~ ~0~ :
i
/a ~;"
= I '
i ,
i~
I
,_
'"
,
i ~ '
'
~ . ' , / &
