alanine aminotransferasealanine aminotransferase (l-alanine : 2-oxoglutarate amino- transferase, ec...

THE JOURNAL OF BIOLOGICALCHEMI~TRY Vol. 242, No. 1, Igsue of January 10, pp. 91-100, 1967

Printed in U.S.A.

Alanine Aminotransferase

I. PURIFICATION AND PROPERTIES*

(Received for publication, June 28, 19tiF)

MILTON H. SAIER, JR., AND W. TERRY JENKINSI

From the Department of Biochemistry, University of Calijornia, Berkeley, California 947,20

SUMMARY

An examination of the subcellular distribution of alanine aminotransferase activity in pig cardiac tissue showed that about 10% of the total activity was bound to particulate material, with the highest specific activity in the sarcosomal fraction.

The soluble enzyme was obtained in a high state of purity, as indicated by sedimentation velocity, starch gel electrophoresis, and spectral analyses. It had a sedimentation coefficient of 6.0 and an estimated molecular weight of about 100,000. Spectral, fluorescent, and optical rotatory properties of the enzyme are described.

The enzyme-substrate interactions were studied by kinetic and spectral methods. Kinetic parameters of the enzyme- catalyzed reaction involving amino transfer from L-alar&e to oc-ketoglutarate were determined at 37’, pH 7.8. At pH 6.9, pyruvate was found to bind effectively to the phosphopyridoxal form of the enzyme and, thereby, competitively inhibit amino transfer from both alanine and glutamate to pyruvate.

A recent examination of the alanine aminotransferase activities associated with soluble and mitochondrial fractions from rat liver has established their isozymal nature (l), and Katunuma et al. (2) have reported that alanine aminotransferase activity appeared in both soluble and particulate fractions of pig heart. It was, therefore, of interest to ascertain whether or not possible mitochondrial isozymes contaminate the soluble enzyme which we have purified.

Alanine aminotransferase (L-alanine : 2-oxoglutarate aminotransferase, EC 2.6.1.2) of pig heart was one of the first amino- transferases to be highly purified (3, 4). Modifications of the early procedures have been presented, but none supplies enough pure enzyme for spectroscopic observation or physicochemical characterization (5-7). The purified enzyme is quite unstable in the absence of protective agents (7). However, some of its spectral properties, described in preliminary communications

* This work was supported in part by Grant 5 ROl HE04417 from the United States Public Health Service.

$ Present address, Department of Chemistry, University of Indiana, Bloomington, Indiana, 47401.

(8, 9), indicate that it is of considerable interest for studies of the mechanism of substrate specificity with phosphopyridoxal enzymes. Therefore, we determined optimal conditions for stability and developed a large scale purification procedure.

Considerable effort has been devoted to the elucidation of the properties of the pig heart alanine aminotransferase (3-9). These investigations have revealed that the enzyme is very sen- sitive to sulfhydryl reagents and carbonyl reagents (5-7). Spec- tral examination of preparations of the phosphopyridoxal form of the enzyme (6, 9) and of sodium borohydride-treated preparations (6, 7) have provided evidence for Schiff’s base linkage between pyridoxal phosphate and an e-amino group of an enzymic lysine residue. Physicochemical and kinetic properties of pig heart alanine aminotransferase are still poorly defined. In this communication we describe both these and the spectral properties in greater detail.

METHODS

Assay Procedures-The assay method routinely used was essentially that of Lenard and Straub (3). The assay concentrations were: 0.1 M L-alanine, 0.01 M cu-ketoglutarate, and 0.04 M Tris base, pH 8.1 (at 25”). Tris buffer was generally used because it had no inhibitory effect on the enzyme. A I-ml reaction mixture was incubated for 10 min at 37” before transamination was terminated by the addition of 1 ml of 60% (w/v) KOH. Salicylaldehyde, 2% in 95% ethanol (0.5 ml) was then added, and the orange color was permitted to develop for 10 min at 37”. Addition of 2.5 ml of ice-water stabilized the color, which was measured at 480 rnp in a Zeiss PMQ spectrophotometer with slit set at 0.01 mm. One unit of activity is defined as that amount of enzyme which produces 1 pmole of pyruvate per min under these conditions. Specific activity is given in terms of units per mg of protein.

For the determination of glutamate-transaminating activity as a function of pH (Fig. 1, Curve B), the salicylaldehyde color reaction was used to measure the decrease in the pyruvate concentration after a IO-min incubation period at 37”. The I-ml reaction mixtures initially contained 0.05 M L-glutamate, 0.01 M

pyruvate, and 0.1 M pyrophosphate. After addition of 5 ml of 40% KOH and 1 ml of 2% salicylaldehyde in ethanol, color was permitted to develop as above before the addition of 7 ml of ice-water.

For determination of Michaelis constants and the pyruvate- phosphopyridoxal enzyme dissociation constant, initial velocities of alanine or glutamate transamination were measured by re-

91

by guest on June 4, 2020http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

ducing the pyruvate or a-ketoglutarate, respectively, formed in in separate experiments with the split beam automatic photo- the reactions with NADH and an excess of lactate dehydrogenase electric scanning absorption system developed by Schachman or glutamate dehydrogenase. With the temperature maintained et al. (15) and Lamers et al. (16). Double sector 12-mm cells at 37”, the decrease in absorbance at 340 rnp was followed with a were used for these experiments, and the rotor speed was 59,780 Gilford recording spectrophotometer. rpm. The sedimentation coefficient at 20” in water was calcu-

Protein was determined routinely by the phenol method (10). lated as described by Schachman (17). However, a modified biuret method (11) was used for protein The molecular weight of alanine aminotransferase was esti- determination in crude fractions. Before the absorbance at 540 mated from the sedimentation equilibrium pattern of the ma- rnp was read, solutions were clarified by shaking with 3.0 ml of terial absorbing at 425 rnp according to the procedure of Schach- acetone and centrifuging. Crystalline bovine serum albumin man and Edelstein (18). These high speed sedimentation was used as a standard for both methods. equilibrium experiments were conducted as described by Yphan-

