effect of methionine sulfoximine on glutathione and amino ...€¦ · mmol/kg), ms0 plus aspartate...

AMERICAN JOURNAL OF PHYSIOL~CY Vol. 231, No. 5, November 1976. Printed in U.S.A.

Effect of methionine sulfoximine on glutathione and amino acid levels in the nephron

JACQUELINE E. BREHE, ARTHUR W. K. CHAN, T. RANDALL ALVEY, AND HELEN B. BURCH Department of Pharmacology, Washington University Medical School, St. Louis, Missouri 63110

BREHE,JACQUELINE E., ARTHUR W.K. CHAN, T. RANDALL ALVEY, AND HELEN B. BURCH. Effect ofmethiorzine sulfoximine on glutkhione and amino acid levels in the nephron. Am. J. Physiol. 231(5): 1536-1540. 1976. -The effect of i-methionine-DL-sulfoximine (MSO) on renal glutathione concentration and aspartic acid transport has been studied by analyses of parts of individual freeze-dried glomeruli, early and late proximal convoluted, early and late proximal straight, and distal straight and convoluted tubules, and patches from thin- limb and papilla areas. Glutathione normally varies threefold along the kidney nephron, being highest in the convoluted and early straight proximal tubule, lowest in the distal straight tubule. Large loads of aspartate cause 20% diminution of glutathione in outer cortex, due entirely to changes in proximal tubule segments. MS0 alone lowers glutathione 90% in all parts of the proximal tubule, with no change elsewhere. MS0 does not affect the large increase in aspartate in proximal tubules caused by saturating aspartate loads, suggesting that glutathione is not directly involved in transport of this amino acid. Aspartate loads cause a large increase in renal glutamine, which is especially marked in the proximal straight tubule. MS0 effectively blocks this increase and depresses tissue glutamine below normal levels.

kidney; aspartate transport; glutamine; glutamate; nephron segments

THE SYNTHESISANDDEGRADATION Of glutathione via the Y-d utamyl cycle has been proposed by Meister (10, 11, 13, 14) as a general mechanism for amino acid transport across cell membranes. The validity of this proposal might be tested with an agent capable of blocking the cycle. (MS0 )

Such an agent is L- methionine- ,nL-sulfoximine , which can decrease glutathione synthesis and

glutathione content in liver and kidney (1’7, 19), pre- sumably by inhibition of y-glutamyl cysteine sy ,nthetase (19). This enzyme has been shown to be decreased in kidney after MS0 treatment (15).

After administration of a large load of aspartate, those segments of the renal tubule that are responsible for reabsorptive amino acid transport accumulate high concentrations of this amino acid (2). It was thought that the validity of the Meister theory, insofar as it applies to aspartate, might be tested by comparing the effects of MS0 on glutathione and aspartate concentrations in different parts of the nephron after giving a large load of aspartate. It also seemed possible that if the theory were correct, amino acid loads which exceed

renal transport capacity might in themselves decrease glutathione levels in transporting segments by seques- tering glutathione components at intermediate steps of the y-glutamyl cycle.

Modest decreases in glutathione in the proximal tubule were in fact observed after giving large loads of aspartate. However, the data to be presented also show that MS0 causes much more drastic decreases in glutathione of the proximal segments of the nephron and yet do not diminish the accumulation of aspartate from an aspartate load. In contrast, the accumulation in proximal tubules of glutamine, which normally accompanies aspartate accumulation, was blocked by MSO. (Gluta- mine synthetase is another enzyme inhibited by the sulfoximine.)

PROCEDURE

Aninkals

Male Sprague-Dawley rats (Zivic-Miller, Inc.), weighing 250-300 g, fed on Purina laboratory chow were used. They were injected with trypan blue (0.1 mg/g), 16-20 h before treatment, as a visual aid in distinguishing proximal convoluted tubules (12). L-Methionine sulfoximine was injected intraperitoneally 100 min prior to removal of kidneys. This was prior to the onset of convulsions, which at the dosage level used (2.5 mmol/kg) began at about 2 h. The amino acid loads were given on a schedule of combined intraperitoneal and subcutaneous injection which provided steady plasma levels for at least 10 min prior to kidney removal (2). When both MS0 and aspartate were given, inulin was also injected in order to permit direct comparison with data from animals in a previous study (2). Blood for plasma was obtained from the tip of the tail 1 min before kidney removal.

