THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 242, No. 10, Issue of May 25, pp. 2319-2324, 1967

Printed in U.S.A.

Further Characterization of Alanine Aminotransferase

of Rat Liver*

(Received for publication, December 20,1966)

PETER W. GATEHOUSE, SARAH HOPPER,$ LILLIAN SCHATZ, AND HAROLD L. SEGAL

From the Biology Department, Xtate University of New York, Bu$aZo, New York i4Sl4

SUMMARY

Rat liver alanine aminotransferase has been obtained in a homogeneous state according to ultracentrifugal, electro- phoretic, and immunological criteria. The molecular weight of the enzyme is 114,000. No physical differences were detected in the enzyme prepared from normal and glucocorti- coid-treated animals.

The enzyme exhibited an absorption maximum at 430 mp at pH 5.0 and at 335 rnp at pH 8.3. After conversion to the pyridoxamine form, a maximum at 325 rnp appeared. Reac- tion of the enzyme with amino-oxyacetate shifted the 430 rnp peak to 370 mp.

The enzyme appeared to exist in several closely related forms of slightly different mobility on acrylamide gel.

In the absence of the reducing agent, P-mercaptoethanol, a series of aggregated forms of the enzyme arose.

Previous reports from this laboratory have described the partial purification of rat liver alanine aminotransferase (EC 2.6.1.2), its specificity, the hormonal control of its synthesis, its half-life in liver, and some of its kinetic and other properties (l-6). En- zyme preparations obtained from normal and corticoid-treated rats were found to be indistinguishable on the basis of kinetic properties, pH optima, behavior. during purification, and im- munological cross-reactivity (1, 3).

The present report describes methods whereby the enzyme has been obtained in an apparently homogeneous state and some properties of the enzyme thus obtained. Further compari- sons of the enzyme from normal and corticoid-treated animals in regard to their electrophoretic and sedimentation behavior sup- port the conclusion that the enzyme from these sources is identi- cal. Conclusive identification of the enzyme as pyridoxal- containing has been achieved by its absorption spectra and pyri- doxal phosphate content.

* This work was supported by Grant AM-08873 from the United States Public Health Service. Part of these results was presented at the Second IUB International Symposium on Chemical and Biological Aspects of Pyridoxal Catalysis, Moscow, USSR, Sep- tember 15 to 22, 1966.

$ Present address, Department of Biochemistry and Nutrition, School of Public Health, University of Pittsburgh, Pittsburgh 13, Pennsylvania.

EXPERIMENTAL PROCEDURE

Definition of enzyme units and enzyme assay were as previ- ously described (Assay Method II) (1). Protein was determined by the method of Lowry et al. (7) or the biuret method (3) with bovine serum albumin as standard or by 278 mp absorbance assuming an extinction coefficient of 1.0 (mg per ml)-1. The ratios of these determinations varied from fraction to fraction; therefore, the basis of the specific activity calculations is specified in each case.

DPNH, lactic dehydrogenase (rabbit muscle, type I), DEAE- cellulose and DEAE-Sephadex were obtained from Sigma. Am- monium sulfate and sucrose were special enzyme grade from Mann. Prednisolone acetate from Pfizer Laboratories, New York, New York, was used for hormone injections.

Acrylamide gel electrophoresis of the enzyme was performed in the E-C Apparatus Corporation, Philadelphia, Pennsylvania, instrument with a 5% gel in buffer containing per liter: 10 g of Tris, 1 g of sodium-EDTA, 0.38 g of HaBOa, and 1W2 mole of @-mercaptoethanol (final pH of 8.9 to 9.1). The enzyme was applied in a volume of 50 ~1 or less. After 1 hour of equilibra- tion, separation was carried out for 2$ hours at 300 volts on a gel slab 17 cm long (milliamperage at the start was 150 to 200 and fell to 100 by the end of the run). Protein staining was carried out in a 0.2 ye solution of Amido black 1OB in a solvent of metha- nol-acetic acid-H20 (5:5:1) for 30 min, and the background was destained electrophoretically in 2y0 acetic acid in water for approximately 14 hours., For elution of the enzyme from the gel, 6-mm segments were taken, chopped fine, placed in a test tube with 1 ml of the electrophoresis buffer, and allowed to stand for 18 hours at 4’. Under these conditions, approximately 70% of the enzyme activity applied could be accounted for.