Subcellular Fractionation oj Alanine Aminotransferase Ac- tis (19) with a rotor speed of 20,000 rpm at 20”. Yphantis cells tivity-Minced pig heart muscle (10 g) was ground with sand in which permitted the simultaneous determination of the sedimen- 20 ml of 0.28 M sucrose-O.01 M EDTA, pH 7.4 (the suspending tation patterns of two enzyme samples were used. Patterns solution). The fluid was squeezed through four layers of cheese- were recorded 17 to 24 hours aft.er speed was attained. cloth, and the residue was suspended three times in 10 ml of Determination of Vitamin BG-Vitamin B6 was hydrolyzed suspending solution, each suspension being followed by removal from the enzyme (specific activity, 340) by autoclaving at 20 of the fluid through cheesecloth. The material which did not p.s.i. in 0.055 N sulfuric acid for 5 hours (20). The vitamin Bc pass through the cheesecloth was resuspended for subsequent content of the hydrolysate was then determined by both micro- assay. The fluid which passed through the cheesecloth (the biological assay (21) and the phenylhydrazine method of Wada homogenate) was used for fractionation essentially according to and Snell with heating (22). Pyridoxal hydrochloride was used the procedure of Cleland and Slater (12). as standard for both methods. The phenol and biuret methods,

aAlanine aminotransferase activity associated with the particles used for determination of protein, gave results which were was freed from soluble alanine aminotransferase by centrifuging in good agreement. the homogenate (from 10 g of wet muscle) at 40,000 x g for 10 Instrumentation--,% Cary model 15 recording spectrophotom- min four times, resuspending the pellet each time in 20 ml of eter was used for the spectral investigations. The pH values suspending solution. The sediment was finally suspended in a were determined with a Beckman model 76 pH meter with a solution that was 0.025 M in phosphate buffer, pH 6.8, and 2 mM Leeds and Northrup glass microelectrode at 25”. The fluores- in cysteine; centrifuged at 500 x g for 3 min in order to remove cent properties of the enzyme were studied with an Aminco- small amounts of sand and residue; and permitted to stand at 4” Bowman spectrophotofluorometer (cat.alogue No. 48100). for 1 hour before being assayed for activity. Preparation of Phosphopyridoxamine Form of Alanine Amino-

Starch Gel Electrophoresis-Electrophoresis was carried out transferase-The aminic form of the enzyme was prepared by essentially as previously described (13), except that a current of dialyzing the enzyme solution against three l-liter volumes of 6 ma was used for 12 hours. 0.1 M L-glutamate, two l-liter volumes of 0.1 M nn-alanine, and,

After completion of the electrophoretic run, the gel was sliced finally, 1 liter of 0.2 M phosphate buffer, pH 6.6. The amount twice horizontally with a nylon thread to give three layers of of the phosphopyridoxal form of the enzyme which remained after gel, each about 1.5 mm in thickness. The top and bottom slices this treatment was less than 5% of the total enzyme, as esti- were stained with Amido black (14), which caused the protein mated spectroscopically. Addition of 0.1 pmole of a-ketogluta- bands to become visible. In order to determine the distribution rate to a solution containing 0.02 pmole of the phosphopyridoxa- of enzyme activity in the slab, two serrated knives, with grooves mine form of the enzyme resulted in complete regeneration of the about 1 mm apart, were mounted in parallel on a plastic plate phosphopyridoxal form. with a j-cm space between the two knives. Nylon threads were Optical Rotatory Dispersion-Optical rotations were measured tightly stretched in parallel across the opening, each thread lying at room temperature with a Cary model 60 spectropolarimeter in a groove of each knife. The protein in the center slice was with the use of a 3-ml micropolarimeter cell with a 0.5-cm path located by comparison with the observable bands in the stained length (Applied Physics Corporation). A single enzyme prep- top and bottom slices, and the entire region was cut out. This aration was used for these investigations, so that the curves piece was placed on the taut, evenly spaced nylon threads wit,h obtained with different forms of the enzyme are comparable. the threads perpendicular to the direction of migration, that is, The absorbance ratio, A217:11425, of the enzyme preparation at parallel to the protein bands. The gel was sliced into many pH 5.5 with the enzyme totally in the phosphopyridoxal form rectangles of equal volume (about 1 x 1.5 X 15 mm) by care- was 5.6, and alanine aminotransferase was the only chromophoric fully pressing it through the thread grid. Each rectangle was protein present in the preparation as indicated by spectral analy- placed in a separate tube, frozen, thawed, suspended in 0.50 ml ses. Protein concentrations were varied between 4 and 10 mg of water, and homogenized. The supernatants (20 ~1) were per ml, and were determined spectrophotometrically by meas- used for assay by the salicylaldehyde procedure. uring the absorbance at 277 mb.

Ultracentrifugal Studies-The enzyme solutions used for these The optical rotatory power, [a]~, was calculated from the investigations contained 20 rnivr EDTA, 20 mM phosphate buffer equation (pH 6.6), 10 mM mercaptoethanol, 100 mM KCl, and from 4 to 10 mg of protein per ml. The sedimentation velocity of the 100 protein was determined with a Spinco model E ultracentrifuge

lollh = -‘ah 1.c

equipped with a phase plate as a schlieren diaphragm. The sedimentation velocity of the chromophoric protein was followed where I is the cell length in decimeters, c is the protein concen-

92 Alanine Aminotransferase. I Vol. 242, No. 1


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Issue of January 10, 1967 Al. H. Saier, Jr., and W. T. Jenkins 93

tration in grams per 100 ml, and (YA is the observed rotation corrected for the solvent blank.

Exchange Transamination-In order to separate radioactive alanine from radioactive pyruvate, lo-p1 aliquots containing about 15,000 dpm were removed periodically from the reaction mixtures and spotted on paper strips previously moistened with 89% formic acid to stop the reaction. Electrophoresis and scintillation counting wTere performed as described previously

(23).

MATERIALS

Crystalline chicken heart lactate dehydrogenase was a gift of A. C. Wilson. Crystalline glutamate dehydrogenase was pur- chased from Boehringer. Calcium phosphate gel was prepared by the method of Keilin and Hartree (24). Sephadex resins were obtained from Pharmacia. Other materials, commercially obtained, were of the highest purity available.

