THB JOURNAL OF Bm~ooxx~ CHEMISTRY Vol. 249, No. 8, Issue of April 2.5, pp. 2554-2661, 1974

Printed in U.S.A.

Isolation and Properties of a New Glutamine

Transaminase from Rat Kidney

(Received for publication, September 4, 1973)

ARTHUR J. L. COOPER AND ALTON MEISTER

From the Department of Biochemistry, Cornell University Medical College, New York, New York 10021

SUMMARY

A glutamine transaminase has been purified from the soluble portion of rat kidney homogenate; this enzyme, designated as kidney glutamine transaminase K, has different catalytic and physical properties from those previously found for the purified soluble glutamine transaminase of rat liver (liver glutamine transaminase L). Liver glutamine transaminase L is highly active toward methionine, gly- oxylate, pyruvate, and several other substrates; kidney glutamine transaminase K is very active toward methionine, phenylalanine, tyrosine, and the cr-keto acid analogs of these amino acids and exhibits relatively little activity toward glyoxylate and pyruvate. Evidence was obtained that the K and L forms of glutamine transaminase are both present in liver and kidney. In addition, both forms of these en- zymes are present in these tissues as mitochondrial as well as soluble isozymes. There are therefore at least four separate glutamine-cu-keto acid transaminases in liver as well as kidney. Rat liver w-amidase also occurs in both soluble and mitochondrial isozymic forms.

Early studies on the glutamine transaminase activities of rat tissues indicated that the liver contains the highest concentration of this activity and that the kidney exhibits less activity (1). However, in a recent study of glutamine transaminase, Kupchik and Knox (2, 3) concluded that rat kidney exhibits a much higher level of glutamine transaminase than does the liver. In con- sidering these apparently conflicting results, we noted that Kup- chik and Knox (2, 3) determined enzyme activity by measuring the rate of glutamine-phenylpyruvate transamination, while as- says of the glutamine-pyruvate transamination reaction had been used in the earlier work (1). We therefore determined the gluta- mine transaminase activities of rat liver and kidney homogenates using several a-keto acid substrates. It was found that kidney homogenates are about 30% as active as liver homogenates in catalyzing transamination, between glutamine and glyoxylate, and about 55% as active in transamination between glutamine and pyruvate. However, kidney homogenates were found to be about 5 times more active than liver homogenates in catalyzing transamination between glutamine and phenylpyruvate. These observations suggested that rat kidney contains a glutamine

transaminase which has a specificity that is markedly different from that present in liver.

In the present work, we have purified the soluble glutamine transaminase of rat kidney about 120.fold, and have demon- strated that this enzyme differs substantially in its physical and catalytic properties from. the soluble glutamine transaminase previously purified in this laboratory from rat liver (4). The purified kidney enzyme is highly active toward glutamine, methi- onine, phenylalanine, and tyrosine (and the corresponding a-keto acids), but exhibits relatively low activity with glyoxylate and several other a-keto acids that are good substrates for the purified rat liver glutamine transaminase. It is also notable that, in con- trast to the enzyme isolated from rat liver (4-7), the rat kidney enzyme does not act on the y-glutamylhydrazones of oc-keto acids nor is it appreciably active with certain glutamine analogs such as albizziin, 0-carbamylserine, and S-carbamylcysteine. In the course of this work, we obtained evidence for the presence in rat liver of an activity which closely resembles that found in kidney; this activity was separated physically from that previously puri- fied from rat liver. The data also indicate that rat kidney con- tains a small amount of glutamine transaminase which resembles the predominating glutamine transaminase of rat liver. It thus appears that rat liver contains at least two types of glutamine transaminases; we refer here to the activity previously purified (4) and most abundant in rat liver as liver glutamine transami- nase L, while the other liver glutamine transaminase (whose speci- ficity resembles the major activity found in kidney) is referred to as liver glutamine transaminase K, and the less abundant gluta- mine transaminase of kidney as kidney glutamine transaminase L. We have also obtained evidence that glutamine transaminase L occurs in two isozymic forms, i.e. a soluble form and a mitochon- drial form. Although both enzymes appear to have similar catalytic properties they can be separated by polyacrylamide disc gel electrophoresis. These findings are in agreement with those of Yoshida (8), who first showed the presence of a distinct rat liver mitochondrial glutamine transaminase. Similarly, gluta- mine transaminase K and w-amidase from rat liver have been found to occur in soluble and mitochondrial forms, which can be separated by polyacrylamide disc gel electrophoresis. Rat kid- ney glutamine transaminase K also occurs in mitochondrial and soluble forms, which exhibit the same respective electrophoretic mobilities as the rat liver transaminase K isozymes.

EXPERIMENTAL PROCEDURE

Materials-The amino acids, ol-keto acids, and other compounds used in this work were obtained as stated previously (4-6). Rat

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liver glutamine transaminase L was isolated as described (4).

Methods-Transamination between glutamine and phenyl- pyruvate (or p-hydroxyphenylpyruvate) was determined by the method of Kupchik and Knox (2, 3) in which the decrease in absorbance at 300 nm of the complex formed between borate and the enol form of the a-keto acid is determined. The reaction mixtures contained 20 mrvr n-glutamine, 0.4 mM sodium phenyl- pyruvate (or sodium p-hydroxyphenylpyruvate), 300 rnrvr sodium borate buffer (pH 8.5), and enzyme in a final volume of 1 ml. The same procedure was used for following transamination between other n-amino acids and these aromatic cu-keto acids.

