ELSEVIER Biochimica et Biophysica Acta 1207 (1994) 201-207

Btt Biochi~ic~a et Biophysica A~ta

Isolation and properties of mitochondrial aspartate aminotransferase from red muscle of grey mullet, Mugil auratus Risso

Sini~a Petrovi6 *, Mirjana Krajnovi6-Ozreti6, Bartolo Ozreti6 Center for Marine Research, Ruder Bogkovi~ Institute, 52210 Rocinj, Croatia

Received 30 August 1993; revised 11 April 1994

Abstract

Following the chromatographic separation of the grey mullet (Mugil auratus Risso) red muscle extract, two fractions with aspartate aminotransferase activity were detected. One of the anticipated enzymes was purified to homogeneity. The isolated enzyme was a dimeric protein composed of identical subunits with the overall M r of about 65 000. It consisted of three electrophoretically distinct subforms with isoelectric points at pH 8.50, 8.70 and 8.85, respectively. The Michaelis-Menten constants of the substrates L-aspartate and 2-oxogluta- rate were estimated to be 0.29 ___ 0.012 mM and 0.45 + 0.016 mM, respectively. For the reverse reaction, the K m for L-glutamate was 8.57 + 2.1 mM and for oxaloacetate it was 0.13 ___ 0.035 mM. The inhibition of the isolated enzyme by hydroxylamine was of a mixed linear noncompetitive type for L-aspartate as a substrate, whereas with 2-oxoglutarate hyperbolic uncompetitive inhibition was observed. The inhibition by aminooxyacetic acid and D,L-glyceraldehyde 3-phosphate was of a mixed linear noncompetitive type with respect to L-aspartate and 2-oxoglutarate. The isolated enzyme was slightly affected by maleate and succinate and no effects were produced by adipate. According to its subcellular distribution, susceptibility to inhibitors, molecular and catalytic properties the isolated enzyme belonged to the mitochondrial form of aspartate aminotransferase.

Key words: Aspartate aminotransferase; Mitochondrion; Enzyme purification; Enzyme characterization; (M. auratus Risso)

I . Introduction

Aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) has been well characterized in different vertebrate species [1-3] where it exists in two molecular forms, one localized in the cytoplasm and the other in mitochondria. In contrast, some bacteria and fungi contain only one enzyme form [4]. In fish, both cytosolic and mitochondrial AAT were detected in rainbow trout liver [2], but they have not yet been isolated in homoge- nous form.

AAT isoenzymes are present in various organs of verte- brate species, where they take part in the general metabolism of aspartate and glutamate. The mitochondrial form contributes to the aspartate-malate shuttle and sup-

Abbreviations: AAT, aspartate aminotransferase; AOA, aminooxy- acetic acid; glyceraldehyde 3-P, D,L-glyceraldehyde 3-phosphate; IEF, isoelectric focusing; mAAT, mitochondrial aspartate aminotransferase; PAGE, polyacrylamide gel electrophoresis; pyridoxal 5-P, pyridoxal 5'- phosphate.

* Corresponding author. Fax: +385 52 813496.

0167-4838/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0167-4838(94)00066-P

plies mitochondria with intermediates of the citric acid cycle [5]. The role of AAT isoenzymes as diagnostic tools in certain human diseases is well recognized [6]. In fish AAT was used as a comparable biochemical indicator of tissue damage and liver distress [7-11]. In spite of its important role in metabolism and relevance in toxicologi- cal studies, little work has been done on the characteriza- tion of AAT in fish [2].

In mammals AAT is the most active in heart tissue, while in fish the red muscle exhibits the highest AAT specific activity [11]. As it is generally known, red muscle in fish performs slow but enduring swimming, mainly powered by oxidative phosphorylation. With respect to the metabolism of carbohydrates and lipids, fish red muscle also resembles some liver functions [12]. The study of the red muscle AAT isoenzymes, involved in different metabolic pathways, can improve the understanding of diverse physiological roles of that muscle. However, a better understanding of fish AATs will also help to asses the usefulness of these enzymes as biochemical indicators of stress.

In this study, two AAT forms were detected in the red

202 S. Petrovi6 et al. / Biochimica et Biophysica Acta 1207 (1994) 201-207

muscle of grey mullet. One of them, which corresponds to the mitochondrial isoenzyme form, was isolated and char- acterized.