For starch gel electrophoresis the enzyme was dialyzed against 0.025 M Tris-chloride, pH 8.6, and subjected to vertical electro- phoresis (9) in the same buffer. The effective voltage across the starch block was 10 volts per cm, and 35 ma of current was applied. After a suitable time period, the starch block was sliced parallel to the slab surface, and one portion was stained for pro- tein in a solution composed of 10 volumes of 50% methanol and 1 volume of a saturated solution of Amid0 black 10B in glacial acetic acid (10). The other portion was cut into l-cm segments which were assayed for enzyme activity. Each segment was homogenized in 4 ml of cold assay medium from which a-keto- glutarate was omitted, centrifuged, and 3 ml of the supernatant solution were transferred to a cuvette. After standing at 37”

2319

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2320 Alanine Aminotransferase of Rat Liver Vol. 242, No. 10

400

%

5 300 I \

if z = 200

G

L z 100 W

35 40 45 50 55

TUBE NUMBER

FIG. 1. DEAE-cellulose chromatography of alanine amino- transferase. Enzyme (11,100 units) was applied to the column in a volume of 37 ml. The total protein was 446 mg (278 nm absorb- ance). The column was then washed with 680 ml of phosphate- mercaptoethanol buffer, which removed 297 mg of protein and 85 units of enzyme. The new buffer containing NazSOd was then introduced and 5.0-ml fractions were collected. Numbering of the tubes begins at this point.

for 5 min, the reaction was started by the addition of cu-keto- glutarate.

RESULTS AND DISCUSSION

Enzyme PurQication

Adult male albino rats were given injections of 2 mg of pred- nisolone acetate daily to induce high levels of the enzyme’ and were killed by decapitation 2 hours after the sixth injection. The livers were homogenized in a Sorvall Omni-Mixer at full speed for 30 set in 7 to 9 volumes of 0.25 M sucrose containing 10m2 M

/3-mercaptoethanol. After centrifugation of the homogenate at 10,000 x g for 30

min at 0”, the supernatant fluid (crude extract) was heated at 55” and then the pH was lowered to 5.0 as previously described (1). Denatured protein was removed by centrifugation, and to the clear solution (heated extract) 275 mg of solid ammonium sulfate were added per ml. The precipitate was separated by centrifugation and dissolved in a small volume of 10m2 M potas- sium phosphate buffer, pH 5.7: containing lo* M /3-mercapto- ethanol. The solution was dialyzed against several changes of the same buffer until the dialysate failed to give a precipitate with an acid solution of Bach. The enzyme solution was then cen- trifuged at 50,000 rpm for 2 hours at 0” (Ammonium Sulfate I Fraction). This procedure sediments a brownish inactive pellet (ferritin?).

For a preparation from a typical group of 25 to 35 rats a DEAE-cellulose column (60 x 2 cm) was prepared as follows.

1 The activity of the enzyme in livers of rats pretreated with glucocorticoids is elevated some 3- to 5-fold above normal (11). The elevated tissue level has been demonstrated to be the result of an increased rate of synthesis of the enzyme (4-6).

2 This pH and phosphate concentration are critical for the DEAE-cellulose step which follows.

The adsorbent (coarse mesh) was suspended in water, and the fines were decanted several times. It was then suspended in 10” M potassium phosphate buffer, pH 5.7, containing lo+ M

@mercaptoethanoP and poured into the column. The column was washed with the buffer at room temperature until the pH of the effluent was 5.7, then placed in a 4” room and washed further with cold buffer. The enzyme solution was placed on the column in a volume of 25 to 40 ml. Buffer was allowed to flow through at a rate of about 300 ml per hour until the 278 rnE.c absorbance fell to 0.04 (about 800 ml). The buffer was then replaced with one as above but containing 0.025 M Na2S04 in addition, and 4- to 5-ml fractions were collected. A typical chromatogram is shown in Fig. 1. About two-thirds of the total activity recov- ered from the column of the best specific activity was pooled (DEAE-cellulose fraction) (tubes 39 through 42 in Fig. 1).

To the pooled material, 1 ml of a Ca3(PO& suspension (200 mg per ml) was added. After stirring for about 5 min, the Ca3(PO& was removed by centrifugation and discarded. This procedure was repeated once or twice more, until the specific activity (278 rnp absorbance) was about 270 (Ca3(POJ2 fraction); then 300 mg of ammonium sulfate were added per ml of solution to pre- cipitate the enzyme (Ammonium Sulfate II Fraction). The en- zyme was frequently stored at 4” in this form and pooled with subsequent preparations before proceeding to the next step.

