159- 164 ( 1985)
Purification of Three Porcine Heart Mitochondrial Citrate Synthase, Aspartate Aminotransferase, and Malate Dehydrogenase’
J.McEvILY,~ of Chemistry,
Received July 6, 1984 The mitochondrial enzymes citrate synthase, malate dehydrogenase, and aspattate aminotransferase were purified to homogeneity from porcine hearts by use of Bio-Rex 70, carboxymethylcellulose CM32, and At&Gel blue chromatography. This procedure provides relatively rapid, large-scale preparation of the three enzymes based on their differential binding to commercially available cation-exchange resins followed by a final affinity chromatography step. 0 1985 Academic KEY
WORDS: chromatography; citrate synthase; malate dehydrogenase; aspartate aminotrans-
The mitochondrial enzymes citrate (si)synthase [(CS),5 citrate oxaloacetate-lyase (pro-34CH2C00 - acetyl-CoA), EC 188.8.131.52, aspartate aminotransferase [(mAAT), L-aspartate:2-oxoglutarate aminotransferase, EC 184.108.40.206, and malate dehydrogenase [(mMDH), L-malate:NAD+ oxidoreductase, EC 220.127.116.111 are metabolically related by the common substrate (product), oxaloacetate (OAA). The three enzymes define a metabolic branchpoint whereby OAA, the product of mMDH catalysis, may enter either the tricarboxylic acid (TCA) cycle by condensation with acetyl-CoA to form citrate as catalyzed by CS or the malate-aspartate shuttle upon transamination by mAAT. Physical interactions between these
enzymes have been demonstrated by various techniques ( l-6). To further investigate these protein-protein interactions and the possibility of substrate channeling between the enzymes it became necessary to develop a large-scale, convenient purification scheme to provide all three enzymes in good yield and in a high degree of purity. After attempting several previously described techniques for the purification of the individual enzymes (7- 11), the procedure outlined below was developed eliminating the need for three separate purification schemes by exploiting chromatographic behavior common to all three enzymes.
’ This work was supported by the National Institutes of Health (Grant HL-12585). 2 Supported by a National Science Foundation Fellowship (Grant XB- 111). 3 Recipient of a Sigma Xi Undergraduate Research Award. 4 To whom correspondence should be addressed. ‘Abbreviations used: CS, citrate synthase; mAAT, mitochondrial aspartate aminotransferase; mMDH, mitochondrial malate dehydrogenase; OAA, oxaloacetate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Materials. Unless otherwise noted all chemicals were purchased from Sigma. BioRex 70 and Al&Gel blue were obtained from Bio-Rad and CM32 carboxymethylcellulose was purchased from Whatman Chemical Separations Ltd. Dialysis tubing, ammonium sulfate, and mono- and dibasic phosphate were obtained from Fisher Scientific. Fresh pig hearts packed in ice were purchased from Goodmark Foods.
Copyright B 1985 by Academic Press. Inc. All rights of reproduction in any form reserved
Enzyme assays. Citrate synthase activity was assayed as previously described (12). mMDH activity was assayed according to the procedure of Gregory et al. (9). mAAT activity was determined using a coupled assay with mMDH (8). All assays were performed on a Gilford 250 spectrophotometer thermostatically controlled to 25 “C and interfaced with a UNC microcomputer. Protein determination. The concentration of the purified enzymes was measured spectrophotometrically at 280 nm utilizing the extinction coefficients tiz = 2.53 for mMDH, 14.0 for mAAT, and 17.8 for CS. Specific activities of impure enzymes were expressed as units/A2s0. Polyacrylamide gel electrophoresis. SDSPAGE was carried out as previously described ( 13). Protein bands were developed with silver stain (14).
Preparation of heart tissue. Hearts were trimmed of fat and connective tissue, cut into 2- to 3-cmdiam strips and stored at -20°C. Storage for several months had virtually no effect on yields; however, increased yields of CS were obtained from frozen rather than fresh hearts. Extraction of CS, mAAT, and mA4DH. The overall purification scheme is outlined in Fig. 1. All procedures were performed at 4°C. The day prior to extraction, 3 kg of frozen pig hearts was placed in a refrigerator and allowed to thaw slowly. After the heart tissue was passed through a meat grinder, approximately 1.5 kg of the ground tissue was placed in a Waring blender (Cliter capacity) with 2 liters 10 mM Pi, 10 pM phenylmethylsulfonyl fluoride (PMSF), pH 6.8. Care should be taken at this point due to the toxicity of PMSF. The material was blended for 1 mitt, and then placed in an ice-water bath and allowed to cool below 10°C. Blending and cooling were alternated eight times. The remaining 1.5 kg of heart was then processed in the same manner. The two
1 CS/flDH 1 CM32
3 Affigel-Blue I Jr
1 mMDH , J Affigel-Blue
FIG. 1. Flow diagram of the purification procedure for mitochondrial malate dehydrogenase, aspartate aminotransferase, and citrate synthase.