Transamination between glutamine and glyoxylate was deter- mined in reaction mixtures (final volume, 0.1 ml) containing 20 m&r n-[U-14C]glutamine, 20 IXIM sodium glyoxylate, 50 mM Tris-HCl buffer (pH 8.4), and enzyme by the procedure previously described in which the formation of or-keto[14C]glutaramate is measured (4).

Transamination between various amino acids and glyoxylate was carried out in reaction mixtures (final volume, 0.1 ml) con- taining 20 mM n-amino acid, 20 mM sodium [1-l%]glyoxylate, 50 mM Tris-HCl buffer (pH 8.4), and enzyme; the formation of [“Clglycine was determined as described (4).

Transamination between albizziin and glyoxylate was carried out in reaction mixtures containing 20 mM n-albizziin, 20 rnM sodium glyoxylate, 100 mM sodium borate buffer (pH 8.5), and enzyme in a final volume of 0.1 ml. After alkalinization, the formation of 2-imidaxolinone-4-carboxylate was determined from the absorbance at 280 nm (6).

Transamination between phenylalanine and a-keto acids was carried out in reaction mixtures (final volume, 0.4 ml) containing 10 rnM n-phenylalanine, 20 mM Lu-keto acid (sodium salt), 100 mM sodium borate buffer (pH 8.5), and enzyme. The formation of phenylpyruvate was determined as described by Wellner and Meister (9).

A unit of enzyme activity is defined as the amount of enzyme that catalyzes the formation of 1 Hmole of product per hour at 37”; this refers to transamination between glutamine and glyoxyl- ate for the liver-type enzyme, and between glutamine and phenyl- pyruvate for the kidney-type enzyme.

Protein was determined by the method of Lowry et al. (10). Ammonia was determined using a Conway diffusion apparatus

(11); color was developed with Nessler’s reagent. The calculations of apparent K, and V,,, values were kindly

performed by Dr. Paul Trotta of this laboratory; these values were calculated from a linear least squares analysis of a weighted Lineweaver-Burk plot using a computer program written by Dr. R. H. Haschemeyer of this department.

Polyacrylamide gel electrophoresis was carried out in 7% gels at pH 8.9 under the conditions previously described (Method A (4)). Polyacrylamide gel electrophoresis in sodium dodecyl sulfate was carried out by the procedure of Weber and Osborn (12) using the modifications previously given (4).

RESULTS

Purification of Soluble Glutamine Transaminase K from Rat Kidney

Unless otherwise stated, the following procedures were carried out at 4’; centrifugations were performed at 45,000 x g for 10 min.

Step I-Fresh or freshly frozen kidneys (12 g) from male Sprague-Dawley rats were homogenized in 3 volumes of ice-cold water in a Waring Blendor for 1 min. The homogenate was centrifuged and the sediment was discarded.

Step d-The supernatant solution obtained in Step 1 was heated at 60-62” for 10 min, and then cooled in ice and centri- fuged. The pellet was discarded. The supernatant solution exhibited no w-amidase activity.

Step S---Solid ammonium sulfate (20 g/100 ml) was added with

standing 20 min, the precipitate which formed was collected by centrifugation and dissolved in the minimal amount (about 5 ml) of ice-cold water. This solution was dialyzed for 24 hours against 10 liters of 5 mM potassium phosphate buffer (pH 7.2) containing 5 mM 2-mercaptoethanol. An inactive precipitate was formed during dialysis and this was removed by centrifugation.

Step &The dialyzed solution was applied to the top of a col- umn (2 x 14 cm) of DE52 previously equilibrated with 5 mM potassium phosphate buffer (pH 7.2). The enzyme was eluted from the column using a linear gradient established between 200 ml of 5 mM potassium phosphate buffer (pH 7.2) and 200 ml of 80 mM potassium phosphate buffer (pH 7.2). The elution diagram is given in Fig. 1; Fractions 23 to 26 were pooled.

Step b--The pooled fractions were dialyzed against 10 liters of 5 mM potassium phosphate buffer (pH 7.2) containing 5 mM 2-mercaptoethanol. This solution was added to the top of a col- umn (1 x 9 cm) of hydroxyapatite previously equilibrated with the same buffer. The enzyme was eluted from the column using a linear gradient established between 150 ml of 5 mM potassium phosphate buffer (pH 7.2) and 150 ml of 100 mM potassium phos- phate buffer (pH 7.2); both buffers contained 5 mM 2-mercapto- ethanol. The enzyme eluted from the column in Fractions 7 to 9 (42 to 54 ml) (Fig. 2) ; the fractions containing the enzyme were pooled and concentrated by ultrafiltration to a volume of 2 ml using a Diaflow XM 50 membrane.

The isolation procedure, which is summarized in Table I, led to a purification of about 120.fold. The first three steps are similar to those used by Kupchik and Knox (2,3), who obtained about a a-fold purification of the enzyme.

The present preparation is devoid of w-amidase, glutamate-

FRACTION NUMBER

FIG. 1. Chromatography of kidney glutamine transaminase K on DE-52 (see the text, Step 4 of the purification). The arrow marks the point at which the phosphate gradient was started; fractions of 8 ml were collected.

