Biochimica et Biophysica Acta 870 (1986) 181-184 181 Elsevier

BBA 32447

Kinetics of inhibition of rat liver and kidney arginases by proline and branched-chain amino acids

Nelson Carvajal * and Stephen D. Cederbaum Departments of Psychiatry and Pediatrics and the Mental Retardation Research Center, UCLA Center for the Health

Sciences, Los Angeles, CA 90024 (U.S.A.)

(Received July 12th, 1985)

Key words: Arginase inhibition; Proline; Branched-chain amino acid; (Rat liver, kidney)

The effects of proline, leucine, isoleucine and valine on kidney and liver arginases were studied. At pH 7.5 and at nearly physiological concentrations, the branched-chain amino acids caused a significant inhibition of liver arginase A 1 and only minor effects on kidney arginase A 4- Kidney arginase was, however, much more sensitive to inhibition by proline than the liver enzyme. The inhibition of liver and kidney arginases by branched-chain amino acids was partial, indicating the existence of allosteric sites on both enzymes. The function of kidney arginase in proline biosynthesis and a possible role of branched-chain amino acids in the hydrolysis of arginine in liver is discussed.

Introduction

Mammalian arginase (L-arginine amidino- hydrolase, EC 3.5.3.1) exists in multiple forms [1-3]. The main form (more than 95%) in the liver is the cytosolic arginase A 1 whereas the mitochondrial enzyme m 4 is the main form (about 90%) in the kidney [4]. These forms also differ in charge and immunological properties [1,2,4], but in man, are identical in Km, K i for lysine and ornithine, heat stability and cation requirement for activation [5]. Kidney arginase may be augmented about 5-fold in patients with deficiency of the liver form of arginase and hyperargininemia [6]. It has been suggested that kidney arginase A 4 is involved in proline synthesis rather than in urea cycle [2,7]. A similar function was suggested for the enzyme in lactating mammary gland [8].

It has been reported that kidney arginase is

* To whom correspondence should be addressed (present address): Departamento de Biologia Molecular, Facultad de Ciencias Biolbgicas y de Recursos Naturales, Universidad de Concepci6n, Casilla 2407, Concepcibn, Chile.

more inhibited by proline and less inhibited by branched-chain amino acids than the liver enzyme [7]. In these studies, however, only high concentra- tions of the amino acids were used, the pH was 9.5 and a detailed kinetic study of the inhibitions was not presented. Considering the possible different functions of liver and kidney arginases, the effects would be of special interest, because of the pre- cursor-product relationship between arginine and proline and the involvement of branched-chain amino acids in the sparing of tissue nitrogen [9]. The present study was therefore undertaken to investigate the effects of these compounds in greater detail and under more physiological condi- tions of pH than those usually employed.

Materials and Methods

Liver arginase A 1 was purified by the method of Schimke [10], and kidney arginases A 1 and A 4

were partially purified through the DEAE-cel- lulose step of the method described by Skrzypek- Osiecka et al. [4].

0167-4838/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

182

Arginase activity was determined by measuring the production of urea [11] from arginine. The buffers were 50 mM Tris-HC1 (pH 7.5) and 50 mM glycine NaOH (pH 9.5). Before the assay, the enzymes were activated with 1 mM MnC12. Kinetic data were analyzed by double-reciprocal plots and by replots of slopes and intercepts against inhibi- tor concentrations. Nomenclature and equations used were according to Cleland and co-workers [12,13].

All chemicals were obtained from commercial sources (most from Sigma) and were the purest available.

Results

Inhibition by proline At pH 7.5, kidney arginase A 4 was 50% in-

hibited by about 4 mM proline, whereas at least 20 mM proline was required for similar inhibition of liver arginase (Fig. 1). The inhibition was linear non-competitive for both enzymes. From the data obtained with kidney arginase (Fig. 2), a K i value of 4.5 mM was calculated.

That the effect caused by proline is rather specific was indicated by the much lower effect caused by hydroxyproline. In fact, 20 mM hy-

lOO

~ 8o

F-- N 6o

< hi ~ 4 0 < Z

I 0 \0

- I

\

\ \

\

2O

1

°o 4

\

\ "O

O~

"" O .,. "O AI

"~" • ..-..~ - e All

I I I I

8 t2 16 20

PROLINE, mM

Fig. 1. Effect of proline on the activity of arginase A 1 from rat liver and A 4 from rat kidney. The buffer was 50 mM Tris-HCL (pH 7.5) and the concentration of arginine was 25 mM.

20raM PROLINE

30 RKA

l/v 20 / ~ pH 7.5

~ ~ T R O L .

