Phosphate-independent glutaminase from rat kidney. Partial purification and identity with gamma-glutamyltranspeptidase.

Phosphate-independent glutaminase can be quantitatively solubilized from a microsomal preparation of rat kidney by treatment with papain. Subsequent gel filtration and chromatography on quaternary aminoethyl (QAE)-Sephadex and hydroxylapatite yield a 200-fold purified preparation of this glutaminase. The purified enzyme also hydrolyzes gamma-glutamylhydroxamate and exhibits substrate inhibition at high concentrations of either glutamine or gamma-glutamyhydroxamate, which is partially relieved by increasing concentrations of maleate. Rat kidney phosphate-independent glutaminase reaction is catalyzed by the same enzyme which catalyzes the gamma-glutamyltranspeptidase reaction. The ratio of glutaminase to transpeptidase activities remained constant throughout a 200-fold purification of this enzyme. The observation that the phosphate0independent glutaminase and gamma-glutamyltranspeptidase activities exhibit coincident mobilities during electrophoresis, both before and after extensive treatment with neuraminidase, strongly suggests that both reactions are catalyzed by the same enzyme. This conclusion is strengthened by the observation that maleate and various amino acids have reciprocal effects on the two activities. Maleate increases glutaminase activity and blocks transpeptidation, whereas amino acids activate the transpeptidase but inhibit glutaminase activity. In contrast, the addition of both maleate and alanine resulted in a strong inhibition of both activities. Both activities exhibit a similar distribution in the various regions of the kidney. Recovery of maximal activities in the outer stripe region of the medulla is consistent with previous quantitative microanalysis which indicated that this glutaminase activity is localized primarily in the proximal straight tubule cells. The glutaminase and transpeptidase activities have different pH optima. Examination of the product specificity suggests that decreasing pH also promotes glutaminase activity and that below pH 6.0, this enzyme functions strictly as a glutaminase. Because of the localization of this activity on the brush border membrane, these resuts are consistent with the possibility that the physiological conditions induced by metabolic acidosis could convert this enzyme from a broad specificity transpeptidase to a glutaminase. Therefore, this enzyme could contribute to the increased renal synthesis of ammonia from glutamine which is observed during metabolic acidosis.


SUMMARY
Phosphate-independent glutaminase can be quantitatively solubilized from a microsomal preparation of rat kidney by treatment with papain. Subsequent gel filtration and chromatography on quaternary aminoethyl (QAE)-Sephadex and hydroxylapatite yield a ZOO-fold purified preparation of this glutaminase.
The purified enzyme also hydrolyzes y-glutamylhydroxamate and exhibits substrate inhibition at high concentrations of either glutamine or y-glutamylhydroxamate, which is partially relieved by increasing concentrations of maleate.
Rat kidney phosphate-independent glutaminase reaction is catalyzed by the same enzyme which catalyzes the y-glutamyltranspeptidase reaction.
The ratio of glutaminase to transpeptidase activities remained constant throughout a 200-fold purification of this enzyme. The observation that the phosphate-independent glutaminase and y-glutamyltranspeptidase activities exhibit coincident mobilities during electrophoresis, both before and after extensive treatment with neuraminidase, strongly suggests that both reactions are catalyzed by the same enzyme.
This conclusion is strengthened by the observation that maleate and various amino acids have reciprocal effects on the two activities.
Maleate increases glutaminase activity and blocks transpeptidation, whereas amino acids activate the transpeptidase but inhibit glutaminase activity.
In contrast, the addition of both maleate and alanine resulted in a strong inhibition of both activities. Both activities exhibit a similar distribution in the various regions of the kidney. Recovery of maximal activities in the outer stripe region of the medulla is consistent with previous quantitative microanalysis which indicated that this glutaminase activity is localized primarily in the proximal straight tubule cells.
The glutaminase and transpeptidase activities have different pH optima. Examination of the product specificity suggests that decreasing pH also promotes glutaminase activity and that below pH 6.0, this enzyme functions strictly as a glutaminase.
Because of the localization of this activity on the brush border membrane, these results are consistent with the possibility that the physiological conditions induced by metabolic acidosis * This investigation was supported in part by Research Grant AM 16651 from National Institute of Arthritis, Metabolism, and Digestive Diseases.