Kidney Samples

Rats were briefly anesthetized with ether and the kidneys removed and dropped within 1 s into Freon-12 (CCl,F,) chilled to its freezing point (-160°C) with liq- uid N,. The frozen tissue was stored at -60°C.

For preliminary studies, a cone of kidney was cut freehand at -2OOC into four major regions as described by Waldman and Burch (25). These were outer cortex, inner cortex plus outer stripe of medulla, inner stripe of medulla, and papilla. HClO, extracts of these regions were made at -20°C as described previously (2).

1536

by 10.220.33.5 on May 8, 2017

http://ajplegacy.physiology.org/D

ownloaded from

http://ajplegacy.physiology.org/

MS0 AND GLUTATHIONE AND AMINO ACIDS IN NEPHRONS 1537

The general technique for isolating and weighing small identified freeze-dried samples has been described in detail (8). The specific procedure for kidney and the identification of various parts of the nephron have also been described (2).

Materials.

Most of the enzymes and biochemicals, including L- methionine sulfoximine, were from Sigma Chemical Co., 5,5’-dithiobis(2nitrobenzoic acid) (Ellman reagent) was from Aldrich Chemical Co., 6-P-gluconate-dehydrogenase was from Boehringer Mannheim.

Analytical Methods

Glutathione analysis. The glutathione method will be described in detail elsewhere. It is based on the method of Tietze (23) and depends on the ability of glutathione and glutathione reductase (EC 1.6.4.2 yeast) to catalyze the oxidation of NADPH by Ellman’s reagent (5). The method does not distinguish between oxidized and reduced glutathione. For each mole of glutathione (as GSH), about 100 mol of NADP+ are formed. For larger samples (5-50 pmol) from extracts of gross kidney regions, the NADP+ was directly determined fluorometri- tally. (Excess NADPH was first destroyed with acid, after which the NADP+ was reduced back to NADPH with glucose-6-P plus glucose-6-P-dehydrogenase, and the fluorescence measured.) For smaller samples (0.03- 0.5 pmol) from individual nephron segments, initial steps were made in aqueous droplets in oil wells (9) and the sensitivity increased by enzymatic cycling.

Aspartate, glutamate, and glutamine measurement. The same procedures were used as described previously for aspartate (2), glutamate (2), and glutamine (3). In the case of glutamine, the procedure was modified to a) increase useful sensitivity (by reducing volumes), b) diminish loss of glutamine (by heating at a lower tem- perature in the first step), and c) insure complete removal of glutamate by including H,O, in the second step (8). Step 1. S amples are added to 0.1 ~1 of 5 mM HCl in an oil well and heated 20 min at 50°C. Step 2. 0.1 ~1 is added of 100 mM Tris-HCl, pH 8.4, containing 1 mM NAD+, 0.6 mM ADP, 200 pg/ml glutamic dehydrogenase (EC 1.4.1.3) and 0.06% H,O,. Step 3. After 30 min at 20-25”C, 0.05 ~1 is added of 5 pg/ml solution of catalase (EC 1.11.1.6) in 20 mM Tris-HCl, pH 8. Step 4. After 10 min at 20-25°C 0.1 ~1 is removed (for glutamate assay) and to the remainder is added 0.05 ~1 of 0.15 N HCl. Step 5. After a few minutes, 0.2 ~1 is added of 25 ,ug/ml glutaminase (EC 3.5.1.2, E. coli) in 100 mM Na acetate buffer, pH 4.9, containing 0.4 mM EDTA, and O.i% BSA. Step 6. After 30 min at 20-25°C 0.2 ~1 is added of glutamate reagent which contained 120 mM Tris-HCl buffer, pH 9.4, and all the other substances as in step 2 except lower NAD+, 0.2 mM, and H,O, was omitted. Step 7. After 30 min at 20-25°C 0.3 ~1 of 0.1 N NaOH is added. Step 8. After 30 min at 80°C 10 ~1 of cycling reagent are added (6) with 50 pg/ml of alcohol dehydrogenase and 5 pg/ml of malic dehydrogenase. Step 9. After 60 min at 20-25OC. 2 ul of 0.5 N NaOH are added.

