The Reversible Inhibition of Gluconeogenesis in Kidney Cortex by Diazenedicarboxylic Acid...

Eur. J. Biochem. 79, 73-83 (1977)

The Reversible Inhibition of Gluconeogenesis in Kidney Cortex by D iazenedicar boxy lic Acid Bi s( N , N-dimet h ylamide) Dennis PILLION, Frederick H. LEIBACH, and Hector ROCHA

Department of Cell and Molecular Biology, Medical College of Georgia, Augusta, Georgia

Francis J. VON TERSCH and Joseph MENDICINO

Department of Biochemistry, University of Georgia, Athens, Georgia

(Received November 15, 1976/April 5 , 1977)

Gluconeogenesis in rat kidney cortex slices was reversibly inhibited by diazenedicarboxylic acid bis(N,N-dimethylamide). The rate of formation of glucose from pyruvate, malate, succinate and D-fructose decreased at least 3-fold in the presence of this inhibitor, commonly called diamide. The extent of inhibition of gluconeogenesis from pyruvate was dependent on the concentration of diamide and closely correlated with the degree of inhibition of protein kinase. The inhibition of gluconeogenesis and protein kinase were completely reversed after removal of the inhibitor. The effects of diamide on particulate enzymes which may be involved at rate-limiting steps in transport processes which support gluconeogenesis in renal tissue was examined. Several enzymes present in kidney brush border and microsomal membrane preparations were inhibited by diamide. Na+, K +-dependent ATPase and glucose-6-phosphatase were inhibited, but Mg2 +-dependent ATPase, alkaline phosphatase and y-glutamyl transpeptidase, which are also present in these same subcellular fractions, were not inhibited by diamide. Based on the results obtained in these studies it is proposed that diamide may inhibit gluconeogenesis at rate-limiting steps catalyzed by znzymes which are regulated by protein kinase. These enzymes, in turn, appear to play a role in the transfer of substrates across the plasma membrane.

The mechanism of inhibition of protein kinase by diamide was also investigated. Protein kinase present in a wide variety of rat tissues as well as phosphorylase kinase and pyruvate dehydrogenase kinase was inhibited by diamide. Kinetic studies showed that the inhibition of protein kinase by diamide was non-competitive with respect to Mg2+, ATP, protein substrates and cyclic AMP. Sedimentation studies in sucrose-density gradients containing histone and cyclic AMP showed that diamide inhibited both the intact and dissociated forms of protein kinase. These results support earlier observations which showed that diamide did not bind at the active site of protein kinase, but instead formed inactive complexes by binding to hydrophobic areas on the surface of the enzyme.

Previous studies in our laboratories have established that diamide inhibits the transport of amino acids and sugars in renal cortical slices [l-31. Di- amide also inhibits RNA and protein synthesis [4- 61, cation transport in the lens [7], Ca2 +-activated-ATPase in sea urchin eggs [S] and it stimulates the utilization

methylamide) ; cyclic AMP, adenosine 3' : 5'-monophosphate. Trivial Name. Diamide, diazenedicarboxylic acid bis(N,N-di-

of glucose in fat cells [9]. Although this inhibitor has been widely used in studies in vivo, its mechanism of action and its effects on the catalytic properties of specific enzymes have not been extensively examined.

In a more recent study we have further shown that the extent of inhibition of the active transport of amino acids and a-methylglycoside by diamide could be directly related to the degree of inhibition of endo-

14 Inhibition of Gluconeogenesis by Diamide

geneous protein kinase in these tissue slices [3]. Diamide also inhibited purified swine kidney protein kinase [lo]. These findings suggest that protein kinase may be involved at a rate-limiting step in amino acid and sugar transport. Dibutryladenosine 3' : 5-monophosphate increases the rate of accumulation of sugars and amino acids in kidney slice preparations [l 1,121. It has been proposed that phosphorylation reactions involving protein kinases may play a role in transport processes in the renal medulla [13]. Isolated brush- border membranes possess protein kinase activity and an endogenous protein substrate which can be phosphorylated by protein kinase and dephosphory- lated by phosphoprotein phosphatase [14]. Collec- tively these observations indicate that diamide may specifically inhibit metabolic processes in kidney which are regulated by cyclic-AMP-dependent protein kinases.

Gluconeogenesis has been studied in renal cortical slices for many years, but the mechanism by which this process is controlled has yet to be conclusively established. There is some evidence that cyclic AMP and protein kinases may be involved in the regulation of gluconeogenesis in kidney [15 - 181. If cyclic- AMP-dependent protein kinases are involved at rate- limiting steps in gluconeogenesis, then it seemed likely that diamide might inhibit gluconeogenesis from p$ru- vate and amino acids. It was therefore of some interest to examine the effects of diamide on the rate of gluconeogenesis in kidney cortex slices to determine if inhibition occurred and whether the extent of inhibition was related to the inhibition of protein kinase under the same conditions. The effects of diamide on gluconeogenesis from pyruvate and a number of other substrates were examined in the present study. In efforts to further elucidate the site of action of diamide in renal tissue and obtain a clearer under- standing of the nature of the interaction of diamide and protein kinases we also investigated the effects of the inhibitor on the kinetic properties of this enzyme.

