The two forms of rat-liver aspartate transaminase

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The two forms of rat-liver aspartate transaminase Two forms (isoenzymes) of aspartate transaminase (L-aspartate: a-ketoglutarate aminotransferase, EC 2.6.1.i.) are present in rat-liver homogenates. The two forms can be separated from one another by zone electrophoresis or ion-exchange chromatography, and the separated forms have been found to possess distinctly different substrate affinities and p H - a c t i v i t y profiles l-a. It appears that one form of the enzyme is associated with the mitochondria, while the other form is associated with the "supernatant" fraction or differentially removed from the mitochondria during isolation. Both of these forms can be partially purified (each one about 2oo-fold) by means of the procedure outlined below. The purified forms retain the characteristic electrophoretic and kinetic differences of the crude forms. Both of the purified forms are irreversibly denatured in 8 M urea, but the rate of denaturation is much greater for the "mitochondrial" form of the enzyme than for the "supernatant" form of the enzyme. The purification procedure involves the following steps for lOO g of rat liver: (a) homogenization for 3 min in a Waring Blendor in o.o 5 M sodium phosphate (pH 6.8), (b) rapid heating with stirring from 4 to 60 ° after having made the homogenate o.o8 M with respect to a-ketoglutarate, maintenance at 6o ° for 1 min, rapid cooling to 4 °, centrifugation, and dialysis of the supernatant liquid against o.o 5 M sodium phosphate (pH 6.8), (c) ammonium sulfate fractionation in the presence of o.o2 M sodium lauryl sulfate and dialysis of the o.6-o.8 saturated ammonium sulfate fraction against o.oo5 M Tris-phosphate (pH 7.5), (d) chromatography on DEAEcellulose. Two elution peaks of transaminase activity are obtained from the chromatographic step: Peak 1 is eluted with 0.005 M Tris-phosphate (pH 7.5), while Peak 2 is eluted with 0.03 M NaCl-o.oo5 M Tris-phosphate (pH 7-5). Peak I contains about 20 mg of the enzyme of specific activity IiO, and Peak 2 contains about 3 mg of specific activity 60 (/,moles D P N H oxidized/min/mg protein). The enzyme is assayed at 25 ° with excess pyridoxal 5-phosphate and excess rat-liver malate dehydrogenase (EC 1.1.1.37 ) by a slight modification of the KARMEN coupled malate dehydrogenase-DPNH assay method 5. Each of the two peaks from the column is about 90% as active when assayed without added pyridoxal 5-phosphate as when assayed with it. In order to demonstrate that this purification procedure actually furnishes both forms of rat-liver aspartate transaminase, the enzyme in Peaks I and 2 is examined by zone electrophoresis, with granular potato starch as the supporting medium and with the starch and the sample equilibrated against 0.05 M sodium citrate (pH 5.6). The starch block is 0.5 × 2.0 × 48 cm; 300 V are applied for 24 h at 4 ° (5-8 mA). Under these conditions, the enzyme activity in Peak I moves toward the cathode, whereas the activity in Peak 2 moves slightly toward the anode. With the same electrophoretic conditions, about 2/3 of the activity of a water extract prepared from a single rat liver moved toward the cathode, while 1/3 of it moved slightly toward the anode. As already mentioned, the mitoehondrial and supernatant forms of rat-liver aspartate transaminase are quite different from one another in their p H - a c t i v i t y dependencies 3, and this is true also of the partially purified forms. The ratio of the Biochim. Biophys. Acta, 65 (1902) 358-359

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activity at p H 7.5 to the activity at p H 6.0 is 0.85 for Peak I (cationic) and 4.0 for Peak 2 (anionic). The irreversible denaturation in 7.9 M urea of the enzyme of Peaks I and 2 is shown in Fig. I. The t e r m "irreversible denaturation" is used here to mean that the enzyme activity is not regained upon a 3o-fold dilution of the urea solution containing the enzyme. The assay method could be used for studying this denaturation because the amount of urea carried over into the assay system was not enough to

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Time (rain)in 7.gM urea

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Fig. I. U r e a - d e n a t u r a t i o n study. 25 °. Curve I: Cationic a s p a r t a t e t r a n s a m i n a s e f r o m Peak i, DEAE-cellulose. Curve 2: Anionic a s p a r t a t e t r a n s a m i n a s e f r o m Peak 2, DEAE-cellulose.

affect the malate dehydrogenase in the assay system. The samples were added to freshly prepared solutions of urea, and at the times indicated, aliquots were removed and diluted (luring the assay procedure. Care was taken to use freshly prepared solutions of urea in order to minimize the concentration of ammonium cyanate 6. The data show a clear difference between the partially purified aspartate transaminase isoenzymes with respect to urea sensitivity and offer an additional criterion to be applied in comparisons of the properties of isoenzymes.

Biochemistry Division, Department of Chemistry and Chemical Engineering, University of Illinois, Urbana, Ill. (U.S.A.)

ROBERT H . HOOK"* CARL S. VESTLING*

1 0 . ROSENTHAL, S. K. THIND AND N. CONGER, Abstract No. 26, Div. of Biol. Chem., Am. Chem. Soc., New Y o r k Meeting, S e p t e m b e r I i 16 (196o). 2 B. W. MOORE AND R. H. LEE, J. Biol. Chem., 235 (196o) 1359 . 3 j. W. BovD, Biochem. J., 81 (1961) 434. 4 R. 17I. H o o K AND C. S. VESTLING, Federation Proc., 21 (1962) 254. 5 A. NARlUEN, J. Clin. Investigation, 34 (1955) 131. 6 R. D. COLE, J. Biol. Chem., 236 (1961) 2670.

Received April I3th, 1962 * S u p p o r t e d b y U.S. Public H e a l t h Service G r a n t No. C-I856. ** Present address: D e p a r t m e n t of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Mich. (U.S.A.),

Biochim. BiophTs. Acta, 65 (1962) 358-359