Step 10. After 15 min at 95”C, 10 ~1 are transferred into a fluorometer tube containing 1 ml of indicator reagent (25 mM 2-amino-2-methyl-l-propanol-HCl buffer, pH 10.0, with 200 PM NAD+, 10 mM glutamate, and 5 cg/ ml of malic dehydrogenase, and 2 pg/ml of aspartate aminotransferase (EC 1.1.1.37, 2.6.1.1). Final reading is made after the reaction is complete (about 8 min). Glu- tamine standards (0.3-1.5 pmol) are incorporated in the first step reagent (HCl).

RESULTS

Glutathione Distribution in Kidney and Effect of MS0

Among the four gross subdivisions of normal kidney, glutathione is highest in the outer cortex and decreases progressively to about 40% of the maximum in the papilla (Fig. 1). Examination of different parts of single nephrons (Fig. 2) shows that this unequal distribution is due to the existence of peak glutathione levels in the proximal convoluted and early straight segments, intermediate levels in late proximal straight and distal convoluted segments, and low levels elsewhere. (Note that the data in Fig. 2 are on a dry weight basis.)

MS0 causes a large decrease in glutathione in outer cortex with successively smaller decreases in deeper gross zones, so that the resulting levels were about the same in all gross subdivisions (Fig. 1). This is seen (Fig. 2) to be due to a profound depression of glutathione in all parts of the proximal tubule, with little or no change elsewhere. The ratio between glutathione in the early part of the proximal straight tubule to that in the distal convoluted tubule falls from 1.4 on the controls to 0.1 after MSO.

GLUTATHIONE

l

ASPARTATE

plasma asp

IC IS PAP OC IC IS PAP-

FIG. 1. Effect of methionine sulfoximine and aspartate on glutathione and aspartate in gross regions of kidney. Animals were given 2.5 mmol/kg MS0 ip 100 min prior to kidney removal and 1 h prior to first dose of aspartate when this was given (see METHODS). Total aspartate load was either 2.7 or 4.5 mmol/kg (MS0 + Asp 2.7 or MS0 + Asp 4.5). Saline control received neither aspartate nor MSO. Regions of kidney are abbreviated: OC, outer cortex; IC, inner cortex and outer stripe of outer medulla; IS, inner stripe of outer medulla; and PAP, papilla or inner medulla. Points are average t SE (verti- cal bars) of 3-11 animals except for MS0 + Asp 2.7, which are averages of 2. Plasma aspartate concentrations (mM) at time of kidnev removal are shown.

by 10.220.33.5 on May 8, 2017


ownloaded from


I I 1 1 I I 1 J

G PCT, PCT, PST, PST, TL DST DCT PAP

FIG. 2. Effect of MS0 and aspartate on glutathione in different nephron segments. Animals were treated as described in procedure above. Animals received either MS0 (2.5 mmol/kg) or aspartate (4.5 mmol/kg), MS0 plus aspartate (2.5 and 4.5 mmol/kg, respectively), or saline on schedule given for Fig. 1. Averages + SE for 4 saline control or MSO-Asp-treated rats and for 3 MS0 or 2 Asp-treated animals are shown. For each rat, 3-8 samples (usually 4) of each segment type were analyzed. Abbreviations are: G, glomerulus; PCT,, proximal convoluted tubule near the glomerulus (stained dark blue); PCTL, proximal convoluted tubule, further from glomerulus (stained light blue); PSTE, proximal straight tubule in medul- lary ray; PSTL, proximal straight tubule in outer stripe of outer medulla; TL, an area of inner stripe of outer medulla (devoid of distal straight tubule tissue); DST, distal straight tubule in inner stripe of outer medulla; DCT, distal convoluted tubule, near glomerulus; PAP, area in inner medulla and Asp, aspartate.