I , 5 0 IO 20 30 40 d

FRACTION NUMBER

constant stirring to the supernatant solution obtained in Step 2. After standing for 20 min the precipitate which formed was re-

FIG. 2. Chromatography of kidney glutamine transaminase K

moved by centrifugation. The supernatant solution was treated on hydroxyapatite (see the text, Step 5 of the purification). The arrow marks the point at which the phosphate gradient was

with additional solid ammonium sulfate (40 g/100 ml), and after started; fractions of 6 ml were collected.

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TABLE I PuriJication of soluble kidney glutamine transaminase K

1. Crude homogenate” 2. Heat treatment.. . 3. Ammonium sulfate fractionation.. 4. DE52 chromatography.. 5. Hydroxyapatite chromatography

and ultrafiltration..

Volume Protein Total‘

ml

37 32

8.5 35

w

910 385 119 10.5

1.1

zrnirs

1800 1450

870 590

2.0 243

Specific activity

units/mg % 1.98 100 3.77 81 7.30 48

56.2 33

221

Yield

13.5

Purification Albizziin-gly- factor oxylate unit9

1 1.9 3.7

28

113

40 35 21 12

5.5

D Glutamine-phenylpyruvate transaminase activity was determined as described (3). b Albizziin-glyoxylate transaminase activity was determined as described (5). c From 12 g of rat kidneys.

aspartate transaminase, and glutamate-oxalacetate transaminase activities. Although there is evidence for the presence in kidney homogenates of glutamine transaminase L (see below), the puri- fied preparation described above appears to be free of this activ- ity. It was observed previously that liver glutamine transami- nase L is unstable at 60” in the absence of an added a-keto acid (4) ; in contrast, kidney glutamine transaminase K is stable at 60” under the conditions employed in the presence or absence of added cr-keto acid. The heating step (Step 2) probably destroys kidney glutamine transaminase L activity.

Physical Properties of Purijied Enzyme

Examination of the purified enzyme preparation by polyacryl- amide gel electrophoresis at pH 8.9 revealed a major protein com- ponent which represented (in our best preparations) about 80% of the total protein, as well as three smaller protein bands. Un- stained gels run in parallel were sliced into 2-mm sections and added directly to the assay system for determination of activity; only the major band exhibited activity. The enzyme exhibited a mobility (relative to that of a bromthymol blue marker) of 0.58. This mobility is substantially different from that exhibited by the purified rat liver-soluble glutamine transaminase L preparation previously described (4), which was 0.30 under the same condi- tions. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed a major band (about 80% of the total protein), which exhibited the same mobility as that found under the same conditions with the purified rat liver glutamine transaminase L preparation. Previous studies indicated that the liver enzyme is a dimer possessing apparently identical subunits of molecular weight 55,000; the molecular weight of the purified kidney enzyme is close to 110,000 as determined by gel filtration on Sephadex G-50. It therefore appears that the kidney and liver glutamine transaminases have similar molecular weights and subunit structures.

The purified kidney glutamine transaminase exhibited absorb- ance maxima at 278 and 415 nm (0.55 mg of protein per ml in 5 mM potassium phosphate, pH 7.2; 25”) ; the ratio of the absorb- antes at 415 and 278 nm is 0.08; the corresponding value for the purified rat liver enzyme is 0.12 (4). The purified kidney enzyme exhibits an absorbance at 280 nm of 0.78 for a solution of the enzyme containing 1 g per liver, compared to a value of 0.65 for the purified rat liver enzyme (4).

Evidence for Presence of Pyridoxal 6’-Phosphate in Kidney and Liver Glutamine Transaminases

Previous findings on purified soluble liver glutamine transami- nase L suggested that this enzyme contains tightly bound pyri-

doxal 5’-phosphate (4). At no time during the purification of this enzyme or that of rat kidney-soluble glutamine transaminase K was activation observed on addition of pyridoxal5’-phosphate. Furthermore, dialysis (12 hours; 4”) of the purified liver and kidney enzymes against 20 DIM L-glutamine or L-cysteine did not lead to loss of activity. Evidence for the presence of pyridoxal 5’-phosphate in both enzymes was obtained by application of the method described by Dempsey and Christensen (13). Thus, 0.5 to 2 mg of the purified enzymes was dissolved in 1 ml of 0.1 M

sodium acetate buffer (pH 4.2), and treated with 1 ml of a aqueous solution (1% w/v) of NaBH4. After 1 hour, the solu- tions were dialyzed successively against 50 mM sodium acetate buffer (pH 4.2) and water. Solutions of the proteins (about 1%) were hydrolyzed in 6 N HCl at 100” in sealed tubes for 16 hours. The solutions were then subjected to flash evaporation and the residues were dissolved in 50 ~1 of water and subjected to ascend- ing paper (Whatman No. 3) chromatography in the dark with a solvent consisting of water, methanol, ethanol, benzene, pyridine, and dioxane (25 :25: 10 : 10 : 10 : 10; v/v), e-Pyridoxyllysine was identified by its fluorescence under ultraviolet light in the hy- drolysates of both enzymes. The RF values (0.31 to 0.33) agreed with those of a standard compound prepared by treatment of bovine serum albumin with pyridoxal 5’-phosphate (13).

