Eur. J. Biochem. 75, 405 - 409 ( 1 977)
Urea as a Selective Inhibitor of Argininosuccinate Lyase JBnos M t N Y H A R T and Jozsef G R O F Cell Biochemistry Research Laboratory, Sernrnelweis Medical University, Budapest (Received October 28, 1975/January 4, 1977)
The effect of urea on various ornithine cycle enzymes has been investigated. It was demonstrated that argininosuccinate lyase was the only ornithine cycle enzyme inhibited by urea in a competitive manner. Based on the data presented the possible role of urea in maintaining a physiological range of intracellular and extracellular urea concentration by controlling hepatic ureogenesis was discussed.
Caldwell and McHenry reported in 1954 [ l ] that isolated liver slices derived from B6-vitamin-deficient rats disclosed lowered rate of urea formation presumably due to the elevated serum urea level characteristically present in B6-vitamin deficiency. Previous studies from this laboratory have also demonstrated that urea production by rat liver slices incubated in the presence of ammonia and carbon dioxide was markedly suppressed if urea was also included in the incubating medium [2]. The extent of inhibition was strictly dependent on the concentration of urea : 2 and 30 mM urea elicited 35 and 95 "/,inhibition in urea production, respectively. Among mechanisms potentially responsible for the urea-dependent suppression of ureogenesis, the inhibition by urea of one or perhaps several ornithine cycle enzymes should also be considered. The present report is concerned with experiments aimed at revealing the effect of urea on the activities of various ornithine cycle enzymes.
MATERIALS A N D METHODS Measurement of' Citrulline Synthesis in Isolated Rut Liver Mitochondria Rat liver mitochondria were isolated by the method of Weinbach [3] with minor modification. The rate of mitochondria1 citrulline synthesis was estimated by measuring citrulline produced in and released from the mitochondria in a 30-min interval [4]. The assay system contained the following constituents in a final volume of 1.O ml : 15 mM KCI ; 4 mM MgCh ; 50 mM
_ _ ~ Lnz,,nw.s. Carbamoylphosphate synthase (ammonia) (EC 2.7.2.5); ornithine carbamoyltransferase (EC 2.1.3.3); argininosuccinate lyase (EC 4.3.2.1); arginase (EC 3.5.3.1); argininosuccinate synthetase (EC 6.3.4.5). ~~
Tris-HCI buffer; 1 6 m M K H C 0 3 ; 10mM NH4CI; 3 m M ATP; 1 0 m M L-ornithine; 1 0 m M sodium glutamate; 5 mM potassium phosphate buffer pH 7.4 and 25 mM sucrose. Incubation was carried out at 37 "C in small medicine bottles tightly closed with rubber stoppers. Before the onset of incubation the bottles were gassed for 10 min with a mixture of 95 0 2 5 "/, C02. Citrulline synthesis was started by the addition of freshly prepared mitochondria with the aid of a microsyringe inserted through the rubber stoppers. Incubation lasted for 30 min. The reaction was stopped with 1.O ml 35 "/, perchloric acid and any precipitated protein was removed by centrifugation. Perchloric acid in the supernatant was transformed by KOH into insoluble potassium perchlorate and then removed by subsequent centrifugation. Citrulline contained in the supernatant was separated on a 1 x 5 cm column prepared from Dowex-50 WX8 cation exchange resin (400- 800 mesh) [5]. Citrulline content of eluted fractions was assayed by the method of Archibald [6] as modified by Ceriotti [7].
+
Measurement of' Arginine Synthesis Arginine synthesis from citrulline was assayed in 1.O ml medium containing the following ingredients [8]: 5 mM ATP; 5 mM L-aspartate; 50mM potassium phosphate buffer pH 7.4; citrulline content varied occasionally between 1 - 5 mM. The enzyme needed for arginine synthesis were added to the system in the form of a cetyltrimethylammonium bromide extract (2 mg protein) prepared from rat liver according to Brown and Cohen [8]. Incubation was carried out at 37 "C for 30 min. After the inucbation had been terminated the protein was precipitated by heat denaturation (100 "C for 2 min) and removed by centrifugation. The supernatant was assayed for both citrulline and arginine content, respectively. Citrulline
406
Urea as a Selective Enzyme Inhibitor
was determined as described above. For arginine determination a modification [9] of the original Sakaguchi reaction [lo] was used. Activities of argininosuccinate synthetase and argininosuccinate lyase enzymes were determined simultaneously by measuring rate of citrulline removal (argininosuccinate synthetase) and that of arginine production (argininosuccinate lyase) in the same assay system.
