Separate enzymatic microassays for aspartate aminotransferase isoenzymes

Biochimica et Biophysica Acta 925 (1987) 175-184 Elsevier

175

BBA 22775

Separate enzymatic microassays for aspartate aminotransferase isoenzymes Judy A. Parli, Donald A. Godfrey and C. David Ross Department of Physiology, Oral Roberts University, Tulsa, OK(U.S.A.) (Received 3 December 1986) (Revised manuscript received 20 March 1987)

Key words: Aspartateaminotransferase; Isoenzyme;fl-Methylene-DL-aspartate;Kinetics; pH dependence; Microassay; (Rat) The properties of the cytosolic and mitochondrial isoenzymes of aspartate aminotransferase were studied using a commercial preparation of the cytosolic isoenzyme, a mitochondrial preparation, and whole brain homogenate. Based on these properties, microassays were developed and shown to be highly specific and quantitatively accurate for measuring the activity of either the cytosolic or mitochondrial isoenzyme in microgram quantities of tissue. The assays have been successfully applied to homogenates of a wide variety of tissues. They can be used to measure the activities of aspartate aminotransferase isoenzymes in sub-microgram samples of freeze-dried tissue.

Introduction

The enzyme aspartate aminotransferase (EC 2.6.1.1), also called glutamate-oxaloacetate transaminase, exists as two isoenzymes, cytosolic and mitochondrial aspartate aminotransferase. Although these isoenzymes catalyze the same reaction, namely the interconversion of glutamate and oxaloacetate with aspartate and a-ketoglutarate, they differ in chemical and physical properties [1-6]. Subforms of each of the two isoenzymes have also been isolated and characterized [3,7,8]. Because of the importance of glutamate and aspartate in brain metabolism, determination of the activity of each isoenzyme is of considerable interest. Immunohistochemical labeling procedures [9-11] and quantitative immunoassays [12] have been developed which discriminate between

Correspondence: J.A. Parli, Department of Physiology, Oral Roberts University,7777South Lewis,Tulsa, OK 74171,U.S.A.

the cytosolic and mitochondrial forms. However, no enzymatic assays have been developed which distinguish between their individual activities. In particular, to enable direct comparisons of quantitative enzyme activity measurements with immunohistochemical labeling results, enzymatic assays are needed which have sufficient sensitivity for analysis of microscopic-size tissue samples. Nisselbaurn and Bodansky [4] prepared a cytosolic aspartate aminotransferase preparation by heating a crude tissue homogenate in the presence of a-ketoglutarate. The heating destroyed the mitochondrial aspartate aminotransferase activity, while the activity of the cytosolic form was retained when a-ketoglutarate was included as a stabilizer. Boyd [1] and Fonnum [5] noted the different properties of the isoenzymes, especially pH and substrate dependencies. The mitochondrial isoenzyme activity was largely retained at low pH and low L-aspartate concentrations, whereas the cytosolic isoenzyme activity decreased drastically. In the study described here, these previous observations were investigated further using a micro-

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assay for total aspartate aminotransferase activity [13] applied to three tissue preparations: a commercially prepared cytosolic aspartate aminotransferase which has been used as a source to generate antibodies for immunohistochemical labeling [9,10], a mitochondrial preparation, and a homogenate of rat whole brain. From the observations made, microassays were developed and shown to be specific for either the cytosolic or mitochondrial isoenzyme of aspartate aminotransferase. These microassays are suitable for measurement of the isoenzyme activities in submicrogram pieces of freeze-dried tissue [14,15]. A preliminary report of these results has been made [16]. Materials and Methods

Chemicals NAD +, NADH, L-aspartate, alcohol dehydrogenase (EC 1.1.1.1, alcohol:NAD ÷ oxidoreductase, 300 U/mg), and malate dehydrogenase (EC 1.1.1.37, L-malate:NAD + oxidoreductase, 1200 U/mg) were obtained from Boehringer Mannheim Biochemicals, Indianapolis, IN. Bis-tris propane (1,3-bis[tris(hydroxymethyl)methylamino]propane), Mes (4-morpholineethanesulfonic acid), Mopso (/3-hydroxy-4-morpholinepropanesulfonic acid), Pipes (1,4-piperazinediethanesulfonic acid), Trizma acetate, Trizma HC1, Trizma base, a-ketoglutarate and bovine serum albumin were obtained from Sigma Chemical Company, St. Louis, MO. Triton X-100 was purchased from Fisher Scientific, and/~-mercaptoethanol from J.T. Baker. fl-Methylene-DL-aspartate was donated to the project by Dr. A.J.L. Cooper.