2. Materials and methods

2.1. Materials

Adult grey mullets (Mugil auratus Risso) were caught by fishermen along the west Istrian coast near Rovinj (north Adriatic, Croatia). Fish were accommodated in aer- ated, continuous flow seawater aquaria.

CM Sephadex C-50, Sephacryl S-200 Superfine, Poly- buffer exchanger PBE 118, Mono Q HR 5 / 5 column, ampholytes, molecular weight markers for PAGE and gel filtration, pI markers for IEF, PhastGel homogenous and gradient media were purchased from Pharmacia LKB, Uppsala, Sweden. DEAE Cellulose 23 SN, Servalyt Pre- cotes, and dialysis tubing were obtained from Serva, Hei- delberg, Germany. Hydroxyapatite, oxaloacetic acid and L-glutamic acid came from Calbiochem, Luzern, Switzer- land. Ultrafiltration cells and a Centricon-10 microconcen- trator system were products of Amicon, Oosterhout, The Netherlands. L-aspartic acid, 2-oxoglutaric acid, maleic acid, succinic acid, adipic acid, pyridoxal 5'-phosphate, O,L-glyceraldehyde 3-phosphate diethylacetal, Fast Blue BB salt, aminooxyacetic acid, NADH, malic dehydro- genase and 2-oxoglutarate dehydrogenase were supplied by Sigma, Munich, Germany. Hydroxylamine was from Merck, Darmstadt, Germany. All other chemicals used were of analytical grade.

2.2. Enzyme assays and protein content

The activity of AAT in the forward direction was measured by the method of Karmen [13] which was opti- mized for measurements in plasma and tissues of grey mullet [10]. The final, 1 ml reaction mixture contained 50 mM potassium phosphate buffer (pH 7.8), 40 mM L- aspartate, 15 mM 2-oxoglutarate, 0.15 mM NADH, 7.5 U of malic dehydrogenase and the appropriate amount of the enzyme. The decrease of absorbance due to the oxidation of NADH was followed at 340 nm and 25°C in 1 cm light path thermostated cells of the Cecil model 6700 spectro- photometer. The activity in the reverse direction (aspartate forming) was measured by coupled assay of McCormick et al. [14]. The enzyme activity was expressed in units (U) which correspond to the synthesis of 1 p, mol of product per min under the specified conditions. Branched-chain amino acid aminotransferase activity was determined as described by Korpela and Saarinen [15]. Tyrosine amino- transferase and phenylalanine aminotransferase activities were assayed by the method of Chang-Won and Des- mazeaud [16]. For the measurement of alanine aminotrans- ferase activity, the method of Bergmeyer and Bernt [17]

was used. The AAT activity in polyacrylamide gels was identified by incubation of gels in 50 mM Tris-HC1 buffer (pH 7.5), containing 50 mM L-aspartate, 12 mM 2-oxo- glutarate and 1.5 mg/ml Fast Blue BB salt at 25°C.

During chromatographic separation, the protein concen- tration was monitored by measuring the absorbance at 280 nm. The protein concentration in samples was determined by the dye-binding method of Bradford [21] with bovine serum albumin as a standard.

The kinetic and inhibition constants were determined by using the coupled assays described above. The values for initial velocities were averages of at least four determina- tions and they were fitted to the Lineweaver-Burk plot using the least squares method. The abscissa and ordinate intercepts and slopes computed from the Lineweaver-Burk plots, were then replotted following the methods of Velick and Vavra [18], Cleland [19] and Segel [20]. The intercepts (_+ S.E.) of these replots represent true kinetic or inhibition constants. All calculations and statistical analyses were done using a commercial PC program (SYSTAT 5.01 for Windows, SYSTAT Evanston, IL, USA).