$ To whom correspondence concerning this manuscript should be addressed. could convert this enzyme from a broad specificity transpeptidase to a glutaminase.
Therefore, this enzyme could contribute to the increased renal synthesis of ammonia from glutamine which is observed during metabolic acidosis.
y-Glutamyltranspeptidase has been extensively purified from hog kidney (1) and from beef kidney (2). It has been characterized as an enzyme of very broad specificity.
It can use a variety of y-glutamyl compounds, such as glutathione or glutamine, as substrates.
It transfers a y-glutamyl moiety to a large number of amino acids and peptides which serve as acceptors. Recent interest in this enzyme has been stimulated by its clinical use as an index of hepatic function (3) and its proposed roles in formation of mercapturic acids (4) and in amino acid transport (5, 6).
Histochemical characterization has indicated that in rat kidney the y-glutamyltranspeptidase is localized primarily on the brush border membrane of the proximal tubule cells (7,8). The rat kidney phosphate-independent glutaminase, originally described by Katunuma et al. (9), has recently been characterized as also being located in the brush border membrane (10). This finding prompted us to investigate the possibility that both of these reactions are catalyzed by the same enzyme.

Materials
White male Sprague-Dawley rats (200 to 400 g) were obtained from Zivic Miller and were maintained on Purina rat chow. Glutamic dehydrogenase in 50% glycerol was obtained from Boehringer.
Sephadex G-200 and quaternary aminoethyl (QAE)-Sephadex were products of Pharmacia and hydroxylapatite and acrylamide were obtained from Bio-Rad.
All other biochemicals were obtained from Sigma.

Methods
Enz2/me Assays-Phosphate-independent glutaminase was assayed by measuring the amount of glutamate formed (11). Except where indicated in the text the standard assay conditions (20 mM glutamine, 60 mM maleate, 0.2 mM EDTA, pH 6.6) were used. u-Glutamyltranspeptidase was assayed (1) by following the appearance of p-nitroaniline at 25" from a solution containing 5 mM min at 3" and was %en centrifuged at 20,000 X g for 10 min. Sufficient solid ammonium sulfate was then added to the supernatant solution to make it 95% saturated (20.5 g/100 ml). After standing for 15 min at 3", the solution was centrifuged at 20,000 X 9 for 10 min.
The resulting pellet was resuspended in 10 ml of buffer and dialvzed for 4 hours against two a-liter changes of buffer. The sample was then applied t% a hydroxylapatite column (2 X 25 cm  II Comparison of rates of ammonia and glutamate synthesis from glutamine by puri$ed phosphate-independent glutaminase The rate of glutamate formation was determined by adding enzyme to 100 pl of a solution containing 50 mM imidazole, 20 mM cY-ketoglutarate, 0.2 mM EDTA, 0.25 rnM ADP, and the indicated amounts of glutamine and maleate adjusted to pH 7.2 and incubated at 26". After 10 min, the reaction was stopped by addition of 10 ~1 of 2 N HCl, and then glutamate was determined as described previously (9). The rate of ammonia formation was determined under the same conditions except that the assay mixture also contained 0.15 mM DPNH and 250 pg per ml of glutamic dehydrogenase.
The complete mixture was preincubated for 10 min to remove endogenous ammonia.
Then enzyme was added and the linear decrease in absorbance at 340 nm was recorded. Data was corrected for a slight, nonenzymatic ammonia formation from glutamine by subtracting the decrease in absorbance which occurred in a sample in which glutaminase was omitted. tion to higher concentrations of glutamine and reduced the per cent inhibition observed at 50 rnti glutamine. A similar profile was observed when y-glutamylhydroxamate was used as substrate.
Using the purified phosphate-independent glutaminase, the rates of ammonia and glutamate production from glutamine were compared (Table II).
At 5 mM glutamine, in the absence of maleate, this enzyme produces more ammonia from glutamine than it produces glutamate, suggesting that some ammonia is produced by a reaction other than glutaminase.