Tissue preparations Tissue homogenates were prepared from male Sprague-Dawley albino rats, killed by decapitation. The tissues were manually homogenized on ice, using Ten Broeck tissue grinders (0.15 mm clearance), in 50 mM potassium phosphate buffer (pH 7.0) at a concentration of 100 g/l, then apportioned among tubes and stored at - 8 0 ° C. Heated brain homogenate was prepared by heating whole brain homogenate in the presence of 3.7 mM a-ketoglutarate [2,4] for 15 min at 70 ° C. A commercially prepared cytosolic enzyme (glutamate-oxaloacetate transaminase, 200 U/mg),

from pig heart, was obtained from Boehringer Mannheim Biochemicals. A mitochondrial preparation isolated from mouse liver and supernatant and mitochondrial fractions isolated from rat liver by standard centrifugation techniques [17] were donated to the project by Dr. David Jones.

Assay procedures The assay procedure for measuring total aspartate aminotransferase activity was based on a previously published method [13]. Table I shows the procedures for measurement of total aspartate aminotransferase, cytosolic aspartate aminotransferase, and mitochondrial aspartate aminotransferase activities. In each case, the reaction was run in the direction of formation of glutamate and oxaloacetate, and was started by addition of one of the substrates, aspartate, along with the cofactor (for the malate dehydrogenase reaction) NADH. The reaction was pulled to completion by reduction of the oxaloacetate product to malate, with concomitant oxidation of NADH, through inclusion of malate dehydrogenase during the incubation. The malate dehydrogenase was added with the substrate medium so that it would not be inactivated by the heating step in the cytosolic aspartate aminotransferase assay. Two aspects of the assay procedures were specifically included because of their intended application to small samples of freeze-dried tissue: the sonication step and the method of heating in the cytosolic aspartate aminotransferase assay. Ultrasonic disintegration is necessary to release the tightly bound mitochondrial aspartate aminotransferase isoenzyme in serum [1] and freeze-dried tissue [13], although it did not sizeably increase the activities of the tissue preparations used in the present study. Heating of freeze-dried tissue in the presence of a-ketoglutarate is optimally done in the preincubation medium. Brain homogenate can either be heated in the same way or prepared as a stock preheated in the presence of 3.7 mM a-ketoglutarate; the two methods were found to give the same results. Each assay included reagent blanks (no tissue included) and calibrated NAD + standards in the total amounts of 2.1, 3.9 and 7.7 nmol. The reagent blank was approximately equal to 1.0 nmol NADH. The fluorometer readings for the samples

177 TABLE I ASSAY P R O C E D U R E S F O R T O T A L A S P A R T A T E A M I N O T R A N S F E R A S E A N D I S O E N Z Y M E ACTIVITY Procedure

Total

Cytosolic

Mitochondrial

Preincubation:

30 #1 of preincubation medium consisting of 99 m M Tris-acetate buffer (pH 7.4), 3.7 m M a-ketoglutarate, 1.3 g / l bovine serum albumin, and 1.5 m l / l Triton X-100 were added to all blank, standard, and sample tubes.

Same as for total aspartate aminotransferase

Same as for total aspartate aminotransferase except buffer: 99 m M sodium acetate buffer (pH 5.1).

Sonication:

All tubes sonicated for 5 min in a Branson ultrasonic (50 kHz) cleaner.

Same as for total aspartate aminotransferase

Same as for total aspartate aminotransferase

Blank, standard, and sample tubes heated at 7 0 ° C for 15 min.

Destruction of mitochondrial isoenzyme: Incubation:

Reaction initiated by addition of 10/~1 of substrate medium consisting of 160 m M L-aspartate, 1.4 m M N A D H , and 1.5 m g / l malate dehydrogenase. As substrate was added, each tube was mixed lightly and placed in the 3 8 ° C incubation bath, at approximately 5 s intervals between tubes. Incubation time was 30 min.

Same for total aspartate aminotransferase

Same as for total aspartate aminotransferase except for aspartate concentration: 8 m M L-aspartate.

Termination of incubation:

40 btl of 0.7 M HC1 were added to each tube as it was removed from the incubation bath, at approximately 5 s intervals between tubes. Each tube was mixed, then a few minutes were allowed for destruction of the N A D H in the acidic medium.