Specificity of Kidney-soluble Glutamine Transaminase K

As indicated in Table II, the purified kidney glutamine trans- aminase K, like the liver glutamine transaminase L previously studied (4), catalyzes transamination between glutamine and a wide variety of a-keto acids. The purified kidney transaminase K preparation is most active with phenylpyruvate, a-keto-y- methiolbutyrate, p-hydroxyphenylpyruvate, and cY-ketoglutara- mate. Data obtained on liver glutamine transaminase L are in- cluded in Table II for comparative purposes. It is clear that there are substantial differences between the specificities of these enzymes. Thus, the kidney enzyme is highly active toward phenylpyruvate and p-hydroxyphenylpyruvate, while it exhibits relatively low activity toward glyoxylate and pyruvate. These findings thus resolve the apparent discrepancies cited above in relation to the results reported by Kupchik and Knox (2, 3) and those obtained earlier (1). There are also some differences be- tween these enzymes with respect to their specificities for other a-keto acids. It is notable that cr-keto-y-methiolbutyrate, ac-ketoglutaramate, a-ketoglutarate-y-ethyl ester, andp-hydroxy- pyruvate are more active with the liver enzyme than with the kidney enzyme. It is of interest that a-ketoisovalerate, which exhibits very low activity with the liver enzyme, is much more active with the kidney enzyme.

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TABLE II Specificity of glutamine transaminuses with respect to a-keto acids

a-K&o acid

Relative activities

Kidney Ka Liver L (4)

Phenylpyruvate. [100]* 17; <1 (0.4 mM) a-Ketor-methiolbutyrate (5 mM) 75 240 a-Ketoglutaramate (80 mM) 40 134 p-Hydroxyphenylpyruvate.. 65* <1 (0.4 rnM)

Glyoxylate 20 Wl Pyruvate........................ 20 28 a-Ketobutyrate.. 21 24 a-Keto-n-valerate. 27 a-Keto-n-caproate.. _. 42 24 8-Hydroxypyruvate. 28 50 a-Ketoisovalerate.. 22 2 a-Ketoisocaproate. 18 3 a-Keto-&carbamidovalerate.. 18 10 a-Ketoglutarate-r-ethyl ester. . 23 77 a-Ketoglutarate. _. 3 3 Oxalacetate...................... 3 4 Trimethylpyruvate <O.l <O.l (D and L)-a-Keto-,%methylval-

erate. _. _. _. <O.l <O.l

0 The assay system (except as noted) contained 20 mM a-keto acid, 20 mM L-[14C]glutamine, 50 mM Tris-HCl buffer (pH 8.4), and purified enzyme in a final volume of 0.1 ml. The rates of transamination were determined at 37” by measuring the forma- tion of a-keto[14C]glutaramate (4).

5 Assayed by the method of Kupchik and Knox (2); the a-keto acid concentration was 0.4 mM.

TABLE III Apparent K, and V,,, values for soluble kidney glutamine

transaminase K

Substrate Kn

Glyoxylate” Pyruvate* u-Ketobutyrateb a-Keto-n-valerate”. a-Keto-n-caproateb.. a-Keto-r-methiolbutyrateb a-Ketoglutaramate*. Phenylpyruvatec. p-Hydroxyphenylpyruvatec L-Glutamined L-Methionined. L-Ethionined. L-Phenylalanine:.

?&%I

4.8 27 18

7.8 8.7 0.92 0.10

<O.l <O.l

1.4 4.2 4.4 0.58

I-

(L Micromoles per hour per unit of enzyme.

V,,X”

0.35 0.39 0.26 0.32 0.64 1.7 1.2 1.7 1.1 1.9 3.4 2.7 0.97

* Determined in reaction mixtures (final volume, 0.4 ml) con- taining 100 mM sodium borate (pH 8.5), 10 mM L-phenylalanine, a-keto acid, and enzyme (0.36 unit); phenylpyruvate formation was determined (9).

c Determined as described under “Methods” with 20 mM L-glu- tamine; disappearance of phenylpyruvate or p-hydroxyphenyl- pyruvate was measured (2).

d Determined as described under “Methods” (2) with 0.4 mM phenylpyruvate.

e Formation of phenylpyruvate was measured (9) with a-keto- glutaramate (150 mM); the K, value was calculated for the con- centration of the open chain form of cu-ketoglutaramate (0.3% of

Comparisons of the relative rates of transamination of glu- tamine with glyoxylate and with phenylpyruvate catalyzed by liver homogenates and by purified liver glutamine transaminase L suggested that liver might also contain an enzyme that ex- hibited a specificity similar to that of kidney transaminase K. In order to investigate this possibility, WC carried out a prepara- tion of soluble glutamine transaminase L from rat liver according to the procedure previously described (4) ; this fractionation was followed by determinations of the glutamine-glyoxylate and glutamine-phenylpyruvate transaminase activities. In this work, 40 g of rat liver were processed according to the procedure previously described (4) ; the two transaminase activities were purified in parallel fashion through Step 4 of the procedure. During Step 5 of the procedure (DE-52 chromatography), it was noted that the glutamine-glyoxylate transaminase activity was eluted earlier than the glutaminc-phenylpyruvate transaminase activity (Fig. 3). The fractions containing glutamine trans- aminase (Fractions 14 to 25) were pooled and dialyzed against 5 mM potassium phosphate buffer (pH 7.2) containing 5 mM 2- mercaptoethanol; this material was subjected to hydroxyapatite chromatography as described in Step 6 of the published purifica- tion procedure (4). As indicated in Fig. 5, the chromatography on hydroxyapatite led to separation of the two activities; thus, the liver glutamine transaminase K is eluted earlier from the column than is liver glutamine transaminase L. Fractions 6 to 9 (Fig. 4) contained 250 units of glutamine-phenylpyruvate trans- aminase and 40 units of glutamine-glyoxylate transaminase; Fractions 10 to 20 (previously taken as liver glutamine trans- aminase L (4)) contained 10 units of glutamine-phenglpyruvate transaminase and 605 units of glutamine-glgosylate transami- nase. These observations indicate that liver contains a glutamine