2.3. Molecular weight and pI determination

The relative molecular weight (M r ) of the native en- zyme was estimated by gel filtration on a Sephacryl S-200 column (95 × 1.2 cm) calibrated with molecular weight markers (LMW calibration kit, Pharmacia LKB) and eluted at a flow rate of 5.6 ml /h with 50 mM potassium phos- phate buffer (pH 7.5), containing 100 mM NaC1. The M r was also determined by the PhastSystem AGE using the PhastGel gradient 8-25% plates under nondenaturing con- ditions (pH 4.2) (PhastSystem Separation Technique File No. 300): The enzyme subunits were detected by SDS- PAGE on PhastGel homogenous plates (7.5%) (PhastSys- tern Technique File No. 111). For the SDS-PAGE, samples were treated with 2.5% (w/v) SDS and 5% (v /v) /3-mer- captoethanol for 5 min at 100°C. The isoelectric point was estimated by IEF on the FBE-3000 apparatus (Pharmacia LKB) using ultrathin polyacrylamide gel plates 3-10 (Servalyt recotes) and pI markers for pH range 3-10 (Pharmacia LKB). Proteins on the gels were stained by Coomassie brilliant blue G-250.

2.4. Differential centrifugation

Mitochondria were prepared from fresh, unfrozen red muscles of grey mullets according to the procedure of Moon and Johnston [22]. The mitochondrial fraction was then sonicated (5 periods of 10 s at 25/zm amplitude using the MSE Soniprep 150 desintegrator) and centrifuged (60 min at 100000 X g) to obtain soluble mitochondrial en- zymes. The supematant was collected, concentrated and used in IEF experiments. Lactate dehydrogenase and gluta- mate dehydrogenase were used as cytoplasmic and mito- chondrial marker enzymes, respectively.

s. Petrovi£ et al. / Biochimica et Biophysica Acta 1207 (1994) 201-207 203

2.5. Enzyme purification

A typical purification scheme of the mitochondrial AAT from grey mullet red muscle is described below. All separation-steps were carried out at 4°C.

the enzyme was eluted using a linear gradient (1000 ml) of 0 to 300 mM NaC1 in the same buffer. The active fractions were pooled, concentrated by ultrafiltration and dialyzed against 15 mM Tris-HC1 buffer (pH 8.8) containing 0.025 mM pyridoxal 5-P.

2.6. Preparation of homogenate 2.10. DEAE Cellulose 23 SN chromatography

Red muscle from freshly sacrificed grey mullets was cut into small pieces and homogenized (Polytron) in four volumes of cold 50 mM potassium phosphate buffer (pH 7.0), containing 1 mM EDTA, 1 mM 2-oxoglutarate and 0.05 mM pyridoxal 5-P. Fats were removed in a separating funnel and the homogenate was clarified by centrifugation for 20 min at 1000 × g. To ensure a complete solubiliza- tion of mitochondrial enzymes, the homogenate was soni- cated for 4 periods of 15 s at the maximum energy output (MSE sonifier), and then centrifuged for 20 min at 12000 x g .

2. 7. Ammonium sulfate fractionation

Supernatant from above (16524 mg protein) was frac- tionated by addition of solid ammonium sulfate, and the precipitated protein fraction (45-85% w / v (NH4)2SO 4 saturation at 0°C) was collected by centrifugation (10000 x g, 30 min). The precipitate was dissolved in a small volume of 10 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA and 0.05 mM pyridoxal 5-P, and dialyzed against the same buffer. The dialyzed solution was clarified by centrifugation. The resulting sample (9417 mg protein) was separated into four batches and stored at 4°C.

2.8. Hydroxyapatite chromatography

The batches (2354 mg protein each) were successively loaded on a hydroxyapatite column (5 X 6.5 cm) equili- brated with 10 mM potassium phosphate buffer (pH 7.0, 1 mM EDTA). The column was washed with the same buffer and then with 80 mM potassium phosphate buffer (pH 7.0, 1 mM EDTA). A material, subsequently eluted with 200 mM potassium phosphate buffer (pH 7.0), con- taining 1 mM EDTA (Fig. 1 Fraction II), was recovered as a 50-85% (w/v ) ammonium sulfate precipitate and sub- jected to further purification.