(We have shown by means of thin layer chromatography that y-glutamylglutamine is formed under these conditions, but we have not quantitated its rate of \synthesis.)2 Upon addition of 60 mM maleate to the 5 mM glutamine, the cneyme now produces almost stoichiometric amounts of glutamate and ammonia; indicating that under these conditions the enzyme is acting strictly as a glutaminase.
Because of substrate inhibition, the rate of glutamate formation at 20 mM glutamine is less than at 5 mM glutamine, but the rate of ammonia production is 2.3-fold greater. Therefore, substrate inhibition of the phosphate-independent glutaminase may be due to increased binding of glutamine at a second site which alters product formation.
Addition of 60 mM maleate at the higher glutamine concentration causes a 7.5-fold increase in the rate of glutamate formation and a 2.6-fold increase in ammonia formation; indicating that in the presence of maleate a greater proportion of the glutamine is hydrolyzed via the glutaminase pathway.
Presumably, if greater concentrations of maleate were added, the enzyme would again act solely as a glutaminase.
Identity with y-Glutamyltranspeptidase--A comparison of the specific activities of phosphate-independent glutaminase and y-glutamyltranspeptidase in various preparations is shown in Table III.
In crude kidney homogenates, the y-glutamyltranspeptidase activity, assayed with glutathione, is 20 times greater 2 N. P. Curthoys, unpublished results. a Numbers in parentheses are the ratio of y-glutamyltranspeptidase to glutaminase activity.

FIG. 3. Elution
of phosphate independent, glutaminase and r-glutamyltranspeptidase activities from polyacrylamide gels. The purified preparation of phosphate-independent glutaminase was subjected to polyacrylamide gel electrophoresis before (A) and after (B) extensive treatment with neuraminidase.
One gel was stained for protein and a duplicate gel was sliced and assayed for both glutaminase and r-glutamyltranspeptidase activities. r-Glutamyltranspeptidase was assayed with 5 mM r-glutamyl-pnitroanilide in the presence of 40 mM methionine at pH 8.4. Both activities are expressed as lo2 X micromoles min+ slice-l. than phosphate-independent glutaminase activity. The microsomal preparation was obtained by differential centrifugation (10) and was heated to 50" for 10 min; such conditions selectively destroy any phosphate-dependent glutaminase activity (9). The ratio of y-glutamyltranspeptidase to phosphate-independent glutaminase activit'ies remains constant in all of these preparations and at all of the steps throughout the purification of phosphate-independent glutaminase. As shown in Fig. 3A, polyacrylamide gel electrophoresis of the purified phosphate-independent glutaminase yields one major protein band, preceded by a region of diffuse staining.
Analysis of an identical gel indicated that the phosphate-independent glutaminase and y-glutamyltranspeptidase activities migrated as a single coincident band, which corresponded to the region which stained diffusely for protein.
Following treatment with neuraminidase to extensively remove sialic acid, the phosphate-independent glutaminase preparation was again subjected to polyacrylamide gel electrophoresis (Fig. 3B). The major protein FIG. 4. Effect of maleate on product specificity of purified phosphate-independent glutaminase using r-glutamyl-p-nitroanilide as substrate.
Sufficient enzyme was added to various 5 mM r-glutamyl-p-nitroanilide solutions, pH 7.2, containing either no maleate (-MALEATE) or 60 mM maleate (+MALEAZ'E) so that when the reactions were stopped with acid, about 50% of the substrate was converted to product. The various solutions also contained either no amino acid (-), 10 mM alanine (ALA ) or 10 mM methionine (MET). Unreacted r-glutamyl-p-nitroanilide solutions (middle), 5 mM glutamate (GLU), and 5 mM r-glutamylalanine (yGA) were spotted as standards.
In all positions, 15 ~1 of sample were spotted. Chromatography was carried out as described under "Experimental Procedure." p-Nitroaniline migrated with the solvent front, but because of the filter used during photography to improve contrast, these spots are not shown. band migrated slightly slower, but at this time the diffusely stained region was replaced by three bands of significantly slower mobility.
Again the phosphate-independent glutaminase and y-glutamyltranspeptidase activities exhibited a coincident mobility but now they peaked in the region of the three slowly moving bands.
The chromatogram shown in Fig. 4 indicates that maleate also affects the product specificity with y-glutamyl-p-nitroanilide as a substrate.