RESULTS

Subcellular Distribution of Alanine Aminotransferase Activity

Table I shows that about 8% of the total alanine aminotransferase activity in the homogenate was associated with sediment- able particles, the highest specific activity being located in the sarcosomal fraction. This fraction also contained the highest specific activity of succinate dehydrogenase, an enzyme known to be associated with mitochondria. Activity associated with the residue and the microsomes may be caused by mitochondrial contamination. More than 90% of the total activity present in the 10 g of muscle used for the fractionation was present in the homogenate. Of that which did not pass through the cheesecloth, most remained in the supernatant solution after centrifugation at 40,000 X g for 10 min.

The sarcosomal activity could be caused by contamination, adsorption of the soluble enzyme onto mitochondrial mem- branes, or the presence of a true mitochondrial isozyme. After isolation of the particles (containing 10% of the total activity of the homogenate), it was found that neither a 24-hour incubation period at 4” in 2 mM cysteine and phosphate buffer, pH 7.0, nor a 15-min incubation period at 60” in acetate buffer, pH 4.0,

TABLE I

Centrifugal fractionation of subcellular components from pig heart Alanine aminotransferase activity was determined with the use

of the salicylaldehyde assay. Turbid assay solutions were clarified by centrifugation immediately before the absorbance at 480 rnp was read. Protein was measured as described in “Methods.” Reported values are the averages of duplicate determinations.

Fraction Activity Protein

% 7%

Homogenate.............................. 100° loo

Residue, 706 X g for 3) min.. 2.5 11 Sarcosomes, 7,506 X g for 74 min. 4.5 15 Microsomes, 105,000 X g for 4 hours. 1 12 Soluble, supernatant of 105,006 X g.. 93 65

Total recovery............................ 101 103

a For this determination, lOCYj$ activity equals 60 units from 10 g of fresh muscle.

PH

FIG. 1. Curves for activity with respect to pH. Curve A, alanine + a-ketoglutarate + pyruvate + glutamate (soluble alanine aminotransferase); Curve B, glutamate + pyruvate + a-ketoglutarate + alanine (soluble alanine aminotransferase) ; Curve C, alanine + a-ketoglutarate --) pyruvate + glutamate (particulate alanine aminotransferase). All tubes contained 0.1 M pyrophosphate buffer. The salicylaldehyde assay procedures were used with conditions as described in “Methods.” Experimental points represent the averages of duplicate determinations.

I I I I I ’ 1OmM

Buffer: Acetate Phosphate Tris-HCI Bogte ---- I I I I I I

4.5 5.5 6.5 7.5 a.5 9.5

PH

FIG. 2. Stability of purified alanine aminotransferase as a function of pH under various conditions. The phosphopyridoxal form of the enzyme (20 units; specific activity, 40) was incubated at 4” in 0.5 ml of 0.1 M buffer for 1 week. Buffers and additions were as indicated in the figure. Tubes were assayed for remaining activity by the salicylaldehyde assay.

resulted in significant solubilization. Cross contamination was, therefore, probably not responsible for the results reported in Table I.

The curves for activity with respect to pH for the partially purified soluble enzyme (specific activity, 40) and the particulate enzyme preparation in pyrophosphate buffer are shown in Fig. 1. The pH optimum of the soluble enzyme is 8.0 for both the forward and reverse reactions. The more rapid decrease in the glutamate transamination activity, as the pH is lowered from the optimum, may be due to pyruvate inhibition (see below). The curve for the particulate activity with respect to pH differs from that for the soluble enzyme in that the pH optimum is slightly lower and the curve is less broad.

Stability

The stability of the enzyme depended on both the degree of purification and the conditions. Fig. 2 shows that loss of activity of a partially purified preparation was probably due to metal-catalyzed air oxidation of protein sulfhydryl groups, a


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


conclusion consistent with the high sensit.ivity of the enzyme to sulfhydryl reagents (5, 6). In the presence of sufficiently high concentrations of mercaptoethanol and EDTA, complete pro- tection at neutral pH was observed. Cysteine and glutathione were rapidly oxidized and therefore showed little protective capacity, but 1 mM dithiothreitol (Cleland’s reagent) was as effective as 10 rnM mercaptoethanol over the pH range investigated. Pyrophosphate (20 mM) was almost as effective as 20 mM EDTA. None of the four substrates protected against loss of activity. The phosphopyridoxamine form of the enzyme was no less stable than the phosphopyridoxal form. Sensi- tivity to repeated freezing and thawing was not observed, and in the presence of EDTA and mercaptoebhanol at neutral pH, purified frozen enzyme preparations (-IO’) retained full activity for at least several months.

Aggregation and precipitation of the active enzyme from solution was frequently observed when the pH was lowered after prolonged incubation at pH values greater than 8. The reagents which protected against inactivation also appeared to hinder aggregation.

Sorvall SS3 centrifuge with a GSA rotor. The precipitate was then suspended in about 500 ml of 0.1 M phosphate buffer containing 10 mM a-ketoglutarate and dialyzed twice against 20- liter volumes of water for periods longer than 8 hours.

Xtep S-The suspension was treated for 15 min with a concentrated solution of Keilin and Hartree (24) calcium phosphate gel (50 mg per ml), sufficient on the basis of a trial experiment to leave less than 15% of the enzyme in the supernatant solution, which was discarded. The precipitate of gel and adsorbed protein was collected by centrifugation in a Sorvall centrifuge operated for 20 min at 12,000 rpm and then suspended in 1 liter of water in a Waring Blendor. The blendor was fitted with a voltage regulator and was thereby operated slowly, to avoid foaming. The washed gel was collected as before, and the aminotransferase was eluted in two or three l-liter washes of 0.05 M potassium phosphate, pH 6.8, in the same manner as before.

PzlriJcalion of Enzyme

Step I-Two 40-pound batches of fresh pig heart ventricles were ground in a meat grinder and dispersed with an equal volume of 5 mM EDTA-0.1 M acetate buffer, pH 3.8, in a large Waring Blendor operated for 1 min at top speed. These procedures were carried out at room temperature as quickly as possible. The suspension was then heated to 60” in the same manner used for aspartate aminotransferase (L-aspartate : 2-oxoglutarate aminotransferase, EC 2.6.1.1) (25), with the water bath maintained at 72”. When the temperature of the homogenate reached 60”, that of the bath was reduced to 62” by the addition of ice, and denaturation was allowed to proceed at 60” for 15 min. The hot suspension was filtered for at least 6 hours through four layers of cheesecloth (made into a bag) in the cold room at 4”, the initial filtrate being refiltered to obtain a clear solution. All subsequent procedures were conducted in the cold room at 4”.