2.9. CM Sephadex C-50 chromatography

The precipitate collected from four batches of the previ- ous step was then dialyzed against 10 mM potassium phosphate buffer (pH 7.1) containing 0.025 mM pyridoxal 5-P. The enzyme solution (260 mg protein) was applied to a CM Sephadex column (2.6 x 25 cm) pre-equilibrated with the same buffer without pyridoxal 5-P. After initial washing with 10 mM potassium phosphate buffer (pH 7.1),

The enzyme solution (123 mg protein) was then loaded on a deae cellulose column (2.6 X 24 cm) pre-equilibrated with the dialysis buffer from above without pyridoxal 5-P. The column was washed with 15 mm Tris-HCl buffer (pH 8.8) and eluted with a linear gradient of NaC1 (0 to 200 mm in a total volume of 1000 ml) in the same buffer.

2.11. Polybuffer exchanger PBE 118 chromatofocusing

The AAT active fractions were pooled, concentrated (17.5 mg protein) and further purified by chromato- focusing on a PBE 118 column (1.0 X 17.5 cm) previously equilibrated with 25 mM triethylamine-HC1 buffer (pH 11.0). The pH gradient was developed with 155 ml of Pharmalyte 8.0-10.5 (1:45 dilution) adjusted to pH 8.0. The active fractions, eluted in the pH range 8.6-9.0, were then concentrated and passed through a Sephacryl S-200 column pre-equilibrated with 50 mM Tris-HC1 buffer (pH 9.0) to remove ampholytes.

2.12. Fast protein liquid chromatography (FPLC)

Some minor impurities, still present in the enzyme preparation, were eliminated by FPLC on a Mono O column. After sample application (1.56 mg protein) and washing with 50 mM Tris-HCl buffer (pH 9.0), the column was eluted with a linear gradient (50 ml) of 0 to 200 mM NaC1 in the same buffer. The fractions with AAT activity were collected, concentrated by a Centricon-10 microcon- centrator, and the buffer was changed to 25 mM potassium phosphate (pH 7.5) containing 0.025 mM pyridoxal 5-P and 50% glycerol (v/v) . The enzyme solution was stored at 20°C until used.

3. Results

The proteins extracted from grey mullet red muscle and precipitated with ammonium sulfate were separated by column chromatography on hydroxyapatite. Two protein fractions with AAT activity were eluted from the column as illustrated in Fig. 1. The analysis of these fractions by IEF revealed the presence of an AAT with pI in the acidic region (Fraction I) and an AAT with pI in the basic region of the gel (Fraction II).

The enzyme from Fraction II was further purified, at least 260-fold with respect to the crude extract, as summa- rized in Table 1. The enzyme preparation thus obtained

204 S. Petrovi~ et aL / Biochimica et Biophysica Acta 1207 (1994) 201-207

1.5

E t -

O oo 1.0

0 0 ¢,-

0.5 O

,<

0.0

I ~ ~ II

A B

1000

Volume (ml)

15 I

O

to E

g # ._~ ~6

5 ~ 0

E

i

2000

Fig. 1. Hydroxyapatite column chromatography of one dialyzed ammo- nium sulfate batch (2354 mg protein). The column was washed with 10 mM potassium phosphate buffer (pH 7.0; 1 mM EDTA) and eluted by a stepwise gradient as indicated by arrows at a flow rate of 70 ml /h . (A) 80 mM potassium phosphate buffer (pH 7.0; 1 mM EDTA); (B) 200 mM potassium phosphate buffer (pH 7.0; 1 mM EDTA); (O) absorbance at 280 n m ; ( * ) AAT activity. The bars with Roman numerals denote collected active fractions.

gave a single protein band upon PAGE under native conditions and was judged to be homogeneous. To confirm the origin of the purified enzyme, mitochondria from red muscle were isolated as described and the mitochondrial enzymes thus obtained were subjected to IEF in parallel with the purified enzyme. After specific staining the AAT activity in both cases was detected in the basic region of the gel (Fig. 2).