In the absence of maleate, the major product formed migrates more slowly than y-glutamyl-p-nitroanilide and is probably y-glutamyl-y-glutamyl-p-nitroanilide.
But, in the presence of maleate, the major product is glutamate.
Addition of maleate has the same affect if the reaction is carried out in the presence of methionine or alanine, it inhibits the formation of dipeptides and promotes glutamate formation.
Therefore, maleate binds to this enzyme and blocks transpeptidation, making it a specific glutaminase.
If maleate promotes glutaminase activity by blocking transpeptidation then maleate should also inhibit amino acid activation of the transpeptidase reaction.
As shown in Fig. 5, the addition of increasing concentrations of amino acids results in increasing y-glutamyltranspeptidase activity of this enzyme preparation.
The addition of 100 mM alanine or glycine causes approximately a 2-fold activation, whereas the presence of 100 ells methionine causes a 5-fold activation.
In the absence of any amino acids, the addition of 60 mM maleate caused a slight increase in the rate of p-nitroaniline formation.
As shown in the previous figure, the other product formed under the latter conditions was glutamate.
The addition of increasing concentrations of methionine in the presence of maleate still resulted in a slight activation.
But, the presence of maleate greatly reduced Glutamate formation from 20 mM glutamine, pH 7.2, was assayed in the presence of increasing concentrations of methionine (0, 0 ), glycine (W, cl), and alanine (A, A). The assays were performed both in the absence and the presence of 60 mM maleate. Enzyme activities are expressed as micromoles min-1 ml-l. the degree of activation observed.
In contrast, the presence of maleate not only blocked activation by glycine or alanine, the addition of increasing concentrations of these two amino acids resulted in a decrease in the rate of p-nitroaniline formation, As shown in Fig. 6, the addition of amino acids has a reciprocal effect on glutaminase activity (glutamate formation from glutamine).
In the absence of maleate, the phosphate-independent glutaminase is inhibited by increasing concentrations of either alanine, glycine, or methionine.
The effect of increasing amino acids is probably similar to that of increasing glutamine concentration.
In the presence of 60 mM maleate, the three amino acids still act as inhibitors of glutaminase activity and the degree of inhibition is similar to that observed with y-glutamyl-pnitroanilide as substrate.
Increasing concentrations of methionine result in only a slight inhibition.
Glycine produces a greater inhibition, but alanine is a very potent inhibitor of glutaminase activity.
The fact that these amino acids show the same order of potency as inhibitors with either glutamine of y-glutamyl-pnitroanilide as substrate supports the conclusion that both reactions are catalyzed by the same enzyme.
HoinTever, an understanding of why glycine and alanine are such potent inhibitors in the presence of maleate will require further investigation.
Previous reports (7, 8), using histological staining have indicated that ths y-glutamyltranspeptidase is localized in the brush border membrane of the proximal convoluted tubules in the rat kidney.
In contrast, the quantitative microanalysis of Curthoys and Lowry (19) has shown that the phosphate-independent glutaminase is localized primarily in the proximal straight tubule cells. This represents the only inconsistency, which we could find in the literature, suggesting that the two reactions are not catalyzed by the same enzyme. To investigate this apparent difference in localization, activities of various brush border marker enzymes were assayed in the various regions of kidney tissue (Fig. 7). A close correlation between phosphate inde- 7. Regional distribution of brush border membrane marker activities in rat kidney. A cone of kidney tissue was cut in such a way that its base consisted solely of cortical tissue and its apex consisted solely of papillary region. Progression from the base of the cone to its apex was associated with progression from cortex, through outer stripe and inner stripe regions of medulla, and finally into papillary region. Consecutive slices were then cut from the base of the cone, homogenized in 0.33 M sucrose, 25 rnllr Tris, and 0.2 mM EDTA buffer, pH 7.5, and assayed for phosphateindependent glutaminase (PZG),r-glutamyltranspeptidase (rGT), and alkaline phosphatase (AZ. Phos) activities, and for protein concentration.
Specific activities are expressed as micromoles min-i mg-l. 8. pH profiles of glutaminase and r-glutamyltranspeptidase activities.