Same as for total aspartate aminotransferase

Same as for total aspartase aminotransferase

Indicator step:

1.0 ml of indicator medium consisting of 95 rnM Tris-HCl buffer (pH 8.5), 860 m M ethanol, 2 m M fl-mercaptoethanol and 11.6 mg/1 alcohol dehydrogenase was added and each tube mixed well.

Same as for total aspartate aminotransferase

Same as for total aspartate aminotransferase

Fluorescence measurement:

After a 15 min period during which virtually all N A D + was converted to N A D H , the fluorescence of each tube was measured with a Farrand Ratio Fluorometer. An excitation wavelength of 360 n m (primary filter C o m i n g No. 5860) and a 460 n m emission wavelength (combination of C o m i n g filters No. 4303 and 3857) were used.

Same as for total aspartate aminotransferase

Same as for total aspartate aminotransferase

in each assay fell within the range of those for the standards, which were linear within this range. Amounts of tissue assayed were 0.2-2.2/~g for the tissue homogenates, 0.04-0.09/~g protein for the supernatant and mitochondrial preparations of mouse and rat liver and 0.4 ng enzyme protein for

the cytosolic enzyme. All tissue homogenate activities were expressed as mol product formed/kg wet wt. per h; activity for the cytosolic enzyme preparation was reported as mol product formed/g enzyme protein per h, and activities for the supernatant and mitochondrial preparations of rat and

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mouse liver were reported as mol product formed/kg protein per h. Protein content was determined by the method of Lowry et al. [18]. Results

Using the mitochondrial preparation from mouse liver as a relatively enriched source of the mitochondrial isoenzyme of aspartate aminotransferase and the commercially prepared cytosolic isoenzyme (glutamate oxoacetate transaminase), several experiments were performed to examine the specific properties of the isoenzymes in comparison to those of the rat brain homogenate.

Destruction of the mitochondrial isoenzyme by heating The optimal temperature for destruction of the mitochondrial isoenzyme without loss of cytosolic activity was determined by heating the different samples at various temperatures for 15 min in the presence of 3.7 mM a-ketoglutarate (Table II). When heated at or above 70 ° C, the mitochondrial preparation lost virtually all of its activity. At 70 ° C, the cytosolic enzyme retained essentially

total activity, whereas an increase in temperature to 75 °C resulted in partial loss of activity. The activity of the rat brain homogenate dropped to about 45% of its total at 65 ° C, remained relatively constant between 65 °C and 70 ° C, then gradually decreased to 38% of its total at 80 o C. Next, to determine a suitable duration of the heating procedure, the preparations were heated at 70 °C for various time periods (Table III). When heated for 10 rain or longer, the activity of the mitochondrial preparation was reduced to about 3% of that of the unheated preparation, while the cytosolic enzyme exhibited no loss of activity through 25 min of heating. The activity of the brain homogenate dropped to about 42% of total by 10 min of heating and then remained constant through 25 rain of heating. The chosen duration of 15 min (Table I) fits within the range of times tested. In a separate experiment, it was determined that the activity of the rat brain homogenate heated at 70°C for 15 min in the absence of a-ketoglutarate was a third that of rat brain homogenate heated in the presence of a-ketoglutarate.

Activity vs. pH The pH-dependence curves for the various preparations (cytosolic enzyme, mitochondrial preparation, brain homogenate, heated brain homogenate, and the calculated difference between brain

TABLE II EFFECT OF HEATING TEMPERATURE ON ASPARTATE AMINOTRANSFERASE ACTIVITY OF THREE PREPARATIONS The total aspartate aminotransferase assay procedure is as presented in Table I. Preparations are abbreviated as: RBH, rat whole brain homogenate; MLH, mitochondrial preparation from mouse liver homogenate; GOT, commercially prepared cytosolic aspartate aminotransferase (glutamate oxaloacetate transaminase) from pig heart. Activities are the means ± S.E, of three separate determinations in one experiment and are expressed as percentages of the activities for unheated aliquots of the preparations. The duration of heating was 15 rain. Temperature

RBH

MLH

TABLE III EFFECT OF HEATING DURATION ON ASPARTATE AMINOTRANSFERASE ACTIVITY OF THREE PREPARATIONS The total aspartate aminotransferase assay is as presented in Table I. Abbreviations as in Table II. Activities are the means ± S.E. of three separate determinations in one experiment and are expressed as percentages of those for unheated aliquots. The temperature of heating was 70 o C.