Measurement of Arginase Activity Arginase activity was assayed in a system containing the following components in a final volume of 2.0 ml [8]: 0.25 mM MnClz; 25 mM sodium glycinate buffer pH 9.5; arginine content varied between 0.5 and 2.0 mM depending on the experimental conditions. Argininase was added as a cetyltrimethylammonium bromide liver extract [8] previously preincubated at 50 “C for 30 min in the presence of 0.25 mM MnCb [ll]. Incubation at 37 “C lasted for 30 min. Deproteinisation was carried out by the addition of 4.0 ml of 0.4 trichloroacetic acid followed by centrifugation. Arginine was determined in the supernatant by method described above [9]. Arginase activity was estimated by measuring rate of arginine removal from the assay system.
RESULTS
The Effect sf’ Urea on Citrulline Synthesis Citrulline synthesis from ammonia and COz taking place within the ornithine cycle is a process of intramitochondrial localisation [I 21. The two-step process is carried out by carbamoylphosphate synthetase and ornithine carbamoyltransferase respectively. Factors affecting activity of one or each of the two enzymes should evidently be mirrored in corresponding changes in the rate of mitochondrial citrulline synthesis as well. In experiments presented in Fig. 1 mitochondrial citrulline synthesis is shown to remain unaffected by urea applied either in 17 mM or in 83 mM concentration, respectively. This is an indication that none of the two enzymes involved in mitochondrial citrulline synthesis are affected by urea.
Time (min)
Fig. 1. C‘itru//irzr s ~ n t h r s i sh j ’ isohted rut liver mitochontlria in the presence and in the absence qf’ureu. Abscissa: time of incubation in min. Ordinate: citrulline produced in and released from the mitoControl; (0) 16.6 mM urea; chondria in the assay system. (0) (a)83.0 mM Urea
second enzyme involved in the synthetic process. Under the conditions employed the citrulline content of the assay system could be solely affected by the activity of argininosuccinate synthetase. This was the reason why the rate of citrulline removal could be used for measuring argininosuccinate synthetase activity. On a similar ground argininosuccinate lyase activity was assayed by measuring the rate of arginine production in the assay system.
Ejject of Urea on Argininosuccinate Synthetase Activity Fig. 2 demonstrates the effect of urea on argininosuccinate synthetase activity as measured by the rate of citrulline removal. As it is shown argininosuccinate synthetase activity was left essentially unaltered by urea in both concentrations tested.
The Eflect of Urea on Arginine Synthesis
Effect of Urea on a Argininosuccinate Lyase Activity
Arginine synthesis from citrulline is carried out extramitochondrially by the so-called ‘arginine synthetase system’ [8], the ‘over-all system’ of Ratner [13]. Two enzymes are involved in the synthetic process. Argininosuccinate synthetase is responsible for the condensation of citrulline and aspartate into argininosuccinate, the immediate precursor of arginine. Argininosuccinate generated in this step is split into fumarate and arginine by argininosuccinate lyase, the
Urea effect on argininosuccinate lyase activity measured by the rate of arginine production is shown in Fig.3. Urea was found to be a potent inhibitor of argininosuccinate lyase in all concentrations tested in these experiments. The extent of inhibition depended at any substrate concentration on the concentration of urea: e.g. at an initial substrate citrulline concentration of 5 mM and in the presence of 8.3, 16.6 and 83 mM urea, respectively approximately 18, 35 and
401
J. Menyhart and J. Grof
m
c
B
'0 [Citrulline] (rnM)
Fig. 2. Urea cqj;ct on the argininosuccinute qxthetase. Enzyme activities were estimated in the presence and in the absence of urea by measuring the rate of citrulline removal at various substrate concentrations. Abscissa: initial substrate concentrations in mM. Ordinate: rate of citrulline removal expressed in pmol citrulline I - ' min-I. Experimental conditions are described in Materials and Control; (0) 16.6 mM urea; (@) 83.0 m M urea Methods. (0)
90 inhibition of the original argininosuccinate lyase activity could be detected. The inhibition by urea of argininosuccinate lyase proved to be a competitive one. This is shown in Fig. 4 where Lineweaver-Burk plots as well as K, and V values are presented as obtained in the presence of varying concentration of urea. The percentage of urea-dependent inhibition of argininosuccinate lyase detected at a single concentration of the substrate (1 mM citrulline) and in the presence of increasing urea concentrations are shown in Fig.5. It is of considerable interest that moderate but well detectable inhibition of enzyme activity could be observed at hardly higher than physiological urea concentration (8.3 mM) already. Urea dependency of enzyme inhibition is clearly seen on the graph, approaching maximal inhibition around 4.15 mM urea concentration. It should be stressed that this is an urea concentration occasionally detected under certain pathological conditions accompanied by azotemia ( e g . uremia) [14,15].