For DEAE-Sephadex chromatography, the precipitated enzyme was centrifuged and dissolved in about 1 ml of 0.04 M potas- sium phosphate buffer, pH 7.3, containing 10m2 M P-mercapto- ethanol and dialyzed against the same buffer. The DEAE- Sephadex (A-50, medium mesh) was suspended in water, and the fines were decanted several times. It was then washed on a Buchner funnel with 0.5 N HCl, 0.5 N NaOH, 0.5 N HCl, and the above buffer several times. The gel was poured into a 1.5-cm column at room temperature to a height of 30 cm and washed overnight in the cold with the buffer. The flow rate was 2 to 4 ml per hour. The enzyme was applied to the column, washed in with 1 ml of 0.04 M buffer, then eluted with a linear gradient of buffer produced with 150 ml each of 0.04 M and 0.10 M potas- sium phosphate, pH 7.3, containing lop2 M /Lmercaptoethanol (DEAE-Sephadex Fraction).4 A typical chromatogram is shown in Fig. 2.

A complete purification protocol is presented in Table I.

Gel Electrophresis

Acrylamide gel electrophoretic patterns of material from five stages of the purification procedure (Table I) are presented in Fig. 3. The enzyme was eluted and assayed from parallel runs on the same slab of gel shown in Fig. 3A. The results are in Fig. 4. The arrow in Fig. 3A indicates the location of the enzyme activity. Only one peak of enzyme activity was observed in any of the stages.

Starch gel electrophoresis of crude enzyme and of enzyme of specific activity corresponding to the DEAE-Sephadex fraction of Table I again yielded one band of enzyme activity and in the latter case one protein band of corresponding mobility migrating toward the anode (3 cm and 9 cm from the origin after 2 hours

3 Buffers containing B-mercaptoethanol exhibited a gradual increase in 278 rnp absorbance (disulfide formation?) and were therefore not retained beyond 1 week.

4 In more recent experiments, somewhat higher specific activi- ties have been obtained by developing the chromatogram entirely with 0.04 M phosphate.

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Tssue of May 25, 1967 Gatehouse, Hopper, Schafz, and &gal 2321

240

15 20 25 30 35 TUBE NUMBER

FIG. 2. DEAE-Sephadex chromatography of alanine amino- transferase. Enzyme (6200 units) was applied to the column in a volume of 2.3 ml. The total protein was 16.5 mg (278 rnp absorb- ance). The elution was developed with a linear gradient of phos- phate buffer as described in the text. A small protein peak was observed at tube 6 in this chromatogram (not shown). In other chromatograms a protein peak rather than the shoulder observed here appeared on the leading edge of the enzyme peak. Specific activity rose to a maximum between tubes .a/; and 30 with an aver- age value of 552 i 12 (S.E.). Pi concentration was determined by conductivity. The volume of the fractions was 3.7 ml.

TABLE I Purification of rat liver alanine aminotransferase

Fraction

Crude extract. 35,250 Heated extract. . . 32,550 Ammonium Sulfate I. 13,400 DEAE-cellulose. . . 5,270 Caa(POd)n. . . 4,390 Ammonium Sulfate II 6,260” DEAE-Sephadex. . . 3,715

-.

Total units

-

-

-

Speci6c activity

units/mg protein

0.88 2.33

23.8 28.4 233 272

231 380 309 552

Yield

%

100 87 38 15 12.5

7.4=

0 An Ammonium Sulfate II Fraction of the same specific activity from an earlier preparation was combined with the present frac- tion prior to the DEAF-Sephadex step. The final yield has been corrected appropriately.

and 74 hours, respectively). No differences were observed in the mobility of the enzyme from normal as opposed to glucocorti- coid-treated livers, either in the crude extracts or in the highly purified preparations.