GOT

(°C)

Time (min)

RBH

MLH

GOT

25 60 65 70 75 80

0 5 10 15 20 25

1~.0±0.6 45.6±3.2 41,5±0.1 42,2±0.4 43,8±1.2 41.3±0.4

1~.0±2.7 5.5±1.2 3.2±0.2 3.4±0.0 2.8±0.3 2.8±0.2

1~.0±3.3 107.5±1.8 107.4±1.0 109.4±1.9 106.1±1.6 108.6±2.2

1~.0±1.8 87.6±1.4 46.9±1.8 43.5±0.4 41.1±0.4 38.4±0.3

1~.0±1.9 86.4±1.2 46.8±0.4 2.8±0.3 1.9±0.2 2.3±0.1

1~.0±1.9 94.7±1.3 92.9±3.0 92.3±0.5 78.9±0.5 75.1±0.6

179

homogenate and heated brain homogenate) were compared (Fig. 1). Optimum activity for all preparations was in the pH range 7.0-8.5. Both the cytosolic enzyme and heated brain homogenate activities decreased drastically at reduced pH, falling to less than 20% of their optimum activities at pH 5. In contrast, the mitochondrial preparation

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retained over 70% of its o p t i m u m activity at p H 5. The p H curve for brain homogenate activity fell between those of the cytosolic enzyme (and heated brain homogenate) and mitochondrial preparation. The calculated total-minus-heated brain homogenate gave a relationship to p H similar to that of the mitochondrial preparation, except below p H 5, where its activity decreased much more than that of the mitochondrial preparation. In contrast to the results for lower pH, at p H above 8.5 the activity of the mitochondriat preparation decreased more than those of the cytosolic enzyme and heated brain homogenate. A p H - d e p e n d e n c e study using combinations of other organic buffers (Bis-tris propane, Mes, Mopso, and Pipes) gave results similar to the p H curves in Fig. 1.

Activity vs. substrate concentrations Substrate-dependency studies (Figs. 2 - 5 ) further demonstrated the similar properties of the

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Fig. 1. Dependence of aspartate aminotransferase (AAT) activity on pH of incubation. The different preparations are represented by different symbols and lines as shown in the key: MLH, mitochondrial preparation from mouse liver homogenate; RBH, rat whole brain homogenate; hRBH, heated rat whole brain homogenate; RBH- hRBH, calculated difference between the activities of brain homogenate and heated brain homogenate; GOT, commercially prepared cytosolic aspartate aminotransferase from pig heart. Activities for each tissue preparation are the means+S.E, of three separate determinations in one experiment and are expressed as percentages of the activity at standard conditions (pH 7.4). The total curve for each tissue preparation was combined from two separate experiments, with one overlapping data point (pH 7.1) to align the two parts of the curve. The buffers used were sodium acetate, pH 4.7-5.5; potassium phosphate, pH 5.5-7.2; Trisacetate; pH 7.2-8.6; and sodium carbonate, pH 8.6-9.4.

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Fig. 2. Dependence of aspartate aminotransferase (AAT) activity at pH 7.4 on the incubation concentration of L-aspartate, with a-ketoglutarate concentration at 2.8 mM. Identification of symbols and lines shown in the key, as in Fig. 1.

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Fig. 4. Dependence of aspartate aminotransferase (AAT) activity at pH 5.1 on the incubation concentration of L-aspartate, with a-ketoglutarate concentration at 2.8 mM. Identification of symbols and lines in key as in Fig. 1.

brain homogenate were similar to those for the cytosolic enzyme, and the values for total-minusheated brain homogenate were similar to those for the mitochondrial preparation (Table IV). De7o

cytosolic enzyme and heated brain homogenate, as well as those of the mitochondrial preparation and total-minus-heated brain homogenate. As would be expected, the brain homogenate values consistently fell between the cytosolic and mitochondrial activities. Only slight activities for the various preparations were obtained at 0 mM concentrations of L-aspartate and a-ketoglutarate, as shown in Figs. 2 and 3. Even at an incubation pH of 5.1, the substrate dependencies of the cytosolic enzyme and heated brain homogenate were similar, as were the mitochondrial preparation and total-minus-heated brain homogenate (Figs. 4 and

5). As determined by double-reciprocal (Lineweaver-Burk) plots, the apparent K m values for t-aspartate and a-ketoglutarate for the heated

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