1
2
3
5
4
6
[Citrulline] (rnM)
Fig. 3. Effbct ($urea (it1 argininosucciwte lyase. Enzyme activity was assayed both in the presence and in the absence of urea at various initial citrulline concentrations. Abscissa: initial concentrations of citrulline in mM. Ordinate: rate of arginine production expressed in pmol arginine 1- min I . Details of the assay system are described in Materials and Methods. (0)Control; ( + ) 8.3 mM urea; (0) 16.6 mM urea; (e) 83.0 m M urea
/
PP lo
*'
J
l/[Cltrullinel j m ~ ~ ' )
Fig. 4. Linebrcwver-Burk plol of tho r~~iiclioi~ s/ioirti in Fig. 3 ii.s ohrained in the presence of various urea concentrations and in rht. ahsetlc~o OJ' urea. Calculated values were; V = 172 pmol x 1 - x inin-' ; K,,, = 1.22 m M ; K,,, (apparent at 8.3 mM urea = 2.44 mM. at 16.6 mM urea = 4.90 mM, at 83 mM urea = 32.86 mM. ( 0 )Con16.6 mM urea; (0)83 m M urea trol; (+) 8.3 mM urea; (0)
The Eflect of Urea on Arginase Activity
DISCUSSION
As it is seen in Fig.6, arginase activity remained essentially unaffected regardless of whether urea was or was not included in the incubating medium.
The present finding that argininosuccinate lyase was the only ornithine cycle enzyme susceptible to urea inhibition lends considerable support to the suggestion
Urea as a Selective Enzyme Inhibitor
408
trations tested in the experiments cited above should be mentioned. In addition, however, intracellular ornithine concentration might also differ considerably in different preparations leading to substantial differences in susceptibility to urea-dependent enzyme inhibitory action observed at various urea concentrations. At such a low range of urea concentration as was tested in isolated liver preparation [16] ureadependent inhibition of argininosuccinate lyase might be easily counteracted by the elevated steady-state concentration of argininosuccinate induced by the comparatively high tissue level of ornithine presumably present in isolated and corectly perfused liver preparation. It is fully recognised that most of the assay systems used in these experiments was far from being strictly specific. The fact however that the rate of citrulline removal remained unaffected while that of arginine production became substantially depressed in the presence of urea provides a sufficiently strong argument to substantiate the suggested inhibition of argininosuccinate lyase, regardless of whether the system used was or was not strictly specific. Data presented in the results showed that urea at a nearly physiological concentration already exerted a detectable inhibition on argininosuccinate lyase. This observation, together with previous data indicating that urea at a similarly low concentration inhibited ureogenesis in rat liver slices [1,2], and also the supposition that the argininosuccinate-lyase-catalysedtransformation of the argininosuccinate molecule is the rate-limiting step within the ornithine cycle [19,20], all support the physiological significance of the observed inhibition. This, on the other hand, raises the possibility that urea might be involved also in the regulation of hepatic ureogenesis in vivo by controlling the rate of hepatic urea production. This idea of course is in sharp contradiction to the view that urea is a metabolically inert and biologically insignificant molecule devoted to excretion as the end product of protein catabolism in ureotelic organisms. The