I I

o 2'0 40 6'o 8'o 12o -1 1/ARGININE, M

Fig. 2. Non-competitive inhibition of rat kidney arginase A 4 (RKA) by proline at pH 7.5. The concentrations of proline were those indicated by the numbers on the lines.

droxyproline caused nearly a 50% inhibition of A 4 and had only a minor, if any, effect on A~.

At pH 9.5, 25 mM proline caused about 50% inhibition of kidney arginase A 4 and about 20% inhibition of liver arginase A~. At this pH, the inhibition of kidney arginase was linear competi- tive and the inhibition of the liver enzyme was linear non-competitive.

Inhibition by branched-chain amino acids Branched-chain amino acids were more strongly

inhibitory to the liver form of arginase, with the

II v

4 0

o,,i..11---------'5 RLA

/o o /

RKA 10 . '8' ~ ° -'----'-~° - - - - - ' - - °

. s O /

EISOLEUCINE],mM Fig. 3. Partial inhibition of liver arginase A 1 (RLA) and kidney arginase m 4 (RKA) by isoleucine at pH 7.5.

100 mM Isoleu 1 /V / 7 5

80 50

06 2 5

o4 4o

o2 ! l o

°o ~.5 5o ~ 2o e _ / J . ~ mM Isoleu ~ ~ ~ - - - - ' r 0

'

0 40 I

1/ARGININE

Fig. 4. Kinetics of inhibition of rat liver arginase A] by isoleucine. The numbers on the lines indicate the concentra- tions of inhibitor used. In the inset, the slopes of lines from the double-reciprocal plot are plotted against isoleucine concentra- tions. The buffer was 50 mM Tris-HC1 (pH 7.5).

effects decreasing in the order isoleucine > leucine > valine. Differences between the effects of iso- leucine and valine were, however, no greater than 10%. Fig. 3 shows that, at pH 7.5, a 50% inhibition of liver arginase A l was caused by 0.50-0.75 mM isoleucine, whereas more than 20 mM of the amino acid was required for a similar inhibition of kidney a r g i n a s e A 4. Fig. 3 also shows that isoleucine is a partial inhibitor of both enzymes. This indicates that the inhibitor is bound to a site different from the active site and that complexes of arginase and isoleucine are enzymatically active [13]. The inhibi- tion was found to be S-hyperbolic-I-hyperbolic non-competitive for kidney arginase and partial competitive for the liver enzyme. From the data in Fig. 4, we calculated a value of 0.4 mM for the dissociation constant of isoleucine from the en- zyme, and an increase of about 20-times in the K,, value for arginine as a consequence of the binding of the inhibitor to arginase A 1.

At pH 9.5, arginase A] was also more inhibited than kidney arginase, but the effects on both en- zymes were markedly lower than those observed at pH 7.5.

The inhibitions caused by all three branched chain amino acids were of the same type and no differences were observed between arginases A t isolated from liver and kidney. With respect to the

183

effect of other amino acids, there were no signifi- cant differences between the liver and kidney en- zymes.

Discussion

It has been suggested that the kidney supplies a considerable larger proportion of the arginine needed for body protein synthesis than the liver [14] and that the role for kidney arginase is not in the urea cycle but in the synthesis of proline [7]. These suggestions are based on the lower ratio of arginase to the arginine-synthesizing systems in the kidney [15] and the very low activity, if any, of the enzymes carbamoyl-phosphate synthetase I and ornithine carbomoyltransferase in this organ [16]. The function of arginase in the biosynthesis of proline in the k idney is also favored by the mitochondrial localization of arginase and ornithine aminotransferase [17] and the marked susceptibility of kidney arginase A 4 to inhibition by proline. One would speculate that the inhibi- tion would be of regulatory significance. A mecha- nism of this kind in which proline would exert a feedback inhibition of arginase was also suggested for the enzyme from mouse mammary tumor [18].

In contrast to kidney arginase A 4, the liver enzyme A t is inhibited by relatively low con- centrations of branched-chain amino acids. Fur- ther work is necessary to elucidate the physiologi- cal significance of the inhibition. However, certain relationships emerge from our data and available information, which seem helpful in formulating a hypothesis for future studies. The levels of branched-chain amino acids are increased after ingestion of protein and also during fasting, and there is evidence for anabolic effects of these com- pounds on muscle and liver proteins [9,10]. The synthesis of urea by rat liver in vitro is inhibited by leucine and the effect has been attributed to the stimulation of glutamate dehydrogenase, which re- suits in increased glutamate synthesis [20]. Due to the partial inhibition caused by branched-chain amino acids on arginase and the large excess of this enzyme in the liver [21,22], the hydrolysis of arginine would be not the rate-limiting step in urea cycle, even at high concentrations of the inhibitors. This would explain the failure of leucine to inhibit the production of urea from citrulline in liver

184

extracts [20]. However , under condi t ions where the flux of ammon ia to the urea cycle is decreased by leucine, the inhibi t ion of arginase may help to prevent the unnecessary loss of arginine genera ted outs ide the urea cycle.