Step C-Ammonium sulfate, 400 g per liter, was added to the pooled phosphate buffer eluates, and the precipitated protein was collected by centrifugation. The precipitate was then washed with 400 ml of 1.4 M ammonium sulfate by stirring gently for 10 min with a magnetic stirring bar. After recentrifugation, the precipitate, which contained all of the enzymic activity, was taken up in a solution containing 5 mM EDTA and 10 mM mercaptoethanol, dialyzed against the same solution in order to remove ammonium sulfate, and then dialyzed against 10 liters of a 5 mM EDTA-10 mM mercaptoethanol-20 rnrvr Tris-HCl (pH 7.0) solution (Buffer A).

Step %-To the combined filtrates (about 30 liters) were added 250 g of purified ammonium sulfate per liter. The precipitate was collected by centrifugation at 10,000 rpm for 5 min in a

Xtep 5-The clear yellow solution was transferred to a DEAE- Sephadex A-50 column (4 x 40 cm) which had been pre-equilibrated with Buffer A, and the column was washed with this solution until the 280 mp absorbance of the eluate was less than 0.1 absorbance unit. The enzyme was eluted with a linear gradient with 1 liter of Buffer A in the mixing bottle and 1 liter of this solution, containing 0.13 mole of KCl, in an identical reservoir. Tubes containing enzyme were pooled. The protein was concentrated by precipitation with ammonium sulfate (400 g per liter), taken up in a minimum of a 5 mM EDTA-10 mM mercaptoethanol-60 mM acetate (pH 5.5) solution (Buffer B), and dialyzed twice against 10 liters of the same solution.

Azso

2

1.6 A480

1 0.8

Step B-The enzyme was permitted to pass through a carboxymethyl Sephadex (A-50) column (2 x 15 cm) which had been pre-equilibrated with Buffer B. Much of the enzyme passed straight through the column, but some remained on the column with most of the protein.1 Tubes containing the bright yellow color (due solely to the presence of alanine aminotransferase) were pooled, and the protein was concentrated by ammonium sulfate precipitation (400 g per liter) to a volume of 5 ml. This was dialyzed against a 5 mM EDTA-10 mM mercaptoethanol-100 ells Tris-HCl (pH 8.0) solution (Buffer C).

0 0 10 15 20 25 30 35

TUBE NUMBER

Step r-Buffer C was used for pre-equilibration of and elution from a Sephadex G-100 column (1.5 x 110 cm) of two 2.5-ml portions of the enzyme eluted from the carboxymethyl Sephadex column. The elution pattern is shown in Fig. 3. Those tubes underneath the bar, which contained the highly purified enzyme, were pooled and concentrated by ammonium sulfate precipitation as before.

A summary of the purification procedure is given in Table II.

FIG. 3. The elution pattern from a Sephadex G-100 column 1 The enzyme which remained on the column was not eluted (Step 7 in the purification procedure). Fractions (2 ml) were by washing briefly with 0.69 M acetate. The spectra of the enzyme collected. The salicylaldehyde assay was used to measure rela- which passed through the column and that which was adsorbed tive enzyme concentrations. appeared to be the same at pH 5.5.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Issue of January 10, 1967 M. H. Saier, Jr., and W. T. Jenkins 95

TABLE II

PuriJication of pig heart alanine aminotransferase

step

1. Heat step and filtration.

2. Ammonium sulfate precipitation.

3. Calcium phosphate gel adsorption..

4. Ammonium sulfate back ex- traction.

5. DEAE-Sepha- dex column.

6. Carboxymethyl Sephadex column .

7. Sephadex G-100 column.

Vdllme

ml

30,000

1,000

2,500

400

50

5

Total activity

‘nils x 10-a

Total protein

- I Specific

activity

g 1 mits/mg

125 140 0.9

loo 40 2.5

63 12 5.2

63 7 9

45 1 40

27 0.17 160

19 0.056 340

Purity and Physicochemical Properties of Enzyme

_-

These studies indicated that aggregation occurred in older samples.

Fig. 6 shows a plot of the logarit’hm of the concentration against Yield the square of the distance from the center of the rotor obtained - with a fresh enzyme preparation. The mdlecular weight calcu- % lated from the slope was 115,000 f 15,000. Error was esti-

100 mated by analyzing several tracings of the sedimentation equilibrium pattern at different degrees of magnification.

Vitamin Bs Content-The prosthetic group was found to be

80 tightly bound to the apoenzyme. Attempts to convert the

50

50

36

21 1.;

15 .I A 480

.l

Specific Activity-Investigators who have recently purified ( _ alanine aminotransferase from pig heart have defined one unit of act,ivity as that amount of enzyme which, when the lactate dehydrogenase-coupled assay is used under the experimental conditions described by Wroblewski and LaDue (26), leads to a decrease in the absorbance at 340 rnp of 0.001 in 1 min (6, 7). With the use of t,his assay at 25.5”, our enzyme preparation had a specific activity of 430,000 units per mg of protein. This is the highest value yet reported.

-w

Starch Gel Electrophoresis-Fig. 4 shows the starch gel elec- trophorctogram of the purified enzyme preparation. There existed a major and a minor protein zone, with some smearing. Analysis of the gel for activity revealed that enzyme activity, as well as protein, was smeared. However, distinct peaks of activity were not observed, indicating that the second zone was, in part, a protein impurity rather than an alanine aminotransferase isozyme. The major protein peak coincided in position with the activity peak. Analysis of the activity in other starch gel slabs always showed considerable smearing, but more than one distinct peak was never observed.

Xeclimentation Coeficient-Essentially all of the protein in a freshly purified enzyme preparation migrated in an analytical ultracentrifuge as a single component (Fig. 5A). The sedimentation coefficient of the protein peak, calculated for water at 20”, was 6.0 S. Fig. 5B shows that all of the material absorbing at 425 mM sedimented in the ultracentrifuge with a single boundary. The sedimentation coefficient of the yellow material was found to be about 6, but it could be determined only approximately because a constant temperature could not be maintained through- out the experiment.