supposition that urea is in fact a metabolically active component of the mammalian organisms gains further support from direct and indirect evidence abundantly found in the pertinent literature. It was repeatedly shown e.g. that urea in higher than physiological concentrations exerts a large number or unfavourable effects upon cellular metabolism [21- 351. In the light of these data the existence of a regulatory mechanism entrusted with keeping urea concentration within normal limits in different fluid compartments of the organisms appears to be a physiological necessity. More than one factor may be involved in such a regulation, each being responsible for exerting control at different level of regulation including production, metabolisation, excretion etc. Urea-dependent inhibition of argininosuccinate lyase described in
3 .O
'0
8.316.6
41.5
83
[Urea] (mM1
Fig. 5. Inhibirion of ar-ginino,siic,crrlat~Iyase as obtained in the presence of I m M citrulline and varying concentrations of urea. The assay system was identical with that used in the experiment represented in Fig. 3. Abscissa: urea concentration; Ordinate: enzyme inhibition
[Arginine] (mM)
Fig. 6. c//ffecrof urea on ar.gina.se. Ewyme activities were determined both in the presence and in the absence of urea by measuring the rate of arginine production at various initial substrate concentrations. Abscissa : initial arginine concentrations. Ordinate: rate of arginine removal expressed in pmol arginine I-' min-'. Experimental details are described in Materials and Methods. (0)Control; (0) 16.6 m M urea; (e)83.0 m M urea
that this effect of urea may be causally related to the urea-dependent suppression of hepatic ureogenesis observed in isolated liver slices as reported previously [1,2]. These observations however appear to be in contradiction to data obtained in isolated perfused liver [16], and isolated hepatocytes [17,18] indicating a linear rate of ureogenesis over a wide range of urea concentrations, The discrepancy may be due to several factors. First of all the lower range of urea concen-
409
J . Mcnyhirt and J. Grof
this paper may be part of the suggested regulatory system exerting control on extracellular and intracellular urea concentration at the level of its hepatic production.
REFERENCES 1. Caldwcll, E. F. & McHenry, E. W. (1954) Acta Biochem. Biop h ~ . .48, ~ . 50 - 54. 2. Menyhirt, J., Horvith, A. & Rosta, A. (1971) Acta Biuchini. Biophys. Acad. Sci.Hung. 5, 145- 147. 3. Wcinbach, H. L. (1961) Anal. Biochem. 2, 335-342. 4. Charles, R., Tager, J. M. & Slater, E. C. (1967) Biochem. Biophj~s.Acta, 131, 29-41. 5. Wats, D. C., Wats, R. L. & Baldwin, A. (1965) Biochem. J . 96.
60 - 70. 6. Archibald, R. M. (1944) J . B i d . Chem. 156, 121 - 142. 7. Ceriotti, G. (1973) Clin. Chim. Acta, 47, 97- 105. 8. Brown, G. W. &Cohcn, P. P. (1959)J. Biol. Cheni. 234,17691774. 9. Gilboc, D. D. & Williams, J. N . (1955) Proc. Soc. E . Y ~ B . id. Met/. 91, 535-536. 10. Sakaguchi, S. (1950) Biochem. J . 37, 231-235. 11. Hcllerman, L. M. & Perkins, E. (1935) J . Biol. Chmz. 112, 175 180. 12. Grisolia, S. & Cohcn, P. P. (1953)J. Biol. Chrm. 204,753-757. 13. Ratncr, S. (1955) Methods Enzymol. 2, 359. 14. Kennedy, A. G., Linton, A. L. & Eaton, J. C. (1962) Lancet, I I , 410-411. 15. Schacknian, R.. Holden, A. J., Chisholm, G. B. & Tigott, R . W. (1962) Brit. M6.d. J . (August) 356-358. 16. Chanialaun, R . A. F. M . & Tager, J. M . (1970) Biochim. Biophys. Acta, 222, 1 19 - 134. -