The par t ia l inhibi t ions caused by b ranched chain amino acids are consis tent with the existence of an al losteric site on arginase [13]. The b ind ing of pro l ine to a site separa te f rom the ca ta ly t ic active site is also ind ica ted by the non-compet i t ive inhibi t ions caused by the amino acid. The compe- t i t ive inhibi t ion of k idney arginase A 4 at p H 9.5 would be expla ined by a confo rma t iona l change induced by arginine, which prevents the b ind ing of the inhib i tor to the enzyme. Evidence has been presented for the existence of al loster ic sites on arginase f rom bovine l iver [23], h u m a n liver [24] and rat k idney [7]. The b ind ing to a second site was used to expla in the ac t iva t ion of k idney arginase by arginine, bu t consider ing the high levels of subs t ra te requi red to observe the effect, it was suggested that some other c o m p o u n d would be the physiological effector of the enzyme [7]. I t is t empt ing to speculate that this role would be p l ayed by pro l ine on k idney arginase and by b ranched- chain amino acids on the liver enzyme.

Acknowledgements

This work was suppor t ed in par t by the U C L A Menta l R e t a r d a t i o n Research Center , U S P H S G r a n t s HD-06576 and HD-04612 and by G r a n t 6-428 f rom the March of Dimes Birth Defects Founda t ion . N.C. was suppor t ed by the Silbert In te rna t iona l Scholars Programs at U C L A .

References

1 Bascur, L., Cabello, J., V61iz, M. and Gonzhlez, A. (1966) Biochim. Biophys. Acta 128, 149-154

2 Glass, R. and Knox, W.E. (1973) J. Biol. Chem. 248, 5785-5789

3 Porembska, Z., Baranczyk, A. and Jachimowicz, J. (1971) Acta Biochim. Pol. 18, 77-85

4 Skrzypek-Osiecka, I., Robin, Y. and Porembska, Z. (1983) Acta Biochim. Pol. 30, 83-92

5 Spector, E.B., Rice, S.C.H., Moedjono, S., Bernard, B. and Cederbaum, S.D. (1982) Biochem. Med. 28, 165-175

6 Spector, E.B., Kern, R.M., Haggerty, D.F. and Cederbaum, S.D. (1985) Mol. Cell Biochem. 66, 45-53

7 Kaysen, G.A. and Strecker, H.J. (1973) Biochem. J. 133, 779-788

8 Yip, M.C.M. and Knox, W.E. (1972) Biochem. J. 127, 893-899

9 Adibi, S.A. (1980) J. Lab. Clin. Med. 95, 475-484 10 Schimke, R.T. (1964) J. Biol. Chem. 239, 3808-3817 11 Archibald, R.M. (1945) J. Biol. Chem. 157, 507-518 12 Cleland, W.W. (1972) in The Enzymes (Boyer, P;D., ed.),

3rd. Edn., Vol. II, pp. 1-65, Academic Press, New York 13 Worcel, A., Goldman, D.S. and Cleland, W.W. (1965) 240,

3399-3407 14 Featherston, W.R., Rogers, Q.R. and Freedland, R.A. (1973)

Am. J. Physiol. 224, 127-129 15 Szepesi, B., Avery, E.H. and Freedland, R.A. (1970) Am. J.

Physiol. 219, 1627-1631 16 Jones, M.E., Anderson, A.D., Anderson, C. and Hodes, S.

(1961) Arch. Biochem. Biophys. 95, 499-507 17 Herzfeld, A. and Knox, W.E. (1968) J. Biol. Chem. 243,

3327 18 Kesara Rao, K.V., Rai, S.R. and Bapat, C.V. (1974) Br. J.

Cancer 30, 129-135 19 Tischler, M.E., Desautels, M. and Goldberg, A.L. (1982) J.

Biol. Chem. 287, 1613-1621 20 Mendes-Mourao, J., McGivan, J.D. and Chappell, J.B.

(1975) Biochem. J. 146, 457-464 21 Schimke, R.T. (1962) J. Biol. Chem. 237, 1921-1924 22 Aperia, A., Broberger, D., Larson, A. and Snellman, K.

(1979) Scand. J. Lab. Invest. 39, 329-336 23 Bedino, S. (1977) Ital. J. Biochem. 26, 264-276 24 Carvajal, N., Acoria, M., Rodriguez, J.P., Fernhndez, M.

and Martinez, J. (1982) Biochim. Biophys. Acta 701, 146-148