Effect of Amino Acids, Glycylglycine, and Glucose on Renal Glutathione

Large loads of aspartate caused a modest but statisti- cally significant (P < 0.001) depression of glutathione in the outer cortex (Table 1). The same may be true for glycylglycine and glutamate, but not enough animals were studied to be sure. Palekar et al. (17) observed similar changes in whole kidney with glycylglycine and glutamate. They did not see any effect with a glutamine load of 1.67 mmol/kg. Even the higher loads used here (2 and 4 mmol/kg) do not cause appreciable decreases. The glutathione decrease after aspartate loads is shown . @‘lg tubul

2) to be due exclusively tocha .nges in the proximal .e, invol .ving particularly the convol uted portion.

Glucose loads large enough to exceed the tubular maximum did not cause significant changes in glutathione (Table 1).

Paradoxically, MS0 and a large aspartate load (each of which alone produced a fall in glutathione) when given in combination caused a somewhat smaller decrease in glutathione than MS0 alone (Figs. 1 and 2). Possibly this is due to the concomitant increase in glutamate which has been shown to be capable of reversing the MS0 inhibition of y-glutamyl cysteine synthetase (19) .

Kidney Aspartate and Glutamate Afier MS0 Treatment

MS0 had,essentially no effect on normal renal aspartate or on the aspartate increase after a large load of the amino acid (Figs. 1 and 3). The lack of effect on the

BREHE, CHAN, ALVEY, AND BURCH

proximal tubules after an aspartate load (Fig. 3) clearly indicates that reabsorptive aspartate transport has not been interfered with. This was confirmed by measure- ments based on urinary spillage of approximately threshold aspartate levels. This is also supported by the difference observed in gross subdivisions of kidney between tissue levels after subthreshold and above threshold aspartate loads (Fig. 1). A load above threshold (4.5 mmol/kg) causes accumulation of a great deal of aspartate in the papilla and inner stripe of the medulla due to unabsorbed aspartate in the concentrated tubular urine (2). A subthreshold aspartate load (2.7 mmol/kg) causes no change in inner stripe or papilla because the tubular urine is nearly free of aspartate (2). MS0 does not affect these results (Fig. 1).

As previously shown (2), an aspartate load causes large increases in glutamate in the proximal tubule. This is probably partly due to the large increase which occurs in plasma glutamate. (The kidney is presented with a large glutamate load which is filtered and reab- sorbed by proximal tubules where it accumulates.) Judging from changes in the outer and inner cortex, pretreatment with MS0 decreases this tubular accumulation, even though it does not have much effect on plasma glutamate (Fig. 4). MS0 also decreases gluta-

TABLE 1. Effect of various compounds on regional distribution of glutathione in rat kidney

Compound Load oc IC IS PAP

mmol lkg

Saline (11) 2.78 2.37 1.50 0.89 kO.07 kO.15 kO.05 20.07

Alanine 20 2.89 2.07 1.51 1.07 3.17 1.80 1.42 0.92

Alanine (3) 30 2.97 2.25 1.48 1.25 20.16 ~10.25 20.17 20.33

Aspartate* (3) 4.5 2.233‘ 2.39 1.91 0.80 20.06 20.17 1~0.25 kO.12

Glutamine 2 3.31 2.04 1.42 1.09 2.60 2.15 1.34 0.80

Glutamine 4 2.76 2.49 1.26 0.72 2.21 2.26 1.31 0.79

Glutamate* 4.5 2.22 2.77 2.10 0.76

Glycylglycine 4 2.33 2.23 1.10 0.63 1.85 2.14 1.21 0.76

Glucose* (3) 24 2.52 2.37 1.47 0.69 kO.06 to.16 kO.11 20.02

The amino acids and glucose were given in divided doses, the first by intraperitoneal injection and the subsequent ones by subcutaneous injection. Glycylglycine was given as a single intraperitoneal injection. (see PROCEDURE). The abbreviations are the same as for Fig. 1. The average for the number of rats in parentheses is shown. Standard errors are given for the controls and animals with glucose, aspartate, and alanine loads. * These analyses were done by an unpublished method of Marie Fleming and Oliver H. Lowry adapted to small volumes. It involves the oxidation of GSH in HClO, extracts by H202, the oxidation of NADPH by oxidized glutathione with the aid of glutathione reductase, and subsequent measurement of the strong alkali nroduct of NADP+ bv fluorometry. tP < 0.001.