MATERIALS AND METHODS

Adult female Sprague-Dawley rats, starved for 19 h, were used in all experiments. Renal cortex slices were prepared as described earlier [l- 31 and each reaction mixture contained about 100 mg wet weight of tissue. All incubations were carried out in Krebs- Ringer bicarbonate buffer, pH 7.4, in 25-ml Conical flasks. Gluconeogenesis was assayed by measuring the rate of appearance of glucose in the incubation medium with glucose oxidase. Slices were incubated with or without diamide for 15 min at 4 "C in 2.5 ml of buffer as described previously [2] and they were then incubated with 10 mM substrate at 37 "C. Samples run without substrate served as controls

and the amount of glucose formed in these reaction mixtures were subtracted from the amount formed in the presence of substrate to determine net glucose pro- duction. Glucose formation was measured in 0.5-ml aliquots of the incubation medium. Glucose oxidase analysis on protein-free supernatants were performed using a Glucostat reagent obtained from Worthington Biochemical Co. Results are expressed as pmol glucose formed x g wet weight of tissue-' x h-' unless otherwise noted. After 90 min of incubation, the slices were homogenized in 10 mM Tris-HC1, pH 7.8 and protein kinase activity was determined with the standard assay [19].

The uptake of a-methyl-D-glucoside by rat kidney cortex slices was measured described previously [2,3]. All the results are expressed as distribution ratios, which represent the ratio of the concentration of the transported material in the intracellular fluid to its concentration in extracellular fluid.

Purified swine kidney protein kinase was prepared by the procedure of Abou-Issa et al. [19] and the preparation used in these studies was purified more than 3000-fold by affinity chromatography. Kinase activity was assayed by measuring the rate of transfer of 32P from [Y-~~PIATP to histone. Reaction mixtures were incubated for 10 min at 30 "C and contained in a total volume of 0.35 ml: 30 mM 2(N-morpholino)- ethanesulfonic acid, pH 6.5, 12 mM MgC12, 120 mM sucrose, 0.03 mM cyclic AMP, 9 mM cysteine or 1 mM dithiothreitol, 1 mg calf thymus histone (fraction IIA, Sigma Chemical Co.) and an appropriate amount of purified enzyme or 0.1 ml of tissue homogenate. After a preliminary incubation of 3 min the reaction was started by the addition of 0.12 pmol of [y-32P]ATP, specific activity 2 x lo7 counts x min-' xpmol-'. The reaction was stopped with 2ml of 2.8 N perchloric acid containing 2 % phosphotungstic acid, 10 mM sodium phosphate and 3 mM sodium pyrophosphate. After centrifugation, the pellet was dissolved in 0.5 ml of 1 M NaOH and then washed twice more by this procedure [19]. The amount of 32P covalently bound to the protein substrate was determined. One unit of activity is defined as the amount of enzyme required to transfer 1 pmol of 32P from [Y-~~P]ATP to the protein substrate per min.

Chymotrypsin activity was assayed according to the procedure of Hummel [20] by following the rate of hydrolysis of benzoyl-L-tyrosine ethyl ester at 250 nm in a reaction mixture containing 0.04 M Tris-HCl, pH 7.8, 0.05 M CaCI2, 25% methanol (w/w) and 0.52 mM benzoyl-L-tyrosine ethyl ester. Proteolytic activity was also estimated by measuring the rate of hydrolysis of azocollagen. The standard incubation mixture contained in 1 ml: 12 mg of azocollagen, 0.2 M N-ethylmorpholine acetate, pH 8.6,5 mM CaC12 and enzyme. The reaction mixture was incubated at 37 "C with stirring for 30 min, then 2 ml of HzO

75 D. Pillion, F. H. Leibach, H. Rocha, F. J. Von Tersch, and J. Mendicino

40

35 - c

was added and the mixture was filtered. The amount of hydrolysis was estimated by measuring the increase in absorbance at 520 nm due to the solubilization of azocollagen. Controls lacking enzyme were incubated in each assay.

Hexokinase, phosphofructokinase and pyruvate kinase, isolated from swine kidney [21], and creatine kinase isolated from rabbit muscle, were assayed by standard spectrophotometric procedures by measuring the rate of formation of ADP with pyruvate kinase and lactic dehydrogenase [22]. Since diamide absorbs strongly at 340nm it was necessary to measure the activity of these kinases by another procedure in inhibition studies. After incubation for various times the ADP was isolated from the reaction mixture by chromatography on a small Dowex 1-C1 column (0.38 x 4 cm) by a modification of the procedure of McQuate and Utter [23]. The ADP was then quanti- tatively eluted with NaCl and assayed. Zero-time controls containing all components of the reaction mixture and diamide were included in each assay. Phosphorylase kinase activity was measured by the procedure of Krebs et al. [24] and pyruvate dehydrogenase kinase was assayed by the method of Linn et al. [25]. Other enzymes were assayed by standard procedures [21,22]. Microsomes and brush border membranes were isolated as described in a previous report [26 1. Sucrose density centrifugation was performed according to Martin and Ames [27].

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RESULTS

Inhibition of Gluconeogenesis by Diamide

The influence of diamide on the rate of conversion of pyruvate to glucose by rat kidney cortex slices is shown in Table 1. Incubation of the slices with increasing concentrations of diamide caused a progres- sive decrease in the rate of formation of glucose. The activity of protein kinase in these preparations also decreased. The formation of glucose and protein kinase activity were not significantly affected by the addition of 1 mM diamide. However, in the presence of 5 mM diamide glucose formation decreased and a nearly parallel inhibition of protein kinase activity was observed after correction for the dilution of diamide during homogenization of the tissue. Higher concentrations of diamide, 10 mM, caused still further inhibition. The intracellular concentrations of diamide in these experiments may be nearly the same as that in the medium since the inhibitor is freely permeable under these conditions. These results are similar to those obtained with isolated kidney protein kinases [lo]. Very little inhibition of the purified enzyme was observed at 1 mM diamide and about 50 % inhibition was seen at 5 mM.