Molecular Weight-The sedimentat.ion velocities and sedimentation equilibrium patterns of several enzyme samples were st,udied with the photoelectric scanning absorption system and, by varying the degree of magnification, different portions of a single sedimentation equilibrium patt,ern could be analyzed.

I \ I \ \ I \ \ i f

\ \

\ \

p+&

Origin

I

I

0 10 20 30 TUBE NUMBER

c (Direction of migration)

0

FIG. 4 (upper). The starch gel electrophoretic pattern of the purified alanine aminotransferase preparation. Top, the pattern as revealed by a protein stain. Bottom, the distribution of enzymic activity in a starch gel slab. Methods and conditions were as described in “Methods ”

FIG. 5 (lower). Sedimentation velocity patterns of purified alanine aminotransferase. A, the schlieren pattern taken 96 min after a speed of 59,780 rpm was attained. Sedimentation was conducted at 6.8’ with an initial protein concentration of 10 mg per ml, and is proceeding from the meniscus at the right toward the left. R, the distribution of the material absorbing at 425 rnp, 60 min after speed was attained. This experiment was condncted at about 20” with an enzyme solution having an initial A,,, of 0.75. Sedimentation is proceeding from the meniscus at the left toward the right.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

96 Alunine Aminotransferase. I Vol. 242, No. 1

1.2

z 0.8

5 T Y

3 0.4

0 42.5 43.0 43.5

NJ2 (W2

FIG. 6. A plot of the sedimentation equilibrium pattern of the chromophoric protein in the highly purified alanine aminotransferase preparation in a fashion suitable for molecular weight determination. Y, the needle deflection, was proportional to the absorbance at 425 rnp and hence to the concentration of the enzyme. X is the distance from the center of the rotor. The run was made at 20” with a solution having an initial A 426 of 0.75.

0.8

0.2

0.C

/ I I I 1

[H+l x10*

WAVELENGTH (mp)

FIG. 7. The spectrum of the phosphopyridoxal form of alanine aminotransferase in 56 mM acetate, pH 5.5, 20 mM mercaptoethanol, and 5 mM EDTA. 1nnset, the change in A416 as a function of hydrogen ion concentration in 0.4 M phosphate buffer at room temperature. The negative of the prototropic dissociation constant, -K, is found at the point of intersection on the abscissa.

enzyme to the apoenzyme were not successful, and in no case during purification did activation result from incubation with pyridoxal phosphate.

The vitamin Bs content of the highly purified preparation (specific activity, 340) was 1 mole of vitamin B6 per 85,000 g of protein as determined by the microbiological assay, and 1 mole per 75,000 g of protein as determined by the phenylhydrazine method.

Spectral Properties of Alanine Aminotransjerase

The absorption spectrum of the phosphopyridoxal form of the enzyme at pH 5.5 is shown in Fig. 7. Besides the peak at 425

ml.c, which may confidently be ascribed to a phosphopyridoxal- aldimine group, and that at 277 rnF due to protein aromatic amino acid residues, an absorption maximum at 325 rnp is seen. When the mercaptoethanol concentration was increased, the height of this last mentioned peak increased slightly, while the absorbance at 425 rnp decreased, but in the absence of mercaptoethanol the absorbance at 325 rnp remained. The absorbance ratio, A425:A325, differed with different enzyme preparations, and because it was greatest when the enzyme was prepared in the absence of sulfhydryl compounds, it seems likely that the magnitude of the absorption at 325 rnp increased during prolonged exposure to mercaptoethanol. Because 325 rnp is near the wave length at which the phosphopyridoxamine form of the enzyme and derivatives of the phosphopyridoxal form in which the aldimine bond is saturated absorb (6, 9), it appears reasonable that mercaptoethanol can either add, or induce addition of an enzymic group, across the aldimine double bond.

The absorbance ratio, AzT7: A,,,, frequently used as a measure of purity, was 4.5, lower than has been observed for any other aminotransferase by a factor of 2.

In slightly basic solutions, alanine aminotransferase absorbs at 425 rnp less strongly than in acidic solutions (9). I f a single prototropic dissociation constant, K = [E][Hf]/[EH+], charac- terizes this spectral change, it can be shown that

lH+l -II

AA el - e2

where [E,] is the total enzyme concentration; AA is the absorbance difference between the fully protonated enzyme and the partially protonated enzyme at a particular hydrogen ion concentration, [PI; and el and e2 are the extinction coefficients of the nonprotonated and protonated forms of the enzyme, respectively. The decrease in the absorbance at 425 rnp with increasing pH was found to obey this equation, with pK = 7.4 (inset of Fig. 7).

Concentrated solutions of the purified enzyme in the presence of mercaptoethanol are bright yellow, but when frozen at -lo”, they become colorless. This change is reversible. The reason for this interesting spectral change is not known.

The optical rotatory dispersion curves of the phosphopyridoxal enzyme at various pH values, and of the aminic form of the enzyme, are reproduced in Fig. 8. At pH 6.5, at which the prosthetic group of the phosphopyridoxal enzyme was largely protonated, a distinct positive Cotton effect, having a peak at 465 rnp and a negative shoulder at about 385 rnp, was observed (Curve A). At pH 8.5 (Curve C), only a small positive shoulder, also near 465 rnp, was observed, and at a pH value at which about half of the enzyme was protonated, the anomalous dispersion curve was intermediary (Curse B). These three curves intersect at 425 rnl.c, the wave length at which the protonated form of the enzyme absorbs maximally (Fig. 7). The Cotton effect which was observed for the acidic form of the phosphopyridoxal enzyme was, therefore, probably associated with this absorbance.