3.1. Molecular properties

The relative molecular mass of the isolated AAT was determined by gel filtration on Sephacryl S-200 column

Table 1 Purification of mAAT from grey mullet red muscle

Purification step Volume Total Total Specific Yield (ml) protein activity activity (%)

(mg) (U) (U/mg)

Homogenate a 1600 16524 20380 1.2 - 45-85% (NH4)2SO 4 260 9417 18750 2.0 - Hydroxyapatite

fraction I 1220 3231 7754 2.4 - fraction II b 47 260 6420 24.7 (100)

CM Sephadex 64 123 5048 41.0 79 DEAE Cellulose 5 17.5 2314 132.2 36 Chromatofocusing

Sephacryl S-200 15 1.56 342 219.2 5 FPLC 0.5 0.63 198 314.3 3

The yield is based on the activity of Fraction II eluted from the hydroxyapatite column, since earlier steps contained cytoplasmic isoen- zyme activity as well. a Obtained from 520 g of the grey mullet red muscle wet weight. b Fraction used for further purification.

pl 1

9 . 3 0 -

8 . 6 5 - 8 . 4 5 - W 8 . 1 5 -

2 3 4

7 . 3 5 - - -

6.85 . . . . . . .

6 . 5 5 - " ~

5 . 8 5 -

5 . 2 0 -

4 . 5 5 -

3 . 5 0 -

Fig. 2. IEF of the purified mAAT and mitochondrial extract of the red muscle. Lane 1, p l markers: trypsinogen (pl 9•30), lentil lectin-basic band (pl 8.65), lentil lectin-middle band (pl 8.45), lentil lectin-acidic band (pl 8.15), horse myoglobin-basic band (pl 7.35), horse myoglobin- acidic band (pl 6.85), human carbonic anhydrase B (pl 6.55), bovine carbonic anhydrase B (pl 5.85), /3-1actoglobulin A (pl 5.20), soybean trypsin inhibitor (pl 4.55), amyloglucosidase (pl 3.50). Lanes 2 and 3, purified enzyme 6.5 /zg (314•3 U/mg). Lane 4, mitochondrial extract 126 /zg (17.4 U/mg). Lanes 1 and 2 were stained for proteins and lanes 3 and 4 for AAT activity. Details of the procedures are given in Section 2.

and the values obtained from two experiments were 63 000 and 65 800, respectively. M r estimated by gradient PAGE under nondenaturing conditions was 66 800. SDS-PAGE of pure denatured enzyme displayed the presence of only one protein band in the gel. Two separate determinations of M r by SDS-PAGE gave the values of 36600 and 37700, respectively. These results indicated that the native enzyme was composed of two identical subunits.

Three bands were identified by IEF at pH 8.50, 8.70 and 8.85, either when stained for protein or specifically for AAT activity (Fig. 2), based on three separate enzyme preparations•

The purified enzyme was stable in the pH range from 7.0 up to 10.5 and between 0-40°C, as determined after 1 h of incubation in buffers of different pH and at different temperatures, respectively. Above 40°C its activity was rapidly decreasing and at 55°C the remaining activity was nearly 3% of the control (at 25°C). Freezing reduced the activity for 30%, but the enzyme was stable for long periods at - 20°C if preserved in 50% ( v / v ) glycerol.

S. Petrovi6 et al. / Biochimica et Biophysica Acta 1207 (1994) 201-207 205

Glyceraldehyde 3-P (mM)

A 0.50 1 / 1.0 ~'.~'~ 0.06-°"°8-

°° 1 . / / o 0.30 , ,

0.0 0.25-

g ,

:>~ 0.15'

I I I I I I i I I I i ~ U'U~' -1.5 -0.5

/ -1.0 -0.5 0.0 0.5 1.0

Glyceraldehyde 3-P (mM)

/ C

I I I

4 -3 -2 -1 0 1 2 3 4 0.5 1.5 L-aspartate (mM) -1 Glyceraldehyde 3-P (mM)

Fig. 3. (A) Noncompetitive inhibition of the isolated enzyme by glyceraldehyde 3-P at various concentrations of L-aspartate. The enzyme and inhibitor were incubated in 0.9 ml of 50 mM potassium phosphate buffer (pH 7.4) for 15 min at 25°C, and the reaction was started by the addition of a 0.1 ml mixture of substrates and other assay components. The final concentration of 2-oxoglutarate was 2.5 mM, and of other assay components as it is stated in Section 2. (B, C) Secondary plots of slopes (Km/Vma x) and y-axis intercepts (1/Vma x) against the inhibitor concentrations. The horizontal axis intercepts of these replots gave the K i value and ot parameter, respectively, for the substrate L-aspartate.

