Adaptation of γ-glutamyltransferase to acidosis

Life Sciences, Vol. 26, pp. 1985-1990 Printed in the U.S.A.

Pergamon Press

ADAPTATION OF ~-GLUTAMYLTRANSFERASE TO ACIDOSIS Proveen D. Dass and T. C. Welbourne Department of Physiology and Biophysics Louisiana State University Medical Center School of Medicine in Shreveport Shreveport, Louisiana 71130 (Received in final form April 4, 1980)

Summary This study established the following points: (l) the rat kidney contains a mitochondrial (PDG) and extramitochondrial glutamine u t i l izing enzymes; (2) this extramitochondrial a c t i v i t y exhibits a moleCblar weight of 70,000 identical with y-glutamyltranspeptidase (y-GTP) and is capable of hydrolyzing both isomers of glutamine; (3) this pathway adapts to metabolic acidosis consistent with a significant contribution to in vivo renal ammoniagenesis. Renal glutamine u t i l i z a t i o n occurs by either the well established mitochondrial (PDG)=glutamate dehydrogenase pathway (1) and, or, an extramitochondrial y-glutamyltransferase (2). Existenceof the second pathway was inferred from significant ammonia production from D-glutamine (3) despite the stereospecificity of the mitochondrial PDG (4) and the microsomal location of this D-glutamine u t i l i z i n g a c t i v i t y (5). In chronic metabolic acidosis, increased glutamine u t i l i z a t i o n is predominantly coupled to the mitochondrial pathway activation ( l , 2). However, an adaptive increase in the extramitochondrial pathway, although quantitatively not as significant as the PDG activation, takes place (2, 3, 5). Therefore, these studies were designed to reveal the identity of the extramitochondrial a c t i v i t y and to determine whether the acidosis induced adaptation appears on the subcellular level. The results to follow demonstrate the existence of a second glutamine u t i l i z i n g enzyme which exhibits a molecular weight of approximately 70,000 and co-purifies with y-glutamyltransferase. In addition, this extramitochondrial pathway undergoes an adaptation during acidosis consistent with its playing a significant role in in vivo renal ammoniagenesis. Materials and Methods Kidneys were removed from control and chronically acidotic, NH4CI induced, male Sprague-Dawley rats weighing 350 to 400 grams. After one week on NH4CI, 1.5% in their drinking water, 24 hour ammoniumexcretion rose from 833±95 to 7,612±925umolesday. The animals were anesthetized, sodium pentobarbital 30mg/Kg, their kidneys removed, and homogenized in 0.44 M sucrose (pH 7.45 with HEPES, 5 mMand 5 mM MgCl2 - l gram kidney/9 voI. homogenizing solution) with a Potter-Elvehjemhomogen]zer using a motor driven teflon pestle (6 strokes at 1400 RPM). Subcellular fractionation was carried out according to the method of de Duve et al (6); aliquots of the homogenate, lysosomal and mitochondrial, L + M, and postmitochondrial supernatants, PMS, were employed in the localization studies. In these studies, succinate cytochrome c reductase (7) was followed as a mitochondrial marker; y-glutaFEyltranspeptidase was assayed by the release of p-nitroanilide from L-y-glutamyl-p-nitroanilide (8); protein content was determined by the method of Lowry et al (9). Ammoniaformation was assayed using 20 mM glutamine under either of the following conditions: 0024-3205/80/231985-06502.00/0 Copyright (c) 1980 Pergamon Press Ltd

1986

y-glutamyltransferase in Acidosis

(I)

D-glutamine

(II)