Acrylamide gel electrophoretic patterns of several fractions around the peak from the DEAE-Sephadex chromatography (Fig. 2) are shown in Fig. 5. As observed on several occasions, the early fractions moved slightly behind the later ones and on some gel runs the enzyme band was seen to split into two sharp closely moving sub-bands of about equal intensity. To test the possibility that these resulted from the use of pooled material

from a large number of animals, enzyme from a single rat was purified through the Ca3(PO& stage. Electrophoresis of this preparation also yielded sub-bands at the location of the enzyme. In view of the relative constancy of the specific activity of the DEAE-Sephadex fractions at the peak and the homogeneity of the pooled fractions during sedimentation (see below), it can be concluded that the sub-bands observed on electrophoresis repre- sent closely related sub-species of the enzyme.

FIG. 3. A&amide gel electrophoresis of alanine aminotrans- ferase at different stages of purification. Migration wea from the top under conditions described in the text. A, from left to Tight preparations were crude extract, heated extract, and Ammonium Sulfate I (Table I). Enzyme, 1 unit, was applied in each case containing 1.4 mg, 0.5 mg, and 0.075 mg of protein (Biuret), re- spectively. The arrow indicates the location of enzyme activity (Fig. 4). B, from left to right the preparations were Ammonium Sulfate I, Cat(POSz, and DEAE-Sephadex (Table I). Enzyme, 20 units, was applied in each case.

o.6 ;RUDE EXTRA’ $TED EXTRA1 CT

1

AS I

I 0 36 72 0 36 72 0 :

DISTANCE FROM ORIGIN (MM)

FIG. 4. ‘Localization of alanine aminotransferase after gel elec- trophoresis. Enzyme was eluted from 6-mm segments as de- scribed in the text after electrophoresis on the same slab as in Fig. 3A. The volume of gel was estimated to be 0.5 ml for cal- culation of enzyme units per tube. AS I, Ammonium Sulfate I.

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2322 Ala-nine Aminotransjerase of Rat Liver

Absorption Spectra

Vol. 242, No. 10

+ .I501 I

The absorption spectra of the enzyme in the pyridoxal form at pH 5.0 and pH 8.3 and in the pyridoxamine form at pH 7.0 are shown in Fig. 6. As isolated the enzyme is in the aldehyde form and is bright yellow at pH 5 with a maximum at 430 rnp and a broad minimum from 320 to 360 mp. At alkaline pH the main peak is at 335 rnp with a lesser maximum in the 400 to 450 rnp region. The pyridoxamine form of the enzyme exhibited a maxi- mum at 325 mp. Upon addition of 10m4 M amino-oxyacetate (a potent inhibitor of the enzyme, competitive with the amino acid substrates and uncompetitive with the keto acid substrates (2)) to the pyridoxal enzyme at pH 5, the peak at 430 rnl.c disappeared and was replaced by one at 370 rnH (Fig. 7).

FIG. 5. Acrylamide gel electrophoresis of fractions from the DEAE-Sephadex column. A, approximately 20 units each of enzyme from tubes 22, W,94,26,R8, and 30 in Fig. 2 (top to bottom of A) were electrophoresed as described in the text. Migration was from right to left. B, electrophoresis of tubes .@ (right) and 30 (left) was repeated in adjacent channels.

.300

.200

A

.I00

.ooc

X (millimicrons)

FIG. 6. Absorption spectra of alanine aminotransferase. Curves marked 6.0 and 8.3 are of the pyridoxal form at the respective pH values. Curve marked Pyr NH2 is from another enzyme prepara- tion after dialysis against 0.1 M glutamate, pH 7.0. Data from both preparations were normalized to a 278 rnF absorbance of 1.00.

AA

.ooo-

-.I50 I 300

I 8 340 380 420 460

X (millimicrons)

FIG. 7. Effect of amino-oxyacetic acid on the absorption spec- trum of alanine aminotransferase. Amino-oxyacetic acid to a final concentration of lo-” M was added in a negligible volume to a solution of the pyridoxal form of the enzyme at pH 5. The difference spectrum shown is the spectrum after aminooxyacetic acid addition minus that before amino-oxyacetic acid addition.

These spectra aie closely similar to those reported for heart aspartic aminotransferase (12). Reported spectra for heart alanine aminotransferase (13, 14) differ from those observed here but the preparations employed were of a lesser degree of purity. The spectrum of the enzyme in the presence of amino-oxyacetate (which may be considered a homologue of hydroxylamine) is pre- sumably that of an oxime of pyridoxal (13, 15, 16). A similar spectral shift was obtained upon additions of isonicotinic acid hydraside to heart aspartic aminotransferase (17).