3.2. Catalytic properties and inhibition studies

The isolated mAAT obeyed a 'Ping Pong Bi Bi' mecha- nism [23] in both directions of the transamination reaction. Michaelis constants for L-aspartate and 2-oxoglutarate were 0.29 + 0.012 mM and 0.45 + 0.016 mM, respectively, with Vma x of 5.46 + 0.2 mU for the forward reaction. In the direction of L-aspartate formation, the Vma x was 17.36 +__ 2.4 mU. The g m values were 8.57 + 2.1 and 0.13 + 0.035 mM for L-glutamate and oxaloacetate, respectively. No transamination activity was detected using L-alanine, L- phenylalanine and L-leucine as amino-group donors,

whereas the transamination rate of L-tyrosine was only 1.7% of the rate obtained with L-aspartate.

The relative inhibitory effects of three dicarboxylic acids, acting as substrate analogues, on the mAAT from grey mullet were determined by coupled assay using 10 mM L-aspartate and 2.5 mM 2-oxoglutarate at pH 7.5. Maleate and succinate at 50 mM inhibited the enzyme by 43% and 24%, respectively, whereas adipate at the same concentration did not affect the enzyme activity.

Three other inhibitory substances were examined. The inhibition of the isolated enzyme by glyceraldehyde 3-P, with respect to L-aspartate and 2-oxoglutarate as substrates,

0.32 -

0.24 -

0.16 -

0.08

0.00

0.0

A Hydroxylamine (pM) 20.0 0.3

. ~ ~ 5 . o 7--

2 ' 8 v

0.1

i i i r i i i

0.5 1.0 1.5 2.0 -0.01 0.00 0.01 0.02 2-oxoglutarate (raM) ~ Hydroxylamine (mM)

Fig. 4. (A) Hyperbolic uncompetitive inhibition of the isolated enzyme by hydroxylamine when 2-oxoglutarate was a varied substrate. The final concentration of L-aspartate was 10 mM and other assay conditions were as described in Fig. 3. (B) The replot of the y-axis intercepts (1/Vma x) versus the inhibitor concentrations.

206 S. Petrovi~ et al. / Biochimica et Biophysica Acta 1207 (1994) 201-207

Table 2 Inhibition constants of tested substances for the isolated mAAT with respect to the substrates L-aspartate and 2-oxoglutarate

Inhibitor Substrate K i ( / z M + S.E.)

L-aspartate 2-oxoglutarate

Aminooxyacetic acid 0.7 _+ 0.08 1.4 ___ 0.09 ot =2.9, /3=0 a =1.6,/3=0

Glyceraldehyde 3-P 450.0 + 20 470.0 + 40 ot = 2.6, /3 = 0 a =1.4, /3=0

Hydroxylamine 1.0 + 0.2 5.8 + 0.45 ot = 4.2, 13 = 0 or,/3 = 0.44

The K i values, ot (affinity of the enzyme for the substrate when inhibitor is present) and /3 (the effect of inhibitor on Vma x) parameters, were derived by the method of Segel [20] from linear replots of slopes and intercepts of the Lineweaver-Burk plots against the inhibitor concentra- tions. The method of Cleland [19] was applied for a hyperbolic replot.

was of a mixed linear noncompetitive type [20]. It is illustrated for L-aspartate as varied substrate by plots in Fig. 3. The same type of inhibition was observed by A O A for both substrates and by hydroxylamine when L-aspartate was a varied substrate. Hyperbolic uncompetitive inhibi- tion was obtained by hydroxylamine for 2-oxoglutarate as a varied substrate (Fig. 4). The inhibition constants, a and /3 parameters of the examined substances are listed in Table 2.

4. Discussion

Fractionation of the grey mullet red muscle extract demonstrated the presence of two protein components with AAT activity. One of them had p I in the acidic region of IEF gel, a characteristic of cytoplasmic isoenzymes [1,2]. The other one had p I in the basic region, a well known property of the mitochondrial AAT isoenzymes [2]. The mitochondrial AAT was isolated in an electrophoretically homogenous form, although with a low yield due to the enzyme instability during the final steps of purification. Similar instability, but in the early stages of isolation and upon electrophoresis, was reported for mitochondrial AAT from rainbow trout liver [2].