L-glutamine

Tris 50 mM pH 7.45 MgCI2 lO mM > PO4 150 mM pH 8.2 >

NH3

+

NH3

Vol. 26, No. 23, 1980

+

D-glutamate

L-glutamate

The reaction was stopped a f t e r 30 minutes with 15 percent T r i c h l o r a c e t i c Acid and the proteins removed by c e n t r i f u g a t i o n . Ammonia was measured in the protein free supernatants by e i t h e r the microdiffusion (I0) or enzymatic ( I I ) methods; results were s i m i l a r with the two methods as reported (12). L-glutamate was determined enzymatically (13); formation of D-glutamate was not quantitated. Recoveries of L-glutamate were carried out on each sample and were q u a n t i t a t i v e . Thin layer chromatography, cellulose plates, was carried out on the above supernatants using a n-butanol:pyridine:H20 ( I : I : I , V/V) solvent system; ninhydrin reacting spots were compared with standards f o r glutamine, L-glutamate and L-y-glutamylglutamine (SIGMA). P u r i f i c a t i o n of the extramitochondrial a c t i v i t y from the PMS was carried out by u l t r a c e n t r i f u g a t i o n , 105,000xg for lh. on a Beckman L3-50 u l t r a c e n t r i f u g e , to obtain the microsomal p e l l e t . The microsomes were then resuspended in homogenizing solution, plus 1 mM d i t h i o t h r e i t o l , and treated with papain, O.25mg/ml, for 2 hours at 37°C. After incubation, the suspension was immediately cooled to 0 ° and r e c e n t r i fuged at 105,000xg for 60 min. Suitable aliquots of the supernatant were then applied to a gel column, 2.6 X lOOcm, housed in the cold and packed with Sephadex G-200 (Pharmacia). The column had been previously e q u i l i b r a t e d and then eluted with b u f f e r , Tris 50 mM, NaCl 130 mM, MgCI2 5 mM, at pH 7.4 and a flow rate of 24mi h- I c o l l e c t i n g 1.5ml f r a c t i o n s on an LKB ultrarac f r a c t i o n c o l l e c t o r . The fractions were assayed f o r the formation of ammonia from D-glutamine and y-glutamyltranspeptidase a c t i v i t y described above. Resul ts Two d i s t i n c t renal glutaminases are c l e a r l y demonstrable on the basis of subc e l l u l a r l o c a l i z a t i o n and substrate s p e c i f i c i t y (Table I ) . TABLE I.

SUBCELLULARLOCALIZATION OF TWO RENAL GLUTAMINASESt L-Glu'ase (PDG)

Succ' Cyto' c Reductase

Homogenate

95"(100)** ! 12

Mitochondrial

161(71) ± 19 22(8) 5

Post-Mitochondrial Supernatant

±

40(100)

D-GIn -NH3

~-GTP

23(100) ± 4

1,680(100) ± 309

119(70)

28(24) ± 6

1,980(22) ± 396

8(9)

32(66) ± 8

2,310(71) ± 492

t

Results from 6 rats.

*

nmoles min -1 mg protein -1, X ± S.D.

**

number in parenthesis represents percent of total fraction.

homogenate a c t i v i t y in each

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y-glutamyltransferase in Acidosis

!987

The L-glutaminase, assayed under PDG conditions (see Methods, glutaminase I f ) is localized predominantly, 71 percent, within the mitochondrial fraction. The mitochondrial marker, cytochrome c reductase, exhibits an identical pattern with only 8-I0 percent of the mitochondrial enzymes carried over into the post-mitochondrial supernatant, PMS. In contrast, the NH3 l i b e r a t i o n from D-glutamine is predominantly in the PMS, 66 percent, with only 24 percent in the mitochondrial fraction. This reflects an overlap of the a c t i v i t i e s in the mitochondrial fraction rather than u t i l i z a t i o n of D-glutamine by the stereospecific PDG (4). In fact, the D-glutamine u t i l i z i n g a c t i v i t y d i s t r i b u t i o n is s t r i k i n g l y similar to that of the brush border localized y-glutamyltranspeptidase. In terms of a c t i v i t y , this extramitochondrial ammonia l i b e r a t i n g a c t i v i t y exhibits considerable a c t i v i t y , being one-fourth as active, 23±4, as the extremely potent, 95±12nmoles min -I mg protein - l , PDG (this a c t i v i t y is assayed under the highly a r t i f i c i a l conditions, 150 mM P04 and pH 8.2 in contrast te the simple assay, pH 7.45, for the D-glutamine). Replacing D-glutamine with the L-isomer results in a near doubling, to 42±6nmoles, of the extramitochondrial glutamine u t i l i zing a c t i v i t y and the appearance of y-glutamylpeptides (autotranspeptidation); however, with D-glutamine the y-glutamyl moiety appears mainly as D-glutamate (hydrolysis) (Figure I ) .

A

FIGURE I.