by 10.220.33.5 on May 8, 2017


ownloaded from


MS0 AND GLUTATHIONE AND AMINO ACIDS IN NEPHRONS 1539

fi PSTE PSTL TL DST DCT PAP

FIG. 3. Effect of MS0 on aspartate in individual nephron segments. Animals injected with ,MSO are those described in Fig. 1. Those injected with aspartate alone and saline controls are from a previous study (2). Except for omission of MSO, they were treated same way. Abbreviations are as for Fig. 2.

mate in the absence of an aspartate load. Similarly, this probably cannot be explained by a lowering of plasma glutamate because the amount of glutamate filtered in this case is very low, with or without MSO. It is also improbable that this is due to an effect on glutamate transport, since aspartate and glutamate are well known to share the same transport mechanism. A more likely explanati .on is that in both cases decreased tissue glutamate is secondary to a profound and tissue glutamine (see following)

decrease in pl .asma

Distribution of Renal Glutamine and Effect of Aspartate and of MS0

An aspatate load causes large increases in glutamine in all four gross subdivisions of the kidney (Fig. 4B). The increase is especially marked in the proximal straight tubule (Fig. 5). This probably cannot be explained by the small increase in plasma glutamine (about 30%), but could be secondary to the increase in renal glutamate (see above and Fig. 4&. There is no increase in patches of tissue from the thin-limb area, and only modest increases occur in the distal tubule and papilla. MS0 not only prevents the rise in renal glutamine , but also depresses the tissu .e levels be low those seen normally in the absence of an amino acid load (Figs. 4B and 5). MS0 1 a so profoundly depresses plasma glutamine levels, with or without an aspartate load (Fig. 4B), but th is d oes not appear to be the cause of the tissue glutamine changes since tissue levels do not cor- relate with plasma levels. As pointed out, an aspartate load in normal animals causes a large increase in tissue glutamine without a comparable rise in plasma glutamine. Similarly, in the presence of MSO, aspartate causes, without essential change in plasma glutamine, a tissue glutamine increase which is large percentage- wise, although small in absolute terms. MS0 is known to inhibit glutamine synthetase (7, 16, 18, 20). It seems likely that MS0 is severely inhibiting this enzyme both in the liver (to account for the fall in plasma glutamine) and in the kidney (to explain the fall in tissue gluta-

mine). It has long been known that MS0 causes a profound decrease in brain glutamine (24).

DISCUSSION

There was found to be absolutely no diminution in aspartate accumulation in the transporting tubule cells when MS0 had reduced glutathione to extremely low levels (l&25% of normal, or 0.4-0.8 mmol/kg wet wt). Therefore, the data do not support any theory of aspartate transport which involves participation of glutathione. The same is true for glutamate, since there is ample evidence that aspartate and glutamate are transported by the same system. The data do not rule out a

GLUTAMATE

oc IC PAP OC PAP

FIG. 4. Effect of MS0 and aspartate on glutamate and glutamine in gross regions of kidney. Animals were given by intraperitoneal injection 2.5 mmol/kg L-methionine sulfoximine neutralized to pH 7. After 60 min either 2.7 or 4.5 mmollkg aspartate were injected in divided doses as described above in procedure. Data for rats given 4.5 mmol/kg Asp loads and saline control from previous experiments (2) are shown for comparison. Abbreviations are as in Fig. 1. Points are averages of regions from 3 or 4 rats 2 SE, except that only 2 animals given MS0 + 2.7 mmoltkg aspartate were analyzed. Plasma glutamate and glutamine concentrations (mM) at time of kidney removal are shown.

