serum AST in human serum involves use of the cytoplasmic isoenzyme isolated from human erythrocytes. (5) in lyophilized serum pools as the reference.
CLIN. CHEM. 27/2,
232-238
(1981)
Isolationand Purificationof AspartateAminotransferaseIsoenzymesfrom HumanLiverby Chromatographyand IsoelectricFocusing Fred V. Leung and Arthur R. Henderson To purify cytoplasmic and mitochondrial isoenzymes of aspartate aminotransferase (EC 2.6.1.1) from human liver, we used heat treatment, ammonium sulfate precipitation, anion- and cation-exchange chromatography, affinity chromatography, and isoelectric focusing. Final preparations of the isoenzymes were homogeneous, with specit Ic activities of 198 and 208 kU/g for the cytoplasmic and the mitochondrialenzymes, respectively. The mitochondrial isoenzyme focused as a single band with a p1 value of 9.60, whereas the cytoplasmic isoenzyme had subforms withp1valuesof 5.22,5.42,and 5.62 at 4 #{176}C. In Tris HCI buffer, both isoenzymes have an activity maximum at pH
7.8. In [bis(2-hydroxyethyl)amino]tris(hydroxymethyl)methane (Bistris) buffer, however, the mitochondrial isoenzyme also showed an optimum pH of 6.7. AdditionalKeyphrases: anion- and cation-exchange chromatoraphy . affinity cfromatoraphy . interspecles values compared . isoenzyme distribution in human liver #{149}reference materials Aspartate aminotransferase (AST; L-aspartate-2-oxoglutarate aminotransferase, EC 3.6.1.1)1 exists in two isoenzyme forms, one localized in the cytoplasm (AST-1, C-AST) and the other in the mitochondria (AST-2, M-AST) of cells from mammalian tissue (1). The measurement of these isoenzymes in human serum reportedly is of clinical significance in assessing tissue damage in diseases, including myocardial infarction (2) and hepatitis (3). The need to develop and assess specific analytical methods for these isoenzymes requires the use of reference materials of high purity. The purity of the isoenzymes used to induce antibodies for immunological methods influences the specificity of these assay procedures (4). Interlaboratory evaluation of the International Federation of Clinical Chemistry (IFCC) recommendations for a reference method for measuring total serum AST in human serum involves use of the cytoplasmic isoenzyme isolated from human erythrocytes (5) in lyophilized serum pools as the reference material (6). Throughout the purification sequence described for the isolation of the cytoplasmic isoenzyme from human erythrocytes, one band of enzyme activity corresponding to aspartate aminotransferase was detected by disc gel electrophoresis. These gels also detected one or more minor protein bands in the final preparation (7), which indicated that the enzyme preparations were not homogeneous. In our study, we describe a purification of cytoplasmic and
Department of Clinical Biochemistry, University Hospital (University of Western Ontario), London, Ontario, Canada, N6A 5A5. Presented in part at the Joint Congress on Clinical Chemistry, Montreal, Canada, June 1979, and at the joint meeting of the AACC and CSCC in Boston, July 1980. Nonstandard abbreviations: C-AST, cytoplasmic aspartate aminotransferase; M-AST, mitochondrial aspartate aminotransferase; Bistris, (bis(2-hydroxyethyl)amino)tris(hydroxymethyl)methane; Tris, tris(hydroxymethyl)methylamine; IFCC, International Federation of Clinical Chemistry. Received Aug. 22, 1980; accepted Nov. 3, 1980. 232
CLINICAL CHEMISTRY,
Vol. 27. No. 2. 1981
mitochondrial AST isoenzymes from human liver, in which absolute homogeneity is approached. A combination of ionexchange chromatography, affinity chromatography, and isoelectric focusing methods produced separate isoenzymes that were free from minor contaminants. The materials and column sizes described for processing 100-g portions of liver may be scaled-up to isolate larger quantities of the isoenzymes in high purity. The pH optima for these isoenzymes are similar to the pH optima range of 7.6-8.0, as recommended by the IFCC method for serum AST (8).