Table 1. Effect of increasing concentration of diamide on the rate of gluconeogenesis and protein kinase activity in kidney slices Slices of rat kidney cortex, about 70 mg, were prepared as described in the text and incubated with various concentrations of diamide for 15 min at 4 "C. The slices were then incubated for 90 min at 37 "C with 10 mM pyruvate and various concentrations of diamide. Data is expressed as mean pmol glucose formed x g wet wt-' x h-' f S.E.M. for triplicate determindtiom. Protein kinase activity was determined in a homogenate prepared from the tissue at the end of the 90-min incubation period. Results are expressed as pmol 3zP incorporated into protein/min f S.E.M. for triplicate determinations

Diamide Gluconeogenesis Protein kinase activity

mM pmol x g-' x h-' pmol/min

21.4 f 5.8 24.5 f 0.9 0 3 20.0 * 2.2 24.1 f 1.8

14.1 k 2.6b 10 4.1 1.7b 8.0 f 1.6b 5 7.4 f 5.7"

a P < 0.05. P < 0.005.

45 I

/*'

I

30 60 90 3 Time ( rnin)

Fig.l. Efect of time of incubation on the inhibition of gluconeogenesis by diamide. The conditions were similar to those described in Table 1 and the final concentration of diamide was 10 mM

The relationship between the time of incubation and the rate of formation of glucose in the presence of diamide is shown in Fig. 1. The rate of synthesis of glucose by untreated slices increased with time for 90 min. Slices treated with diamide showed a markedly decreased rate of synthesis throughout a 120-min incubation period. It should be noted that the values in Fig.1 represent net glucose formation, since the amount of glucose formed in the absence of 10 mM pyruvate has been subtracted in each case. Slices treated with diamide also formed less glucose from endogenous substrates.


Reversibility of Inhibition by Diamide

The inhibition of amino acid and u-methylglucoside transport by diamide in kidney slices can be reversed by removing diamide [3]. The reversibility of the inhibitory effect of diamide on gluconeogenesis was examined. It is clear from the data presented in Table 2 that this inhibition can also be almost completely reversed by removing the inhibitor from slices previously treated with diamide. The activity of endogenous protein kinase is likewise restored after removal of diamide. The inhibition of the protein kinase is greater than that shown in experiment 2, since intracellular diamide is diluted during preparation of the homogenate before assay. Thus, inhibition by diamide does not appear to be attributable to irreversible damage to the tissue slices, since its effect on gluconeogenesis are completely reversed after removal of the inhibitor.

Effect of Diumide on Gluconeogenesis from Different Substrates

In order to more precisely define the site of action of diamide, its effect on the rate of synthesis of glucose from a number of different gluconeogenic substrates was examined. The pattern of inhibition by diamide is shown in Table 3. The addition of diamide resulted in a 3-4-fold decrease in the rate of formation of glucose from 2-0x0 glutarate, succinate, malate, glycerol, fructose and glutathione. The extent of inhibition of gluconeogenesis was nearly the same in each case, even though the amount of glucose formed from the different substrates varied over a wide range. The results clearly indicate that some process common to the conversion of all of these substrates to extracellular glucose may be inhibited by diamide. The rate of transport of glucose out of the cell may become limiting in the presence of diamide, and hence this step may show the greatest sensitivity to the presence of the inhibitor. If protein kinase regulated a rate- limiting process involved in the transport of glucose from the cell, then it might be expected that diamide would have a qualitatively similar effect on the extent of inhibition of gluconeogenesis from different substrates.

Many of the effects of diamide on slices have been attributed to its ability to cause a transient oxidation of reduced glutathione [6]. Therefore, the effect of reduced and oxidized glutathione on the inhibition of gluconeogenesis by diamide was examined. Surprisingly, both forms of glutathione acted as substrates for gluconeogenesis. As seen in Table 3 , both forms of glutathione were equally effective as substrates. Glutathione is not usually used as a substrate for gluconeogenesis, but the amino acids present in this peptide are excellent gluconeogenic precursors. In addition, the kidney is known to contain enzymes

Table 2. Keversibiliry qf the inhibitory eifects of diamide on gluconeogenesis In experiment 1, slices of rat kidney cortex, 100 mg, were incubated in 2.5 ml of Krebs-Ringer bicarbonate buffer for 15 min at 4 'C. They were then washed and incubated with 2.5 ml of buffer containing 20 mM glutathione for 15 min at room temperature. Finally the slices were incubated for 90 min at 37 "C with 10 mM pyruvate as described in the text. Experiment 2 was carried out under the same conditions except that 5 mM diamide was added to the buffer. In Experiment 3, the slices were treated with 5 mM diamide for 15 min at 4 "C and they were then washed and incubated with buffer containing 20 mM glutathione for 15 min at room temperature. Glucose formation was measured for 90 rnin at 37 "C. Results are expressed as S.E.M. for triplicate determinations

Experiment Gluconeogenesis Protein kinase activity

pmol x g-' x h- ' pmol/min

1. No treatment 34 k 2.0 33 k 1.5 2. Diamide added I f 2.2 18 k 1.5 3. Diamide added then

removed 32 k 2.2 31 f 1.0

Table 3. Effect of diarnide on gluconeogenesis from various substrates Rat renal cortical slices, 100 mg, were incubated under standard conditions with or without 5 mM diamide and 10 mM substrate for 90 rnin at 37 "C. Results are expressed as pmol glucose formed/g wet weight of tissue in 90 min & S.E.M. for triplicate determinations

Substrate Glucose formed from added substrate

minus dcdmide plus diamide

None 2-Oxoglutarate Succinate Malate Pyruvate Glycerol D-Fructose Glutathione Oxidized glutathione

p m o k

4.5 k 7.0 2.0 & 1.2 46.0 ri: 7.8 17.1 k 1.1 28.9 f 4.3 9.0 & 1.0 60.8 8.8 24.4 3.6 57.5 F 7.0 10.6 f 1.0 32.1 k 5.2 9.6 ri: 1.2

30.0 & 9.0 17.1 f 3.1 6.1 ri: 1.0 15.9 f 1.7 3.6 & 0.4

107.2 f 13.5

which degrade glutathione. The addition of diamide inhibited the rate of gluconeogenesis from glutathione to about the same extent as it inhibited this process with most of the other substrates tested.