I T

FIG. 5. Glutamine in segments of nephron after MS0 and aspartate treatment. Animals and abbreviations are those of Fig. 2. Points represent averages with + SE of 4 rats, 4-8 samples for each segment of an individual animal, except that Asp 4.5 represents only3 ani- m

by 10.220.33.5 on May 8, 2017


ownloaded from


1540

glutathione theory for transport of other amino acids, and it is a fact that aspartate is one of the poorest acceptors for y-glutamyl transpeptidase, although this is not the case for glutamate (22).

If it were not for the clear-cut negative result of MS0 on aspartate accumulation, some support for a glutathione theory might be found in the modest decrease in glutathione produced by loads of aspartate and glycylglycine. Under the circumstances, it seems probable that this has some other cause.

The remarkable restriction to the proximal tubule of the MS0 depression of glutathione demands explanation. Two possibilities present themselves which are not mutually exclusive, and which might act in concert. Glutathione may turn over more rapidly in the proximal tubule than elsewhere, and MS0 may be actively transported into the proximal tubule cells and accumulate there, as is true for other amino acids. The’ fact that, in contrast, glutamine depression by MS0 is not limited to the proximal tubule suggests that selective accumulation of MS0 cannot be the whole explanation for the selectivity in regard to glutathione. An unu- sually rapid glutathione turnover in the proximal tubule would suggest an important function for a glutathione synthesis cycle, whether or not this is involved in amino acid transport. Sekura and Meister (21) have shown that glutathione turnover in whole kidney is 5 times faster than in liver.

REFERENCES

1. BURCH, H. B., A. W. K. CHAN, AND 0. H. LOWRY. Direct quanti- tation of amino acid transport and metabolism in segments of individual nephrons. In: Third Symposium on Biochemical As- pects of Kidney Function, edited by U. Schmidt and U. C. Du- bath. Bern: Huber. In press.

2. CHAN, A. W. K., H. B. BURCH, T. R. ALVEY, AND 0. H. LOWRY. A quantitative histochemical approach to renal transport. I. As- partate and glutamate. Am. J. PhysioZ. 229: 1034-1044, 1975.

3. CURTHOYS, N. P., AND 0. H. LOWRY. Glutamate and glutamine distribution in the rat nephron in acidosis and alkalosis. Am. J. Physiol. 224: 884-889, 1973.

4. CURTHOYS, N. P., AND 0. H. LOWRY. The distribution of glutaminase isoenzymes in the various structures of the nephron in normal, acidotic, and alkalotic rat kidney. J. BioZ. Chem. 248: 162-168, 1973.

5. ELLMAN, G. L. Tissue sulfhydryl groups. Arch. Biochem. Bio- phys. 82: 70-77, 1959.

6. KATO, T., S. J. BERGER, J. A. CARTER, AND 0. H. LOWRY. An enzymatic cycling method for nicotinamide-adenine dinucleotide with malic and alcohol dehydrogenases. AnaZ. Biochem. 53: 86- 97, 1973.

7. LARMAR, C., JR., AND 0. 2. SELLINGER. The inhibition in vivo of cerebral glutamine synthetase and glutamine transferase by the convulsant methionine sulfoximine. Biochem; PharmacoZ. 14: 489-506, 1965.

8. LOWRY, 0. H., AND J. V. PASSONNEAU. A FZexibZe System of Enzymatic Analysis. New York: Academic, 1972, p. 229-260.

9. MATSCHINSKY, F. M., J. V. PASSONNEAU, AND 0. H. LOWRY. Quantitative histochemical analysis of glycolytic intermediates and cofactors with an oil well technique. J. Histochem. Cyto- them. 16: 29-39, 1968.

10. MEISTER, A. On the enzymology of amino acid transport. Science 180: 33-39, 1973.

11. MEISTER, A. Glutathione; metabolism and function via the y- glutamyl cycle. Life Sci. 15: 177-190, 1974.

12. OLIVER, H. New directions in renal morphology: a method, its results and its future. Harvey Lectures 40: 102-155, 1944-1945.

