studies on specific enzyme inhibitors · studies on specific enzyme inhibitors viii....

THE JOURNAL OR BIOLOGICAL CHEMISTRY Vol. 239, No. 9, September 1964

Printed in U.S.A.

Studies on Specific Enzyme Inhibitors

VIII. ENZYME-REGULATORY MECHANISMS OF THE ENTRY OF GLUTAMIC ACID INTO METABOLIC PATHWAYS IN KIDNEY TISSUE*

E. KuN,t J. E. AYLING, AND B. G. BALTIMORE

From the Departments of Pharmacology and Biochemistry and the Cardiovascular Research Institute, University of California School of Medicine, San Francisco 24, California

(Received for publication, February 3, 1964)

A kinetic problem of metabolic regulation is exemplified in its elementary form when several enzymes known to be present simultaneously in a biochemical system are exposed to one common substrate. This experimental model appears to be suitable for the testing of predictions based on results of kinetic studies with each isolated enzyme. In the simplest case, e.g. in a purely random system, the sum of kinetic parameters will decide the distribution of the common substrate in various metabolic direc- tions as determined by the nature of the enzymes present. It may be anticipated, however, that in enzyme systems present in biological material, besides primary kinetic and thermodynamic determinants characteristic for each particular enzymatic step, there are more complex regulatory mechanisms that may result in an obligatory coupling or sequence of individual enzyme reactions.

Discovery of specific regulatory mechanisms that modify single enzymatic reactions into physiological processes is difficult, owing to the complexity of such systems. Qualitative and quantitative analyses of enzyme contents of animal tissues are only a first approximation of the metabolic potential of each tissue. There is no a p+-iori reason to believe that all enzymes ex- tractable from all types of cells are fully active at all times when present in their physiological environment, i.e. in the intact cell. The influence of restrictive, i.e. rate-limiting, forces, particularly through reversals of energy-conserving reactions (e.g. oxidative phosphorylations) is not immediately obvious from kinetics and equilibria of individual enzymatic reactions and must be studied in complex systems where these mechanisms operate. Conventional experimental procedures, such as balance studies (i.e. analyses of steady state concentrations of substrates and products), may often not distinguish between alternate enzymatic pathways and ordered sequences, since identical distribution of reactants may occur by either mechanism, as shown in this paper.

We have adopted a systematic experimental approach that should distinguish between these possibilities. By developing specific inhibitors (l-8), we can selectively influence individual enzymes in multienzymatic systems. Application of these inhibitors should enable us to solve problems of multienzyme systems in a unique fashion. Since inhibitors of several enzymes

* Supported by Grants C-4681 and C-3211 from the United States Public Health Service and by National Science Foundation Grant G23739.

t Recipient of the Research Career Award of the United States Public Health Service.

reacting with glutamic acid have become available (see Table I), we are pursuing the problem of metabolic control of glutamate utilization in various tissue preparations. A preliminary report on this work has already appeared (8). The present paper is concerned with the regulation of glutamate metabolism by rat kidney tissue. It deals primarily with the influence of specific inhibitors on the over-all utilization of this substrate, as measured by O2 consumption and amino acid balance. As a consequence of these studies the mechanism of obligatory coupling of glutamic dehydrogenase with transaminases was recognized.

EXPERIMENTAL PROCEDURES

Enzyme Inhibitors-Mono- and difluoro-oxaloacetic acids and ol-monofluoroglutaric acid were synthesized as described earlier (1, 5, 7). Amino-oxyacetic acid was a gift of Dr. D. P. Wallach from the Research Laboratories of Upjohn Company, Kalama- zoo, Michigan.’

Nucleotides-Pyridine and adenine nucleotides were obtained from either Pabst Laboratory (Milwaukee, Wisconsin) or Sigma Chemical Company (St. Louis, Missouri). Their purities were checked by high voltage paper electrophoresis (see below). All other reagents were of analytical purity.

Tissue Preparation-Male Long-Evans rats, 110 to 200 g, were used in all experiments. After exsanguination, the kidneys were immediately removed and immersed in 0.25 M sucrose solution (pH 7.4 containing 0.05% EDTA) that had been chilled previously to O-4” in an ice bath. The kidney cortices were homoge- nized in 0.25 M sucrose-Versene (at 0”) in a Potter-Elvehjem homogenizer fitted with a Teflon plunger. Mitochondria were prepared according to Schneider and Hogeboom (9). Suspend- ing medium for homogenates was 0.15 M KC1 adjusted to pH 7.4.

Analytical Procedures-Protein was determined by a modified biuret procedure (10). Amino acids were analyzed after precipi- tation of protein with perchloric acid at 0” (final concentration, 3%). Perchlorate ions were removed by addition of equivalent amounts of solid KHCOa, and the supernatant fluids were kept frozen at - 15” until the time of analysis. Aliquots of 20 ~1 were analyzed by paper electrophoresis (Pherograph apparatus, Brinkman Company, Menlo Park, California) in sodium formate buffer, pH 3.6 (I’/2 = 0.05; current density, 50 ma; potential gradient, 33 volts per cm) for 1.5 hours at 0”. The amino acids were developed with cadmium-ninhydrin (11) , extracted with methanol, and measured spectrophotometrically (12). Adenine