17. Williamson, R., Mcijer, A. J. & Ohkawa, K. (1974) in Regulation of Hepatic Metabolism (Lundquist, F. & Tygstrup, N., cds), pp. 457-479, Munksgaard, Copenhagen. 18. Tager, J. M., Zunrcndonk, P. F. & Akerboom, T. P. M. (1943) in Inborn Errors of’Metabolism (Hommes, F. A. & Van Den Berg, C. J., eds), pp. 177-200, Academic Press, London. 19. Krebs, H. A. & Hcnselcit, K. (1932) Hoppe-Seyler’s Z . Phy.~io/. Chenz. 210, 33-45. 20. Freedland, R. A. & Sodikoff, C. H. (1962) Proc. Sac. E Y ~ Biol. . Med. 109,394- 396. 21. Giordano, C., Bloom, I. & Merill, I. P. (1967)J. Lab. Clin. Met/. 59, 396-401. 22. Rayagapolan, K., Fridowich, K. V. & Handler, P . (1960) Fetl. Proc. 19,49- 50. 23. Withycombe, W. A. (1965) Biochem. J . 94, 384-389. 24. Harris, J. I . (1956) Nature (Lond.) 177, 471 -473. 25. Obenaus, H. & Guidoux, R. (1969) E.xperientia (Busel), 25, 1064-1067. 26. Lascclles, P. T. & Taylor, W. H. (1966) Clin. Sci. (O.t:/:) 31. 403-409. 27. Robinson, J. R. (1957) J . Physiol. (Lorid.) 137, 21-29. 28. Robinson, J. R. (1962) J . Pliysiol. (Lond.) 164, 552-558. 29. Bccker, E. & Koch, F. (1925) Dtsch. Arch. K1in. Med. 148, 7885. 30. Pankow, D. & Pohle, K. (1968) Z. Klin. Chenz. 6,369-374. 31. Gregory, M . & Robinson, J. R . (1965) J . Physiol. (Lond.) 177. 122- 131. 32. Baur, M. (1932) Areh. e.Yp. Pathol. Pharmacol. 167, 104- 112. 33. Grollman, E. F. & Grollman, A. (1959) J . Clin. Invest. 38, 749 - 754. 34. Hutching, R. H., Hcgstrom, R. M . & Scribner, B. H. (1966) Ann. Int. Med. 65, 275-283. 35. Horowitz, H. L. & Boston, M. D. (1970) Areh. hlt. Mrcl. 120. 823 - 826.
J. Mcnyhirt and J . Grof, Scjtbiokemiai Kutato Laboratorium, Semmclwcis Orvostudominyi Egyetem, Urologiai Klinika, Ulloi ut 78/b, H-1082 Budapest, Hungary
Urea as a Selective Inhibitor of Argininosuccinate Lyase JBnos M t N Y H A R T and Jozsef G R O F Cell Biochemistry Research Laboratory, Sernrnelweis Medical University, Budapest (Received October 28, 1975/January 4, 1977)
The effect of urea on various ornithine cycle enzymes has been investigated. It was demonstrated that argininosuccinate lyase was the only ornithine cycle enzyme inhibited by urea in a competitive manner. Based on the data presented the possible role of urea in maintaining a physiological range of intracellular and extracellular urea concentration by controlling hepatic ureogenesis was discussed.
Caldwell and McHenry reported in 1954 [ l ] that isolated liver slices derived from B6-vitamin-deficient rats disclosed lowered rate of urea formation presumably due to the elevated serum urea level characteristically present in B6-vitamin deficiency. Previous studies from this laboratory have also demonstrated that urea production by rat liver slices incubated in the presence of ammonia and carbon dioxide was markedly suppressed if urea was also included in the incubating medium [2]. The extent of inhibition was strictly dependent on the concentration of urea : 2 and 30 mM urea elicited 35 and 95 "/,inhibition in urea production, respectively. Among mechanisms potentially responsible for the urea-dependent suppression of ureogenesis, the inhibition by urea of one or perhaps several ornithine cycle enzymes should also be considered. The present report is concerned with experiments aimed at revealing the effect of urea on the activities of various ornithine cycle enzymes.