the total concentration (14)). transaminase which resembles that purified from kidney and

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Kidney glutamine transaminase K was found to be very active in catalyzing transamination between cY-keto acids and phenyl- alanine, methionine, or ethionine. A summary of the apparent Km and V,,, values for several amino and cu-keto acids is given in Table III. The apparent K, value for phenylpyruvate in the glutamine-phenylpyruvate transamination reaction was esti- mated to be less than 0.1 mM; the value could not be determined accurately by the experimental procedure employed. Kupchik and Knox (2, 3) estimated the apparent K, value for phenyl- pyruvate to be about 0.01 mM. In the present studies, we found considerable substrate inhibition with concentrations of phenyl- pyruvate that were greater than 0.4 InM; thus, the initial rate of transamination with 20 mM phenylpyruvate was only 10% of that observed with 0.4 mM phenylpyruvate. The data sum- marized in Table III indicate that the lowest apparent K,

values were obtained with phenylpyruvate, p-hydroxyphenyl- pyruvate, cu-ketoglutaramate, and cY-keto-y-methiolbutyrate, and that the highest V,,, values were obtained with these cr-keto acids.

Kidney glutamine transaminase K was found to be much less active than liver glutamine transaminase L toward several glutamine analogs; thus, as shown in Table IV the kidney en- zyme exhibited very little activity toward r,-albizziin, S-car- bamyl-L-cysteine, 0-carbamyl-L-serine, and y-cyano-n-cr-amino- butyrate. It is of interest that kidney glutamine transaminase K does not interact appreciably with the L-y-glutamylhydra- zones of glyoxylate and phenylpyruvate.

Separation of Soluble Glutamine Transaminases

K and L from Rat Liver

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TABLE IV

Relative rates of transamination between amino acids and phenyl pyruvate, glyoxylate, and a-ketoglutaramate by soluble

glutamine transaminase.P

I- Kidney K Liver K Liver L I

-

Phenyl- PYN- Kite*

3lyoxy. IateC or a-keto- :Iu tara- mateld

Phenyl- PYIU- vat&

( ;1yoxy1- ate (4-6)

WoP

17 63

16 57

<l 125 127

20 30

30

40 15 70

<l <1 <l

10 5

<1

12

35 9

10 a7 50 42 10

2

[371 [351

85 8

Amino acid

L-Glutamine n-r-Glutamylmethyl-

amide................. L-Glutamic acid-r-

methyl ester L-y-Glutamyldimethyl-

amide................. L-Methionine n-Ethionine r.-Methionine-SR-sulfoxi.

mine.................. L-Methionine-SR-sulfox-

ide.................... L-Methionine sulfone. S-Methyl-L-cysteine.. L-Albizziin.............. S-Carbamyl-L-cysteine 0-Carbamyl-L-serine DL-Homoserine (40 mM) L-Cysteine y-Cyano-n-a-aminobuty-

rate. L-Phenylalanine. . L-Tyrosine.............. L-r-Glutamylhydrazone

of glyoxylatee.. L-y-Glutamylhydrazone

of phenylpyruvate”.

20

<I 145 120

31

47 15 85

<1 <1 <l

6 5

<l

1

20

2

2

20 22

2

]3Z] I361

1 120

15

FRACTION NUMBER

FIG. 3. Chromatography of glutamine transaminase from rat liver on DE-52. Rat liver (40 g) was purified by the procedure of Cooper and Meister (4). The arrow marks the point at which the gradient was started; fractions of 10 ml were collected. Glu- tamine transaminase L is maximal in Fraction 14, and glutamine transaminase K is maximal in Fraction 19.

elf , $; , p-.: , , / / + \lo 10 IO 20

FRACTION NUMBER

FIG. 4. Chromatography of glutamine transaminases from rat liver on hydroxyapatite (Step 6 of the published purification procedure (4)). The arrow marks the point at which the gradient was started; fractions of 5 ml were collected. Glutamine trans- aminase L fractions under A. Glutamine transaminase K frac- tions under B.

a The values are expressed relative to those found for glutamine- phenylpyruvate transamination with the kidney K (specific ac- tivity, 243 units per mg) and liver K (specific activity, 120 units per mg) enzymes (given as (100)); and to those found for glu- tamine-glyoxylate transamination for the liver L enzyme (specific activity, 300 units per mg).