1 We are greatly indebted to Dr. D. P. Wallach for this courtesy.

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September 1964

Inhibitor

Difluoro-oxaloacetate

Monofluoro-oxaloacetate

cu-Fluoroglutarate

Amino-oxyacetic acid

E. Kun, J. E. Ayling, and B. G. Baltimore

TABLE I

Summary of prc

Enzyme

Glutamic-aspartic transaminase

Malate dehydrogenase

Glutamic-aspartic transaminase

Glutamate dehydrogenase

Glutamate-alanine transaminase

erties of inhibitors

Substrate

Aspartate a-Ketoglutarate

Malate Oxaloacetate Fz-oxaloacetate* Aspartate a-Ketoglutarate

Glutamate

Alanine a-Ketoglutarate

Type of inhibition

Noncompetitive Competitive

Competitive Competitive Noncompetitive Noncompetitive Competitive

Competitive

Competitive Uncompetitive

.- 3.3 x 10-d 2.4 X lo+

7.6 X 10-e 2.8 x 10-S 3.3 x 10-S 6.9 X 10-5

1.6 X 10-S

3.3 x 10-d

6 X 10-S

-

I .-

-

2897

<eference

5 5

1 1 5

:

7

13

* Fz-oxaloacetate is difluoro-oxaloacetate. t Assay conditions were the same as described earlier (2,5). Soluble mitochondrial extracts, used as sources of transaminase, were

prepared by sonic disruption of mitochondria followed by removal of particles by centrifugation (105,000 X g for 60 minutes), as pub- lished previously (4, 6).

nucleotides were separated by electrophoresis in citrate buffer of pH 6.2 (I’/2 = O.l), eluted, and spectrophotometrically analyzed.

Kinetic Measurements-Rates of O2 consumption were used as an over-all measure of the sum total of a multiplicity of enzymatic reactions in the presence and the absence of externally added glutamate and inhibitors. Two distinct types of rate measurements were routinely employed: (a) conventional manometry, a system simulating pseudosteady state conditions; and (b) polarography for initial rate studies. The latter method (cf. Reference 6) was improved by the use of a reaction chamber (2-ml capacity) equipped with an internal magnetic stirrer.

RESULTS

I. Manometric Experiments with Kidney Mitochondria

A. Apparent Ineffectiveness of Transaminase Inhibitors- Mono- and difluoro-oxaloacetates are inhibitors of glutamate-aspartate transaminase (2, 5). In long term experiments mono- and difluoro-oxaloacetates were used simultaneously, since in complex enzyme systems they have a stabilizing effect upon each other (5).

It was found in preliminary experiments that these inhibitors in the presence of glutamate and 2.3 X 10e3 M ATP did not influence the over-all rate of 02 uptake of kidney mitochondria except for rapidly vanishing inhibition, which was detectable in the first 5 minutes of manometric readings. Similar observations were made with amino-oxyacetate, whereas the inhibitory effect of fluoroglutarate was influenced by ATP to a lesser extent (see Section C). The following experimental facts argue against the possibility that permeability barriers are the cause of this apparent ineffectiveness. (a) Under appropriate conditions instant inhibitory effects can be observed. (b) Direct spectro- photometric measurements have previously shown that fluoro- oxaloacetates cause an instantaneous reoxidation of intramitochondrial reduced pyridine nucleotides (5). Since both keto acids are substrates of intramitochondrial malate dehydrogenase (1,5), these reactions demonstrate that no measurable permeability barriers exist in mitochondria against these inhibitors. (c) Glutamate consumption and aspartate formation were dimin-

ished by inhibitors that had only small effect on O2 uptake. (d) In recent experiments (14) using 32P turnover as an indicator of the metabolism in vitro of excised hemidiaphragms of rats, we found that these inhibitors readily penetrate even muscle tissues.

There is at present no reason to believe that enzymatic reactions exist, beyond the ones already determined (1,2,5), capable of inactivation of these inhibitors. The apparent ineffectiveness of transaminase inhibitors in intact kidney mitochondria under given experimental conditions must therefore indicate that in this system glutamate-aspartate and alanine transaminases do not play a rate-limiting role in glutamate metabolism, as measured by over-all consumption of 02.

B. Experimental Conditions that Influence Effectiveness of Inhibitors-It was subsequently recognized that the major factor that determined the apparent effectiveness of inhibitors on O2 uptake was the initial concentration of ATP in the mitochondrial reaction system. Two extreme conditions were henceforth defined experimentally. “High ATP” conditions were created by 2.3 X 10m3 M initial concentration of added nucleotide. This concentration of ATP was intended to simulate intracellular physiological conditions. “ATP depletion” was obtained artifi- cially by incubating mitochondria in the presence of excess amounts of yeast hexokinase and glucose. The added nucleotide in this case was 2.3 x 10m3 M ADP. ATP levels were kept below 10m5 M in this “ATP-depleted” system. Oxygen uptake of mitochondria, with glutamate or glutamine as substrate, was stimulated 50% by hexokinase and glucose ( +ADP) above the rates obtained in the presence of added ATP alone. The exact reason for this relatively small stimulation of O2 uptake by the added phosphate acceptor system is not known. It may be that under given experimental conditions (in the presence of added ATP) glutamine synthesis results in a sufficiently rapid formation of ADP to cause some stimulation of respiration even in the absence of an added phosphate acceptor system. Furthermore, kidney tissue contains K+- and Na+-activated ATPase (15,16), and it may be assumed that in a system containing relatively high concentrations of K+ (see the legend to Table II) activation of this enzyme could influence the respiratory control mechanism. Under “initial rate” conditions, respira-


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2898 Studies on SpeciJic Enzyme Inhibitors. VIII Vol. 239, No. 9

TABLE II

Effects of inhibitors on kidney mitochondria. I

Each respirometer flask contained kidney mitochondria (10 mg of protein in 1 ml of 0.25 M sucrose + 0.05% Versene), 1OF M

glutamate, 3 X lo+ M phosphate (pH 7.4), 10e3 M Mg++, and 0.15 M KC1 to a volume of 3.0 ml. CO2 was absorbed by KOH. The gas phase was air, and the substrate was glutamate. In experiments designated “high ATP,” 2.3 X 10-a M ATP was added. In those designated “ATP-depleted,” 2.3 X 10-3 M ADP + 0.03 M glucose + crystalline yeast hexokinase (0.21 mg of protein) was added. The inhibitors used were: 1, mono- and difluoro- oxaloacetates, 2 X lo+ M each; 2, a-fluoroglutarate, 5 X 10-a M;

and 3, amino-oxyacetate, 5 X 10M6 M. -02 means 02 consumed; -Glu, glutamate consumed; +Asp, aspartate formed; and Glu- NH2, glutamine.