MATERIALS A N D METHODS Measurement of' Citrulline Synthesis in Isolated Rut Liver Mitochondria Rat liver mitochondria were isolated by the method of Weinbach [3] with minor modification. The rate of mitochondria1 citrulline synthesis was estimated by measuring citrulline produced in and released from the mitochondria in a 30-min interval [4]. The assay system contained the following constituents in a final volume of 1.O ml : 15 mM KCI ; 4 mM MgCh ; 50 mM
_ _ ~ Lnz,,nw.s. Carbamoylphosphate synthase (ammonia) (EC 2.7.2.5); ornithine carbamoyltransferase (EC 2.1.3.3); argininosuccinate lyase (EC 4.3.2.1); arginase (EC 3.5.3.1); argininosuccinate synthetase (EC 6.3.4.5). ~~
Tris-HCI buffer; 1 6 m M K H C 0 3 ; 10mM NH4CI; 3 m M ATP; 1 0 m M L-ornithine; 1 0 m M sodium glutamate; 5 mM potassium phosphate buffer pH 7.4 and 25 mM sucrose. Incubation was carried out at 37 "C in small medicine bottles tightly closed with rubber stoppers. Before the onset of incubation the bottles were gassed for 10 min with a mixture of 95 0 2 5 "/, C02. Citrulline synthesis was started by the addition of freshly prepared mitochondria with the aid of a microsyringe inserted through the rubber stoppers. Incubation lasted for 30 min. The reaction was stopped with 1.O ml 35 "/, perchloric acid and any precipitated protein was removed by centrifugation. Perchloric acid in the supernatant was transformed by KOH into insoluble potassium perchlorate and then removed by subsequent centrifugation. Citrulline contained in the supernatant was separated on a 1 x 5 cm column prepared from Dowex-50 WX8 cation exchange resin (400- 800 mesh) [5]. Citrulline content of eluted fractions was assayed by the method of Archibald [6] as modified by Ceriotti [7].
+
Measurement of' Arginine Synthesis Arginine synthesis from citrulline was assayed in 1.O ml medium containing the following ingredients [8]: 5 mM ATP; 5 mM L-aspartate; 50mM potassium phosphate buffer pH 7.4; citrulline content varied occasionally between 1 - 5 mM. The enzyme needed for arginine synthesis were added to the system in the form of a cetyltrimethylammonium bromide extract (2 mg protein) prepared from rat liver according to Brown and Cohen [8]. Incubation was carried out at 37 "C for 30 min. After the inucbation had been terminated the protein was precipitated by heat denaturation (100 "C for 2 min) and removed by centrifugation. The supernatant was assayed for both citrulline and arginine content, respectively. Citrulline
406
Urea as a Selective Enzyme Inhibitor
was determined as described above. For arginine determination a modification [9] of the original Sakaguchi reaction [lo] was used. Activities of argininosuccinate synthetase and argininosuccinate lyase enzymes were determined simultaneously by measuring rate of citrulline removal (argininosuccinate synthetase) and that of arginine production (argininosuccinate lyase) in the same assay system.
Measurement of Arginase Activity Arginase activity was assayed in a system containing the following components in a final volume of 2.0 ml [8]: 0.25 mM MnClz; 25 mM sodium glycinate buffer pH 9.5; arginine content varied between 0.5 and 2.0 mM depending on the experimental conditions. Argininase was added as a cetyltrimethylammonium bromide liver extract [8] previously preincubated at 50 “C for 30 min in the presence of 0.25 mM MnCb [ll]. Incubation at 37 “C lasted for 30 min. Deproteinisation was carried out by the addition of 4.0 ml of 0.4 trichloroacetic acid followed by centrifugation. Arginine was determined in the supernatant by method described above [9]. Arginase activity was estimated by measuring rate of arginine removal from the assay system.
RESULTS
The Effect sf’ Urea on Citrulline Synthesis Citrulline synthesis from ammonia and COz taking place within the ornithine cycle is a process of intramitochondrial localisation [I 21. The two-step process is carried out by carbamoylphosphate synthetase and ornithine carbamoyltransferase respectively. Factors affecting activity of one or each of the two enzymes should evidently be mirrored in corresponding changes in the rate of mitochondrial citrulline synthesis as well. In experiments presented in Fig. 1 mitochondrial citrulline synthesis is shown to remain unaffected by urea applied either in 17 mM or in 83 mM concentration, respectively. This is an indication that none of the two enzymes involved in mitochondrial citrulline synthesis are affected by urea.
