Archives of
Hicrobiology
Arch. Microbioi. 111, 197-198 (1976)
9 by Springer-Verlag 1976
Regulation of Ornithine Transcarbamylase in Rhodotorula glutinis FRANCISCO
JOS]~ MATAIX, M a. JOSEFA FERNANDEZ, and MANUEL RUIZ AMIL
Instituto de Ferrnentaeiones Industriales del Patronato de Investigaci6nCientificay T6cnica "Juan de la Cierva" Consejo Superior de Investigaciones Cientificas (C.S.I.C.), Madrid-6, Spain
Abstract. The regulation ofornithine transcarbamylase (OTC) of Rhodotorula glutinis has been studied, by growing the yeasts in different carbon and nitrogen sources and estimating the enzyme level in crude yeasts extracts. The results show a nutritional repression of OTC by arginine, when added to the culture media as carbon, nitrogen or carbon and nitrogen sources. On the other hand ornithine does not exert any effect in the same experimental conditions. Key words: Enzyme regulation -- Ornithine transcarbamylase - OTC - Rhodotorula glutinis - Repression - Yeast -- Arginine biosynthesis - Citrulline biosynthesis - Metabolic regulation - Amino acid metabolism.
The ornithine transcarbamylase (OTC) (EC 2.1.3.3) enzyme which catalyses the synthesis of citrulline from carbamyl phosphate and ornithine, constitutes the first specific enzymatic step in the biosynthetic pathway of arginine in plants and microorganisms. In Saccharomyces cerevisiae the regulation of OTC synthesis when adding to the culture medium several amino-acids, had been investigated by different authors. Bechet et al. (1962, 1970) reported that arginine exerted a feed-back inhibition and repression. Bechet and Wiame (1965) showed that induced arginase by addition of arginine interacts with OTC. In this way a futile cycle resulting in ATP hydrolysis is avoided (suppressive regulation) (Messenguy and Wiame, 1969). Likewise Ramos et al. (1970) in an extensive work using mutants of S. cerevisiae showed OTC regulation synthesis in relation to the presence of several amino-acids and of the pool of intracellular arginine. The present communication is concerned with the OTC regulation in Rhodotorula glutinis as compared
to S. cerevisiae, using arginine and ornithine as carbon and/or nitrogen sources since R. glutinis is particularly interesting due to its non fastidiousness. The results show a nutritional repression of OTC by arginine in this yeast. The yeast cells were grown aerobically with shaking, using the synthetic medium of Hertz (1943) modified according to Fernfindez et al. (1972) without addition of ammonium sulphate. The carbon and nitrogen sources employed are indicated in each case. The growth of the yeast was determined measuring the absorbance at 660 nm. The cells were harvested at the logarithmic phase by low-speed centrifugation and washed with 0.9 ~o KCI. The yeast extracts were obtained by grinding the later cells in a mortar with twice their weight of alumina (Alcoa, A-305) extracting with twice their weight of cold 50 gM EDTA. After centrifuging for 20rain at 20000xg at 0~ the supernatant was collected. Oi~nithine transcarbamylase activity was tested as described by Jones (1962), measuring citrulline formation according to Archibald's colorimetric method (1944). Enzyme units are expressed as nmoles of citrulline formed per rain (mU) under the conditions of the assay. Protein was estimated by the method of Lowry et al. (1951). As indicated in the Table 1, when R. glutinis was grown in the presence of glucose as the carbon source and arginine as nitrogen source, it exhibits and OTC specific activity of 50 ~ of that obtained when the nitrogen source was ammonium sulphate, a fact which shows a nutritional repression by arginine. Nevertheless no ornithine effects could be observed. The results found in R. glutinis differ from those found in S. cerevisiae by Ramos et al. (1970) who clearly proved a strong repression by arginine and also some repressive effect of ornithine on OTC.
198
Arch. Microbiol., Vol. 111 (1976)
Table 1.