subsequently can undergo transamination with a keto acid to form ol-ketoglutaramate, followed by deamidation to cy-ketoglutarate and NH4+ (18). As shown earlier (5, 7), the inhibitors do not influence glutamine synthesis. The glutamine-specific transaminase is also insensitive to fluoro-oxaloacetates and amino-oxyacetate. A necessary prerequisite for the operation of the “glutamine oxidation” pathway in kidney mitochondria is provided by the experimental results, which show that 02 consumption is augmented to the same extent by both glutamate and glutamine in this system. This is not the case in liver mitochondria. It is

predictable that at a critical low level of ATP, glutamine synthetase will no longer operate efficiently and enzymatic pathways utilizing glutamate will become predominant. Speck (19) and Elliott (20) found that in brain, glutamine synthetase was inhibited 50% when the ATP:ADP ratio was 0.3. A specific regulatory role of ADP is suggested by the fact that this nucleotide is an inhibitor of glutamine synthetase (17).

C. Effects of Inhibitors under Predetermined Experimental Con- ditionsPredictions based on considerations discussed under Sections A and B were tested experimentally. Respiration and amino acid balance were measured with both glutamate and glutamine as substrates under both “high ATP” and “ATP- depleted” conditions. Results are summarized in Tables II and III. In the absence of inhibitors, glutamate was converted almost quantitatively to aspartate. Small amounts of alanine and glutamine were also present (about 0.1 PM). Five- to ten-fold more glutamine was found in “high ATP” conditions than in the “ATP-depleted” system.

In the presence of glutamate, fluoro-oxaloacetates had virtually no effect on 02 uptake under “high ATP” conditions (10% inhibition), but- they lowered glutamate consumption and aspartate formation (48 to 50 y. inhibition), changing the ratio of 02 to aspartate from 1.5 to approximately 3.0. Under “ATP-depleted” conditions, both O2 uptake and glutamate consumption were inhibited to about the same extent (35 to 400/,), an effect predictable from known inhibitory properties of fluoro-oxaloacetates on glutamate-aspartate transaminase (5).

Fluoroglutarate, a specific inhibitor of glutamic dehydrogenase (7), diminished O2 uptake and glutamate consumption under all experimental conditions. In the presence of added ATP (Table II), the degrees of inhibition of O2 uptake and glutamate-aspartate conversion were nearly the same (31 and 26%), whereas in “ATP-depleted” systems, O2 consumption was more inhibited (51 To) than aspartate accumulation (37 %). The ratio of 02 to aspartate was 1.5 in the “high ATP” system and 1.1 in the “ATP-depleted” system.

Amino-oxyacetate is a powerful inhibitor of glutamate-alanine transaminase (13). In concentrations up to 5 X low4 M it has no effect on glutamate-aspartate transaminase or on the oxidation of glutamate by glutamic dehydrogenase. In kidney mitochondria, in the presence of glutamate, this inhibitor had only small influence on O2 uptake (17% inhibition), but it markedly depressed aspartate formation (58 %) under “high ATP” conditions. This effect was increased under “ATP depletion,” where inhibition of 0% uptake was 39% and that of aspartate formation 76%. The influence of an inhibitor of glutamate-alanine transaminase on the over-all metabolism of glutamate is of major significance and, together with other evidence, led us to a revision of currently accepted views on glutamate metabolism.

The effects of inhibitors in the presence of glutamine as substrate are summarized in Table III. Fluoro-oxaloacetates and

T “High ATP” I “ATP-depleted”

-02 -Glu I - I I I +ASP a y& -0 a -Gh

pmoles/mg $5rotein/30 min

1.34 0.96 0.86 0.12 1.9 1.22 1.21 0.56 0.46 0.13 1.25 0.77 0.92 0.73 0.63 0.12 0.93 0.78 1.11 0.47 0.37 0.13 1.07 0.28 0.61 0.33 0.28 0.08 0.42 0.34 1.03 0.35 0.29 0.09 0.97 0.37 0.39 0.28 0.23 0.08 0.37 0.54 0.47 0.37 0.29 0.10 0.33 0.63

Inhibitors - I +A~P 2 EY

2- __

- -

None 1 2 3 1+2 1+3 2+3 l+2+3

1.22 0.04 0.77 0.04 0.78 0.04 0.30 0.01 0.37 0.01 0.39 0.01 0.54 0.04 0.63 0.04

TABLE III

E$ects of inhibitors on kidney mitochondria. II

Experimental conditions are the same as described in the legend of Table II, except that glutamine (1O-2 M) was the substrate added.

Inhibitors

“High ATP” I

‘ ‘ATP-depleted” - -if:- * I -- - 1.01 0.95 0.90 1.12 0.87 0.97 0.79 0.83

-

+Giu~+Asp~ -0x /kz-

pmle/mg protein/30 n&in

- I - +Glu +Asp

0.42 0.61 2.10 0.42 0.55 1.75 0.38 0.55 1.10 0.60 0.55 1.70 0.35 0.54 0.49 0.52 0.47 1.66 0.54 0.28 0.54 0.48 0.38 0.36

1.53 1.35 1.29 1.40 1.18 1.35 1.26 1.20

-

0.46 1.10 0.61 0.78 0.60 0.73 0.86 0.58 0.80 0.42 0.83 0.56 0.82 0.48 0.87 0.37

itochc 3n dria was I ;ti muls tte d by ADP 3- to

None 1.31 1 1.30 2 0.93 3 1.40 1+2 0.75

1+3 1.28

2+3 0.50 l+2+3 0.58

tion of kidney I

5-fold above the rate measured in the presence of ATP; therefore the low respiratory control seems to be a peculiarity of the manometric system. In manometric experiments, in the presence of glutamate, after 30-minutes incubation, about two-thirds of the added ATP could still be recovered, indicating that the equilibrium concentration of ATP in such systems was close to 1.5 X lo-3 M.