Time (min)
Fig. 1. C‘itru//irzr s ~ n t h r s i sh j ’ isohted rut liver mitochontlria in the presence and in the absence qf’ureu. Abscissa: time of incubation in min. Ordinate: citrulline produced in and released from the mitoControl; (0) 16.6 mM urea; chondria in the assay system. (0) (a)83.0 mM Urea
second enzyme involved in the synthetic process. Under the conditions employed the citrulline content of the assay system could be solely affected by the activity of argininosuccinate synthetase. This was the reason why the rate of citrulline removal could be used for measuring argininosuccinate synthetase activity. On a similar ground argininosuccinate lyase activity was assayed by measuring the rate of arginine production in the assay system.
Ejject of Urea on Argininosuccinate Synthetase Activity Fig. 2 demonstrates the effect of urea on argininosuccinate synthetase activity as measured by the rate of citrulline removal. As it is shown argininosuccinate synthetase activity was left essentially unaltered by urea in both concentrations tested.
The Eflect of Urea on Arginine Synthesis
Effect of Urea on a Argininosuccinate Lyase Activity
Arginine synthesis from citrulline is carried out extramitochondrially by the so-called ‘arginine synthetase system’ [8], the ‘over-all system’ of Ratner [13]. Two enzymes are involved in the synthetic process. Argininosuccinate synthetase is responsible for the condensation of citrulline and aspartate into argininosuccinate, the immediate precursor of arginine. Argininosuccinate generated in this step is split into fumarate and arginine by argininosuccinate lyase, the
Urea effect on argininosuccinate lyase activity measured by the rate of arginine production is shown in Fig.3. Urea was found to be a potent inhibitor of argininosuccinate lyase in all concentrations tested in these experiments. The extent of inhibition depended at any substrate concentration on the concentration of urea: e.g. at an initial substrate citrulline concentration of 5 mM and in the presence of 8.3, 16.6 and 83 mM urea, respectively approximately 18, 35 and
401
J. Menyhart and J. Grof
m
c
B
'0 [Citrulline] (rnM)
Fig. 2. Urea cqj;ct on the argininosuccinute qxthetase. Enzyme activities were estimated in the presence and in the absence of urea by measuring the rate of citrulline removal at various substrate concentrations. Abscissa: initial substrate concentrations in mM. Ordinate: rate of citrulline removal expressed in pmol citrulline I - ' min-I. Experimental conditions are described in Materials and Control; (0) 16.6 mM urea; (@) 83.0 m M urea Methods. (0)
90 inhibition of the original argininosuccinate lyase activity could be detected. The inhibition by urea of argininosuccinate lyase proved to be a competitive one. This is shown in Fig. 4 where Lineweaver-Burk plots as well as K, and V values are presented as obtained in the presence of varying concentration of urea. The percentage of urea-dependent inhibition of argininosuccinate lyase detected at a single concentration of the substrate (1 mM citrulline) and in the presence of increasing urea concentrations are shown in Fig.5. It is of considerable interest that moderate but well detectable inhibition of enzyme activity could be observed at hardly higher than physiological urea concentration (8.3 mM) already. Urea dependency of enzyme inhibition is clearly seen on the graph, approaching maximal inhibition around 4.15 mM urea concentration. It should be stressed that this is an urea concentration occasionally detected under certain pathological conditions accompanied by azotemia ( e g . uremia) [14,15].
1
2
3
5
4
6
[Citrulline] (rnM)
Fig. 3. Effbct ($urea (it1 argininosucciwte lyase. Enzyme activity was assayed both in the presence and in the absence of urea at various initial citrulline concentrations. Abscissa: initial concentrations of citrulline in mM. Ordinate: rate of arginine production expressed in pmol arginine 1- min I . Details of the assay system are described in Materials and Methods. (0)Control; ( + ) 8.3 mM urea; (0) 16.6 mM urea; (e) 83.0 m M urea
/
PP lo
*'
J
l/[Cltrullinel j m ~ ~ ' )
Fig. 4. Linebrcwver-Burk plol of tho r~~iiclioi~ s/ioirti in Fig. 3 ii.s ohrained in the presence of various urea concentrations and in rht. ahsetlc~o OJ' urea. Calculated values were; V = 172 pmol x 1 - x inin-' ; K,,, = 1.22 m M ; K,,, (apparent at 8.3 mM urea = 2.44 mM. at 16.6 mM urea = 4.90 mM, at 83 mM urea = 32.86 mM. ( 0 )Con16.6 mM urea; (0)83 m M urea trol; (+) 8.3 mM urea; (0)
The Eflect of Urea on Arginase Activity
DISCUSSION
As it is seen in Fig.6, arginase activity remained essentially unaffected regardless of whether urea was or was not included in the incubating medium.