The appearance of three isoelectric focusing bands of isolated mAAT was probably the consequence of enzyme changes occurred during the isolation procedure, since the fresh mitochondrial extract gave only one, the most basic band (Fig. 2). The grey mullet enzyme was eluted from a gel filtration column as a single, symmetrical peak, and the enzyme preparation was shown to be homogeneous by gradient PAGE under native conditions, indicating that subforms did not arise from the differences in their respec- tive molecular weights. These subforms might have been the result of deamidation process, as it was proposed by Williams and John [24] for AAT from pig heart. The subforms of AAT were detected by electrophoretic tech- niques for cytoplasmic and mitochondrial isoenzymes from

different species [1,2]. The M r of the grey mullet enzyme and its dimeric nature resembled the findings for other AAT enzymes [1,3,25,26].

The observed 'Ping Pong Bi Bi ' mechanism of transam- ination is typical for the aspartate aminotransferase en- zyme. The calculated K m of the isolated enzyme for L-aspartate and 2-oxoglutarate was comparable to the val- ues reported for the mitochondrial isoenzymes from human heart [26] and ribbed mussel, M o d i o l u s demissus , [14]. According to the affinities toward all four substrates, the isolated mAAT probably operated in the forward direction as it was already proposed for homotopic isoenzymes with similar kinetic properties [5,27,28]. The amino-donor specificity of the purified enzyme resembled that of verte- brate species [4].

The mitochondrial AAT from grey mullet was less sensitive toward maleate and succinate than the homotopic isoenzyme from pig heart [5]. The isolated enzyme was different from the cytoplasmic AAT forms that were sensi- tive to adipate and other 5-carbon dicarboxylic acids [5,18].

By affecting the AAT activity, glyceraldehyde 3-P might have contributed to the control of gluconeogenesis, as already proposed by Kopelovich et al. [29]. The inhibition of the isolated AAT by this glycolytic intermediate was of the same type and similar K~ values were obtained as those for mitochondrial isoenzymes from rat brain and liver [27,29].

The inhibition effect of hydroxylamine on AAT was known for the cytoplasmic isoenzyme from pig heart [18], whereas no data were available for mitochondrial forms of AAT. We have shown that hydroxylamine inhibited mAAT from grey mullet in an uncompetitive manner similar to that described by Velick and Vavra [18], when 2-oxogluta- rate was a varied substrate. The obtained hyperbolic replot revealed that hydroxylamine could neither completely block the binding of 2-oxoglutarate, nor the activity of the isolated enzyme. Both were reduced by 56%, as shown by the values of a and /3 parameters. When using L-aspartate as a varied substrate, mixed linear noncompetitive inhibi- tion was observed by hydroxylamine for grey mullet mAAT, in contrast to the competitive inhibition of cyto- plasmic isoenzyme reported by Velick and Vavra [18]. However, the inhibition data of cytoplasmic isoenzyme were analyzed by the Dixon plot that does not unambigu- ously distinguish these two types of inhibition, whereas the Lineweaver-Burk plot do [19,30].

The effect of AOA, the inhibitor of pyridoxal phosphate requiring enzymes, on the activity of the isolated AAT was comparable to that obtained for partially purified enzymes of ribbed mussel [31].

The obtained results suggest that grey mullet has the same AAT isoenzymic system as other vertebrate species thus far described. On the basis of its electrophoretic characteristics, the kinetic properties and the inhibition experiments, the purified enzyme belongs to the mitochon- drial form of AAT enzymes. According to the observed

S. Petrovi£ et al. / Biochimica et Biophysica Acta 1207 (1994) 201-207 207

kinetics and substrate affinities, the m A A T from grey

mul le t may be invo lved in the aspartate-malate shuttle, as

wel l as in the citric acid cycle. However , further invest iga-

tion is needed to elucidate its precise metabol ic role in

mi tochondr ia o f the fish red muscle .

Acknowledgments

The authors wish to thank Dr. Lj. Vitale for reading the

manuscr ipt and Dr. R. Batel for technical support. This

work was f inanced by the Scient i f ic Research Fund of the

Republ ic of Croatia.

References

[1] Braunstein, A.E. (1973) in The Enzymes (Boyer, P.D., ed.), Vol. 9, pp. 379-481, Academic Press, New York.