batches were pooled and centrifuged at 6000g in a refrigerated centrifuge for 30 min. The pellet was then reextracted with three I-min alternate blending and cooling periods and centrifuged, and the supernates from both extractions were pooled after filtering through cheesecloth. The yield for the second extraction was typically 50% of the initial extraction for each protein. Ammonium sulfate fractionation. The extraction supernate pool was brought to 45% (NH&SO4 saturation and allowed to stir for 2 h, after which it was centrifuged as described above for 45 min. The supernate was then brought to 75% (NH4)2S04 saturation and allowed to stir overnight. The 75% sat (NHJ2S04 pool was centrifuged as above, and the pellet was dissolved in a minimum of 10 mM Pi, pH 6.8 (-750 ml), and placed in dialysis against 4 liters of the same buffer. The first batch of dialysis buffer contained 10 PM pyridoxal-5’-phosphate and 0.5 mM cY-ketoglutarate to stabilize mAAT. The dialysis buffer was changed twice (total dialysis buffer = 12 liters), allowing at
least 4 h per dialysis. The dialysate was then spun at 10,OOOg to clarify. Bio-Rex 70 chromatography. Supernate from the 45-75% sat (NH&SO4 fractionation was applied to a 5 X 50-cm Bio-Rex 70 cation-exchange column previously equilibrated with 10 mM Pi, pH 6.8. The column was washed with 4 liters of buffer and the eluant was collected in fractions. All fractions with red color were pooled and saved for further purification of cytoplasmic AAT and MDH according to Gregory et al. (9). Proteins were eluted from the column with a linear salt gradient composed of 2 liters of 10 mM Pi, pH 6.8, in the starting buffer and 2 liters of the same buffer adjusted to a conductivity of 10 mmho with NaCl (approx 11 g/liter) in the limiting buffer. Fractions with mAAT specific activity 3 20 were pooled. CS and mMDH coelute; therefore, all fractions with CS and mMDH activity were pooled. The separate pools were concentrated by overnight dialysis against 100% sat (NH&S04. A typical Bio-Rex 70 chromatographic profile is presented in Fig. 2. CM32 carboxymethylcellulose chromatography. The mAAT 100% sat (NH&SO4 di-
alysate was spun at 35,OOOg for 20 min and
the pellet was dissolved in a minimum of 5 mM Pi, pH 6.6, and placed in dialysis against 2 liters of the same buffer. The dialysis buffer was changed twice (total dialysis buffer = 6 liters) allowing 2 h per dialysis. The dialysate was centrifuged at 35,OOOg for 20 min to clarify. The supernate was diluted with buffer until conductivity < 0.7 mmho and then loaded onto a 2.6 X 40cm CM32 column previously equilibrated with 5 tnM Pi, pH 6.6. The column was washed until the AZ80 of the eluate < 0.05 (-400 ml). The protein was eluted with a linear gradient composed of 700 ml 5 mM Pi, pH 6.6, in the starting buffer and 700 ml of 50 mM Pi, pH 6.6, in the limiting buffer. Fractions with mAAT specific activity 340 were pooled and concentrated by dialysis against 100% sat (NHMO4. The CS/mMDH pool from the Bio-Rex 70 column was chromatographed on the CM32 as described for mAAT. Separation of CS and mMDH was achieved at this point. Fractions with CS specific activity 340 were pooled, as were fractions with mMDH specific activity 2500. Both pools were concentrated by dialysis against 100% sat (NH&S04. A
FIG. 2. Bio-Rex 70 chromatography of the 45-7590 (NH&SO4 pool. The column was developed at 250 ml/h as described in the text. The fraction volume was 20 ml. Fractions were monitored for absorbance at 280 nm (-), and for mMDH X10-’ (O), mAAT (O), and CS (Cl) activity as well as conductivity (A).