S

C

Fate of y-glutamyl moiety in: A, PDG assay, C, with D-glutamine, and, B, when L-glutamine is substituted for the D-isomer. Homogenate was added to assay media I or media I I (PDG); aliquots of the reaction supernatant were then spotted along with authentic standards for glutamine 1 , glutamate 2 , and 3 y-glutamylglutamine.

The p u r i f i c a t i o n steps for the isolation of the extramitochondrial glutamine hydrolyzing a c t i v i t y are shown in Table 2.

D, L-

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y-glutamyltransferase in Acidosis

TABLE 2.

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PURIFICATION OF THE EXTRAMITOCHONDRIAL D-GLUTAMINEUTILIZING ACTIVITY

D-G1utamine Hydrolysis Total Activity

S.A.

Homogenate

2,640

Microsomes

T-Glutamyltranspeptidase

Yield

Total Activity

S.A.

Yield

22

I00

201,840

1,682

I00

1,720

115

65

149,120

9,940

73

Gel Filtration 1,250 (Sephadex G-200)

1,800

47

104,960

!49,940

52

Protein

Over 78 percent of the D-glutamine u t i l i z i n g a c t i v i t y was found in the microsomal fraction while the purified D enzyme, 82 fold, obtained after gel f i l t r a t i o n yielded 47 percent of that present in the starting homogenate. The y-glutamyltranspeptidase activity assayed as described in Methods, exhibited an identical purification, 90 fold, and yield, 52 percent. Furthermore, molecular weight estimation for both the purified D-glutamine u t i l i z i n g a c t i v i t y and ~-glutamyltranspeptidase by gel f i l t r a t i o n , Figure 2, were similar, 70 to 80,000 and close to that reported for the highly purified ~-glutamyltranspeptidase (14).

51

2.5 Ve

Vo 1.9

••B

SA

y , _ G T P / ~

INASE

1.5 104

105 MOLECULAR

FIGURE 2.

I06 WEIGHT

Estimation of the subcellular weight of D, L-glutaminase and y-glutamyltranspeptidase (y-GTP) by gel f i l t r a t i o n on a Sephadex G-200 column; standards are cytochrome c, MW= 12,000; BSA, MW= 68,000; and urease, MW= 482,000.

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y-glutamyltransferase

in Acidosis

1989

Rat kidneys perfused with 1 mM D-glutamine produced s i g n i f i c a n t amounts of ammonia and in metabolic acidosis the amounts almost double. The fact that this occurs with the D-isomer rules out the well known PDG adaptation ( I ) . I f this adaptation can be observed on the subcellular level, i t would confirm that this pathway produces ammonia under physiological, 1 mM glutamine, conditions. Additiona l l y , an acidosis induced adaptation in both the D-glutamine u t i l i z i n g a c t i v i t y and ¥-GTP would further serve to confirm t h e i r common i d e n t i t y . TABLE 3.

EFFECT OF METABOLIC ACIDOSIS ON D-GLUTAMINASE AND ~-GLUTAMYLTRANSPEPTIDASEACTIVITY Perfused Kidney

Kidney Homogenate

NA*

A

%tt

D-Glutamine (NH3)

16±4"*

29±5

(81)

y-Glutamyltranspeptidase

. . . . . . . .

NA 22±4 1,680±381

*

NA is nonacidotic; A is NH4CI, one week, metabolic acidosis

**

umole h"1 Kd- l

t

nmoles min -1 mg protein " l

~t

Percent A increase over NA

A

%tt

37±7

(68)

2,840±835

(69)