Materials and Methods Materials Succinic anhydride, potassium L-aspartate, Tris, 2-oxoglutarate, pyridoxal phosphate, Bistris, and Triton X-100 were obtained from Sigma Chemical Co., St. Louis, MO 63178. 3,3’-Iminobis(propylamine) was supplied by Eastman Organic Chemicals, Rochester, NY 14650. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide was purchased from Pierce Chemical Co., Rockford, IL 61105. CNBr-activated Sepharose-4B, Blue Sepharose CL-6B, DEAE-Sephacel, and CM-Sephadex C50 were obtained from Pharmacia (Canada) Ltd., Dorval, Quebec, H9P 1 H6. Carrier ampholyte solutions (Ampholine, LKB) and Ampholine PAGplate kits (LKB) were distributed by Fisher Scientific Co., Don Mills, Ontario, M3A 1A9. All other chemicals were of reagent or higher grade. Solutions were prepared with reagent grade water (specific resistance >18 MWcm) obtained by passage through Milli-RO and Mffli-Q purification systems (Millipore Ltd., Mississauga, Ontario, L4V 1L2).
Methods Assay for aspart ate aminotransf erase activity. We measured this according to the modified IFCC method (9), using a GEMSAEC centrifugal analyzer (Electro-Nucleonics, Inc., Fairfield, NJ 07006) as described by Sampson et al. (10) at 30 or we used a LKB 8600 Reaction Rate Analyzer (LKB, Bromma, Sweden) with the Boehringer Mannheim AST kit (cat. no. 191337) at 37 #{176}C (Boehringer Mannheim Canada, St. Laurent, Quebec, H4R 1V8) in the absence of exogenous pyridoxal phosphate. The assay method at 37 #{176}C was a rapid way to screen for active enzyme fractions from chromatographic columns. Specific activities of the enzymes were determined at 30 #{176}C unless otherwise stated. One unit (U) of AST activity is defined as that amount of enzyme which converts 1 mol of 2-oxoglutarate to 1 zmol of L-glutamate per minute per liter of sample, with concurrent oxidation of 1 imol of NADH at 340 mn. Tests for contaminating enzymes. Alanine aminotransferase (cat. no 191345), creatine kinase (cat. no. 124184), and lactate dehydrogenase (cat. no. 191353) were assayed with Boehringer Mannheim kits at 37#{176}C with an LKB 2086 Kinetic Analyzer (Fisher Scientific, Toronto, Ontario, M3A 1A9). Gamma-glutamyltransferase was assayed with a Beckman kit (cat. no. 682303) at 37#{176}C on the System TR Enzyme Analyzer (Beckman Instruments, Inc., Toronto, Ontario, M8Z 5T2). Glutamate dehydrogenase was measured according to the modified method of Schmidt (11), with 2 mmol of ADP per
liter. Malate dehydrogenase was measured at 30 #{176}C with a Unicam SP 1700 spectrophotometer, according to the method of Francis and Coughlan (12). Protein determination. We determined protein concentrations by the method of Bradford (13), using the reagents supplied by Bio-Rad Laboratories Ltd., Mississauga, Ontario, L4X 2A9. A stock standard solution (1.4 g/L) of bovine ‘y-globulin was prepared. Protein estimation by ultraviolet absorbance at 260 and 280 nm was also used to monitor column eluates, according to the method of Layne (14).
