ANALYTICAL

BIOCHEMISTRY

Properties

95, 188- 193 (1979)

of Tyrosine Aminotransferase

KAI-LIN

LEE,

Biology

Division,

LAURENCE Oak Ridge

E. ROBERSON, National

Laboratory,

from Rat Liver1’2

AND FRANCIS Oak

Ridge,

T. KENNEY

Tennessee

37830

Received October 1978 A series of sequential chromatographic procedures which yield essentially homogeneous tyrosine aminotransferase (L-tyrosine:2-oxglutarate aminotransferase, EC 2.6.1.5) from rat livers is described. Analysis of the purified enzyme indicates that its molecular weight is about 100,000, and that it consists of two subunits of identical mass and charge, each bearing one functional site for reaction with pyridoxal phosphate.

The tyrosine aminotransferase (EC 2.6.1.5) of rat liver and of cultured liver and hepatoma cells has become an important model for the study of regulation in mammalian cells because of the sensitivity of its synthesis to a variety of hormonal regulators (l-4) and the rapid turnover of both the enzyme (5) and its messenger RNA (6). Several methods of purification of this enzyme have been described in reports from our laboratory (7,8) and by others (9-15), but the isolation of significant quantities of homogeneous enzyme has remained difficult to attain in a reproducible fashion. We report here a series of sequential chromatographic procedures which we have found will routinely yield essentially homogeneous tyrosine aminotransferase from rat liver. Also, some discrepancy exists in published data on the fundamental structural properties of this enzyme, including molecular weight, subunit structure, and coenzyme content (lo- 15). We have analyzed these properties and present here the results of our determinations. 1 This paper is dedicated to the memory of Dr. Alvin Nason. 2 Research sponsored by the Division of Biomedical and Environmental Research, U.S. Department of Energy under contract W-7405eng-26 with the Union Carbide Corporation. 0003-2697/79/070188-06$02.00/O Copyright All rights

8 1979 by Academic Press, Inc. of reproduction in any form reserved.

EXPERIMENTAL

PROCEDURES

Tyrosine aminotransferase was assayed under conditions described before (1,7) but with the product assay of Diamondstone (16); one unit is equivalent to 1 nmol of phydroxyphenylpyruvate formed per minute. Proteins were determined by the method of Lowry et al. (17). RESULTS AND DISCUSSION

Enzyme Purifcation Initial steps in purification of tyrosine aminotransferase, including preparation of homogenates and soluble extracts, heat treatment, and absorption to and elution from large columns of DEAE-cellulose, were as described before (8). The enzyme eluted by 400 mM KC1 was concentrated by precipitation at 70% (NH4)$04, then dialyzed against buffer A (50 mM potassium phosphate pH 7.6, 5 mM cu-ketoglutarate, 1 mM EDTA and p-mercaptoethanol) containing 10 PM pyridoxal phosphate. DEAE-Sephadex I. The dialyzed enzyme from livers of 100 hydrocortisone-treated rats was applied to a column of DEAE-Sephadex A-50 (22 x 3 cm) equilibrated with buffer A, and the column was washed with this buffer supplemented with 10 pM pyri188

TYROSINE

AMINOTRANSFERASE

189

FROM RAT LIVER

doxal phosphate, mercaptoethanol].

1 mM

EDTA

and p-

CM-Sephadex. The enzyme was applied to a column of CM-Sephadex C-50 (25.5 x 1.6 cm) equilibrated with buffer B. As this exchanger separates the three multiple forms of tyrosine aminotransferase ( 18), no gradient fractionation was made. After loading, the column was washed with 1 bed vol of buffer B, and the enzyme was then eluted by increasing KC1 to 400 mM. At this stage, nearly 80% of the initial activity was refactor of covered, with a purification about 400. DEAE-Sephadex II. The product from CM-Sephadex was diluted with buffer B in 10 20 30 3-ml aliquots to reduce the KC1 concentraFRACTION NO. tion to 100 mM, applied to a 27 x 1.5-cm column of DEAE-Sephadex equilibrated FIG. 1. Final chromatography on DEAE-Sephadex. with buffer B, and eluted with a linear gra(closed circles) Enzyme activity, (open circles) protein, (semiclosed circles) specific activity. Fractions (9 ml dient of KC1 in buffer B from 100 to 400 mM, each) indicated by the shaded bar were pooled and 400 ml of each, taking lo-ml fractions. Peak concentrated to constitute the purified enzyme. fractions (50 ml) were combined and concentrated to 2.2 ml in a Diaflo ultrafiltration doxal phosphate and 100 mM KCl. The en- apparatus with a PM-10 membrane. zyme was then eluted with a linear gradient Sephadex G-200. The concentrated en(100 to 400 mM KCl, 300 ml each) in lo-ml zyme was applied to a 54 x 2.3-cm column fractions. Those of highest specific activity of Sephadex G-200 which had been equiliand containing more than 85% of the ac- brated with buffer A and calibrated with tivity applied were pooled, and the enzyme marker proteins. The enzyme was eluted was precipitated with ammonium sulfate as with buffer A, and peak fractions were above. This preparation was dialyzed against pooled and concentrated by ultrafiltration. buffer B [50 mM potassium phosphate (pH Specific activity of the peak fractions was 6.5). 2.5 mM a-ketoglutarate, 10 PM pyri- nearly constant but analysis of heavily