FLINT, AND HARRISON
typical CM32 chromatographic profile for a single polypeptide band on SDS-PAGE. the CS/mMDH pool is presented in Fig. 3. The results of a typical purification of the Afi-Gel blue chromatography. The fol- three enzymes are presented in Table 1. lowing procedure applies to the three separate enzyme pools. The CM32 100% sat DISCUSSION (NH&SO4 precipitate was centrifuged and the pellet was dissolved in a minimum of 10 The purification scheme presented here mM Pi, pH 7.0 (- 100 ml). Dialysis and has several distinct advantages over previously centrifugation were performed as for the described procedures. By exploiting chroCM32 column but in 10 mM Pi, pH 7.0. matographic behavior common to all three The pool was diluted to approximately 10 enzymes, the need for three separate purifimg/ml protein and applied onto a 1.5 X 60- cation schemes is eliminated. The procedure cm AI&Gel blue column previously equiliis relatively short, allowing large quantities brated in the phosphate buffer. The column of the proteins to be brought to homogeneity was washed until the AzgO of the eluate was in high yield. ~0.01. A concave gradient of 250 ml 10 mM Heart tissue was not extracted with acetone as an initial step in the purification as dePi, pH 7.0, in the starting buffer and 500 ml 10 mM Pi, 1 M NaCl, pH 7.0, in the limiting scribed by Gregory et al. (9), allowing greater buffer was used to elute the protein. All recovery of mMDH, mAAT, and CS activity directly from frozen hearts and eliminating fractions with high specific activity (mAAT and tedious part of the > 150, CS B 80, and mMDH 2 1000) were a time-consuming pooled and concentrated by dialysis against procedure. 100% sat (NH&S04. All three enzymes were All resins used are commercially available. In the purification of CS as outlined by stored as suspensions in 100% sat (NH&S04 at 4°C. The average yields were 500 mg Beeckmans and Kanarek (10,ll) the pyromMDH, 200 mg mAAT, and 250 mg CS mellitic acid (PMA) affinity column has to from 3 kg hearts. Each protein exhibited a be synthesized in the lab. Once constructed high specific activity which agreed well with the resin is labile to buffers used in the previously published results and migrated as preparation (i.e., Tris) and therefore has a
FIG.3. CM32 chromatography of the post-Bio-Rex 70 CS/mMDH pool. The column was developed at 150 ml/h with a fraction volume of 10 ml. Fractions were monitored for absorbance at 280 nm (-), and for mMDH X10-l (@) and CS (0) activity as well as conductivity (- - -).
TABLE 1 PURIFICATION
OF MIT~CHONDRIAL MALATE DEHYDROGENASE, AND CITRATE SYNTHASE FROM 3 kg PORCINE
Extraction mMDH mAAT cs
45-75%(NH4)2S0, mMDH mAAT cs
Bio-Rex 70 mMDH mAAT cs
ASPARTATE AMINOTFUNSFERASE, HEART MUSCLE
Total units (x10-q
Specific activity (unW&d
487 146 100
2.5 0.75 0.5 1
47 100 100 100
20 450 125 79
19 5.3 3.3
92 86 79
1145 1030 1145
1.15 1.24 1.15
232 50 66
176 39 50
62 63 66
CM32 mMDH mAAT cs
310 454 466
0.60 1.76 1.44
192 48 55
1030 60 82
51 60 55
Affi-Gel blue mMDH mAAT cs
0.67 (478)’ 1.33 (19O)C 2.13 (320)’
1485 (375)d 158 (220)d 92 (164)d
48 53 52
’ mMDH and mAAT yield corrected for separation from cytoplasmic isozymes following Bio-Rex 70. b Determined before concentrating. ’ Numbers in parentheses represent total mg pure enzyme based on Azso. d Numbers in parentheses represent specific activity as units/mg protein.
limited lifetime. In addition, the PMA column, due to its high concentration of carboxy1 groups, has the potential to behave as a cation-exchange resin. Upon attempting this procedure, CS was found to elute in low yield when using the prescribed eluting buffer while CS activity remained associated with the resin. The second chromatographic step in the Beeckmans and Kanarek CS preparation also requires synthesis of the resin to be used, in this case Blue Sepharose according to the procedure of Ryan and Vestling (15). Commercially available A&Gel blue was substituted for the Blue Sepharose and was found to bind CS quantitatively. ‘Simply removing citrate from the column buffer did not cause elution of CS from the Affi-Gel blue as
described in the former procedure with Blue Sepharose; therefore a salt gradient was employed providing >90% yield of enzyme for this step. The purification of mAAT as outlined by Barra et al. (8) calls for a QAE-Sephadex column for the final step of purification. The procedure requires running the column in 10 mM 2-amino-2-methylpropan1,2diol HCl, pH 9.4. The high pH may account for the low yield afforded from this column, typically 40-50%. Al&Gel blue chromatography, which avoids exposing the enzyme to high pH, was found to bind mAAT quantitatively, and upon elution provided pure enzyme in high yield (>85%). A combined preparation for AAT and MDH was described by Glatthaar et al. (7),
but it involves numerous ion-exchange and gel-filtration steps for each enzyme with very low overall yields for the mitochondrial MDH and AAT (21 and 12%, respectively). The procedure outlined in this paper eliminates the need for gel filtration, is less time consuming, and provides higher yields of mMDH and mAAT in addition to CS. ACKNOWLEDGMENTS We are grateful to Elizabeth Leininger-Zapata and Mary A. Lapadat for their assistance in purification of the enzymes presented herein.
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