lsoenzyme
Purification
1. Homogenization: Cytoplasmic and mitochondrial human AST was prepared from human liver obtained at autopsy and stored at -70 #{176}C until used. The tissue (100 g/200 mL of medium) was homogenized in a Multi-Mix (Canlab) stainlesssteel homogenizer in cold Tris - HC1 buffer, pH 7.4, 25 mmol/L, containing, per liter, 5 mmol of 2-oxoglutarate, 40 imol of pyridoxal-5’-phosphate, 20 mmol of 2-mercaptoethanol, and 2 mL of Triton X-100. The homogenate was then centrifuged (4000 X g, 4 #{176}C, 30 mm) and the supernatant fluid decanted. 2. Heat treatment: The supernat.ant solution was incubated in a water bath at 55 #{176}C for 20 mm with gentle shaking. After cooling to 4 #{176}C, the denatured protein cont.aminants were removed by centrifugation (4000 X g, 4 #{176}C, 60 mm). 3. (NH4)2SO. fractionation: The supernatant fluid from the heat-denaturation treatment was decanted and ammonium sulfate added to it, to 45% saturation at 4 #{176}C (300 g/L). This solution was stirred in an ice/water bath for 45 mm and centrifuged (10 000 X g, 30 mm, 4 #{176}C). The pellet was discarded and additional ammonium sulfate was added to the supernatant fluid to 85% saturation at 4 #{176}C (610 g/L). The final pellet, collected by centrifugation (10 000 X g, 60 mm, 4 #{176}C), was dissolved in Tris- HCI buffer (pH 7.8, 10 mmol/L, and containing 5 mmol of 2-oxoglutarate and 40 imol of pyridoxal-5’-phosphate per liter) and was dialyzed against 35 volumes of this buffer for 18 h at 4 #{176}C. 4. DEAE-Sephacel chromatography: The dialyzed solution was concentrated with an Amicon Ultrafiltration system (YM 10 membranes; Amicon Canada Ltd., Oakville, Ontario, L6H 2B9) and placed on a DEAE-Sephacel column (25 X 250mm). The column was washed with 10 mmol/L Tris - HC1 buffer, pH 8.0, then eluted with a step-wise gradient of sodium chloride from 50 to 350 mmol/L. Two main fractions of aspartate aminotransferase activity were resolved. The first peak, eluted in the load/wash fractions, corresponded to the mitochondrial isoenzyme; the second peak of activity was eluted with the 250 mmol/L sodium chloride and contained the cytoplasmic isoenzyme. These two peaks of enzyme activity were separately pooled and concentrated by ultrafiltration for subsequent purification.
Mitochondrial
Aspartate
Aminotransferase
5. Sepharose-4B-aspartate chromatography of M-AST: The first fraction of enzyme activity from the ion-exchange column was further purified by affinity chromatography on aspartate-coupled Sepharose. This gel was prepared essentially by the method of Lo and Bewick (15), but in place of the laboratory activation of agarose beads with cyanogen bromide, we used the less hazardous CNBr-activated Sepharose-4B (Pharmacia) preparation. About 15 g of the activated gel was swollen and washed with 3 L of 1 mmol/L HCI solution on a glass filter. After a final wash with 1 L of cold water, the gel was suspended in a solution that contained 10 mL of 3,3’iminobis(propylamine) in 40 mL of cold distilled water, the pH of which had been adjusted to 10 at 4#{176}C with 5 mol/L HC1 before the gel was added. This suspension was stirred slowly for 16 h at 4 #{176}C. The ligand-coupled Sepharose was washed
with 2 L of cold distilled water. In about 40 mL of distilled water, 7.5 g of solid succinic anhydride was added to the gel at 4 #{176}C and the pH was increased and maintained at pH 6.0 by titration with 5 mol/L NaOH. When this pH was stabilized, the suspension was slowly stirred for 5 h at 4 #{176}C. The succinate-bound gel was washed with cold distilled water to remove unbound reagent and then suspended in 30 mL of water. A solution of 17.12 g of potassium L-aspartate dissolved in 20 mL of water and adjusted to pH 5.0 with 5 mol/L HCI was then added to the gel suspension. Then 3.75 g of 1-ethyl-3(3-dimethylaminopropyl)carbodiimide in 22 mL of water was added to the mixture over a 15-mm period. The reaction was allowed to proceed at room temperature for 20 h, with slow stirring. The final gel was washed with 4 L of water in a BUchner glass filter funnel. The aspartate-coupled Sepharose was packed in a column and equilibrated with 50 mmol/L Bistris buffer, pH 6.5. M-AST isoenzyme was loaded onto this column and unbound protein was washed free with the Bistris buffer containing 5 mmol of succinate per liter. The major isoenzyme peak was eluted with 20 mmol of succinate per liter of Bistris buffer. 6. Isoelectric focusing of M-AST: For preparative electrofocusing we used a linear 1.36-0 mol/L sucrose density gradient in a 110-mL LKB 8101 Electrofocusing Column. M-AST was focused in 2% Ampholine (LKB), pH range 8-11 (equal mixture of 8-9.5 and 9-11 pH range Ampholines) with a final voltage of 600 V and a current of 1.5 mA at 4 #{176}C for 48 h. We used a peristaltic pump at a flow rate of 48 mL/h to elute the column and collected 2-mL fractions. Sucrose and Ampholines were removed from these fractions by continuous ultrafiltration (diafiltration) in an eight-cell ultrafiltration unit (Amicon) with the YM-lO membranes. Analytical thin-layer isoelectric focusing was facilitated by the use of ready-made polyacrylamide gels, LKB Ampholine PAGplate, pH range 4.0-6.5 and pH 3.5-9,5, and the LKB Multiphor Apparatus.