Levels ~ of ornithine transcarbamylase in Rhodotorula glutinis grown on different carbon and nitrogen sources
Carbon source
Glucose
Glucose
Glucose
Arginine
Ornithine
Arginine
Ornithine
Nitrogen source
(NH4)2SO4
Arginine
Ornithine
(NH4)2SO4
(NH4)2SO4
Arginine
Ornithine
202 214 196
101 92 98
220 212 191
154 144 157
228 230 163
126 130 119
196 221 212
204
97
207
151
207
125
209
Averages
Expressed as mU per mg protein
Respecting the absence of any effect in R. gtutinis OTC synthesis when ornithine is added to the culture medium, this result could be explained as a partial inhibition of the entry or ornithine into the cell by glucose, as happens with pyruvate in this yeast (Medrano et al., 1969). Notwithstanding the fact that the growth is very similar when this yeast uses ammonium sulphate, arginine or ornithine as nitrogen source, it allows us t o conclude that ornithine does not exert any regulatory effect on R. glutinis OTC synthesis. On the other hand when R. glutinis used arginine as carbon source and ammonium sulphate as the nitrogen source a lesser decrease than in the case of glucose-arginine of OTC specific activity can be observed. No effect was observed when ornithine was added to the culture medium in the presence of ammonium sulphate as nitrogen source as is the case for the glucose-ornithine cultures. The minor repressive effect of arginine when used as carbon source on the levels of OTC in R. glutinis, could be explained according to Grenson (1966) as an inhibition of a specific permease for each amino acid by ammonium ion. F r o m the results obtained when arginine or ornithine are both carbon and nitrogen sources for R. glut&is we can conclude that the arginine acts as a nutritional repressor for OTC in this yeast and that ornithine has neither direct nor indirect influence on the synthesis of this enzyme. The studies about growth of R. glutinis on arginine or ornithine as the only nutritional source flows us to discard the above mentioned inhibition of the entry of both amino acids into the cell, by ammonium ion, because in those conditions the growth is even slower than when ammonium ion is also present. The behaviour of R. glutinis due to OTC repression by arginine and ornithine as compared to S. cerevisiae might be due to a major catabolic activity ofR. glutinis
for these amino acids. This could determine a lesser accumulation of them in the cell either attenuating or suppressing its repressive effects.
REFERENCES Archibald, R. M, : Determination of citrulline and allantoin and demonstration of citrulline in blood plasma. J. biol. Chem. 156, 121 - 142 (1944) Bechet, J., Grenson, M., Wiame, J. M.: Mutations affecting the repressibiIity of arginine biosynthetic enzymes in Saccharomyces cerevisiae. Europ. J. Biochem. 12, 3 1 - 39 (1970) Bechet, J., Wiame, J . M . : Indication of a specific regulatorybinding protein for ornithine transcarbamylase in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 21,226 - 2 3 4 (1965) Bechet, J., Wiame, J. M., DeDeken, R., Grenson, M. : Regulation de la synth+se de l'ornithine transcarbamylase chez Saccharomycescerevisiae. Arch. int. Physiol. Biochim. 70, 564-570 (1962) Fernandez, M.J., Garcia, M.V., Llaguno, C., Garrido, J. : Metabolisme de l'6thanol par les 16vures de
Hicrobiology
Arch. Microbioi. 111, 197-198 (1976)
9 by Springer-Verlag 1976
Regulation of Ornithine Transcarbamylase in Rhodotorula glutinis FRANCISCO
JOS]~ MATAIX, M a. JOSEFA FERNANDEZ, and MANUEL RUIZ AMIL
Instituto de Ferrnentaeiones Industriales del Patronato de Investigaci6nCientificay T6cnica "Juan de la Cierva" Consejo Superior de Investigaciones Cientificas (C.S.I.C.), Madrid-6, Spain
Abstract. The regulation ofornithine transcarbamylase (OTC) of Rhodotorula glutinis has been studied, by growing the yeasts in different carbon and nitrogen sources and estimating the enzyme level in crude yeasts extracts. The results show a nutritional repression of OTC by arginine, when added to the culture media as carbon, nitrogen or carbon and nitrogen sources. On the other hand ornithine does not exert any effect in the same experimental conditions. Key words: Enzyme regulation -- Ornithine transcarbamylase - OTC - Rhodotorula glutinis - Repression - Yeast -- Arginine biosynthesis - Citrulline biosynthesis - Metabolic regulation - Amino acid metabolism.