The conversion of pyruvate to glucose was inhibited more severely than the other substrates, and a 5 - 6-fold decrease was usually observed. These results suggested that another site of inhibition by diamide might be between pyruvate and phosphoenolpyruvate. In other experiments [l -'4C]pyruvate and [2-14C]- pyruvate were incubated with kidney cortex slices in the absence and in the presence of diamide. The 14C02 formed was trapped in alkali during a 90-min incubation period. In the presence of diamide the slices

I1 D. Pillion, F. H. Leibach, H. Rocha, F. J. Von Tersch, and J. Mendicino

formed more I4CO2 than untreated samples and this effect was completely reversed when diamide was removed. These results demonstrated that diamide stimulated pyruvate oxidation under conditions in which gluconeogenesis was inhibited. The inhibition of pyruvate dehydrogenase kinase, which inactivates pyruvate dehydrogenase, by diamide could explain these effects.

The addition of 5 mM diamide to reaction mixtures containing kidney cortex mitochondria increased the rate of oxidation of pyruvate by about 20% over controls. The presence of inhibitor increased the rate of uptake and utilization of pyruvate by mitochondria. These findings further support the suggestion that the inhibition of mitochondria1 protein kinase may decrease the steady rate level of the phosphorylated form of pyruvate dehydrogenase. Since the dephospho form of this enzyme is catalytically active [25], these results are consistent with the increased rate of oxidation of pyruvate to COZ after the addition of diamide.

EfJ; t of Diamide on the A T P levels in Kidney Cortex Slices

Diamide did not reduce the steady-state concentration of ATP in kidney cortex slices. Furthermore, it did not inhibit respiration nor uncouple oxidative phosphorylation of isolated kidney cortex mitochondria. Either of these effects could cause an inhibition of gluconeogenesis in slices. The concentration of ATP, ADP and AMP in kidney cortex slices in the presence of 10 mM diamide was compared to controls lacking the inhibitor under the conditions described in Table 1. After incubation for 90 min at 37 "C the concentration of adenine nucleotides in the slices was determined by the procedure of Faupel et al. [28]. The concentration of AMP, ADP and ATP in tissue slices treated with diamide was 30,640 and 4,100 nmol per g wet weight, respectively. The corresponding values in the control samples were 45, 670 and 3,800 nmol per g wet weight. These values were averages of triplicate determinations. The concentration of AMP in several samples was found to be as low as 10 nmol per g of tissue. Since the steady-state concentration of AMP is highly sensitive to inhibition of oxidative processes as well as stress, these results agree with previous observations which showed that diamide did not cause irreversible damage to tissue slices and that its effects could be completely reversed by removing it from the tissue. These results were further verified by adding 10 mM diamide to reaction mixtures containing kidney cortex mitochondria oxidizing pyruvate under conditions described previously [22]. The rate of respiration increased by about 20 % and the rate of esterification of Pi was essentially unchanged after the addition of diamide The results are in agreement with similar observations

Table 4. Influence of cyclic A M P und diamide on the transport of cc-methylglucoside Slices of rat kidney cortex were prepared as described in the text. In experimcnt 1 the slices were incubated in 2.5 ml of buffer for 15 min at 4 "C and then they were incubated at 37 "C for 90 min with 1.8 mM a-methyl-D-glucoside (0.1 Ci/mol). Experiment 2 was performed under the same conditions except that 3.0 mM diamide was added. In experiment 3, 1 mM cyclic AMP was added to the incubation medium, and experiment 4 was carried out with the addition of both 3.0 mM diamide and 1 mM cyclic AMP. Data represent mean distribution ratios & S.E.M. from four determinations

Experiment Additions Distribution ratio ~

1 2 3 4

None 3.3 0.2 Diamide 2.3 0.1" Cyclic AMP 4.4 0.05 Cyclic AMP plus diamide 2.9 0.3h

a P < 0.01 compared to experiment 1. P < 0.01 compared to experiment 3.

reported by other workers, who showed that even high concentrations of diamide did not effect the ATP levels maintained by isolated rat liver mitochondria ~ 9 1 .

Influence of Cyclic A M P on the Inhibition of Transport by Diamide

Since the principal sites of action of diamide appeared to involve rate-limiting steps regulated by protein kinases, it was of interest to examine the effects of diamide and cyclic AMP on sugar transport in kidney cortex slices. Cyclic AMP increases the rate of uptake of a-methylglucoside in cortex slices [12], presumably by increasing the activity of protein kinase. The results of experiments summarized in Table 4 show that diamide inhibits the transport of a-methylglucoside, whereas cyclic AMP stimulates the rate of accumulation of a-methylglucoside. How- ever, the percentage inhibition by diamide compared to controls is nearly the same in the presence of and in the absence of cyclic AMP, about 30% in each case. In other experiments it was observed that the extent of inhibition by diamide was independent of the concentration of cyclic AMP. These results are in agreement with those obtained with isolated protein kinases, which showed that the degree of inhibition by diamide was not influenced by the addition of cyclic AMP [lo]. They further support the view that diamide may act at rate-limiting steps in transport processes by virtue of its inhibitory effect on protein kinase.