Cytoplasmic
Aspartate
Aminotransferase
7. CM-Sephadex C50 chromatography: Fraction II from the DEAE-Sephadex column was dialyzed against a solution containing 20 mmol of sodium acetate, 5 mmol of 2-oxoglutarate, and 40 mol of pyridoxal phosphate per liter, pH 6.0. The enzyme solution was applied to a column of CM-Sephadex C50 (25 X 200 mm) that had been equilibrated with a solution of sodium acetate buffer containing the same composition and concentrations as the dialysis fluid. The column was washed with this buffer to elute the active enzyme fractions, which were pooled and concentrated by ultrafiltration. 8. Blue Sepharose CL-6B chromatography: The concentrated C-AST solution from the CM-Sephadex C50 column was applied to a 25 X 60mm column of Blue Sepharose CL-6B equilibrated with 20 mmol/L sodium acetate buffer, pH 6.0. The column was washed with 200 mL of the equilibration buffer and the fractions containing the isoenzyme activity were eluted with the 100 mmol/L NaC1 in this acetate buffer. These fractions were pooled, concentrated by ultrafiltration, and dialyzed against 20 mmol/L acetate buffer, pH 5.3. 9. Sepharose-4B-aspartate chromatography of C-AST: The dialyzed cytoplasmic isoenzymes were loaded onto a 25 X 150 inns column of aspartate-coupled Sepharose equilibrated with 20 mmolfL sodium acetate buffer, pH 5.3. The column was washed with 100 mL of the equilibration buffer and with 100 mL of this buffer containing 2.5 mmol of succinate per liter. The major peak of isoenzyme activity was displaced from the resin with 20 mmol/L sodium acetate buffer, pH 6.0, containing 10 mmol of succinate per liter. 10. Isoelectric focusing of C-AST: The active enzyme fractions eluted from the affinity column of aspartate-coupled CLINICAL
CHEMISTRY.Vol.
27. No. 2, 1981
233
SUCCINATE 5 mmoi/L
.#{149},
pH
0
65
SUCCINATE 20 m,rd / L pH 65
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>-
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FRACTIONNUMBER
FRACTION NUMBER
Fig. 1. Elution profile of aspartate aminotransferase from DEAE-Sephacel About 3700 U of aspartate amlnotransferase from the dialyzed ammonltmi sulfate was applied onto the column as described In Methods. After washing the column wIth 10 mmol/L TrIs - HCI buffer, pH 8.0(23 #{176}C), we used step-wIse adIents of MaCI in this buffer, as indIcated by the a,rows, to elute the protein and enzyme Into 10-mL fractIons. 0, protein; #{149} #{149} enzyme activity
fractions
Fig.
3. AffinIty chromatography
profile
of mitochondrial
as-
partate aminotransferase from Sepharose . 4B aspartate The main enzyme activIty was eluted wIth succlnate in Sistris buffer (50 rriTlol/L pH 6.5 at 23 #{176}C) as Indicated by the arrows. 0, protein; S, enzyme activity
Sepharose were combined and dialyzed against Tris . HC1 buffer, pH 7.4, containing 5 mmol of 2-oxoglutarate and 40 smol of pyridoxal phosphate per liter. The C-AST fractions were concentrated by ultrafiltration and were focused in a preparative isoelectric focusing column (LKB, 110 mL) in 2% Ampholine pH 4-6. Focusing was conducted for 48 h at 600 V, with a final current of 2 mA. The fractions were collected and treated by diafiltration as described for the mitochondrial isoenzyme. The concentrated fractions with enzyme activity were further analyzed by thin-layer isoelectric focusing.
Results Purification
A.