The marked influence of ATP levels on the effectiveness of transaminase inhibitors of glutamate oxidation points to an alter- native glutamate pathway controlled by adenine nucleotides. This alternate pathway is provided by the enzymatic amidation of glutamate in the presence of ATP and NH4+ (17). Glutamine


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September 1964 E. Kun, J. E. Ayling, and B. G. Baltimore

amino-oxyacetate had no effects on glutamine oxidation under “high ATP” conditions. Under “ATP-depleted” conditions, these inhibitors caused some reduction of O2 consumption, and amino-oxyacetate in addition markedly diminished aspartate formation.

Under “high ATP” conditions, with glutamine as substrate, fluoroglutarate inhibited only 02 uptake (29%). With glutamate as substrate (Table II), under conditions favoring glutamine synthesis, the inhibitory effect of fluoroglutarate can be readily attributed to an inhibition of glutamate dehydrogenase (7), which is necessary for the generation of NH4+ for glutamine synthesis and also plays a critical role in the metabolism of glutamate itself. It is not clear, however, whether the inhibitory effect of fluoroglutarate on glutamine oxidation under “high ATP” conditions is due to an inhibition of glutamate dehydrogenase or whether it has an additional inhibitory effect on an enzyme participating in the glutamine pathway (Mechanism 1). Interpretation of effects of fluoroglutarate on glutamate metabolism in complex systems would not be overcomplicated by such an additional effect, since this predictably occurs only under conditions in which glutamine synthesis becomes rate-limiting for glutamate utilization (under “high ATP” conditions). The inhibition pattern obtained with glutamine as substrate under “ATP-depleted” conditions reflects an increased deamidation of glutamine.

From the stoichiometric relationship observed with glutamine as substrate, under “high ATP” conditions a metabolic pathway of glutamate, defined as “glutamine shunt,” can be formulated (see Fig. 5). This pathway involves the participation of glutamine-specific transaminase, deamidation of cY-ketoglutaramate, and oxidation of a-ketoglutarate to oxaloacetate and pyruvate. This mechanism (shown below) is based on the following experimental results: ratio of glutamate to aspartate = 0.7, of 02 to aspartate = 2.0, of CO2 t0 02 = 0.84.

2 Glutamine + 2 glutamate + 2 NH,+

3 Glutamine + 3 oxaloacetate +

Cl)2

(2) 3 aspartate + 3 a-ketoglutaramate

I Glutamine + pyruvate + alanine + a-ketoglutaramate (3)

3 a-Ketoglutaramate + 4.5 02 ---t (4)

3 oxaloacetate + 3 CO, + 3 NH*+

1 a-Ketoglutaramate + 1.5 O2 + pyruvate + 2 CO2 + NHd+ (5) Sum: 6 Glutamine + 6 02 +

2 glutamate + 3 aspartate + 1 alanine + 5 CO2 + 6 NH4+

Mechanism 1

Combined effects of inhibitors on mitochondrial glutamate and glutamine metabolism were also tested. Each inhibitor was present at a concentration previously employed. With glutamate or glutamine as substrate under “ATP-depleted” conditions, approximately additive effects were observed when transaminase inhibitors were combined with fluoroglutarate. Less than additive effects were found when only transaminase inhibitors were present. It is of interest to note that amino-oxyacetate, which by iteslf has little or no effect on glutamate or glutamine oxidation under “high ATP” conditions, markedly

2 Reaction 1 may be a direct deamidation, or a cyclic transamination with a-ketoglutarate.

TABLE IV

E.#‘ect of inhibitors and DPN+ on glutamate metabolism of kidney homogenates

Each Warburg respirometer flask contained 1 ml of kidney homogenate (19 mg of protein) prepared in 0.15 M KCl, ADP (2.3 X 10e3 M), Mg++ (1 X 10-s M), phosphate (1.3 X low3 M, pH 7.4), and glutamate (1 X 10m2 M). DPN+ was added to alternate flasks at a concentration of 1 X 10-S M. The final volume of 3.0 ml was made up with 0.15 M KCI. CO2 was absorbed by KOH. The temperature was 30”, and the time of incubation, 30 minutes. Results are expressed as micromoles of 02 and glutamate consumed, and aspartate and glutamine formed per mg protein per 30-minute experiment. Inhibitors 1 and 2 are as defined in the legend to Table II; 3, here, is 1 X 10-b M amino-oxyacetic acid.

-02 -Glu = +Asp Glutamine Inhibitor

-DPN I

+DPN I I

-DPN +DPN 1 I

-DPN +DPN

!mmles/mg protein/30 min

None 1.07 1.09 0.83 0.77 0.16 0.16 1 0.59 0.86 0.45 0.58 0.17 0.18 2 0.73 0.75 0.58 0.56 0.15 0.15 3 0.77 1.00 0.51 0.65 0.13 0.16 1+2 0.25 0.49 0.36 0.43 0.16 0.19 1+3 0.48 0.85 0.40 0.54 0.16 0.15 2+3 0.49 0.70 0.52 0.54 0.16 0.17 1+2+3 0.23 0.50 0.34 0.40 0.17 0.14

increases the inhibitory effect of fluoroglutarate. This finding suggests that glutamine and a-ketoglutaramate may also react with glutamate-alanine transaminase under conditions in which the glutamine-specific pathway is inhibited (by fluoroglutarate).