All forms of the enzyme exhibited peak absorbances at 278 rnp.

Molecular Weight a.nd Pyridbxal Phosphate Content

The sedimentation coefficient of the enzyme at a concentration of 0.5 mg per ml was determined to be 6.3 S at 20”, corrected to water. Molecular weight determinations of two separate prep- arations by the equilibrium sedimentation method of Yphantis (18) gave a value of 114,000 f 5,000, extrapolated to zero con- centration (Fig. 8). A partial specifmvolume of 0.74 ml per mg was assumed. The preparations employed in these experiments appeared to be homogeneous in both the sedimentation velocity and equilibrium experiments.

As in the starch gel electrophoresis experiments, sucrose density gradient centrifugation of crude extracts from normal or gluco- corticoid-treated livers gave a single peak of activity at the same location in both cases. Calculation of the sedimentation coeffi- cient and molecular weight by comparison with alcohol dehy- drogenase in the gradient, according to the method of Martin and Ames (19), gave values of 6.2 S .and 111,000, respectively. These are remarkably close to the figures obtained with the analytical ultracentrifuge. On the other hand, comparison with the considerably heavier catalase molecule gave too high a value of 7.0 s.

This procedure was also used on a preparative scale with mod- erate success as the last step in the purification procedure prior to adoption of DEAE-Sephadex chromatography.

Preliminary analyses of the pyridoxal phosphate content by two different methods (20, 21) gave somewhat scattered results but revealed in all cases the presence of substantial quantities of

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Issue of May 25, 1967 Gatehouse, Hopper, Schatz, and Segal 2323

bound pyridoxal phosphate. Experiments are in progress to ob- tain more precise values.6

Enzgm Aggregation

A phenomenon of interest was observed when the fractionation procedure (Table I) was carried out in the absence of &mercapto- ethanol. Chromatography on Sephadex G-200 under these cir- cumstances led to a marked spreading of the enzyme activity

s Note Added in Proof--A value of 2 pyridoxal phosphate groups per enzyme molecule can now be reported (to be published).

I.11 I

of3\ 0.5 1.0 1.5 2.0

c (mg/ml)

FIG. 8. Equilibrium sedimentation analysis of alanine amino- transferase by the method of Yphantis (18). Ordinate is the re- ciprocal of apparent molecular weight at a series of radii within the cell, and abscissa is the concentration of protein at these points from fringe displacement (average of 5 at each point). Vertical bars represent standard deviations. Points, standard deviations, and intercepts were calculated by computer. Data from two separate cells are shown at loading concentrations of enzyme of 0.230 mg per ml (0 ) and 0.092 mg per ml (O), respec- tively (278 u absorbance). The enzyme was dissolved in 0.04 M potassium phosphate, pH 7.3, containing 10M2 M P-mercaptoeth- anol. The identity of the two curves is a criterion of homogeneity of the enzyme preparation.

with a major portion appearing in the excluded front. In the presence of @mercaptoethanol, on the other hand, the enzyme was retarded and moved as a single peak. Similarly, upon acrylamide gel electrophoresis of enzyme prepared without the reducing agent a large fraction failed to enter the gel, and the re- mainder was spread out up to the point where enzyme prepared with fl-mercaptoethanol migrated. When the crude extract was subjected to electrophoresis and the enzyme location was deter- mined by elution and assay, a single band of activity appeared even in the absence of the thiol compound. Finally, in sucrose density gradient centrifugation experiments with highly purified preparations which had been stored for extended periods in the absence of P-mercaptoethanol, enzyme activity was frequently observed in regions below the main peak. On the other hand, in

120 ,9.0

ENZYME (ML) FIG. 9. Precipitin curve of alanine aminotransferase with rabbit

antienzyme serum. Tubes contained in 1.0 ml, 0.1 ml of 0.1 M potassium phosphate, pH 7.3, 0.33 ml of antiserum, and varying quantities of an Ammonium Sulfate I enzyme preparation (30 units per ml), as shown on the abscissa. After centrifugation and washing twice with 0.9% NaCl, the protein content of the pre- cipitates (according to the method of Lowry) (0) and the enzyme activity remaining in the supernatant solutions ( l ) were deter- mined.