Glutaminase activities were determined by measuring glutamate formation from 20 mr.r glutamine either in the absence (open $gures) or presence (half-shaded $gures) of 60 mM maleate. r-Glutamyltranspeptidase activity was determined by measuring p-nitroaniline formation from 5 mM r-glutamylp-nitroanilide in the presence of 40m~ methionine (shadedfigures). pH was maintained by using the following buffers: 50 mM piperazine (circles), 50 mM imidazole (ttiangles), 50 mM Tris (squares), or 50 mM bicarbonate (X). Enzyme activities are expressed as micromoles min+ ml-l. pendent glutaminase and y-glutamyltranspeptidase activities was observed.
Both activities were maximal in the outer stripe region of the medulla, consistent with the idea that both enzyme activities were localized primarily in the proximal straight tubule cells. In contrast, alkaline phosphatase activity was found to be maximal in both cortex and outer stripe regions.
E$ects of pH----The y-glutamyltranspeptidase and glutaminase activities exhibit different pH profiles (Fig. 8). With glutamine as substrate, the glutaminase activity in the absence of maleate shows a broad pH optimum between pH 6 and 7. The addition of 60 mM maleate causes a dramatic activation of the glutaminase activity and shifts the optimum to a sharp peak at pH 7.4. FIG. 9. Effect of pH on product specificity of purified phosphate-independent glutaminase using r-glutamyl-p-nitroanilide as substrate.
Sufficient enzyme was added to 100 ~1 of 5 mM r-glutamyl-p-nitroanilide at each of the various pH values so that when the reaction was stopped with acid about 50% of the substrate was converted to product.
In all positions, 10 ~1 of sample were spotted. Chromatography was carried out as described under "Experimental Procedure." In contrast, the y-glutamyltranspeptidase activity, assayed with y-glutamyl-p-nitroanilide in the presence of 40 mM methionine, exhibits maximal activity at pH 8.6. If an amino acid is not added, a similar profile, but with the activity reduced 3-fold, is observed.
Using y-glutamyl-p-nitroanilide as a substrate, and following only the appearance of p-nitroaniline, one cannot distinguish whether the enzyme is catalyzing a glutaminase-or transpeptidase-type reaction.
Comparison of the pH profiles suggests that a decrease in pH may promote glutaminase activity over transpeptidation.
Chromatographic analysis of products formed from y-glutamyl-p-nitroanilide as a function of pH is shown in Fig. 9. From pH 8.0 to 7.0, the enzyme functions primarily as a transpeptidase (the major product formed is probably y -glutamyl -y -glutamyl -p -nitroanilide) . With decreasing pH the per cent formation of this product decreases and an increasing amount of glutamate is formed.
Below pH 6.0, this enzyme preparation functions exclusively as a glutaminase.

DISCUSSION
The phosphate-independent glutaminase activity is extremely stable. A dilute preparation (less than 1 mg per ml) of the purified phosphate-independent glutaminase has been stored at 4' for 6 months without any loss of activity.
All of the chromatographic steps during the purification are conducted at room temperature without encountering any sizable loss of activity. The enzyme is also extremely resistant to inactivation by papain. Rat kidney microsomal preparations can be incubated up to 16 hours in a solution containing 0.25 mg per ml of papain without any loss of activity.
The enzyme solubilized by this procedure exhibits the same kinetic properties in terms of glutamine saturation and maleate activation as the particulate enzyme. These observations suggest that papain does not cause any proteolytic degradation in the active site region of the phosphate-independent glutaminase. Polyacrylamide gel electrophoresis shows that the preparation of phosphate-independent glutaminase is not pure. But, neuraminidase treatment of the glutaminase preparation decreases its mobility on polyacrylamide gels from an RF of 0.25 to one of 0.10. This is consistent with the removal of negatively charged sialic acid residues and strongly suggests that this enzyme is a glycoprotein.
Staining of duplicate gels for protein with Coomassie blue and for carbohydrate with periodic-acid Schiff reagent (20) produces identical banding patterns; this result suggests that the major contaminating protein is also a glycoprotein.
The finding that the phosphate-independent glutaminase exhibits substrate inhibition at high glutamine concentrations suggests that this enzyme contains more than one site which binds glutamine.