Influence of Diamide on the Activity of Enzymes Present in Kidney Membrane Fractions

Since the pattern of inhibition of gluconeogenesis by diamide indicated that transport processes might be affected, we decided to examine the direct effects


Table 5. Effect of diamide on the activity of bound enzymes present in renal brush border and microsomal membrane fractions The subcellular fractions were prepared from fresh rat kidneys as described in the text. When indicated, diamide was added to the incubation mixture before initiating the reaction with substrate. Results are expressed as pmol of product formed x min-' x mg of protein-', except those for protein kinase which are expressed as pmol x min-' x mg protein-'

Enzyme Diamide Subcellular fraction present

brush border microsomes

mM pmol x min-' x mg protein-'

Protein kinase none 18.2 f 3.1 15.4 f 2.3 1.5 17.5 k 2.3 11.9 f 0.7 4.5 9.1 f 1.3 7.3 f 0.9

pmol x min-' x mg protein-'

Glucose-6- none - 8.2 f 0.2 phosphatase 1 - 7.8 f 0.5

5 - 4.1 f 0.3

Alkaline phosphatase none 4.5 f 0.3 14.3 f 1.0 1 4.7 f 0.5 16.2 f 1.8 8 4.3 f 0.2 13.7 f 0.9

Na+,K+-ATPase none - 35.5 f 2.9 0.5 - 30.1 f 2.4 3.0 - 20.7 f 4.3

MgZf-ATPase none 6.8 f 0.9 157.8 12.7 0.5 6.7 f 0.7 150.2 & 11.0 3.0 6.4 f 0.3 149.7 f 12.7

transpeptidase none - 43.1 f 3.7 0.5 - 41.0 f 3.6 5.0 - 44.2 f 4.3

y-Glutamyl-

of the inhibitor on enzymes present in different membrane fractions prepared from kidney cortex. The data presented in Table 5 show that bound protein kinase present in brush border and microsome membrane fractions was inhibited by diamide. The charac- teristics of the inhibition were similar to those observed with the soluble enzyme [lo]. Only a slight inhibition was observed at 1.5 mM diamide, whereas about 50 % inhibition was obtained with 4.5 mM diamide. Glucose-6-phosphatase and Na' , K +-dependent ATPase were also inhibited. These enzymes are thought to play a role in transport processes. Other enzymes present in these particulate fractions were not inhibited by diamide. Thus, the activities of alkaline phosphatase, Mg2 +-dependent ATPase and y-glutamyl transpeptidase were not altered by the addition of diamide. Clearly, the inhibitor shows some specificity for enzymes present in these membrane fractions. It is not certain whether diamide is acting directly on glucose-6-phosphatase and Na', K+- dependent ATPase or whether the observed decreases are indirectly related to the inhibition of protein kinase

Table 6. Injluence of diamide on the activity of dgferent protein kinases The standard assay conditions were used and the concentration of diamide was 6 mM with the purified soluble enzymes and 16 mM with crude tissue extracts. Triplicate determinations were carried out with each tissue. Fresh extracts were prepared from a 10% homogenate of each tissue in 0.01 M Tris-HC1, pH 7.8. The super- natant solution obtained by centrifugation at 25000xg for 30 min was used in the assay. The activity of the extracts are expressed as pmol of 32P incorporated per ml of extract

Inhibition Enzyme or tissue tested Activity

minus plus diamide diamide

Protein kinase Pyruvate dehydrogenase

Phosphorylase kinase Kidney Spleen Liver Lung Heart Brain Fat pad

kinase

pmol/min x 90 50

85 34 105 56 131 34 113 63 121 49 107 38 109 37 103 39 11.3 3.7

44

40 45 74 44 60 64 66 62 67

in these preparations. It should be noted that the extent of inhibition of these enzymes at different concentrations of diamide were similar to those observed for bound protein kinase. Diamide may inhibit transport by interfering with sodium gradient which is maintained by Na+, K+-dependent ATPase and by de- creasing the activity of glucose-6-phosphatase which appears to be required for the secretion of glucose formed during gluconeogenesis.

Specificity of Diamide in the Inhibition of Purified Soluble Enzymes

Incubation of highly purified soluble swine kidney protein kinase at 30 "C and pH 6.5 in the presence of diamide results in a loss of enzymatic activity as seen in Table 6. The extent and rate of inactivation is not dependent on preliminary incubation of the enzyme and diamide, since essentially identical inhibitions are observed when the reaction mixture is incubated with diamide for various times before initiating the protein kinase reaction by the addition of ATP. The extent of inhibition is directly proportional to the concentration of enzyme and the addition of 6mM diamide caused a 44 %inhibition under the conditions described in Table 6. Purified protein kinases isolated from rabbit muscle [30], beef heart [31], and brain [32] were also inhibited by diamide. Phosphorylase kinase and pyruvate dehydrogenase kinase, are inhibited to about the

D. Pillion, F. H. Leibach, H. Rocha, F. J. Von Tersch, and J. Mendicino 79

same extent as swine kidney protein kinase under these conditions. The data summarized in Table 6, further shows that diamide inhibits the protein kinases present in kidney, spleen, liver, lung, heart, brain and fat pad. At a concentration of 16mM, diamide inhibited the activity in rat kidney extracts by 74%. The enzymes present in the other tissues were inhibited from 44-67% by diamide. However, enzymes such as pyruvate kinase, hexokinase, phosphofructokinase and creatine kinase, which act on low molecular weight substrates, are not inhibited even when high concentrations of diamide, 18 mM, are added to the reaction mixture. A large number of other enzymes, many of which contain essential sulfhydryl groups have also been examined. Phosphoglucose isomerase, aldolase, triosephosphate isomerase, glyceraldehyde- 3-phosphate dehydrogenase, phosphoglycerokinase, phosphoglycerate mutase, enolase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phos- phogluconate dehydrogenase, glutathione reductase and glutathione peroxidase are not inhibited by diamide [33].