FIG. 10. Double diffusion immunochemical analysis of alanine aminotransferase. The plates contained a 1.2% solution of Agar- ose agar (Bausch and Lomb) in a medium containing per liter, 6.8 g of NaCl, 1.48 g of Na*HPO+ 0.43 g of KHzPO,, and 29 mg of NaNa. The wells were 3 mm in diameter and 3 mm apart. A, the center well contained 0.24 units of “pure” antiserum K and well 6 contained 0.15 unit of “pure” antiserum J. Wells 6, 6, and 6 contained 0.21 unit, 0.05 unit, and 0.16 unit, respectively, of an Ammonium Sulfate I enzyme fraction (Table I). WeZZ 1 was inad- vertently overloaded with enzyme and well 4 was empty. B,

the center well and well 6 contained 0.3 units of “pure” antiserum K. Wells 1 and 6 contained 1.5 units and 3.8 units, respectively, of a DEAE-Sephadex enzyme fraction (Table I). Wells 6, 4, and b contained 1.5 units, 15 units, and 3.8 units, respectively, of an Ammonium Sulfate I enzyme fraction. C, the center well and well 4 contained 0.3 unit of “pure” antiserum K. Wells 6 and 6 contained 0.3 unit of “crude” antiserum E. Well 1 contained 15 units of an Ammonium Sulfate I enzyme fraction. Wells 6 and 6 contained 15 units and 3.8 units, respectively, of a DEAK-Sepha- dex enzyme fraction.

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2324 Alanine Aminotransferase of Rat Lzver Vol. 242, No. 10

freshly prepared crude extracts only one activity peak appeared in the usual position.

A reasonable conclusion from these observations is that an ag- gregation of the enzyme occurs under certain conditions giving rise to a spectrum of active oligomers. This aggregation can be prevented by the presence of P-mercaptoethanol.

Immunochemical Experiments

For the preparation of “crude” and ‘(pure” antisera, enzyme fractions at the Ammonium Sulfate I and DEAE-Sephadex stages of purity, respectively, were used (Table I). Injections, treat- ment of sera, and incubations were as previously described (3), with approximately 200 units of enzyme in each injection.

In the quantitative precipitin reaction (22) with “pure” anti- serum and an enzyme preparation at the Ammonium Sulfate I stage of purity (Fig. 9)6 the equivalence point of the titration was at the zone of maximum precipitation, a finding consistent with immunochemical homogeneity of the antiserum. At this point 6.2 units of enzyme, equivalent to 21 pg of protein (Lowry), were present in the total precipitate of 100 pg. Thus, there were present in the precipitate 3.8 pg of antibody per pg of enzyme, and the antiserum contained 0.24 mg of antienzyme per ml.

Further confirmation of immunochemical homogeneity was ob- tained by the double diffusion procedure of Ouchterlony (23). The results are shown in Fig. 1O.7 In Fig. 10.4 there was a single band of precipitation between the crude Ammonium Sulfate I en- zyme fraction and two different pure antisera (center well against wells 2, S, and 5 and well 6 against well 5). In Fig. 1OB this single precipitation band (center well against wells 2, 4, and 5) showed a line of identity with the precipitation band produced by the homogeneous DEAE-Sephadex enzyme (center well against wells 1 and 6). In Fig. 1OC the homogeneous DEAE-Sephadex enzyme gave a single precipitation band with crude antiserum (well 5 against well B), which shows a line of identity with the precipitation band with pure antiserum (well 5 against center

well). That the crude antiserum was heterogeneous may be seen from the multiple bands in the reaction with the crude Ammonium Sulfate I enzyme fraction (well 6 against well 1).

6 The technical assistance of Miss Conception Gonzalez-Lopez in these experiments is acknowledged.

7 We are indebted to Dr. Noel Rose for advice and the use of his acilities in these experiments.

The single precipitation band formed by interaction of DEAE- Sephadex enzyme and crude antiserum is evidence for the homo- geneity of the enzyme, as is its ability to elicit antiserum which reacts specifically with the enzyme in a crude liver extract.

Aclcnowledgments-The experiments with the analytical ultra- centrifuge were performed by Dr. David A. Yphantis and Mr. Dennis Roark to whom we wish to express our sincere apprecia- tion.

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Peter W. Gatehouse, Sarah Hopper, Lillian Schatz and Harold L. SegalFurther Characterization of Alanine Aminotransferase of Rat Liver

1967, 242:2319-2324.J. Biol. Chem. 

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