Fig. 2. Analytical isoelectric focusing of aspartate aminoaminotransferase fractions from DEAE-Sephacel chromatography Pattern on left shows multiple bands, some of which contained cytoplasmic Isoenzyme activity. Pattern on right shows multipie cathodic bands with mitochon&ial Isoenzyme activity in the L4,permOst dense band. Ampholine PA(late, pH range 3.5-9.5; cathode at top 234
CLINICAL CHEMISTRY,
Vol. 27, No. 2, 1981
of M-AST
lsoenzyme
from Human Liver
Table 1 summarizes the yield and purification obtained for a typical preparation from 100 g of human liver. DEAE-Sephacel chromatography (step 4) is illustrated in Figure 1, in which peak I (fractions 3-9) contained the mitochondrial isoenzymes recovered in the early fractions. This peak is free from cytoplasmic isoenzyme activity, but comprises several cathodic protein components as shown by isoelectric focusing on the thin-layer Ampholine PAGplate, pH 3.5-9.5 (Figure 2). The next stage of this purification is to pass the isoenzyme through an affinity column of aspartate coupled to Sepharose-4B (Figure 3). The isoenzyme was bound to the ligand with 50 mmol/L Bistris buffer, pH 6.5, and was eluted as a sharp peak by the addition of succinate, 20 mmol/L to this buffer system. The presence of a single protein band, corresponding to the isoenzyme activity peak from the affinity column, indicates that the inactive protein components were removed, as evaluated by analytical isoelectric focusing. Because of enzyme degradation from storage with freezing and thawing and the loss of selectivity by the affmity column after regeneration of the gel, we occasionally detected a second band of protein near the the usual cathodic band on the analytical isoelectric focusing plate. In such cases, we used a preparative isoelectric focusing density gradient system to remove the enzymically inactive protein component and to produce a single band of mitochondrial aspartate aininotransferase isoenzyme (Figure 4). The p1 of this cathodic band of enzyme activity is 9.60 ± 0.02 (n = 4) at 4 #{176}C; this was confirmed with analytical isoelectric focusing.
Table 1. PurifIcation of Human Liver Mitochondrial and Cytopiasmlc Aspartate Aminotransferase from 100 g of Human Liver Total acty, U
protein,
mg
Spec. acty.. U/mg
4560 4286 3690 1718 1040 542
10260 4590 888 32 5.8 2.6
0.44 0.94 4.16 53.7 179.3 208.5
100 94 81 38 23 12
1 2 9.5 122 407 474
3977 3580
8853 3314
0.45 1.08
1080 60
30 20 20
1071 486 285
42 14 4
2.30 19.0 25.5
1 2.4 5
30
2484 1140
100 90 62
Vol Step
mL
Mitochondrial 1. Liver homogenate
190
2. Heated supernate 3. 4. 5. 6.
135 74 20 20 4
Ammonium sulfate resIdue DEAE-Sephacel pooled eluateI Sepharose-4B-aspartate pooled eluate Isoelectrlc focusIng
Total
R.cov.ry, %
Purification factor
Cytoplasmic 1. LIver homogenate 2. Heated supernate 3. Ammonlum sulfate resIdue 4. DEAE-Septiacel pooled eluate Il
185
123 42
5. CM.-Sephadex C50 6. Blue Sepharose CL-6B 7. Sepharose-4B-aspartate
8. Isoelectrlc
focusing
4
Purification of C-AST lsoenzyme Table 1 also summarizes the purification sequence and the yield of cytoplasmic isoenzyme. Steps 1 to 4 of the isolation procedure are the same as that used for the extraction of the mitochondrial isoenzyme except that the cytoplasmic isoenzymes are bound on the DEAE-Sephacel column with 10 mmol/L Tris - HCI buffer, pH 8.0. After an initial wash with
this buffer containing
198
1
The cytoplasmic
34.7 71.2
198
fractions
29 27
42 56
12 7
77 157
5
440
(peak II; fractions
17-23 in Fig-
ure 1) from the DEAE-Sephacel column were often contaminated with hemoglobin and other plasma proteins as noted on analytical isoelectric focusing (Figure 2). By the application of the weakly acidic cation exchanger, CM-Sephadex C50 (Figure 5), a large proportion of the contaminant hemoglobin
50 mmol of NaCI per liter, the active
cytoplasmic isoenzyme fractions were eluted with Tris buffer containing 250 mmol of NaCl per liter. In a typical elution profile (Figure 1) fractions 17 to 23, having the highest specific activity, were pooled, concentrated by ultrafiltration, and dialyzed against 20 mmol/L sodium acetate buffer, pH 6.0, containing coenzyme and substrate stabilizers as described in Methods.