Other enzymes which act on protein substrates were also tested. Trypsin and chymotrypsin are inhibited by diamide. More than 90% inhibition of these proteases was observed at 6 mM diamide with azocollagen as the protein substrate. The activity of trypsin with benzoly-L-tyrosine ethyl ester as the substrate was also inhibited 86% at 6mM diamide. The surfaces of these proteases contain hydrophobic areas which appear to be required for activity [34], and the interaction of diamide with hydrophobic groups in these enzymes may be the cause of the observed inhibition. Known serine protease inhibitors such as diisopropyl fluoro- phosphate and phenylmethylsulfonyl fluoride, which act at the active site of trypsin and chymotrypsin, did not inhibit protein kinases, even at concentrations as high as 1 mM. It is apparent that the mechanism of inhibition of proteases, and possibly protein kinases, by diamide is different from that of serine protease inhibitors which react at the active site proteolytic enzymes. It should also be emphasized that the binding interaction between these enzymes and diamide is completely reversible, whereas serine protease inhibitors react in an irreversible fashion.

Effect of p H and Magnesium Ion on the Inactivation of Protein Kinase by Diamide

The percentage inhibition of protein kinase by diamide was completely independent of pH in a range from 6.0 to 8.5. The pH optimum, 7.5, was the same in the presence or absence of diamide. Since diamide does not ionize in this pH range, it may be concluded that changes in the degree of ionization of different groups in the protein do not significantly influence its interaction with diamide. These results are in agree-

l/[Histone] (FM-’)

Fig.2. Infruence of increasing concentrations of diamide on the activity of protein kinase as a function of the concentration of histone. The standard assay procedure was used with the concentrations of histone shown in the figure. Each reaction mixture contained 4.5 Fg of purified protein kinase. A molecular weight of 16000 for calf thymus histone was used to calculate the molarity of the protein substrate. (I) Without diamide; (11) 1 mM diamide; (111) 6 mM diamide; (IV) 12 mM diamide; (V) 18 mM diamide

ment with other observations which suggest that diamide may be selectively interacting with hydrophobic areas on the surface of the enzyme.

Protein kinase requires magnesium ion for activity [19]. The possibility that diamide might inhibit protein kinase by chelating magnesium ion was examined by varying the concentration of MgCL between 1 mM and 24 mM at concentrations of 3 mM, 6 m M and 9mM diamide. The same percentage of inhibition by diamide was observed at each concentration of magnesium ion. Moreover, the binding con- stant of the enzyme for magnesium ion was unaltered by the addition of diamide.

Effect of Protein Substrates, ATP and Cyclic A M P on the Inhibition of Protein Kinase

The activity of purified protein kinase was inhibited to nearly the same extent when histone, protamine or casein was used as the protein substrate. When the observed velocities for the inhibition of protein kinase by diamide as a function of the concentration of histone are analyzed, Fig.2, it is apparent that the inhibition is non-competitive with respect to histone. But, it is also clear from the double reciprocal plots in Fig. 2, that the observed velocities bear a distinctly non-linear relationship to the diamide concentration. Thus, as observed in earlier studies [lo], the presence of 1 mM diamide causes little or no inhibition, whereas 6 mM diamide results in about 50 % inhibition and at still higher concentrations, 12 mM and 18 mM, the I j V intercepts do not change in a linear fashion with

80

plus

Inhibition of Gluconeogenesis by Diamide

0 0.05 0.10 l/[ATP] (~LLM-')

Fig. 3 . Influence of increasing concentrations of diamide on the activity ofprotein kinase as a function of the concentration of ATP. The standard assay procedure was used with the amounts of ATP shown in the figure and 4.5 pg of purified enzyme. (I) No diamide; (11) 1 mM diamide; (111) 6 mM diamide; (IV) 12 mM diamide; (V) 18 mM diamide

the concentration of inhibitor. Clearly, the inactivation is not a simple second-order kinetic process. The data show that saturation of the kinase by reversibly bound diamide occurs only at very high concentrations of the inhibitor. At intermediate concentrations of diamide, 2 mM to 8 mM, plots of the observed l / V as a function of diamide concentration are nearly linear. Similar results were obtained with protamine and casein as substrates. Although the kinetics of the inhibition are complex, it is possible to conclude from these experiments that the inhibition by diamide is non-competitive and is not dependent on the nature of the protein substrate used. Therefore, the data do support the earlier conclusion that diamide is reacting with groups in the enzyme rather than with protein substrates. They further suggest that diamide does not influence the binding of the substrates to the enzyme.

The effect of ATP on the activity of protein kinase at different concentrations of diamide is shown in Fig. 3. The inhibition by diamide is non-competitive with respect to ATP. The kinetic properties are very similar to those observed with protein substrates. The K, for ATP, 7 pM, was not altered by the addition of diamide and the 1/V values changed in a non- linear fashion with respect to the concentration of inhibitor.