II. Manometric Experiments with Kidney Homogenates

The metabolism of glutamate by fresh kidney homogenates in the presence of 2.3 x low3 M ATP was depressed by all inhibitors studied. Analyses for adenine nucleotides at the end of incubation revealed that the concentration of ATP had dropped below 10m6 M, probably owing to the action of Na+- and Ktactivated ATPase (15, 16). It is therefore expected that in such a system glutamate is metabolized by the same pathway as that observed in mitochondria when it is preincubated with glucose and hexokinase.

Since it is believed that in tissue homogenates endogenous pyridine nucleotide levels tend to decrease owing to the activity of degrading enzymes, the influence of added DPNf on endogenous respiration and glutamate oxidation was also determined in the absence and the presence of inhibitors. Externally added DPN+ greatly stimulated *endogenous respiration but had no influence on the rate of 02 uptake in the presence of glutamate as substrate (Tables IV and V). This observation indicated that in the presence of added glutamate no rate-limiting deficiency in DPNf occurs.

A striking influence of DPN+ was observed on the inhibitory effects of fluoro-oxaloacetates. Inhibition caused by fluoro- oxaloacetates was largely counteracted when DPN+ was added. The fact that DPN+ almost abolishes the depression of endogenous respiration caused by fluoro-oxaloacetates points to a mechanism that already operates at the “endogenous substrate” level. The time course of this effect is shown in Fig. 1. The depression of endogenous respiration by amino-oxyacetate is also countered by DPN+. This reversal of inhibition occurs also


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2900 Studies on Specijic Enzyme Inhibitors. VIII Vol. 239, No. 9

TABLE V

Efects of inhibitors and DPN+ on endogenous respiration of kidney homogenates

The conditions of this experiment were the same as those described in the legend of Table IV, except that no glutamate was added.

None 0.29 0.46 0.037 0.031 0.110 0.089 0.089 0.121 1 0.14 0.33 0.047 0.037 0.042 0.079 0.105 0.105 2 0.23 0.22 0.047 0.047 0.074 0.068 0.116 0.126 3 0.20 0.4 0.052 0.026 0.089 0.105 0.100 0.100

500 -

400 -

300 -

ON

5

200 -

p?de/mg )rotein/30 min

-

0 IO 20 30

MINUTES

I FIG. 1. Experimental conditions are the same as described in the legend of Table IV. F, + Fz, mono- and difluoro-oxaloacetates, each at 2 X 10m4 M.

in the presence of added glutamate. On the other hand, the inhibitory effect of fluoroglutarate on both endogenous respiration and glutamate oxidation was unaltered by added DPNf. Externally added DPN+ did not reverse the inhibitory effect of transaminase inhibitors on the respiration of isolated mitochondria metabolizing glutamate under “ATP-depleted” conditions. This is in contrast to results obtained with homogenates. It could be ascertained in preliminary experiments that addition of a crude cytoplasmic fraction to mitochondria resulted in a reap- pearance of the “DPN+ effect,” as manifested by a reversal of inhibitory effects of transaminase inhibitors. A detailed analysis of the contribution of cytoplasmic components to mitochondrial respiration will be presented elsewhere.

Since it has been shown that there is no deficiency of DPN+, the apparent reversal of inhibition of glutamate metabolism by added DPN+ is probably due to a direct activation of glutamate dehydrogenase. This apparent activation, however, requires some additional cytoplasmic component that has not yet been identified. TPN+ is almost without effect under conditions in which DPN+ exhibits a “reversal” of inhibition caused by transaminase inhibitors.

III. Initial Rate Xtudies

The effectiveness of inhibitors was also determined in short term experiments of 1 to 3 minutes’ duration with the aid of the polarographic technique. When the relative rates of initial O2 uptake (determined polarographically) were plotted against the negative logarithm of inhibitor concentrations (Fig. 2), sigmoid curves simulating straight lines of approximately the same slope were obtained, indicating that each enzyme inhibitor alone is able to block glutamate metabolism completely. These observations were found to be valid also under manometric conditions, although there were some differences in the amounts of inhibitors required.

In manometric experiments representing pseudosteady state conditions, sufficient time is available for the distribution of substrates derived from added glutamate between various possi- bly sequentially arranged enzyme systems, each contributing to 0% uptake in a kinetically dependent manner. Under initial rate conditions (in the presence of 2 X 10e3 M ADP, 5 X 10” M Pi, and 2 x lop3 M glutamate), however, it can be assumed that the rate of 0% consumption depends primarily on the catalytic function of enzymes that initiate glutamate metabolism. Experi- mental conditions employed during initial rate measurements were essentially the same as in the manometric experiments, when glucose and hexokinase were used to deplete the system of ATP. Under these circumstances, during short experiments (1 to 3 minutes) there is not a sufficient accumulation of ATP for glutamine synthesis to occur. It is therefore not surprising that the inhibition pattern obtained is in general agreement with that found in manometric experiments under “ATP-depleted” conditions (Table VI). With glutamine as substrate, respiration was virtually insensitive to difluoro-oxaloacetate and amino- oxyacetate. A small inhibition of glutamine oxidation by mono-

Vi/V

FIG. 2. Experimental conditions were the same as those described in the legend of Table VI. O-O, Monofluoro-oxaloacetate; O-O, difluoro-oxaloacetate; A-A, amino-oxyacetate; A-A, monofluoroglutarate. The ordinate represents the relative velocity (vi/a, where vi is the rate in the presence of inhibitor and v is the control rate). The abscissa represents the negative logarithm of the inhibitor concentration.