*The assay mixtures contained 20 mM L-amino acid, 300 mM sodium borate buffer (pH 8.5), 0.4 mM phenylpyruvate, and from 2 to 20 units of enzyme in a final volume of 1.0 ml; incubated at 37”. The rate of disappearance of phenylpyruvate was deter- mined (2).

c The reaction mixtures contained 20 mM [W]glyoxylate, 20 mM L-amino acid, 50 mM Tris-HCl buffer (pH 8.5), and 1 to 10 units of enzyme in a final volume of 0.1 ml. After incubation at 37” [i4C]glycine formation was determined (4).

d The assay mixture contained 20 mM L-phenylalanine or 5 mM

n-tyrosine, 100 mM sodium borate buffer (pH 8.5), 150 rnM a-keto- glutaramate, and 2 units of enzyme in a final volume of 0.4 ml. After incubation for 30 min the formation of phenylpyruvate or p-hydroxyphenylpyruvate was determined (9).

e The reaction mixtures contained 20 mllr cY-keto acid r-glu- tamylhydrazone in 0.1 ml of a solution containing 100 mM sodium borate buffer (pH 8.5); 1,4,5,6-hydroxytetrahydro-6-pyrida- zinone-3-carboxylate formation was determined (5). No free a-keto acid was added.

which can be physically separated from the glutamine trans-

aminase previously purified from liver.

Specificity of Liver- and Kidney-soluble Glutamine Transaminase K

As indicated in Table IV, the values for the rates of trans-

amination between phenylpyruvate and a variety of amino acids are substantially the same with kidney glutamine transaminase

K and liver glutamine transaminase K. In addition, both en- zymes catalyze similar rates of transamination between a-keto-

glutaramate and phenylalanine (or tyrosine). It is of interest

that kidney glutamine transaminase K catalyzes albizziin-

glyoxylate transamination to a small but significant extent. This activity is evidently a property of transaminase K since

this activity co-purifies with the glutamine-phenylpyruvate transaminase activity in kidney (Table I).

Evidence for Isozymic Forms of w-ilmidase, and

Glutamine Transaminases L and K

The glutamine transaminase previously purified from rat liver (transaminase L (4)) was isolated from the soluble fraction of

rat liver homogenates. In the present work, we have obtained

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TABLE V

Tissue distribution of transaminase and w-amidase activities0

Tissue

Liver (soluble). Liver (mitochon-

drial)

Kidney (soluble) Kidney (mitochon-

drial)

Brain (soluble). Brain (mitochon-

drial)

Cardiac muscle (soluble).

Cardiac (mitochon. drial)

w-An& dasd

340

135

390

40

4

4

74

8

Glutamine-a-k& acid transaminase

25 83 165 35

10 27 40 10

135 24 105 20

12 3 10 3

<2 5 10 6

<2 <2 5 3

6

3

5

<2

12

5

4

3

-K&o-y nethiol. butyr-

at&

Pyru- vated

1 I

1

-

/-Gluta- nY1 dY- oxylate hydra-

zone reactione

89

20

4

<2

5

<2

5

<2

0 The tissues were homogenized in 5 volumes of 0.25 M sucrose and the mitochondrial and cytosol fractions were prepared by the method of Remmer et al. (15). The mitochondria were suspended in an equal volume of 0.25 M sucrose and sonicated at 20 kc for 5 min at 0” using a Sonifi-r S-75 (Branson Instruments Inc.). The values given (micromoles of product formed at 37” per hour per g of tissue) are the averages of several determinations on the pooled tissues of four animals.

* The reaction mixtures contained 100 mM sodium borate buffer (pH 8.5), 5 mM sodium or-ketoglutaramate, and 0.1 ml of tissue ex- tract in a final volume of 1 ml. After incubation for 10 to 60 min, ammonia formation was determined (11). Skeletal muscle, testis, spleen, pancreas, lung, and intestine were found to have 23, 12,38, 30,25, and 30 units of w-amidase per g of tissue, respectively. No glutamine transaminase activity was detected in these tissues using the a-keto acids given in the table.

c The reaction mixtures contained 20 mM L-glutamine, 0.4 mM sodium phenylpyruvate, 300 mM sodium borate buffer (pH 8.5), and 0.1 ml of tissue extract in a final volume of 1.0 ml. Disap- pearance of phenylpyruvate was measured by the interrupted assay procedure of Kupchik and Knox (2).

d The assay system consisted of 20 mM L-[U-Wlglutamine, 20 mM sodium pyruvate or sodium glyoxylate, or 5 mM sodium 01. keto-r-methiolbutyrate, 100 mM sodium borate buffer (pH 8.5), and 40 ~1 of tissue extract in a final volume of 0.1 ml. The forma- tion of a-keto[r%]glutaramic acid was determined by the method of Cooper and Meister (4).

6 The assay system consisted of 20 mM glyoxylate r-glutamyl- hydrazone, 100 mM sodium borate buffer (pH 8.5), and 40 ~1 of tissue extract in a final volume of 0.1 ml. At various intervals aliquots of 20 ~1 were withdrawn and the protein was precipi- tated by addition of 20% trichloroacetic acid. The mixture was treated with 0.96 ml of 2 N HCl, and after standing at 25” for 40 min, the precipitated protein was removed by centrifugation, and the increase in absorbance at 267 nm due to the formation of 1,4,5,6-tetrahydro-6-py;idazinone-3-carboxylic acid was deter- mined (5).

evidence that there is a distinct mitochondrial glutamine trans- aminase L; this finding is in agreement with the work of Yoshida (8). The first step of our purification procedure (4) involves homogenization of rat liver; after centrifugation of the homoge- nate we found that considerable glutamine transaminase ac-

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tivities (L and K) remained associated with the pellet. These activities could be solubilized by rapid freeze-thawing or by sonication as stated in Table V.