[2] Porter, P.B., Barra, D., Bossa, F., Cantalupo, G., Doonan, S., Martini, F., Sheehan, D. and Wilkinson, S.M. (1981) Comp. Biochem. Physiol. B 69, 737-746.

[3] Doonan, S., Barra, D., Bossa, F., Porter, P.B. and Wilkinson, S.M. (1981) Comp. Biochem. Physiol. B 69, 747-752.

[4] Kagamiyama, H., Kondo, K. and Yagi, T. (1984) Prog. Clin. Biol. Res. 144B, 293-302.

[5] Michuda, C.M. and Martinez-Carrion, M. (1970) J. Biol. Chem. 245, 262-269.

[6] Rej, R. (1978) Clin. Chem. 24, 1971-1979. [7] Racicot, J.G., Gaudet, M. and Leray, C. (1975) J. Fish Biol. 7,

825-835. [8] Casillas, E., Myers, M. and Ames, W.E. (1983) Aquat. Toxicol. 3,

61-78. [9] Casillas, E. and Ames, W. (1986) Comp. Biochem. Physiol. C 84,

397-400.

[10] Krajnovi6-Ozreti6, M. and Ozreti6, B. (1987) Dis. Aquat. Org. 3, 187-193.

[11] Krajnovi6-Ozreti6, M. and Ozreti6, B. (1992) in Proceedings of the FAO/UNEP/IOC Workshop on the Biological Effects of Pollutants on Marine Organisms, Malta, September 1991 (Gabrielides, P.G., ed.), pp. 165-175, MA Technical Reports Series No. 69. UNEP, Athens.

[12] Bilinski, E. (1974) in Biochemical and Biophysical erspectives in Marine Biology (Malins, D.C. and Sargent, J.R., eds.), Vol. 1, pp. 239-288, Academic Press, New York.

[13] Karmen, A. (1955) J. Clin. Invest. 34, 131-133. [14] McCormick, A., aynter, K.T., Brodey, M.M. and Bishop, S.H.

(1986) Comp. Biochem. hysiol. B 84, 163-166. [15] Korpela, T.K. and Saarinen, R. (1985) J. Chromatogr. 318, 333-341. [16] Chang-Won, L. and Desmazeaud, M.J. (1985) J. Gen. Microbiol.

131,459-467. [17] Bergmeyer, H.U. and Bernt, E. (1974) in Methods of Enzymatic

Analysis (Bergmeyer, H.U., ed.), Vol. 2, pp. 752-758, Academic Press, New York.

[18] Velick, S.F. and Vavra, J. (1962) J. Biol. Chem. 237, 2109-2122. [19] Cleland, W.W. (1963) Biochim. Biophys. Acta 67, 173-187. [20] Segel, I.H. (1975) Enzyme Kinetic, Behavior and Analysis of Rapid

Equilibrium and Steady-State Enzyme Systems, pp. 170-192, Wi- ley, New York.

[21] Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. [22] Moon, T.W. and Johnston, I.A. (1981) J. Fish Biol. 19, 653-663. [23] Cleland, W.W. (1963) Biochim. Biophys. Acta 67, 104-137. [24] Williams, J.A. and John, R.A. (1979) Biochem. J. 177, 121-127. [25] Barra, D., Bossa, F., Doonan, S., Fahmy, H.M.A., Martini, F. and

Hughes, G.J. (1976) Eur. J. Biochem. 64, 519-526. [26] Teranishi, H., Kagamiyama, H., Teranishi, K., Wada, H., Yamano,

T. and Morino, Y. (1978) J. Biol. Chem. 253, 8842-8847. [27] Magee, S.C. and Phillips, A.T. (1971) Biochemistry, 10, 3397-3405. [28] Shrawder, E.J. and Martinez-Carrion, M. (1973) J. Biol. Chem. 248,

2140-2146. [29] Kopelovich, L., Sweetman, L. and Nisselbaum, J.S. (1971) Eur. J.

Biochem. 20, 351-362. [30] Cornish-Bowden, A. (1974) Biochem. J. 137, 143-144. [31] Paynter, K.T., Hoffmann, R.J., Lehman, E.L. and Bishop, S.H.

(1984) J. Exp. Zool. 231, 185-197.