Distinct shoulders in the optical rotatory dispersion curve of the phosphopyridoxamine form of the enzyme were not observed (Curve D). However, the data deviated significantly from the Moffit and Yang equation with X0 = 212 rnp (27), indicating that the dispersion curve was not plain.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


0

-100

-200

-300

-400

350 400 450 500

WAVELENGTH (nwL)

FIG. 8. Optical rotatory dispersion curves of alanine aminotransferase. A, the phosphopyridoxal enzyme, pH 6.5 (0.2 M phosphate); B, the phosphopyridoxal enzyme, pH 7.5 (0.2 M phosphate + 0.1 M Tris); C, the phosphopyridoxal enzyme, pH 8.5 (0.2 ?M phosphate + 0.4 M Tris) ; D, the phosphopyridoxamine enzyme, pH 6.6 (0.2 M phosphate). Present in all solutions were 10 mM mercaptoethanol and 10 mM EDTA.

The curve which was obtained for the oxime enzyme (the phosphopyridoxal enzyme + 2 mM hydroxylamine at pH 6.6 in 0.2 M phosphate) also failed to conform to the Moffit and Yang equation. This curve was the same as that for the aminic form of the enzyme, except that the specific rotation was slightly less negative between 330 and 370 rnp. The absorption spectrum of the colorless oxime enzyme showed two peaks, one at 360 mp and the other at 330 ml.r, with AB,J:ASX = 1.1.

The fluorescence of a solution containing 10 mg of the purified enzyme per ml and 0.1 M Tris-HCl, pH 8.5, was examined in the wave length range from 300 to 600 rnp, the wave length of excita- tion being varied over the same range. Under these conditions, the enzyme was found to be essentially nonfluorescent as com- pared with pyridoxal phosphate.

Kinetic Parameters of Forward Reaction

The equation describing the observed rate of alanine transamination at constant temperature, u, in terms of the maximum transamination velocity, V,,,, the absolute Michaelis constants, Ka and Kg, for alanine and cr-ketoglutarate, respectively, and the substrate concentrations is

v UlBX K-4 KB

__ = ’ ’ [n-alanine] ’ [a-ketoglutarate] v

Ka and Ke were found to equal 28 mM and 0.4 InM, respectively (Fig. 9). For a theoretical interpretation of the meaning of these constants see Reference 28. The estimated maximum turnover number was 1.1 x lo3 mole per set per bound mole of pyridoxal phosphate.

The apparent Michaelis constant for pyruvate in the presence of 20 mM L-glutamate was about 0.3 mM in 0.1 M Tris-HCl, pH 8.1. With a spectrophotometric method (Method I of Refer- ence 29), the glutamate-alanine aminotransferase dissociation constant was estimated as 25 m&f.

Abortive Complex Formation

Radioactive Exchange between Alanine and Pyruvate--In the presence of an amino acid substrate, A, and its analogous keto

V/[L-Alanine]

[L-&ninel(mM) cn

1 I I -12 -10 -8 -6 -4 -2 0 2 4 6

l/bKGI

FIG. 9. Determination of the absolute Michaelis constants for alanine and or-ketoglutarate. The reaction mixtures (2.0 ml) contained 209 pmoles of Tris-HCl (pH 8.1), 0.3 mg of NADH, variable amounts of the substrates as indicated in the figure, and excess lactate dehydrogenase. A, the slopes are equal to the negative values of the apparent Michaelis constants for alanine, K’a, at the indicated a-ketoglutarate concentrations. The in- verse of the absolute Michaelis constant for alanine is obtained at the intercept on the ordinate of the inset. B, the negative reciprocals of the apparent Michaelis constants for a-ketoglutarate, K’B, at the indicated n-alanine concentrations are obtained at the intercepts on the abscissa. Ke is obtained by extrapolating these values to infinite alanine concentration (inset). Velocity is in arbitrary units.

acid, B, transaminase equilibria which are thought to exist involve only binary complexes, as shown below.

B A

+ Kl K2 + A + R Tp.===d EX e Ez+B

II K3 II K4 EIB &A

El represents the phosphopyridoxal form of the enzyme, and Ez, the phosphopyridoxamine form. K1 = [A] [E,]/[EX]; KZ = [B] [EMEX]; KS = [Bl L%1I[E~Bl; and K4 = [Al LW[E2Al. The complexes E2A and EIB are thought to be “abortive complexes.” [EX] represents equilibrium concentrations of binary complexes, some of which are obligatory intermediates in the interconversion of El and Ez, With these equilibria as a basic assumption, it can be shown that


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


[Al 1 -=- V V

+ K1 + RK, (1) msx

1 + j$ + K$ 3 4

where v is the exchange transamination velocity and V,,, is defined as the theoretical maximum velocity, that which would be observed if the enzyme existed totally in the form EX. R is defined as the ratio of the amino acid concentration to the keto acid concentration, [A] : [B].

By appropriate integration of the fundamental rate equation for radioactive exchange,

Ld!+(g-~)

the equation

-ln [A,*1 - IA,*1 Lb*1 - [A,*] >

= gl (1 + R)

is obtained. Here [A t*] is the concentration of radioactive amino acid at time t, [A,*] is its concentration at equilibrium, and [Ao*] is its concentration before the reaction is initiated by introduction of enzyme into the reaction mixture. Combining Equations 1 and 2 gives

Ml t(l + R) -=- V In [At*1 - &,*I

[Ao* - &*I

+ Kl+ RKz

A plot of

Il~ [At*1 - [A,*1 [&*I - &,*I >

against time should give a straight line whose slope can be used

0 4 8 12 16 20 24 28 2 0 2 4 6 8 10

Minutes AlanIne (d)

FIG. 10. Amino transfer from radioactive alanine to pyruvate at 37”. Reactions were initiated by introduction of enzyme into the 0.50-ml assay mixtures which contained 0.10 M phosphate buffer, pH 6.9, and substrates at the concentrations indicated in the figures. A, the decrease in the radioactivity in alanine as a function of time at four alanine concentrations with the alanine to pyruvate concentration ratio, R, at 0.10. A* is the concentration of radioactive alanine. Subscripts are as indicated in the text. Values of the exchange transamination velocity, v, were calculated from the slopes. B, the rate of exchange transamination as a function of substrate concentration. The graph is analogous to a Lineweaver-Burk, S/v against S, plot.

II -5 0 lo 20 30 40 50 60

I/R FIG. 11. The reciprocal of the apparent maximum velocity

plotted against the reciprocal of the concentration ratio. Data were obtained from the slopes in Fig. 10B.

for the calculation of [Al/v. The experimental data which were obtained with R = 0.1 are shown in Fig. IOA.