Hersh reported that more than 90% of rat liver w-amidase can be recovered in the cytoplasmic fraction after removal of the mitochondria and microsomes (14). We prepared rat liver mitochondria by the procedure (15) used by Hersh, and deter- mined the w-amidase activity. While the total activity of a suspension of the intact mitochondria was very low (about 2% of that recovered from the soluble portion), after the mitochon-

dria were subjected to freeze-thawing or sonication, the total activity increased to about 40% of that found in the soluble fraction (Table V).

As shown in Fig. 5, the soluble and mitochondrial forms of liver w-amidase, glutamine transaminase K, and glutamine transaminase L exhibit significantly different mobilities. We also found that soluble transaminase K isolated from kidney exhibited’mobility that was ident,ical with that of the soluble K enzyme from liver. Similarly, the mitochondrial form of glu- tamine transaminase K from rat kidney had the same relative mobility on gel electrophoresis as that prepared from liver mito- chondria. We have not purified the mitochondrial glutamine transaminase; however, Yoshida (8) has published on the puri- fication of a mitochondrial glutamine transaminase, which ex- hibited electrophoretic mobility different from that of the soluble enzyme, and which had a substrate specificit,y similar to that of the soluble enzyme.

Glutamine Transaminase Activity of Other Tissues

Various tissues were removed from male Sprague-Dawley rats and homogenized in 5 volumes of 0.25 M sucrose. The cytoplasmic and mitochondrial fractions were prepared as de- scribed (15) ; the mitochondria were sonicated and both the soluble and mitochondrial fractions were assayed for w-amidase and various glutamine transaminase activities (Table V). No glutamine transaminase activity was detected in homogenates

of skeletal muscle, testis, spleen, pancreas, lung, and intestine. However, some activity was found in brain and cardiac muscle. Sugiura (17) reported the presence of glutamine-pyruvate trans- aminase activity in rabbit brain. Our data indicate that rat brain also contains low levels of glutamine transaminase, which is probably of the L type. However, detailed studies on trans- amination of glutamine in the brain are needed. The w-amidase activity of brain is relatively low compared to that of liver and kidney.

DISCUSSION

These studies show that rat liver and kidney contain at least two types of glutamine transaminase and that each of these types occurs in soluble and mitochondrial forms. Glutamine transaminase K is highly active toward methionine, phenyl- alanine, tyrosine, and the cr-keto analogs of these amino acids; glutamine transaminase L is also active with methionine, but is more active with glyoxylate, pyruvate, and certain other cu-keto acids than it is with phenylpyruvate or p-hydroxyphenyl- pyruvate. Transaminases L and K are further distinguished by the much greater activity of the former toward y-glutamyl- hydrazones of cy-keto acids and certain glutamine analogs (e.g. albizziin).

While the metabolic function (or functions) of these enzymes is not yet certain, we feel that our previous suggestion (4) that glutamine transaminase may serve as a “salvage” enzyme is worthy of serious consideration. Thus, transamination of glu-

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FIG. 5. Behavior of the isozymes of rat liver glutamine trans- aminases L and K and w-amidase on polyacrylamide disc gel electrophoresis. The liver was homogenized in 5 volumes of 0.25 M sucrose and the isolated mitochondria (prepared as de- scribed (15)) were taken up in equal volume of 0.25 M sucrose and subjected to sonication. Samples (50 ~1) of the soluble and sonicated mitochondrial preparations containing 2.5 and 1.5 mg of protein, respectively, were applied to the top of 7% gels and subjected to electrophoresis at 37”. (Conditions: upper buffer, 0.5 M Tris-glycine (pH 8.9); lower buffer, 0.10 M Tris-HCl (pH 8.1); gel buffer, 0.37 M Tris-HCl (pH 8.9); (4).) After the brom- thymol blue marker had moved 40 mm the gels were removed and sliced into l-mm sections, which were assayed for the various enzyme activities listed in the figure. Thus, the gels labeled “soluble” were replicate runs, each of which was examined for separate enzyme activities; the same was true for the gels labeled “mitochondrial.” The data show that each of the three soluble activities and each of the three mitochondrial activities of the crude supernatant and sonicated mitochondrial fractions, respec- tively, was separated under the conditions employed. When samples containing mixtures of the crude soluble and mitochon- drial fractions (25 ~1 of each) were subjected to electrophoresis and then assayed for these activities, two peaks of activity OC- curred on the gel for each of the three enzymes. Glutamine transaminase L activity was determined by the albizziin-glyoxyl- ate reaction. Each section was incubated at 37” for 3 hours, after which time the formation of 2-imidazolinone-4-carboxylic acid was measured at 280 nm in the presence of 1 N NaOH (6). Transaminase K was measured by the disappearance of phenyl- pyruvate from the standard assay mixture containing 20 mM L-glutamine and 0.4 rnM phenylpyruvate (2) after incubation at 37” for 3 hours. w-Amidase activity was assayed by measuring the formation of r-glutarylmonohydroxamate with ferric chloride in a solution containing 10 rnM glutarate, 50 mM hydroxylamine, and 100 mM sodium borate buffer (pH 8.5) in a final volume of 0.1 ml. After 3 hours, 0.9 ml of a solution containing 0.2 M tri-

chloroacetic acid, 0.67 M hydrochloric acid, and 0.27 M ferric chloride was added. The increase in absorbance at 550 nm due to the formation of 7.glutarylmonohydroxamate was determined (1’3).