The ornithine transcarbamylase (OTC) (EC 2.1.3.3) enzyme which catalyses the synthesis of citrulline from carbamyl phosphate and ornithine, constitutes the first specific enzymatic step in the biosynthetic pathway of arginine in plants and microorganisms. In Saccharomyces cerevisiae the regulation of OTC synthesis when adding to the culture medium several amino-acids, had been investigated by different authors. Bechet et al. (1962, 1970) reported that arginine exerted a feed-back inhibition and repression. Bechet and Wiame (1965) showed that induced arginase by addition of arginine interacts with OTC. In this way a futile cycle resulting in ATP hydrolysis is avoided (suppressive regulation) (Messenguy and Wiame, 1969). Likewise Ramos et al. (1970) in an extensive work using mutants of S. cerevisiae showed OTC regulation synthesis in relation to the presence of several amino-acids and of the pool of intracellular arginine. The present communication is concerned with the OTC regulation in Rhodotorula glutinis as compared
to S. cerevisiae, using arginine and ornithine as carbon and/or nitrogen sources since R. glutinis is particularly interesting due to its non fastidiousness. The results show a nutritional repression of OTC by arginine in this yeast. The yeast cells were grown aerobically with shaking, using the synthetic medium of Hertz (1943) modified according to Fernfindez et al. (1972) without addition of ammonium sulphate. The carbon and nitrogen sources employed are indicated in each case. The growth of the yeast was determined measuring the absorbance at 660 nm. The cells were harvested at the logarithmic phase by low-speed centrifugation and washed with 0.9 ~o KCI. The yeast extracts were obtained by grinding the later cells in a mortar with twice their weight of alumina (Alcoa, A-305) extracting with twice their weight of cold 50 gM EDTA. After centrifuging for 20rain at 20000xg at 0~ the supernatant was collected. Oi~nithine transcarbamylase activity was tested as described by Jones (1962), measuring citrulline formation according to Archibald's colorimetric method (1944). Enzyme units are expressed as nmoles of citrulline formed per rain (mU) under the conditions of the assay. Protein was estimated by the method of Lowry et al. (1951). As indicated in the Table 1, when R. glutinis was grown in the presence of glucose as the carbon source and arginine as nitrogen source, it exhibits and OTC specific activity of 50 ~ of that obtained when the nitrogen source was ammonium sulphate, a fact which shows a nutritional repression by arginine. Nevertheless no ornithine effects could be observed. The results found in R. glutinis differ from those found in S. cerevisiae by Ramos et al. (1970) who clearly proved a strong repression by arginine and also some repressive effect of ornithine on OTC.
198
Arch. Microbiol., Vol. 111 (1976)
Table 1.
Levels ~ of ornithine transcarbamylase in Rhodotorula glutinis grown on different carbon and nitrogen sources
Carbon source
Glucose
Glucose
Glucose
Arginine
Ornithine
Arginine
Ornithine
Nitrogen source
(NH4)2SO4
Arginine
Ornithine
(NH4)2SO4
(NH4)2SO4
Arginine
Ornithine
202 214 196
101 92 98
220 212 191
154 144 157
228 230 163
126 130 119
196 221 212
204
97
207
151
207
125
209
Averages
Expressed as mU per mg protein
Respecting the absence of any effect in R. gtutinis OTC synthesis when ornithine is added to the culture medium, this result could be explained as a partial inhibition of the entry or ornithine into the cell by glucose, as happens with pyruvate in this yeast (Medrano et al., 1969). Notwithstanding the fact that the growth is very similar when this yeast uses ammonium sulphate, arginine or ornithine as nitrogen source, it allows us t o conclude that ornithine does not exert any regulatory effect on R. glutinis OTC synthesis. On the other hand when R. glutinis used arginine as carbon source and ammonium sulphate as the nitrogen source a lesser decrease than in the case of glucose-arginine of OTC specific activity can be observed. No effect was observed when ornithine was added to the culture medium in the presence of ammonium sulphate as nitrogen source as is the case for the glucose-ornithine cultures. The minor repressive effect of arginine when used as carbon source on the levels of OTC in R. glutinis, could be explained according to Grenson (1966) as an inhibition of a specific permease for each amino acid by ammonium ion. F r o m the results obtained when arginine or ornithine are both carbon and nitrogen sources for R. glut&is we can conclude that the arginine acts as a nutritional repressor for OTC in this yeast and that ornithine has neither direct nor indirect influence on the synthesis of this enzyme. The studies about growth of R. glutinis on arginine or ornithine as the only nutritional source flows us to discard the above mentioned inhibition of the entry of both amino acids into the cell, by ammonium ion, because in those conditions the growth is even slower than when ammonium ion is also present. The behaviour of R. glutinis due to OTC repression by arginine and ornithine as compared to S. cerevisiae might be due to a major catabolic activity ofR. glutinis
for these amino acids. This could determine a lesser accumulation of them in the cell either attenuating or suppressing its repressive effects.
REFERENCES Archibald, R. M, : Determination of citrulline and allantoin and demonstration of citrulline in blood plasma. J. biol. Chem. 156, 121 - 142 (1944) Bechet, J., Grenson, M., Wiame, J. M.: Mutations affecting the repressibiIity of arginine biosynthetic enzymes in Saccharomyces cerevisiae. Europ. J. Biochem. 12, 3 1 - 39 (1970) Bechet, J., Wiame, J . M . : Indication of a specific regulatorybinding protein for ornithine transcarbamylase in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 21,226 - 2 3 4 (1965) Bechet, J., Wiame, J. M., DeDeken, R., Grenson, M. : Regulation de la synth+se de l'ornithine transcarbamylase chez Saccharomycescerevisiae. Arch. int. Physiol. Biochim. 70, 564-570 (1962) Fernandez, M.J., Garcia, M.V., Llaguno, C., Garrido, J. : Metabolisme de l'6thanol par les 16vures de