Table 3 shows that kidneys from chronically acidotic rats exhibit an increased a c t i v i t y of ammonia liberation from D-glutamine from 22 to 37nmole min-I (P<.05) and that this increase, 70 percent, is proportional to this pathway's in situ adaptation, 16 to 29umole h-I kd"I. Moreover, the y-glutamyltranspeptidase, assayed by y-glutamyl-pnitroanilide u t i l i z a t i o n exhibits a 70 percent rise supporting the common identity of the 2 a c t i v i t i e s . Discussion The purpose of this study was to demonstrate the existence of 2 glutamine u t i l izing pathways and the response of the extramitochondrial glutaminase to metabolic acidosis. The results clearly demonstrate a second pathway, located extramitochond r i a l l y (Table l ) , u t i l i z i n g both glutamine isomers (Figure l ) and exhibiting a molecular weight of approximately 70,000 (Figure 2). From the similar subcellular location, co-purification, molecular weights and adaptation to metabolic acidosis (Table 3), we conclude that this a c t i v i t y is identical to y-glutamyltranspeptidase-y-glutamyltransferase (EC 2.3.2.2). The a b i l i t y of hog kidney y-glutamyltranspeptidase to hydrolyze both isomers of glutamine as well as other y-glutamyl donors had previously been shown (15). Ammoniageneration by this reaction is surprisingly active, being 25 to 40 percent of the PDG, at pH 7.45 and in the absence of any activator, i . e . , maleate or y-glutamyl acceptor, which is consistent with this pathway contributing to in vivo renal ammoniagenesis (2, 3, 5). Furthermore, the acidosis induced adaptation of this pathway in both the functioning kidney preparation, l mM glutamine, and on the subcellular level (Table 3) indicates that this pathway plays an increased role in ammoniagenesis during aci!dosis (3,-5, 16). These findings are consistent with the previous studies suggesting that the yglutamyltranspeptidase catalyzed glutaminase may play a significant role in renal ammoniagenesis (17). However, intracellular glutathione is the preferred y-glutamyl

1990

7-glutamyltransferase in Acidosis

Vol, 26, No. 23, 1980

donor" for the ~-glutamyltranspeptidase since perfusing kidneys with an acceptor load, glycyiglycine, results in a f a l l in tissue glutathione levels (18). I f a portion of the enzyme faces the lumenal f l u i d , then i t would seem l i k e l y that glutamine, l mM, would compete favorably with glutathione, less than lO~M, as the y-glutamyl donor. I t seems l i k e l y , in t h i s instance, that the formation of ammonia is merely obligatory to the y-glutamyltransferase a c t i v i t y , i . e . , the formation of y-glutamylpeptides, and that these peptides may act as an economical reservoir of y-glutamyl moieties (18, 19).

Acknowledgements We wish to thank Loretta Bogan for her s k i l l f u l Lorene Rogers for her excellent secretarial service.

technical assistance and

References I. 2. 3. 4. 5. 6. 7. 8. 9. lO. If. 12. 13. 14. 15. 16. 17. 18. 19.

Baruch, S. in "The Kidney" (C. Rouiller and A. F. Muller, eds.), Vol. 3, p. 253, Academic Press, New York (1971). Welbourne, T. C. Med. Clin. North Am. 59, 629 (1975). Welbourne, T. C. Proc. Soc. Exp. Biol. Med. 152, 64 (1976). Sayre, R. W. and Roberts, E. J. Biol. Chem. 233, I128 (1958). Phenix, P. and Welbourne, T. C. Am. J. Physiol. 228, 1269 (1975). de Duve, C., Pressman, B. C., Gianetto, R., Watriaux, R. and Applemans, F. Biochem. J. 60, 604 (1955). Sottocassa, G. L., Kuylenstierna, B., Ernster, L. and Bergstrand, A. J. Cell. Biol. 32, 415 (1967). Orlowski, M. and Meister, A. Biochem. Biophys. Acta 73, 679 (1963). Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. J. Biol. Chem. 193, 265 (1951). Conway, E. J. Microdiffusion Analysis and Volumetric Error. Crosby and Lockwood, London, p. 287 (1950). Tabor, C. W. in "Methods in Enzymology" (S.P. Colowick and N. O. Kaplan, eds.), Vol. 17, p. 137, Academic Press, New York (1974). Diamond, A. J. and Weiner, M. W. Mt. Sinai J. Med. 45, 342 (1978). Bernt, E. and Bergmeyer, H. A. in "Methods in Enzymatic Analysis" (Bergmeyer, H., ed.), p. 1704, Academic Press, New York (1974). Tate, S. S. and Meister, A. J. Biol. Chem. 250, 4619 (1975). Orlowski, M. and Meister, A. J. Biol. Chem. 240, 338 (1965). McFarlane Anderson, N. and Alleyne, G. A. O. FEBS Lett. 79, 51 (1977). Kalra, J. and Brosnan, J. T. Can. J. Biochem. 52, 762 (1974). Welbourne, T. C. Can. J. Biochem. 57, 233 (1979). Samuels, S. J. Theor. Biol. 64, 729 (1977).