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fluoro-oxaloacetate is most likely due to its effect on malate dehydrogenase. In long term experiments, metabolism of monofluoro-oxaloacetate and steady state concentrations of oxaloacetate and malate abolish this effect of monofluoro-oxaloacetate if it is employed alone. Comparable concentrations of combined fluoro-oxaloacetates cause the same degree of inhibition under both manometric and initial rate conditions. This is, however, not the case with amino-oxyacetate, since concentrations far in excess of that required to cause complete inhibition of glutamate-alanine transaminase were required to inhibit glutamate metabolism under initial rate conditions (compare Table II, Fig. 2, and Table VI), whereas in manometric experiments the effectiveness of this inhibitor agrees with predictions made from its Ki with respect to glutamate-alanine transaminase. The reasons for this apparent discrepancy were determined. It was found that amino-oxyacetate in relatively high concentrations (10m3 M) inhibits the reductive amination of pyruvate by TPNH + NH4+, catalyzed by intramitochondrial glutamate dehydrogenase, although the oxidation of glutamate by the glutamate-specific form of the same enzyme is not inhibited by the same concentration of amino-oxyacetate (21). This finding indicates that under initial rate conditions glutamate-alanine transaminase does not participate in reactions leading to O2 uptake. Under these conditions a relatively high concentration of amino- oxyacetate exerts its action on 02 uptake by inhibiting the removal of TPNH + NH4+ by reductive amination of pyruvate, catalyzed by the alanine-specific form of glutamate dehydrogenase (Fig. 4, Reaction 5). This enzymatic reaction precedes glutamate-alanine transaminase (Fig. 3) in the proposed sequence of reactions. However, the net effect of inhibition of either of the two reactions (i.e. reductive amination of pyruvate or transamination of alanine) on the over-all function of the multienzyme system is the same. In contrast to initial rate experiments, under pseudosteady state conditions, glutamate metabolism is highly sensitive to inhibition by amino-oxyacetate (Table II), indicating that this enzyme has a rate-limiting role when quasi- physiological conditions prevail. It is predictable from these considerations that in short term experiments glutamate dehydrogenase plays an initiating role, providing substrates to subse-

TABLE VI

Initial rate studies with kidney mitochondria: inhibitory effects in presence of glutamate and glutamine

Additions to the reaction chamber were made in the following order: 1.6 ml of 0.15 M KU, pH 7.4; 0.05 ml of 0.2 M phosphate, pH 7.4; 0.05 ml of 0.1 M MgC&; 0.10 ml of mitochondria (1.5 mg of protein); 0.05 ml of 0.1 M ADP; and 0.05 ml of 0.1 M glutamate or glutamine. Inhibitors to obtain final concentrations as indicated were added in a volume of 0.05 to 0.1 ml after establishment of control rates for approximately 1 minute. The temperature was 25”, and the duration of experiments, 2 to 3 minutes. Results are expressed as relative rates.

Inhibitors

Substrates

Glutamate Glutamine

None................................. 1.0 1.0 Monofluoro-oxaloacetate, 2 X 1OV M . 0.50 0.75 Difluoro-oxaloacetate, 2 X lo+ M. . . 0.55 0.90 Fluoroglutarate, 2 X 10e3 M.. . . 0.40 0.63 Amino-oxyacetate, 1 X low3 M.. . . . 0.60 0.90

quent enzyme systems (Fig. 3). This prediction is verified by the observation that a+fluoroglutarate, inhibiting glutamate oxidation by glutamic dehydrogenase, is about 2 to 3 times more effective in initial rate experiments than in the manometric ones. None of the inhibitors in the concentrations used above affected O2 uptake of mitochondria when a!-ketoglutarate was the substrate.

DISCUSSION

An interpretation of our results must account for the following observations. (a) Inhibition of glutamate-aspartate or alanine transaminase blocks glutamate metabolism in whole homogenates, but not in isolated mitochondria, unless ATP in the mitochondrial system is converted to ADP. (b) Oxidation of glutamine, which proceeds in kidney mitochondria at the same rate as that of glutamate, is uninfluenced by these transaminase inhibitors, unless “ATP-deficient” conditions are created. (c) Inhibition of glutamate-pyruvate transaminase markedly lowers aspartate formation. (d) Any one of these inhibitors is capable of inhibiting the over-all utilization of glutamate in isolated mitochondria in the presence of ADP and inorganic phosphate. (e) Addition of DPN+ to homogenates counteracts the inhibitory effects of transaminase inhibitors on glutamate respiration.

The first two observations are intimately related. Inhibition of glutamate-aspartate and glutamate-alanine transaminase does not influence 02 uptake in the presence of glutamate as substrate when the “glutamine shunt” (Fig. 5) operates. The controlling function of the intramitochondrial ATP:ADP ratio and factors influencing it will thus decide on the first reactions of glutamate in intracellular multienzyme systems.

Observations c, d, and e are related and suggest a hitherto un- recognized sequential relationship between transaminases and glutamate dehydrogenases. Ever since the proposal of Krebs and Bellamy (22, 23) of the aspartate-glutamate “cycle,” con- siderable uncertainty has prevailed concerning the exact role of this transaminase reaction with respect to other multienzyme systems (24-28). Glutamate dehydrogenase, although clearly recognized as an important enzyme, has been implicated merely as a provider of keto acids for glutamate transaminations. Until now no definite connections between these enzymatic reactions and glutamate transaminations (29-31) have been formulated.