c

TABLE SUMMARY

OF TYROSINE

1

AMINOTRANSFERASE

PURIFICATION

Fraction

(ml)

Total activity (units X 10mR)

Total protein (mg)

Extract Heat treatment DEAE-Cellulose DEAE-Sephadex I CM-Sephadex DEAE-Sephadex II Sephadex G-200 DEAE-Sephadex III

3940 3570 162 70 25 2.2 4.5 4.0

22,944 24,258 22,329 19,602 18.179 10,356 7.352 3,978

74,190 33,915 2.3% 1,022 144.5 48.5 27.9 13.7

Volume

Specific activity (unitsimg protein) 309 715 9.3 19 19.180 125,806 213,526 263,513 290,365

190

LEE, ROBERSON,

AND KENNEY

I-

80

120

ELUTION

160 VOLUME

200 (ml)

0.2 RELATIVE

0.6 0.4 MOBILITY

FIG. 3. Estimations of tyrosine aminotransferase molecular weight. (A) By chromatography on Sephadex G-200. The column was calibrated with marker proteins: (1) rabbit y-globulin, (2) bovine serum albumin, (3) chicken ovalbumin, (4) chymotrypsinogen (bovine pancreas). The open circle represents the elution position of tyrosine aminotransferase. (B) By sedimentation in glycerol gradients. Gradients (5 ml) containing 8 to 35% glycerol and buffer A were centrifuged for 12 h at 65,000 rpm. Designation of marker proteins is as in (A) except (2d) the dimeric form of bovine serum albumin.

FIG. 2. Electrophoretic analysis of the purified enzyme. (A) Intact enzyme (60 pg) was electrophoresed in 7.5% polyacrylamide at 1 mA/tube for 2 h as described by Brewer and Ashworth (19). Mobility of the enzyme relative to bromphenol blue was 0.4. (B) Enzyme subunits (60 pg) in sodium dodecyl sulfate-polyacrylamide gel according to Weber and Osborne (20). The enzyme was dissolved in 1% sodium dodecyl sulfate-2% p-mercaptoethanol at 100% for 10 min, then dialyzed overnight against 0.1% sodium dodecyl sulfate, 1% &mercaptoethanol, and 10 mM phosphate buffer, pH 7.0, before electrophoresis at 8 mA/tube for 5 h. (C) enzyme subunits (50 pg) in urea-polyacryl-

loaded electrophoretic gels of the concentrated product revealed some contaminating proteins. DEAE-Sephadex III. The enzyme was rechromatographed on DEAE-Sephadex as described for DEAE-Sephadex II, except that 300 ml of each of the eluting buffers was used in the gradient. Peak fractions with essentially constant specific activity were pooled as indicated (Fig. 1) and concentrated by ultrafiltration. This preparation contained 17% of the activity of the initial extract, purified about lOOO-fold (Table I), and appeared to be homogeneous on electrophoresis in acrylamide gel (Fig. 2A). Enzyme Structure weight. Gel filtration of the enzyme on Sephadex G-200 indi-

Molecular

purified

amide gel. The enzyme was treated with 8 M urea and 1 mM dithiothreiotol at 37°C for 0.5 h before electrophoresis at 1 mA/tube for 3.5 h in 7.5% gel containing 8 M urea (19).

TYROSINE

AMINOTRANSFERASE

191

FROM RAT LIVER

is again found (Fig. 2C). Thus the subunits appear to be identical in charge as well as size. This analysis also revealed two minor contaminants in the purified enzyme contributing about 1% or less to the total protein content. Coenzyme content. Pyridoxal-P binding to the purified enzyme was determined after resolution of the holoenzyme by treatment with tyrosine followed by (NH&SO, pre04 0.6 0.2 05 0.4 0.6 0.8 cipitation and dialysis; less than 1% of activRELATIVE MOBILITY ity remained when assayed in the absence FIG. 4. Estimations of molecular weight of tyrosine of coenzyme, but it was fully restored by aminotransferase subunits. (A) By sedimentation in 5 to coenzyme supplementation of assay mix20% sucrose gradients containing 0.5% sodium dodecyl tures. In Fig. 5 are presented results of sulfate and 10 mM Tris pH 7.4, centrifuged for 14 h at 65,000 rpm. Designation of marker proteins as in Fig. titrations of the apoenzyme with coenzyme 3A, except 1 h and 1 I, the heavy and light chains, respectively, of rabbit y-globulin. (B) by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Designation of marker proteins as in Fig. 3A, except 5, rabbit muscle glyceraldehyde-3-phosphate dehydrogenase: and 6, egg-white lysozyme.