The present finding that argininosuccinate lyase was the only ornithine cycle enzyme susceptible to urea inhibition lends considerable support to the suggestion
Urea as a Selective Enzyme Inhibitor
408
trations tested in the experiments cited above should be mentioned. In addition, however, intracellular ornithine concentration might also differ considerably in different preparations leading to substantial differences in susceptibility to urea-dependent enzyme inhibitory action observed at various urea concentrations. At such a low range of urea concentration as was tested in isolated liver preparation [16] ureadependent inhibition of argininosuccinate lyase might be easily counteracted by the elevated steady-state concentration of argininosuccinate induced by the comparatively high tissue level of ornithine presumably present in isolated and corectly perfused liver preparation. It is fully recognised that most of the assay systems used in these experiments was far from being strictly specific. The fact however that the rate of citrulline removal remained unaffected while that of arginine production became substantially depressed in the presence of urea provides a sufficiently strong argument to substantiate the suggested inhibition of argininosuccinate lyase, regardless of whether the system used was or was not strictly specific. Data presented in the results showed that urea at a nearly physiological concentration already exerted a detectable inhibition on argininosuccinate lyase. This observation, together with previous data indicating that urea at a similarly low concentration inhibited ureogenesis in rat liver slices [1,2], and also the supposition that the argininosuccinate-lyase-catalysedtransformation of the argininosuccinate molecule is the rate-limiting step within the ornithine cycle [19,20], all support the physiological significance of the observed inhibition. This, on the other hand, raises the possibility that urea might be involved also in the regulation of hepatic ureogenesis in vivo by controlling the rate of hepatic urea production. This idea of course is in sharp contradiction to the view that urea is a metabolically inert and biologically insignificant molecule devoted to excretion as the end product of protein catabolism in ureotelic organisms. The supposition that urea is in fact a metabolically active component of the mammalian organisms gains further support from direct and indirect evidence abundantly found in the pertinent literature. It was repeatedly shown e.g. that urea in higher than physiological concentrations exerts a large number or unfavourable effects upon cellular metabolism [21- 351. In the light of these data the existence of a regulatory mechanism entrusted with keeping urea concentration within normal limits in different fluid compartments of the organisms appears to be a physiological necessity. More than one factor may be involved in such a regulation, each being responsible for exerting control at different level of regulation including production, metabolisation, excretion etc. Urea-dependent inhibition of argininosuccinate lyase described in
3 .O
'0
8.316.6
41.5
83
[Urea] (mM1
Fig. 5. Inhibirion of ar-ginino,siic,crrlat~Iyase as obtained in the presence of I m M citrulline and varying concentrations of urea. The assay system was identical with that used in the experiment represented in Fig. 3. Abscissa: urea concentration; Ordinate: enzyme inhibition
[Arginine] (mM)
Fig. 6. c//ffecrof urea on ar.gina.se. Ewyme activities were determined both in the presence and in the absence of urea by measuring the rate of arginine production at various initial substrate concentrations. Abscissa : initial arginine concentrations. Ordinate: rate of arginine removal expressed in pmol arginine I-' min-'. Experimental details are described in Materials and Methods. (0)Control; (0) 16.6 m M urea; (e)83.0 m M urea
that this effect of urea may be causally related to the urea-dependent suppression of hepatic ureogenesis observed in isolated liver slices as reported previously [1,2]. These observations however appear to be in contradiction to data obtained in isolated perfused liver [16], and isolated hepatocytes [17,18] indicating a linear rate of ureogenesis over a wide range of urea concentrations, The discrepancy may be due to several factors. First of all the lower range of urea concen-
409
J . Mcnyhirt and J. Grof
this paper may be part of the suggested regulatory system exerting control on extracellular and intracellular urea concentration at the level of its hepatic production.
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J. Mcnyhirt and J . Grof, Scjtbiokemiai Kutato Laboratorium, Semmclwcis Orvostudominyi Egyetem, Urologiai Klinika, Ulloi ut 78/b, H-1082 Budapest, Hungary