As shown in Figs. 3, 4, and 5, a connection between these elementary enzymatic reactions of glutamate is proposed. The role of glutamate dehydrogenase has to be redefined, particularly in the light of more recent discoveries. The work of Klingenberg, Slenczka, and Pette (36, 37), makes it very probable that this enzyme utilizes TPN+ rather than DPNf as coenzyme when it is functioning intracellularly. Moreover, the work of Slater and Tager (38) indicates that, in intact mitochondria this enzyme functions more readily in the synthetic than in the catabolic direction. Recent progress in pyridine nucleotide-linked electron transfer mechanisms provides further clues regarding a control of the oxidation-reduction state of the coenzymes in mitochondria. It is now clearly recognized (36, 39) that succinate in phosphorylating mitochondria is a powerful reducing agent of intramitochondrial DPN+, a reaction intimately linked to the first point of electron transfer-linked ATP synthesis. It is also known that in the presence of ATP, or active electron transfer from DPNH or succinate to molecular 02, DPNH quantitatively transfers H to TPN+ (32, 36,40). This in turn can be readily interpreted as an energy-linked functional inhibition of


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2902 Studies on Specijic Enzyme Inhibitors. VIII

PROPOSED MOLECULAR CONTROL OF GLUTAMATE PATHWAYS

[F- GLU,TARATE]

9 f \

1. GLUTAMATE + TPN+ <\j NH; + TPNH + d- KETOGLUTARATE

’ k 4 T

pml[ATP, Or<-------DPNH +co2

\ \

- 1.5 02

[Ar’=j \ \

\ 2. GLUTAMATE t OAA \

Vol. 239, No. 9

ASPARTATE ; d KETOGLUTARATE > PYRUVATE tJ

I I

tco2 co

[ I

-1.502 ? J FOA A \

‘\ ALANINE

F20AA

[

. ~[OAA]

NH2 -OXY-ACE+--------+,

ASPARTATE

FIG. 3. Reaction 1 is the energy-controlled glutamate dehy- Reaction 2 is glutamate-aspartate transaminase. Reaction 3 is drogenase system coupled to oxidative phosphorylations and the reductive amination of pyruvate to alanine by the alanine- transhydrogenations between pyridine nucleotides. N I indicates specific form of glutamate dehydrogenase (see Equation 5 of Fig. an energy-rich intermediate generated by ATP or aerobic oxida- 4) and cyclic coupling of CO2 fixation into pyruvate (cf. Refer- tion of DPNH or succinate, which causes quantitative transfer ences 34 and 35) and transamination of alanine and oxaloacetate, of H from DPNH to TPN (32, 33). The ATP-linked reduction catalyzed by glutamate-alanine transaminase. Oxaloacetate, of DPN by succinate and its alternate oxidation by 02 via the which does not accumulate in this cycle, is shown in brackets (see electron transfer multienzyme system is indicated by dashed lines. Equations 6 and 7 of Fig. 4). Inhibitors acting on specific en- An alternate route of aerobic oxidation of TPNH (via DPN), zymes in this pathway are abbreviated as follows: F-glutarate, which may operate in the absence of energy-linked transhydro- cY-fluoroglutarate; FOAA, monofluoro-oxaloacetate; FzOAA, genation in the opposite direction, is also shown by dashed lines. difluoro-oxaloacetate; and NHZ-oxy-acct., amino-oxyacetate.

,. GLUTAMATE + TPN+ = NH; + TPNH + oL-KETOGLUTARATE Mg++ 4 Glutamate + 4 ATP + 4 NHn+ -+

2. GLUTAMATE + OAA = ASPARTATE + ti-KETOGLUTARATE

3. c(-KETOGLUTARATE + 1.5 oz = OAA + Co2

4. 0f-KETOGLUTARATE + I.5 o2 = PYRuvATE + 2 CO2

5. PYRUVATE + NH2 + TPNH = ALANINE + TPN+

6. PYRUVATE + CO2 = OAA

7. OAA + ALANINE = ASPARTATE + PYRUVATE

SUM : 2 GLUTAMATE + 3 02 = 2 CO, + 2 ASPARTATE

OR: GLUTAMATE + I.5 O2 = CO2 + ASPARTAfE

FIG. 4. Mechanism 2

+2 co2

-1.5 02

TPNH oxidation by DPNf by a thermodynamically favored opposite reaction (i.e. DPNH-TPNf transhydrogenation). Thus two intimately connected, energy-controlled mechanisms-

4 glutamine + 4 ADP + 4 Pi

4 a-ketoglut.aramate I

$3 co2

-4.5 02

4 NHh+

- 4 a-ketoglutarate

FIG. 5. Glutamine shunt

the synthetic tendency of functionally organized glutamic dehy- acetate. Removal of oxaloacetate is, however, not sufficient by drogenase and the unfavorable energetic conditions for indirect itself to “pull” glutamate oxidation. Another mechanism must TPNH oxidation by OS-tend to make the oxidative direction remove the other products, i.e. TPNH and NH4+. On the basis (i.e. from left to right) of this reaction dependent on other of our analytical and kinetic results, we favor a mechanism “pulling” mechanisms. Since the repression of glutamate dehy- offered by recent discoveries concerning the catalytic properties drogenase activity is generated to a large extent by reactions that of glutamate dehydrogenase. It is now recognized that a molec- oxidize cr-ketoglutarate to oxaloacetate, it is logical to anticipate ular variety of glutamate dehydrogenase can reductively a kinetically critical mechanism that efficiently removes oxaloace- aminate pyruvate by TPNH and NH4+ (41-43). Affinity for tate. This mechanism is provided by glutamate-aspartate TPNH is the same in this reaction as in the “glutamate-specific” transaminase via another molecule of glutamate, an enzymatic variety of the purified dehydrogenase, although the rate of reac- reaction that is known to be one of the fastest reactions of oxalo- tion with pyruvate appears to be slower. On the other hand,


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September 1964 E. Kun, J. E. Ayling, and B. G. Baltimore 2903