The velocity of the protein kinase reaction was stimulated about 100 % when cyclic AMP was added to the reaction mixture. However, increasing the concentration of cyclic AMP as a function of the concentra-

Fraction number

Fig.4. Influence of diamide on the rate of sedimentation of protein kinase in sucrose density gradients. In each case, 50 units of protein kinase in 0.1 ml of 20 mM Tris-HCI, pH 7.0, was layered onto a 5 - 20 % linear sucrose gradient (5.0 ml) containing 10 ml Tris-HCI, pH 7.0, 10 mM 2-mercaptoethanol and 1 mM EDTA. The sample was centrifuged at 49000 rev./min for 16 h at 3 "C in a SW 50.1 rotor and afterwards the solution was fractionated as described by Martin and Ames [27]. The activity of the enzyme was assayed by the standard procedure. Tubes containing 0.15-ml fractions were numbered starting from the bottom of the gradient. (I) Obtained with 50 units of the purified protein kinase; (11) obtained when 6 mM diamide was added to the enzyme solution and included in the sucrose gradient mixture; (111) obtained when 0.5 mg histone per ml was added to the enzyme and sucrose gradient mixture; (IV) obtained when 6 mM diamide and 0.5 mg histone per ml were added

tion of diamide did not significantly influence the extent of inhibition. The inhibition was non-competitive with respect to cyclic AMP, which suggested that diamide inhibited both the intact and dissociated forms of protein kinase.

Mechanism of Inhibition of Protein Kinase

To further understand the nature of the interaction between diamide and protein kinase, the sedimentation behavior of the enzyme in the presence of diamide, histone and cyclic AMP was examined by sucrose density centrifugation. The profiles of protein kinase activity after sedimentation in the presence of histone and diamide are shown in Fig.4. In the absence of histone and diamide (Fig.4, curve I), the enzyme is found near the bottom of the gradient (molecular weight, 170000). Histone dissociates the catalytic and regulatory subunits of protein kinase, and when it is added to the gradient the catalytically active component of protein kinase (molecular weight, 120000) is found near the middle of the gradient


(Fig.4, curve 111). When diamide is added in the absence of histone, only one smaller peak (curve 11) in the same position as that of the native enzyme (curve I) was found. Moreover, all of the protein was recovered in this peak which demonstrated that inhibition by diamide could occur without dissociation of the native enzyme.

Alternatively, when diamide and histone were both added, a smaller peak (curve IV) in the same position as the dissociated enzyme (curve 111) was found. Thus, diamide also inhibited the dissociated form of protein kinase. It should be noted that although histone activates protein kinase by dissociating it into its catalytically active subunit (curve I compared to curve 111), the extent of inhibition of the intact enzyme (curve I1 compared to curve I) was essentially the same as that of the dissociated enzyme (curve IV compared to curve 111).

These results were not influenced by the manner in which protein kinase was dissociated, since the same results were obtained when protein kinase was dissociated by the addition of 0.5 mM cyclic AMP to the centrifugation medium. The evidence obtained in these experiments demonstrate that diamide inhibits both intact protein kinase as well as the dissociated form of the enzyme. The data support the observation that diamide interacts with the catalytically active subunit of the enzyme and not with substrates or bound regulatory subunits. The inhibition of protein kinase was reversible and almost all of the activity could be recovered if diamide was removed from the reaction mixture. The reversibility of the inhibition was demonstrated by treating a sample of purified enzyme with a specific activity of 600 pmol x min-' x mg-' with 18 mM diamide under the standard assay conditions. The inhibited sample of purified enzyme with a specific activity xmin-' xmg-' was then dialyzed for 8 h against 4 1 of 0.02 M Tris-HC1, pH 7.0. After dialysis the specific activity of the sample was restored to 570 pmol x min-' x mg-'. When an aliquot of the inhibited reaction mixture was passed into a Sephadex G-25 column (2.2 x 25 cm) and eluted with 0.02 M Tris-HC1, pH 7.0, the specific activity of the reisolated sample was 550 pmol x min-l x mg-'. The removal of diamide by either dialysis or gel filtration results in almost complete recovery of enzyme activity.

DISCUSSION

Data obtained in the present study show that gluconeogenesis in rat kidney cortex slices is reversibly inhibited by diamide. The pattern of inhibition of gluconeogenesis as well as amino acid and sugar transport [3] in many ways bears a striking similarity to the inhibition of endogenous and purified kidney

protein kinases [lo]. Low concentrations of diamide, 1 mM, has little or no effect on these processes, whereas 4-6 mM diamide causes about 50% inhibition. The extent of inactivation by diamide in the presence of cyclic AMP is nearly the same when either sugar transport or protein kinase activity [lo] is measured. The inhibitory effects are reversible in all of these systems. These similarities suggest that decreased gluconeogenesis and transport in kidney cortex slices may result from the inhibition of protein kinase by diamide. The exact role of protein kinases in the regulation of gluconeogenesis is not presently known, however the results obtained in the present study suggest that rate-limiting steps involved in the transfer of substrates in membrane fractions may be a principal site of action of protein kinases and diamide.

Diamide inhibited gluconeogenesis from all the substrates tested, including fructose and glycerol. Other inhibitors of gluconeogenesis have been used in similar studies and in some cases the point of inhibition has been determined. Hypoglycin inhibits gluconeogenesis from pyruvate, malate, glyceraldehyde 3-phosphate and fructose 1,6-bisphosphate, but not from fructose 6-phosphate or fructose ; thus hypoglycin is an inhibitor of fructose 1,6-bisphosphatase [35]. Quinolinate inhibits gluconeogenesis from pyruvate, malate, and 2-oxoglutarate, but not from glycerol, dihydroxyacetone or fructose. Quinolinate is believed to be an inhibitor of phosphoenolpyruvate carboxykinase [36]. The fact that diamide inhibits gluconeogenesis from all of the substrates tested would argue against the possibility that diamide in an inhibitor of one of the gluconeogenic enzymes.