tamine may provide a means for the reamination of a-keto acids formed by other transaminases. It has long been known that liver preparations can catalyze transamination between cr-keto- glutarate and a wide variety of amino acids (18, 19) and rela- tively recent studies indicate that such amino acids as phenyl- alanine, tyrosine, and methionine may be substrates of certain forms of glutamate-aspartate transaminase (20, 21). For example, it has been concluded that rat liver mitochondrial tyrosine transaminase (which is identical with glutamate-as- partate transaminase) also acts on phenylalanine, methionine, and other amino acids (20). Transamination of tyrosine is also catalyzed by a soluble enzyme (20), and the reaction catalyzed by this enzyme is generally believed to be on the normal degra- dative pathway of tyrosine; however, p-hydroxyphenylpyruvate produced in excess in the mitochondria may apparently escape the action of soluble p-hydroxyphenylpyruvate hydroxylase (22). Mitochondrial glutamine transaminase K may function normally in the physiological amination of p-hydroxyphenyl- pyruvate. Transamination of phenylalanine and methionine by soluble and mitochondrial enzymes would lead to formation of ol-keto acids whose dietary essential carbon chains might be lost by excretion and thus be unavailable for protein synthesis; these oc-keto acids are apparently not degraded extensively normally in mammals, and may accumulate in substantial amounts in certain inborn errors of metabolism (e.g. phenyl- pyruvate (23) and oc-keto-y-methiolbutyrate (24)). The ac- cumulation of even relatively small concentrations of such cy-keto acids as ol-keto-y-methiolbutyrate and phenylpyruvate might conceivably produce toxic effects. We postulate that the glu- tamine transaminases may function normally to reconvert the small amounts of such cy-keto acids produced to the corresponding amino acids. In contrast to most transamination reactions, which are readily reversible, transamination between glutamine and cu-keto acids would be expected to be essentially irreversible in vivo; thus, the presence of w-amidase would favor utilization of glutamine and the formation of amino acids.

Administration of large amounts of glutamine to patients with phenylketonuria led to substantially decreased urinary excretion of phenylpyruvate (25) ; this finding is consistent with the oc- currence of increased transamination between glutamine and phenylpyruvate catalyzed by glutamine transaminase K, al- though other explanations cannot be excluded. It has been sug- gested that lowered plasma and tissue concentrations of glu- tamine may be related to the abnormalities found in phenyl- ketonuria ((26), but see also Refs. 27 and 28) ; such depletion of glutamine might occur by increased transamination or by increased utilization of glutamine for phenylacetylglutamine synthesis. It has been proposed that accumulation of certain oc-keto acids may inhibit normal formation or myelin (29). I f such overproduction of a-keto acids is indeed deleterious, ther- apy with large amounts of glutamine might conceivably be useful in metabolic diseases in which oc-keto acids accumulate.

The findings on w-amidase reported here indicate that a sub- stantial fraction (about 30%) of the total liver w-amidase occurs in the mitochondria. Although more than 90% of the soluble liver w-amidase can be obtained in the supernatant solution after removal of the mitochondria by centrifugation (14), considerable amounts of this activity have been shown in the mitochondria; this finding is in general agreement with those of Yoshida (8) who found w-amidase in both soluble and mitochondrial frac- tions. The data thus indicate that the glutamine transamina-

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tion-deamidation pathway can occur in both the cytosol and mitochondria. The question as to whether glutamine trans- aminase and w-amidase may be linked physically in these intra- cellular locations remains to be investigated.

w-Amidase seems to function physiologically to drive the glutamine-ol-keto acid transamination reaction in the direction of amino acid formation. The high w-amidase activity present in liver and kidney suggests that little if any oc-ketoglutaramate accumulates normally. The recent report by Vergara et al. (30) that cr-ketoglutaramate accumulates to the extent of 30 to 100 pmoles per liter in the cerebrospinal fluid of patients with hepatic coma (normal values, less than 5 pmoles per liter) is of consider- able interest. These workers have considered the possibility that a-ketoglutaramate is toxic to the nervous system (30). The activities of the glutamine transaminases and w-amidase are relatively low in rat brain as compared to rat liver and kidney (Table V), and they are evidently also low in rabbit brain (17). However, the possibility must be considered that the increased glutamine formation which may occur in the brain in hepatic coma (in response to increased ammonia formation) may be accompanied by increased transamination of glutamine. If the w-amidase activity of brain is limiting, relative to the glutamine transaminase activities, cu-ketoglutaramate might accumulate and conceivably produce toxicity as suggested by Vergara et al. (30). These considerations suggest that one of the physiological functions of w-amidase may be to prevent a potentially toxic compound, i.e. cY-ketoglutaramate, from accumulating in the brain and other tissues.

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Arthur J. L. Cooper and Alton MeisterIsolation and Properties of a New Glutamine Transaminase from Rat Kidney

1974, 249:2554-2561.J. Biol. Chem. 

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