Frieden found that GTP stimulates the reductive amination of pyruvate by crystalline glutamate dehydrogenase (44), confirm- ing earlier experiments of Tomkins, Yielding, and Curran (43). It seems to us that the complicated molecular control of glutamate dehydrogenase by nucleotides as described by Frieden (44), particularly the activating effect of the pyruvate reaction by GTP, must have a physiological importance in the control of intramitochondrial enzymatic reactions. Under conditions of oxidative phosphorylation exactly when the proposed energy- linked repression of glutamate dehydrogenase activity takes place, GTP concentration should be expected to reach high levels, because of transphosphorylations from ATP. It is therefore probable that the alanine-specific species of the dehydrogenase should find favorable conditions for the synthesis of alanine, thus removing the products (TPNH and NHd+) of the slow glutamate dehydrogenase step of these reaction sequences. Clearly, this type of removal of NHd+ and TPNH would by itself have only negligible influence on the rate of reactions in the forward direction. Glutamate-alanine transaminase, however, can readily remove alanine by a cyclic combination with CO2 fixation into pyruvate by pyruvate carboxylase (34,35) and serve as an additional pulling mechanism, much the same way as glutamate- aspartate transaminase functions for removal of oxaloacetate. The cyclic process of CO2 fixation into pyruvate and alanine- oxaloacetate transamination is shown in combination of Equa- tions 5, 6, and 7 of Fig. 4. In each case aspartate is the end product, and stoichiometry of reactants and products is in good agreement with our analytical data on glutamate and O2 consumption, and on COZ and aspartate formation. This stoichiometry is identical with that proposed by Krebs for the “aspartate- glutamate” cycle (22, 23), yet Krebs’ mechanism does not predict the inhibition pattern found in our experiments. Thus balance studies alone, however useful and necessary, cannot predict complex enzymatic mechanisms in systems in which various types of enzyme-controlling devices, such as oxidative phosphorylation and enzyme modifiers, operate.

The reversal of inhibitory effects of transaminase inhibitors by DPN+, observed in kidney homogenates, also predicts the function of the sequential enzymatic mechanism proposed in Figs. 3, 4, and 5. Apparently through the mediation of a cytoplasmic component, DPN+ can activate glutamate oxidation by OS. This activating effect must be due to an alteration of the energy-controlled inhibition of glutamate dehydrogenase, render- ing its catabolic function independent from pulling mechanisms provided by transaminases. S UC h a mechanism is probable since glutamate dehydrogenase is known to be modified by pyridine and adenine nucleotides (44).

It should be remembered that there is at present at least one other known mechanism that could account for the reductive amination of pyruvate and could in part serve as a “coupling system” between glutamate dehydrogenase and glutamate-ala nine transaminase. Radhakirshnan and Meister (45) and Wellner and Meister (46) showed that reduced amino acid oxidase can readily aminate pyruvate to alanine. If this flavoprotein could be reduced by TPNH generated by glutamate dehydrogenase, such a system might fulfill the role that we presently ascribe to the pyruvate-specific molecular species of glutamate dehydrogenase.

The foregoing working hypothesis predicts all our experimental results obtained by the use of synthetic enzyme inhibitors. It is of importance to recognize that factors influencing enzyme pro-

teins directly (47) and controlling mechanisms predictable from recent knowledge of energy-linked functions of mitochondria (33) find an obligatory role in this proposed biochemical “organi- zation” of elementary enzymatic reactions.

In conclusion, it should be mentioned that earlier experiments carried out with the inhibitory isomer of fluorocitrate (6) already predicted that in kidney mitochondria the citric acid “cycle” in its original interpretation cannot participate in the metabolism of glutamate, since fluorocitrat,e does not inhibit glutamate “oxidation” in this t,issue.

SUMMARY

1. The inhibitory effects of mono- and difluoro-oxaloacetates, fluoroglutarate, and amino-oxyacetate on the metabolism in vitro

of glutamate by rat kidney mitochondria and homogenates were determined.

2. Inhibitors of glutamate-aspartate and glutamate-alanine transaminases did not depress 02 uptake of kidney mitochondria in the presence of glutamate unless the adenosine triphosphate (ATP) concentration was kept low (below lop5 M) by hexokinase and glucose.

3. Glutamine is oxidized by kidney mitochondria at the same rate as glutamate. The oxygen uptake in the presence of 2.3 x low3 ivr ATP and glutamine was uninfluenced by fluoro-oxaloacetates and amino-oxyacetate. Fluoroglutarate had an inhibitory effect on this reaction.

4. It is deduced from enzyme inhibition studies (i.e. from the ineffectivity of transaminase inhibitors in the presence of 1O-3 M

ATP) that synthesis of glutamine and its subsequent transamination play a significant role in glutamate metabolism of kidney tissue.

5. Respiration of kidney homogenates with or without added glutamate was depressed by all inhibitors studied. The inhibition caused by fluoro-oxaloacetates and by amino-oxyacetate was reversed by externally added diphosphopyridine nucleotide; that by fluoroglutarate was not.

6. Determination of the inhibitory effects of fluoro-oxaloacetates, fluoroglutarate and amino-oxyacetate on the initial rate of glutamate oxidation showed that each inhibitor alone was capable of almost completely inhibiting glutamate metabolism.

7. A sequential enzymatic mechanism is proposed that involves an obligatory coupling of glutamic dehydrogenase and glutamate-aspartate and glutamate-alanine transaminases.

Acknowledgments-Some of the kinetic constants were redeter- mined by Mrs. B. Achmatowicz with the Gilford automatic spectrophotometer. We are grateful to Miss Clementina Moya for the typing of the manuscript.

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E. Kun, J. E. Ayling and B. G. BaltimoreMETABOLIC PATHWAYS IN KIDNEY TISSUE

MECHANISMS OF THE ENTRY OF GLUTAMIC ACID INTO Studies on Specific Enzyme Inhibitors: VIII. ENZYME-REGULATORY

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