It appears that maleate increases glutaminase activity by preventing substrate inhibition and by altering the product specificity.
These observations, along with the ability of this glutaminase to hydrolyze y-glutamylhydroxamate and its subcellular localization (lo), suggested to us that the phosphate-independent glutaminase could be a partial reaction catalyzed by y-glutamyltranspeptidase.
The observation that the ratio of y-glutamyltranspeptidase to phosphate-independent glutaminase activities remains constant throughout a 200-fold purification and that these two activities exhibit coincident banding on polyacrylamide gels, both before and after extensive treatment with neuraminidase, strongly suggests that these two activities are catalyzed by the same enzyme. This conclusion is strengthened by the observation that maleate and various amino acids have reciprocal effects on the two activities.
Maleate increases glutaminase activity and blocks transpeptidation, whereas, amino acids activate the transpeptidase but inhibit glutaminase activity.
The regional distribution for alkaline phosphatase activity in both cortical and outer stripe regions is consistent with histological staining for this activity (21) ; this observation suggests that it is localized in the brush border membrane of both the proximal convoluted and proximal straight tubule cells. In contrast, the results of the regional distribution analysis of the y-glutamyltranspeptidase are not consistent with its reported histological localization in the proximal convoluted tubule brush border membrane (7,8). The specific activity of both the phosphate-independent glutaminase and the y-glutamyltranspcptidase are greatest in the outer stripe region.
This observation is consistent with the quantitative microanalysis of the distribution of this glutaminase activity (19). When assayed in individually dissected tubular structures, the phosphateindependent glutaminase activity was found to be lo-fold greater in the proximal straight tubule cells than in any of the other structures of the kidney nephron.
In addition to the numerous functions already proposed (3-6), the phosphate-independent glutaminase-y-glutamyltranspeptidase could also contribute to increased renal ammonia synthesis during metabolic acidosis. In the rat kidney, the largest proportion of the acidification of the urine occurs in the proximal convoluted tubule cell (22). Micropuncture studies in normal rats indicate that the fluid in the lumen at the end of the proximal convoluted tubule cell has a pH of about 6.8 (23). During metabolic acidosis, the decreased bicarbonate concentration in the glomerular filtrate greatly facilitates acidification of the fluid in the proximal convoluted tubule.
Under these conditions, the fluid entering the lumen of the proximal straight tubule is close to pH 6.0. The phosphate-independent glutaminase appears to be localized on the external surface of the brush border membrane of these cells (10). Therefore, this fluid constitutes the physiological medium in which this enzyme functions.
Examination of the products formed from y-glutamyl-p-nitroanilide indicates that pH values in this range promote glutaminase activity, and that below pH 6.0 this enzyme functions strictly as a glutaminase.
Therefore, acidification of the fluid in the tubular lumen during acidosis may convert this enzyme from a transpeptidase to a glubaminase. Maleate also appears to alter the product specificity of this enzyme; it increases glutaminase activity and blocks transpeptidation.
In contrast, alanine activates transpeptidation but inhibits glutaminase activity.
The addition of both maleate and alanine causes a dramatic inhibition of both glutaminase and transpeptidase activities; this finding indicates that the enzyme has separated binding sites for both of these modulators. If maleate had affected the enzyme activity by only competing with amino acid binding at the acceptor site, it would be difficult to explain why addition of both of these modulators results in an effect different from that obtained by the addition of either separately.
It is unlikely that maleate is a physiological activator of this glutaminase activity. But, if maleate binding occurs at a distinct site on the enzyme, this would suggest the possibility of a physiological counterpart to maleate. Katunuma et al. (24) has reported the extraction of a kidney factor which stimulates rat kidney phosphate-independent glutaminase activity and recently Alleyne and Roobol (25) have demonstrated the presence of a factor in serum of acutely acidotic rats which stimulates ammonia synthesis from glutamine in kidney slices. The appearance of such a factor in the tubular lumen in response to acidosis could convert y-glutamyltranspeptidase into a specific glutaminase.
Therefore, the possibility that the phosphate-independent glutaminase-y-glutamyltranspeptidase enzyme may contribute to renal ammonia synthesis in response to metabolic acidosis warrants further investigation.