30 in
b >( -j
20
-
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30 FRACTION
50
NUMBER
Fig. 6. Elution profile of cytoplasmic aspartate aminotransferase isoenzyme from Blue Sepharose CL-6B The main peak of enzyme activity eluted with 20 mmoi/L sodium acetate, pH 6.0. containing
100 mmol of NaCi per liter, as described
10
30
FRACTION NUMBER
in Methods. 0. protein;
#{149}, enzyme activity
was retained on this gel while the cytoplasmic isoenzymes were recovered in the early fractions (4-8) eluted with 20 mmol/L sodium acetate buffer, pH 6.0. Further monitoring of the major enzyme peak by analytical thin-layer isoelectric focusing revealed the presence of several contaminant protein bands. Both C-AST isoenzymes and albumin migrate towards the anode under these electrophoretic conditions. The capacity of bound Cibacron Blue F3GA to selectively remove albumin from other proteins was used as the next stage in this purification. A column of Blue Sepharose CL-6B equilibrated with 20 mmol/L sodium acetate buffer, pH 6.0, was used to bind the cytoplasmic isoenzymes. After washing unbound proteins from this affinity column, the C-AST activity was recovered in the eluate eluted with 100 mmol of NaCI per liter of this acetate buffer (Figure 6). With an elution medium of this ionic strength the enzyme activity was recovered in a sharp peak in fractions 24 to 30, while the albumin was retained by the gel in the column. After concentration and dialysis to reduce the salt content, the active enzyme fraction was loaded onto a column of Sepharose-4B-aspartate. The cytoplasmic isoenzyme was not as strongly bound to this affinity gel, so we used a lower pH and lower ionic-strength buffer to attach the isoenzyme to the column medium. In 20 mmolfL sodium acetate buffer, pH 5.3, the isoenzymes were retained on the column while contaminant proteins were eluted. The cytoplasmic isoenzymes were then recovered by elution with 20 mmolfL sodium acetate buffer, pH 6.0, containing 10 mmol of succinate per liter (Figure 7). The main fractions of isoenzyme activity, with specific activities exceeding 50 kU/g of protein, were monitored for homogeneity by analytical isoelectric focusing. Because several protein bands were detected in these fractions, we used preparative isoelectric focusing to further resolve the components. As illustrated in Figure 8, three fractions of cytoplasmic isoenzyme activity were detected, with p1’s at 5.22 ± 0.02, 5.42 ± 0.02, and 5.62 ± 0.02 (4 #{176}C). These fractions were concentrated 50-fold by “diafiltration” with an Amicon YM1O 236
0
CLINICAL CHEMISTRY, Vol.27,No. 2,1981
Fig. 7. Affinity chromatography of cytoplasmic aspartate aminotransferase isoenzyme on Sepharose-4B aspartate About 500 U of cytoplasmic isoenzyme was applied to the resin in 20 mmol/L sodium acetate buffer, pH 5.3. The main peak of enzyme activity (#{149}). was eluted at 23 #{176}C, with 20 mmoi/L sodium acetate of succinate per liter. The volume of each
buffer, pH 6.0 containing 10 mmoi was 10 mL. 0, protein
fraction
membrane system. Analytical isoelectric focusing (Figure 9) confirmed the presence of the three subforms of the cytoplasmic isoenzymes. These samples were extracted from liver that had been stored frozen, but the purification process did i2
8
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4
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U
20
40
FRACTION NUMBER Fig. 8.
Preparative isoelectric focusing of
cytoplasmic
aspartate
aminotransferase of 1.36-0 mol/L containing 2% pH 4-6 Ampholine was loaded with 285 U of cytopiasmic isoenzyme. Anode at the bottom of the column (fraction 1) with 2-mi.. fractions. 0, absorbance at 280 nm, enzyme activity, and. ..,pHat4#{176}C Sucrose adient
#{149},
CATHODE
______
ANODE Fig. 9. Analytical isoelectric focusing of cytoplasmic aspartate amlnotransferase fractions Fractions from the preparative isoelectric focusing coiunIn (FIg. 8) were focused on Ampholine PAGplate, pH 4-6.5, at 4 #{176}C. The results (left to right): fraction 41 (uriconcd.); fractions 31. 36. and 41 (combined and concd. 50-fold): fraction 41, fraction 36, fractIon 31 individually focused (concd. 50-fold)
not involve further storage of the isolated samples under frozen conditions. The major fraction of enzyme activity was located in the pH 5.6 region. In a separate extraction procedure, in which the enzymes were frozen and thawed at various stages during the purification, the main component of enzyme activity had a p1 at 5.4, and a reduced amount of p1 5.6 isoenzyme fraction was obtained.