13. ORLOWSKI, M., AND A. MEISTER. The y-glutamyl cycle: a possible transport system for amino acids. Proc. NatZ. Acad. Sci. US 67:

BREHE, CHAN, ALVEY, AND BURCH

As shown, glutamine is increased three- to fivefold in the various segments of the proximal straight tubule after a large aspartate load. The same is true for large glutamate loads (data not shown) (1). A noteworthy feature of this phenomenon is that peak levels in proximal straight segments are 4 or 5 times higher than in the immediately preceding late segment or the proximal convoluted tubule, whereas aspartate (as well as glutamate (2) rises at least as much in the late convoluted as in the straight segments (compare Fig. 2 with Fig. 5). It may be presumed that in the proximal straight tubule either glutamine synthesis is especially rapid or degra- dation is especially slow. Compatible with slow degra- dation is the fact that phosphate-dependent glutaminase is exceedingly low in the straight segment (4). On the other hand, the fact that poisoning with MS0 flat- tens out the glutamine distribution curve after an aspartate load (Fig. 5) suggests that the normal differen- tial in glutamine concentration is due to glutamine synthetase. Differentials in glutamine concentrations among proximal segments exist, even in the absence of aspartate loads, although in this case the absolute levels are much lower (Fig. 5).

This work was supported by Public Health Service Grants HD- 03891A and NS-05221 and American Cancer Society Grant BC-4R.

Received for publication 12 February 1976.

14. ORLOWSKI, M., AND A. MEISTER. y-glutamyl cyclotransferase. J. BioZ. Chem. 248: 2836-2844, 1973.

15. ORLOWSKI, M., AND S. WILK. In vivo inhibition of y-glutamyl cysteine synthetase by L-methionine-sulfoximine; influence on intermediates of y-glutamyl cycle. J. Neurochem. 25: 601-606, 1975.

16. PACE, J., AND E. E. MCDERMOTT. Methionine sulphoximine and some enzyme systems involving glutamine. Nature 169: 415-416, 1952.

17. PALEKAR, A. G., S. S. TATE, AND A. MEISTER. Decrease in glutathione levels of kidney and liver after injection of methionine sulfoximine into rats. Biochem. Biophys. Res. Commun. 62: 651- 657, 1975.

18. RAO, S. L. N., AND A. MEISTER. In vivo formation of methionine sulfoximine phosphate, a protein-bound metabolite of methionine sulfoximine. Biochemistry 11: 1123-1127, 1972.

19. RICHMAN, P. G., M. ORLOWSKI, AND A. MEISTER. Inhibition of y- glutamylcysteine synthetase by L-methionine-S-sulfoximine. J. BioZ. Chem. 248: 6684-6690, 1973.

20. ROWE, W. B., AND A. MEISTER. Identification of L-methionine-S- sulfoximine as the convulsant isomer of methionine sulfoximine. Proc. NatZ. Acad. Sci. US 66: 500-506, 1970.

21. SEKURA, R., AND A. MEISTER. Glutathione turnover in the kidney; considerations relating to the y-glutamyl cycle and the transport of amino ‘acids. Proc. NatZ. Acad. Sci. US 71: 2969- 2972, 1974.

22. TATE, S. S., AND A. MEISTER. Interaction of y-glutamyl transpeptidase with amino acids, dipeptides, and derivatives and ana- logues of glutathione. J. BioZ. Chem. 249: 7593-7602, 1974.

23. TIETZE, F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione. AnaZ. Bio- them. 27: 502-522, 1969.

24. TOWER, D. B. The neurochemistry of asparagine and glutamine. In: The Neurochemistry of NucZeotides and Amino Acids, edited by R. 0. Brady and D. B. Tower. New York: Wiley, 1960, p. 173- 204.

25. WALDMAN, R. H., AND H. B. BURCH. Rapid method for study of enzyme distribution in rat kidney. Am. J. Physiol. 204: 749-752, 1963.

1248-1255, 1970.

by 10.220.33.5 on May 8, 2017


ownloaded from


effect of methionine sulfoximine on glutathione and amino ...€¦ · mmol/kg), ms0 plus aspartate...

Documents