A plot of [Al/v against [A] at constant R should give a straight line, the slope of which should be equal to l/V’,,, = [V,,,/ (1 + KI/RKQ + K2R/K4)]-l, the reciprocal of the apparent maximum velocity. Fig. 10B shows the data which were obtained with three values of R: 0.1, 0.05, and 0.02. A plot of l/V’nl,x against 1 /R gave a straight line (Fig. 11). Therefore, K2R/K4 must be negligibly small, and the intercept on the abscissa gives the value, -K3/K1, the magnitude of which is seen to be less than 1. This treatment therefore indicates that, at pH 6.9 in 0.1 M phosphate buffer, pyruvate binds to the active site of the phosphopyridoxal form of the enzyme more tightly than does alanine.

Kinetic and Spectral Vem$cation of Abortive Complex Forma- tion-Pyruvate inhibition of amino transfer from glutamate to pyruvate was demonstrated by coupling the formation of cr-ketoglutarate with NADH oxidation (Fig. 12). Because the Michaelis constant for pyruvate is very small, the role of pyruvate as substrate is not apparent in the concentration range used, and the data reflect only its role as a competitive inhibitor. The reaction conditions used in this investigation differed significantly from those used in the radioactive exchange experiment because a fairly high ammonium sulfate concentration was re- quired for the coupling reaction. However, the value of 30 mM obtained for the abortive complex dissociation constant, KS, was not appreciably influenced by the presence of this salt; doubling its concentration increased KS to 34 mu.

A plot of the intercepts on the ordinate against the reciprocal of the glutamate concentration gave a stright line from which the absolute Michaelis constant of glutamate (18 mM) at this pH was derived (inset, Fig. 12).

Addition of pyruvate to a concentrated solution of the phosphopyridoxal form of alanine aminotransferase resulted in a decrease in the absorption maximum at 425 rnp (upper inset, Fig. 13), and Fig. 13 shows that the absorbance change as a function of the pyruvate concentration resembled a typical titration curve. Assuming the spectral change to be a consequence of abortive complex formation, it can be shown that

[&I , KJpyruvatel + 1 -= AA e2 - el


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


-25 0 25 50 75 DISCUSSION

[PYRUVATEI (mM)

FIG. 12. Kinetic determination of the pyruvate-phosphopyridoxal enzyme dissociation constant. Present in the 2.0-ml reaction mixtures were: 200 pmoles of phosphate buffer (pH 6.9), 100 pmoles of ammonium sulfate, 0.2 mg of NADH, a large excess of glutamate dehydrogenase, and variable amounts of pyruvate and glutamate. The reactions were initiated by addition of alanine aminotransferase after temperature equilibration at 37”. Values were corrected for the slow glutamate dehydrogenase- catalyzed oxidation of NADH by pyruvate, which was observed in the absence of alanine aminotransferase. The graph is in the form of a Dixon plot for competitive inhibitors, l/v against [Z]. Inset, the reciprocals of the maximum velocities of amino transfer from glutamate to pyruvate at three glutamate concentrations were obtained by extrapolating the data in Fig. 12 to zero pyruvate concentration. The absolute Michaelis constant for glutamate, K,, is obtained from the intercept on the abscissa. Velocity is in arbitrary units.

I I I I

1.00 c L) c

0.98 -

Wavelength (mr)

-1 0 1 2

[LOG PYRUVATE] (mM)

FIG. 13. Spectrophotometric titration of the phosphopyridoxal form of alanine aminotransferase with pyruvate. The phosphate concentration was 0.1 M, pH 6.9, and the temperature was 37”. Circles and squares represent experimental points obtained in two titration experiments in which the initial 425 rnp absorbance was 0.3. Lower inset, data were obtained from a third titration experiment in which the initial 425 rnp absorbance was 1.8. A 2 M

pyruvate-0.1 M phosphate, pH 6.9, solution was added to the enzyme solution with a precision micrometer buret (Roger Gil- mont Instruments Inc., catalogue No. S-3100 A). Values were corrected for dilution.

where [Et] is the total concentration of enzyme, AA is the de-

crease in the absorbance at 425 mp, and e’1 and e’z are the extinction coefficients of the free enzyme and the pyruvate-alanine aminotransferase complex, respectively. With the use of this equation, Ks was found to equal 20 mM (lower inset, Fig. 13). This value is in fair agreement with the value determined kinet-

ically with much lower enzyme concentrations in the presence of ammonium sulfate.

Although pyruvate forms an abortive complex with alanine aminotransferase, 50 mM a-ketoglutarate had no detectable effect on the absorbance at 425 rnp at pH 6.9, and only very high oc-ketoglutarate concentrations reduced the rate of pyruvate formation from alanine and a-ketoglutarate at pH 6.9 (phosphate) or pH 8.1 (Tris-HCl). Similarly, high concentrations of the amino acid substrates did not inhibit transamination by

abortive complex formation.

Particulate alanine aminotransferase activity was found to constitute up to 10% of the total activity in pig heart. However, the purified enzyme must originate from the soluble fraction because activity remained with the particles during a heat step

comparable to that of the purification procedure, and particulate material was removed by the subsequent filtration. The ques- tion of isozymes of the soluble alanine aminotransferase arose due to “anomalous” behavior of the enzyme during chromatog- raphy in Step 6 of the purification procedure. Analysis of starch gel electrophoretograms of the. highly purified preparations showed only one distinct peak of enzymic activity, and thus provided no evidence for the presence of isozymes.

The molecular weight of alanine aminotransferase was shown to be about 115,000 by ultracentrifugation, but investigations with calibrated dextran gel columns indicated a lower molecular weight of 90,000 (see also Reference 7 and Fig. 3). Adsorption of the enzyme on the Sephadex beads may have reduced the rate at which it passed through the column to yield an artificially low molecular weight. An alternative explanation is that the ultracentrifugal value is high because the enzyme was partially polymerized during sedimentation.