Apparent
pH Optima
The activities of the two isoenzymes were compared over the pH range 5-9.0 by using two overlapping buffer systems: Bistris, 50 mmol/L (pH 5.0 to 7.5), and Tris. HCI, 50 mmol/L (pH 7.0 to 9.0), at 37 #{176}C. The combined fractions of cytoplasmic isoenzymes showed an activity maximum at pH 7.8. At both pH 7.5 and 8.5 in Tris . HCI, the activities were 13% lower than this maximum. M-AST isoenzyme showed an optimum pH value of 6.7 in the Bistris buffer system and a 20% decrease in activity at pH 7.5. In the Tris- HCI system a second peak of activity was found at pH 7.8 that represented 95% of the pH 6.7 activity peak. This bimodal variation in pH was not the result of contamination by the cytoplasmic enzyme, because this material was homogeneous to isoelectric focusing. The optimum pH depends on the type of buffer and ionic strength used and may contribute to this finding for the mitochondrial isoenzyme. Tris buffer, 80 mmol/L, pH 7.8 at 30#{176}C, is recommended for the serum enzyme analysis of AST (16); these conditions would be suitable for the analysis of both isoenzyrnes, each of which has maximum activity under optimal substrate concentrations according to the IFCC recommendations (9). Other Enzyme
Tests
The isoenzyme fractions were also tested for contamination with alanine aminotransferase, creatine kinase, lactate dehydrogenase, ‘y-glutamyltransferase, glutamate dehydrogenase, and malate dehydrogenase; none of these was detectable in the final preparations.
Discussion Specific activities of 198 kU/g (30 #{176}C) for the cytoplasmic and 208 kU/g (30 #{176}C) for the mitochondrial isoenzymes are slightly higher than, but still comparable with, the activities obtained by Rej (20) of >150 kU/g (30 #{176}C) for both human liver aspartate aminotransferase isoenzymes. Our prepara-
tions have equivalent or higher specific activities than purified preparations of these isoenzymes from liver from other species, such as sheep: C-AST 217 kU/g, M-AST with 182 kU/g (25 #{176}C) (17); rat: cytoplasmic isoenzyme 9.2 kU/g (25 #{176}C), mitochondrial isoenzyme 39 kU/g (25 #{176}C) (18); and chicken: cytoplasmic isoenzyme 74 kU/g (25 #{176}C), mitochondrial isoenzyme 18.8 kU/g (25 #{176}C) (19). We estimate the distribution of the isoenzymes in human liver (by DEAE-Sephacel chromatography) to be 20-25% cytoplasmic to 75-80% mitochondrial, values that agree well with those reported by Rej (20): 19% soluble and 81% mitochondrial isoenzyme in normal human liver. Isoelectric focusing shows that the mitochondrial isoenzyme from human liver exists in a single form but that the cytoplasmic isoenzyme has at least three subforms with distinct isoelectric points. Rej (21) found that such multiple forms of the cytoplasmic enzymes from human liver showed similar immunochemical behavior. Electrophoretically distinct subforms have been detected for liver cytoplasmic aspartate aminotransferase from other species: rat (22), chicken (19), and sheep (23). In the present study, fresh samples that had not been stored frozen during the purification stages yielded a major fraction with p1 = 5.6, whereas freezing and thawing produced a shift in the proportion of the subform to p1 = 5.4. A similar transition was noted by Campos-Cavieres and Munn (23) for aged samples of sheep-liver cytoplasmic isoenzyme repeatedly frozen and thawed. The presence of the y-subform they found was in addition to the a- and /-subforms usually detected in fresh samples in their sheep-liver studies. The cytoplasmic isoenzyme from pig heart muscle also has been reported by Banks et al. (24) to exist in subforms, designated a, 3, and y in order of increasing anionic character. They showed that the f3- and ‘y-forms could be generated from the a-form by lowering the pH from 8.6 to 7.5. John and Jones (25), using sheep heart cytoplasmic AST isoenzymes, demonstrated that the ‘y-subform has two more negative charges and the fl-subform has one more negative charge per dimer than the a-form. They suggested that the mechanism may be due to deamidation of glutamine or asparagine as a covalent modification rather than to a conformational change in the protein structure. Further studies by