cated a molecular weight of 100,000, while glycerol density gradient centrifugation yielded a value of 105,000, both in comparison to standard proteins of known molecular weight (Fig. 3). Subunit structure. A single electrophoretie band is found after sodium dodecyl sulfate denaturation and electrophoresis on polyacrylamide gels containing this detergent (Fig. 2B). This and the other gels shown in Fig. 2 were loaded heavily to assess homogeneity of the enzyme; analysis of smaller amounts (5 to 10 pg) under the conditions described yields a single sharp band. The molecular weight of the enzyme subunit was determined by comparison to marker proteins both by electrophoresis on polyacrylamide gels and by centrifugation in sucrose density gradients (both containing sodium dodecyl sulfate), yielding values slightly greater than 50,000 in both analyses (Fig. 4). When the enzyme was denatured and subjected to electrophoresis in 8 M urea, conditions wherein net charge contributes as a determinant of migration, a single band

PYRIDOXAL

PHOSPHATE

(nmoli

FIG. 5. Titration analyses of coenzyme content. The apoenzyme of tyrosine aminotransferase (3 mg) prepared as described in the text and in 1 ml of 20 mM phosphate buffer pH 7.0 containing 1 mM EDTA and dithiothreitol was titrated at room temperature with increasing amounts of an 800 nM solution of pyridoxal phosphate and absorbancy at 425 nm determined. (Insert) Titration by borohydride reduction of the reconstituted holoenzyme. Apoenzyme (0.089 nmol) was incubated as above at 37°C for 5 min with increasing amounts of pyridoxal phosphate, after which 50 nmol of 3H-labeled NaBH, (170 mCi/nmol, New England Nuclear) was added. Radioactivity associated with the protein was determined in samples pipetted onto filter paper disks and washed as described by Mans and Novelli (21). 3H incorporated in the absence of added coenzyme (ca. 2500 cpm) has been subtracted from the data presented.

192

LEE, ROBERSON,

and measurements of the aldimine formed according to the method of Dempsey and Snell (22). Titration of 3 mg (30 nmol) of enzyme indicates saturation of apoenzyme with 64 nmol pyridoxal-P, or 2.13 mol coenzyme per mol enzyme. Replicate determinations of this sort gave ratios ranging from 1.8 to 2.3. The apoenzyme had little or no absorbance at 425 nm without added coenzyme. A similar titration, requiring only small amounts of enzyme and measuring the Schiff base formed by its capacity to be reduced by 3H-labeled NaBH,, indicated saturation of 0.089 nmol of apoenzyme with 0.17 nmol coenzyme, for a ratio of 1.91 (Fig. 5, insert). The resolved apoenzyme was significantly reduced by NaBH, in the absence of added coenzyme, accepting about 25% as much 3H as the fully reconstituted enzyme. Analyses of fluorescence, by the method of Churchich and Farrelly (23), of pyridoxamine-P formed after reaction with tyrosine are given in Table 2. In the first of these, apoenzyme prepared as described above was found to yield small but significant fluorescence, while the unresolved holoenzyme contained 2.27 mol of coenzyme per mol TABLE FLUORESCENCE

DISCUSSION These data indicate clearly that the aminotransferase is structurally a dimer, consist2 OF COENZVME

CONTENT

Incubation with tyrosinen

Coenzymel enzyme ratio

Functional coenzyme (mol/mol enzyme)

2.46 (apo) 2.46 (holo)

+ +

0.19 5.78

0.08 2.35

2.21

1.24

-

-

0.11 2.81 0.15 5.21

0.09 2.27 0.06 2.10

2.18 2.04

+ +

2.85 6.39 4.14 10.12

1.69 3.78 1.23 3.00

2.09 1.77

1 2a

2.48 2b

enzyme above this level. In the second experiment, measurements were made on two preparations of unresolved holoenzyme before and after reaction with tyrosine. These data reveal variable quantities of coenzyme bound to the purified enzyme, but not functional in reaction with tyrosine. Active coenzyme determined by this procedure ranged from 1.77 to 2.18 mol pyridoxal-P per mol enzyme. The various methods all yield values clustering around 2 mol of functional coenzyme per mol enzyme, indicating that each subunit contains a single functional coenzymebinding site. As isolated, the enzyme also contains variable amounts of nonfunctional coenzyme, which may have been introduced during the purification procedures, perhaps when the preparation is heated in the presence of pyridoxal-P. Binding of coenzyme in an inactive mode to the aspartate aminotransferase of pig heart was reported by Martinez-Carrion et al., who suggested that it was present as a substituted aldimine (24).