Evaluation of the spectral properties of alanine aminotransferase permits a striking correlation to be drawn between this enzyme and the more thoroughly investigated aspartate aminotransferase from pig heart. These two enzymes are unique in that they are the only vitamin RB enzymes so far investigated

which exhibit pH indicator properties. While aspartate aminotransferase is totally colorless in solutions of pH greater than 8, the basic form of alanine aminotransferase retains about one- third of the yellow color observed in acidic solutions. Associated with the 430 rnF absorption maximum of the acidic form of aspartate aminotransferase is a positive Cotton effect (30, 31), and a very similar effect was correlated with the 425 rnp absorption

peak of the corresponding form of alanine aminotransferase. While the Cotton effects produced by other forms of the two enzymes are much less pronounced, quantitative differences appear to exist. For example, the distinct Cotton effect with peak at 350 mp, which is observed for the phosphopyridoxamine form of aspartate aminotransferase (31, 32), is not observed for the corresponding form of alanine aminotransferase. Finally, both enzymes are only very weakly fluorescent, the magnitudes of their fluorescences being at least 100 times less than observed for the same molar concentration of pyridoxal phosphate.

Acknowledgments-We wish to thank Dr. H. K. Schachman and Mr. S. J. Edelstein for the ultracentrifugal analyses with the photoelectric scanning absorption system, and Miss Doris Midgarden for performing the sedimentation velocity experiment with the schlieren scanning method. Thanks are also extended


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


to Dr. B. M. Guirard for the microbiological assays for vitamin B6, and to Mr. I. Givot for assistance with enzyme preparations.

REFERENCES 1. SWICK, R. W., BARNSTEIN, P. L., AND STANGE, J. L., J. Biol.

Chem., 240, 3334 (1965). 2. KATUNUMA, N., MIKUMO, K., MAKOTO, M., AND OKADA, M.,

J. Vitaminol.. (Kyoto), 8, 68 (1962). 3. L&NARD. P.. AND STRAUB. F. B.. Studies Inst. Med. Chem.

Univ.‘&eied., 2, 59 (1942). ’ 4. GREEN, D. E., LELOIR, L. F., AND NOCITO, V., J. Biol. Chem.,

161, 559 (1945). 5. GREIN, L., AND PFLEIDERER, G., Biochem. Z., 330, 433 (1958). 6. TORCHINSKII, Y. M., Biokhimiya, 27, 916 (1962). 7. TURANO, C.,AND RI~A, F., Ital. J. Biochem., 13, 293 (1965). 8. JENKINS. W. T.. Federation Proc.. 20. 978 (1961). 9. JENKINS; W. T.,‘in E. E. SNELL, P: M: FA&LLA,.A. E. BRAUN-

STEIN, AND A. ROSSI-FANELLI (Editors), Proceedings of the symposium on chemical and biological aspects of pyridoxal catalysis, Rome, 1968, Pergamon Press, New York, 1963, p. 139.

10. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem., 193, 265 (1951).

11. MOKRASCH, L. C., AND MCGILVERY, R. W., J. Biol. Chem., 221, 909 (1956).

12. CLELAND, K. W., AND SLATER, E. C., Biochem. J., 63, 547 (1953).

13. MARTINEZ-CARRION, M.; AND JENKINS, W. T., J. Biol. Chem., 240, 3538 (1965).

14. SMITHIES, O., Advan. Protein Chem., 14, 74 (1959).

15. SCHACHMAN, H. K., GROPER, L., HANLON, S., AND PUTNEY, F., Arch. Biochem. Biophys., 99, 175 (1962).

16. LAMERS, K., PUTNEY, F., STEINBERG, I. Z., AND SCHACHMAN, H. K., Arch. Biochem. Biophys., 103, 379 (1963).

17. SCHACHMAN, H. K., in S. P. COLOWICK AND N. 0. KAPLAN (Editors), Methods in enzymology, Vol. 4, Academic Press, New York, 1957, p. 32.

18. SCHACHMAN, H. K., AND EDELSTEIN, S. J., Biochemistry, 6, 2681 (1966).

19. YPHANTIS, D. A., Biochemistry, 3. 297 (1964). 20. RABINOWITZ, J. C., AND SNELL, E. E., Ind. Eng. Chem. Anal.

Ed., 19, 2f7 (1947). 21. SNELL. E. E.. in P. GY~RGY (Editor). Vitamin methods. VoZ. 1.

Academic Press, New York, 1950; P. 438. 22. WADA, H., AND SNELL, E. E., J. Biol. Chem., 236, 2089 (1961). 23. JENKINS, W. T., AND TAYLOR, R. T., J. Biol. Chem., 240, 2907

(1965). 24. KEILIN, D., AND HARTREE, E. F., Proc. Roy Sot. (London),

Ser. B, 124, 397 (1938). 25. JENKINS, W. T., Biochem. Prep., 9, 47 (1962). 26. WROBLEWSKI, F., AND LADUE, J. S., Proc. Sot. Exptl. Biol.

Med., 91, 569 (1956). 27. MOFFIT. W., AND YANG, J. T., Proc. Natl. Acad. Sci. U. S.,

42, 596 (1956). 28. HOPPER. S.. AND SEGAL. H. L.. J. Biol. Chem.. 237,3189 (1962). 29. JENKINS, W’. T., AND D’ARI, L., J. Biol. Chem.; 241,2845 (1966). 30. TORCHINSKII, Y. M., AND KORENEVA, L. G., Biokhimiya, 28,

1087 (1963). 31. FASELLA, P., AND HAMMES, G. G., Biochemistry, 3, 530 (1963). 32. TORCHINSKII, Y. M., AND KORENEVA, L. G., Biokhimiya, 29,

780 (1964).


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Milton H. Saier, Jr. and W. Terry JenkinsAlanine Aminotransferase: I. PURIFICATION AND PROPERTIES

1967, 242:91-100.J. Biol. Chem.

http://www.jbc.org/content/242/1/91Access the most updated version of this article at

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

http://www.jbc.org/content/242/1/91.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/content/242/1/91

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;242/1/91&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/242/1/91

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=242/1/91&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/242/1/91

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/242/1/91.full.html#ref-list-1

http://www.jbc.org/

alanine aminotransferasealanine aminotransferase (l-alanine : 2-oxoglutarate amino- transferase, ec...

Documents