Williams and John (26) provided evidence for deamidation by measuring the ammonia released from pig-heart cytoplasmic enzyme, and by using thin-layer isoelectric focusing to measure the time course of this change in subform movement. This change would lead to only a small decrease in enzyme activity. Another modification suggested by these authors was the loss of binding of the coenzyme to the apoenzyme, with a corresponding loss of enzyme activity. As demonstrated in the study by Delincee and Radola (27) on the effects of different fractionation methods as they influence the purity and homogeneity of horseradish peroxidase isoenzymes, the use of CM cellulose or preparative isoelectric focusing individually did not yield isoenzymes in very high purity as judged by the absorbance ratio of the isoenzymes. Their most effective procedure involved a combination of chromatographic steps and isoelectric focusing both to isolate the isoenzymes and to obtain highly pure isoenzymes. In our study, the combination of DEAE-Sephacel, a specific aspartate-coupled affinity column of Sepharose-4B, and preparative isoelectric focusing similarly allowed extensive purification and separation of the isoenzymes into their single, homogeneous components. In these studies, we used analytical isoelectric focusing, beginning with a different pH range, to satisfy the criterion of carrying out electrophoresis at more than one pH as a test of protein homogeneity (28). For the mitochondrial isoenzyme, the maximum peak at p1 of 9.6 from the preparative column pH range 8-11 was localized sufficiently away from the upper pH 11 limit to isolate the M-AST CLINICAL CHEMISTRY,
Vol. 27. No. 2, 1981
237
protein from other possible contaminating proteins with p1 above the maximum. A single band was noted in the flat bed isoelectric plate at the upper limit of the pH range 3.5-9.5. There were no minor contaminants detected below this band. The M-AST also formed a single band by disc-gel electro. phoresis according to the method of Davis (29), and provided additional evidence of protein homogeneity. For the cytoplasmic isoenzyme (the maximum component), the p1 of 5.6 obtained from the preparative isoelectric focusing column, pH 4-6, refocused as a single band in the analytical isoelectric focusing plate, pH range 4-6.5. These isoenzymes retained their full activity for 6 months, when stored at -20 #{176}C in a solution containing 10 mmol Trig - HC1, 5 mmol of 2-oxoglutarate, and 40 smol of pyridoxal phosphate per liter, pH 7.8. Other methods for stabilizing these isoenzymes in various matrices have been described (5, 30). In addition to the high purity of the isoenzymes isolated from the same tissue source, the high preparative capacity of the system enables sufficient material to be obtained for use as a human reference enzyme or for antibody production.
chromatographic separation of isoenzymes of aspartate aminotransferase. Clin. Chem. 24, 1805-1812 (1978). 11. Schmidt, B., Glutamate dehydrogenase. In Methods of Enzymatic Analysis, 2, Section C, H. U. Bergmeyer, Ed., Academic Press, New York, NY, 1965, pp 752-756. 12. Francis, A. P., and Coughian, M. P., Isolation and characterization of two forms of malate dehydrogenase from human placenta. mt. J. Biochem. 7, 59-65 (1976). 13. Bradford, M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal. Biochem. 72,248-254(1976). 14. Layne, E., Spectrophotometric and turbidimetric methods for measuring proteins. Methods Enzymol. 3,447-454 (1957). 15. Lo, T. C. Y., and Bewick, M. A., The molecular mechanism of dicarboxylic acid transport in Escherichia coli K. 12. J. Biol. Chem. 253, 7826-7831(1978).
16. Bergmeyer, H. V., Scheibe, P., and Wahlefeld, A. W., Optimization of methods for aspartate aminotransferase and alanine aminotransferase. Clin. Chem. 24,58-73 (1978). 17. Orlacchio, A., Campos-Cavieres, M., Pashev, I., and Munn, E. A., Some kinetic and other properties of the isoenzymes of aspartate aminotransferase isolated from sheep liver. Biochem. J. 177,583-593 (1979).
This study was supported by a grant (3-4, 1980-81) from the Ontario Heart Foundation, which is gratefully acknowledged.
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