Coenzyme foundb (nmol)

Enzyme added (nmol)

Experiment number

ANALYSES

AND KENNEY

1.69 3.31

+

a Enzyme was incubated at 37°C for 30 min in 50 mM potassium phosphate (pH 7.6) and 4 mM tyrosine. b Determined at 390 nm, excitation at 325 nm (23).

TYROSINE

AMINOTRANSFERASE

ing of two apparently identical subunits of molecular weight about 50,000, as suggested in previous reports from our laboratory (6, 25) and by others (13- 15). Earlier reports suggesting a tetrameric protein with subunit size about 25,000 (10,ll) were based partially on coenzyme analyses which did not distinguish functional from nonfunctional coenzyme. As indicated in Table 2 the total coenzyme content of some enzyme preparations approaches the value of 4 mol coenzyme per mol enzyme. ACKNOWLEDGMENT

8. Kenney, F. T. (1962) J. Biol. Chem. 237, 16051609. 9. Hayashi, S., Granner, D. K., and Tomkins, G. M. (1967) J. Biol. Chem. 242, 3998-4003. 10. Valeriote, F. A., Auricchio, F., Tomkins, G. M., and Riley, W. D. (1969) J. Biol. Chem. 244, 3618-3625. 11. Auricchio, F., Valeriote, F. A., Tomkins, G. M., and Riley, W. D. (1970) Biochim. Biophys. Actu 221,307-313.

12. Iwasaki, Y.. Lamar, C., Danenberg, K., and Pitot, H. C. (1973) Eur. J. Biochem. 34, 347-357. 13. Roewekamp, W., and Sekeris, C. E. (1977) FEB.5 Lett.

73, 225-228.

14. Belarbi, A., Bollack, C.. Befort, N.. Beek, J. P., and Beek. G. (1977) FEBS Lett. 75, 221-225. 15. Donner, P., Wagner, H., and Kroger, H. (1978) Biochem.

We are indebted to our colleague J. G. Farrelly (now at the Cancer Research Center, Frederick, Md.) for help in various aspects of this work.

REFERENCES 1. Kenney, F. T. (1962) 1. Biol. Chem. 237, 34953498. 2. Holten, D., and Kenney, F. T. (1967) J. Biol. Chem.

242, 4372-4377.

3. Wicks, W. D., Kenney, F. T., and Lee, K.-L. (1969) J. Biol. Chem. 244, 6008-6013. 4. Tomkins, G. M., Thompson, E. G., Hayashi, S., Gelehrter, T., Granner, D., and Peterkofsky, B. (1%6) Cold Spring Harbor Syrnp. Quant. Biol. 31, 349-357.

Proc.

Nat.

Acad.

Sci.

USA

7. Kenney, F. T. (1959) J. Biol. 2711.

73, 2643-2639. Chem. 234, 2727-

Biophys.

Res. Commun.

80,766-772.

16. Diamondstone, T. I. (1966) Anal. Biochem. 16, 395-401. 17. Lowry, 0. H., Rosebrough, N. J., Farr. A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-300. 18. Johnson, R. W.. Roberson, L. E., and Kenney, F. T. (1973) J. Biol. Chem. 248, 4521-4527. 19. Brewer, J. M., and Ashworth, R. B. (1969) J. Chem. Educ. 46, 41-45. 20. Weber, K., and Osborne, M. (1969) J. Biol. Chem. 244, 4406-4412. 21. Mans, R. J., and Novelli, G. D. (1961) Arch. Biothem.

Biophys.

94, 48-56.

22. Dempsey, W. B.. and Snell, E. E. (1963) Biochemistry 6, 1414- 1419. 23. Churchich. J. E., and Farreily, J. G. ( 1969) J. Biol. Chem.

5. Kenney, F. T. (1967) Science 156, 525-528. 6. Stiles, C. D., Lee, K.-L., and Kenney, F. T. (1976)

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FROM RAT LIVER

244,72-76.

24. Martinez-Carrion, M., Turano, C., Chiancone, E.. Bossa, F., Giartosio, A., Riva, F., and Fasella, P. (1967) J. Biol. Chem. 242, 2397-2409. 25. Lee, K.-L., and Nickel, J. M. (1974) J. Biol. Chem. 249, 6024-6026.