The results obtained in a number of studies indicate a direct involvement of cyclic-AMP-dependent protein kinases in the regulation of gluconeogenesis [37- 391. Our present findings suggest that diamide may limit the rate of gluconeogenesis by inhibiting protein kinases which are involved in the regulation of glucose transport from cells in the kidney cortex. Support for this suggestion was obtained in experiments which showed that the cyclic-AMP-stimulated transport of a-methylglucoside was inhibited by diamide. Di- amide also inhibited Na' , K+-dependent ATPase and glucose-6-phosphatase in isolated microsome and brush border membrane preparations. Both of these enzymes are thought to have important functions in transport processes [40]. However, it did not inhibit the alkaline phosphatase nor Mg2 +-dependent ATPase which are also present in these same subcellular fractions. It is not yet certain whether these enzymes are directly inhibited by diamide, or whether the decrease in activity is an indirect result of the inhibition of protein kinase in these preparations. The exact mechanism responsible for the active transport of amino acids and sugars in kidney is not well under- stood. However, the inhibition of protein kinase,


Na+ , K +-dependent ATPase and glucosed-phosphatase by diamide is certainly consistent with the observed decrease in amino acid and a-methylglycoside transport in renal cortical slices [l - 31.

The phosphorylation and dephosphorylation of membrane proteins may play a role in the regulation of transport processes in kidney [13,14]. Our previous studies have demonstrated that renal brush border membranes contain protein kinase and phosphoprotein phosphatase, as well as an endogenous bound protein which can serve as a substrate for these two enzymes [14].

In earlier studies the inhibitory effects of diamide on various cellular processes were generally vaguely interpreted in terms of its ability to specifically oxidize reduced glutathione [l - 71. Diamide oxidizes reduced glutathione stoichiometrically [6,41,42], and causes the rapid depletion of reduced glutathione in tissue slices [5,6]. The reaction of diamide with reduced glutathione is reversible. Although diamide also oxidizes some thiol groups in proteins [41,42], most evidence indicates that it cannot effect the activity of isolated enzymes by this mechanism. In view of these observations it was of interest to examine the influence of different thiols on the extent of inactivation of protein kinase by diamide. Sur- prisingly, the addition of sulfhydryl reagents increased the extent of inactivation of purified protein kinase by diamide [lo]. In the presence of 9 mM dithiothreitol, reduced glutathione or cysteine only 9 mM, 5.4 mM and 4.2 mM diamide, respectively, were required for 50 % inhibition compared to 12.7 mM in the absence of sulfhydryl compounds. These results demonstrated that the inhibition of protein kinase by diamide did not involve the oxidation of sulfhydryl groups in the enzyme. Ordinarily protein kinase is assayed in the presence of 9 mM cysteine and sulfhydryl reagents stabilize the enzyme during storage. The interesting fact which emerges from these studies is that the sulfhydryl reagents, at concentrations in excess of diamide, which would be expected to overcome the effects of the inhibitor, in fact increase the affinity of the enzyme for the inhibitor, leading to an apparent increase in the inhibition. Thus under conditions where most of the diamide is in the reduced form the inhibition of protein kinase is increased which suggests that the reduced form of diamide may be a more effective inhibitor of the enzyme. Clearly, the oxidative reactions usually associated with the action of diamide cannot explain its inhibitory effects on protein kinases.

Studies with purified enzymes demonstrated that diamide inhibits soluble cyclic-AMP-dependent protein kinases, phosphorylase kinase, pyruvate dehydrogenase kinase and particulate protein kinases present in subcellular fractions isolated from kidney homo- genates. Diamide interacts with protein kinases in such a way as to give rise to enzymatically inactive

complexes. Enzymes inhibited by diamide have ex- tensive nonpolar areas in their surfaces which can combine with the inhibitor through hydrophobic binding. The resulting conformational changes can apparently cause a loss of catalytic activity without influencing interactions between bound substrates and functional groups at the active site of the enzyme. The results obtained in the kinetic studies show a complex noncompetitive inhibition with respect to protein substrates, ATP and cyclic AMP. Since no evidence for the dissociation of subunits was observed in sucrose density centrifugation studies, the inhibitor does not act by causing the dissociation of the enzyme into inactive subunits. These data further suggest that the enzyme can bind protein substrates and the inhibitor simultaneously to form inactive ternary complexes.

This investigation was supported by United States Public Health Grant AM 13150 from the National Institute of Arthritis, Metabol- ism and Digestive Diseases and USPHS-NIH Grant FR 5363. We wish to thank Dr Robert A. McRorie of the Department of Bio- chemistry, the University of Georgia, for his assistance in the assay of the proteolytic enzymes.

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D. Pillion, F. H. Leibach, and H. Rocha, Department of Cell and Molecular Biology, Medical College of Georgia, Augusta, Georgia, U.S.A. 30902

F. J. Von Tersch and J. Mendicino*, Department of Biochemistry, Graduate Studies Research Center, University of Georgia, Athens, Georgia, U.S.A. 30602

* To whom correspondence should be addressed.

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The Reversible Inhibition of Gluconeogenesis in Kidney Cortex by Diazenedicarboxylic Acid...

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