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Regulation of sugar utilization by Aspergillus nidulans
491
PRELIMINARY NOTES BBA 2 1 2 2 2
Regulation of sugar utilization by Aspergillus nidulans Mutants of micro-organisms which lack pyruvate dehydrogenase activity (pdh; pyruvate:lipoate oxidoreductase (acceptor-acetylating), EC 1.2.4.1) require acetate for growth; when glucose is added to cultures of such mutants, pyruvate is excreted into the growth medium 1. When this experiment was performed with a pdh- mutant of Aspergillus nidulans R46, it was unexpectedly observed that little or no pyruvate was produced from added glucose as long as acetate remained in the growth medium, although pyruvate excretion began immediately if such cultures were incubated with glucose in the absence of acetate. The same effect of acetate on pyruvate formation was noted when fructose was used instead of glucose. However, acetate did not retard the onset of pyruvate formation from sucrose (Fig. I). These findings suggest that acetate, or a product of acetate metabolism, inhibits the utilisation of some sugars; since this effect is observed with hexoses but not with sucrose, the inhibition appears to be exerted on the uptake of the sugars rather than on the activity of a glycolytic enzyme. It is the purpose of this communication to present evidence in support of this view. Fg. "B
P,O I/-
14
/
12
d 8
~4 0
0
I 5
I 10
I 15
I 20
5
10
15 20 Tirne(~)
Fig. I. Effect of a c e t a t e on p y r u v a t e f o r m a t i o n from v a r i o u s s u g a r s b y a pdh- m u t a n t of A. nidulans. Cells, g r o w n for 44 h on m e d i a c o n t a i n i n g IOO mM a c e t a t e as sole c a r b o n source, w e re h a r v e s t e d , w a s h e d a n d r e s u s p e n d e d in b a s a l m e d i u m plus t h e c a r b o n sources i n d i c a t e d . A. N o a c e t a t e w a s a d d e d . B. i o o mM a c e t a t e w a s a d d e d . P y r u v a t e f o r m a t i o n from t h e c a t a b o l i s m of 5 ° mM glucose (O), 5 ° mM fructose ( A ) or 25 mM sucrose ( × ) w a s m e a s u r e d on s a m p l e s of t h e i n c u b a t i o n med ia, b y t h e m e t h o d of FRIEDEMANN AND HAUGEN 2.
When wild-type A. nidulans grew on glucose as sole carbon source, in media containing the hexose at initial concentrations of 4.5, 9 or 18 mg/ml, the glucose content of the media fell linearly with increase in cell mass. At these concentrations of the hexose, the formation of i mg dry weight of cells was accompanied by the utilisation of 2. 9, 3.2 and 3.0 mg of glucose, respectively. Growth of similar cultures, in media of the same glucose content but also containing ioo mM sodium acetate, proceeded at similar rates and to a similar extent; however, under these conditions, 0.7, i . i and I . i mg of glucose, respectively, were removed from the media for each increase of I m g dry weight of cells, The presence of acetate thus decreased the utilisation of glucose for cellular syntheses by nearly 70 %. That this effect was due largely to an exclusion of glucose from the cells was shown by studies with [14C6]glucose. When [14C6]glucose was added (at I . i ffC/ml of medium; 0.04 izC/ffmole) to a culture of A. nidulans growing on 25 mM glucose, 14C Biochim. Biophys. Acta, I58 (I968) 4 9 1 - 4 9 3
492
PRELIMINARY NOTES
was rapidly incorporated into cellular components: in IO h, the specific activity of the organisms rose to about 16ooo counts/rain per mg of dried cells. In contrast, a similar culture growing on 25 mM glucose and IOO mM acetate took up only a small amount of [~4CG]glucose: the specific activity rose to only 2ooo counts/rain per mg of dried cells in the first 2 h, and was maintained at this low level throughout the duration of the experiment (Fig. 2). These effects were noted also with washed cells of A. ,nidula~s, which had been grown on media containing either glucose or glucose and acetate. In these experiments, the cells were incubated for only 15 rain with I mM [14C61glucose (o.II ffC/ffnaole), F,q. 3
O
O
"! 5C --
/
Fig.2
c~ (3
O~O--O--O I I I 2
4
4C --
zx
I
e 8 1O T i m e (h)
[irrl<~ ' rrl;r~
Fig. 2. I n c o r p o r a t i o n of I~C from [14Cejglucose (I.I ttC/ml) by A. nidulans growing on 2 5 mM glucose (O) or 25 mM glucose and ioo mM acetate ( 0 ) - Samples of the growing cultures were filtered on Millipore filters, which were washed with cold basal m e d i u m and were assayed for 14C, whilst suspended in BRAY'Sa fluid, with a P a c k a r d scintillation counter. Separate samples of the cells were dried to c o n s t a n t weight and weighed. Fig. 3- I n c o r p o r a t i o n of 14C from [laCe]glucose (O,I I ffC/inl) by washed suspension of A. nidulans, Cells were g r o w n for 18 h on basal m e d i u m containing 5 ° mM glucose, with or w i t h o u t ioo mM acetate as indicated. T h e y were harvested, washed w i t h 5 ° mM s o d i u n l - p o t a s s i u l n p h o s p h a t e (pH 7.0) and resuspended in this buffer. The incorporation of tiC from ~14Celglucose was m e a s u r e d as described in tile legend of Fig. 2, except t h a t the glucose c o n t e n t of all media was only I mM, O, glucose-grown cells, no added acetate; O , glucose-grown cells incubated with IOO m3I acetate; 2x, cells grown on glucose and acetate, no acetate added to incubation m e d i u m ; Jk, cells grown on glucose and acetate, incubated with ioo mM acetate.
in the presence or absence of i00 mM acetate. Again, the presence of acetate reduced the rate of isotope incorporation by glucose-grown cells to less than 2o % of that noted in the absence of acetate. Although cells which had been grown on medium containing glucose and acetate contained only about one-third of the glucokinase activity (EC 2.7.I.2) of cells grown on glucose as sole carbon source, such cells manifested but a relatively slight reduction in the rate of isotope incorporation from [14Ce]glucose, and the presence of acetate again caused a dramatic inhibition of this incorporation (Fig. 3)No effect of acetate on glucose utilisation was observed with a mutant 4 of A. nidulans devoid of acetyl-CoA synthetase activity (EC 6 . 2 . 1 . 1 ) , which shows that it is a metabolic product of acetate and not acetate itself that inhibits hexose utilisation. The results presented here suggest that, in A. nidulans, acetyl-CoA, which is 13iochim. Biophys. Acta, 158 (1968) 491-493
PRELIMINARY NOTES
493
the major end-product of glycolysis, m a y control the rate of glycolysis b y regulating the entry of some sugars into the cell. We thank Dr. C. F. ROBERTS for advice on the care and feeding of A. nidulans and of its mutants, and Miss JOAN FARR for technical assistance. This work was performed during the tenure by A. H . R . of a Senior Postdoctoral Research Fellowship of the National Science Foundation.
Department of Biochemistr% University of Leicester, Leicester (Great Britain) 1 2 3 4
A. T. G. D.
A. H. ROMANO H. L. KORNBERG
D. GOUNARIS AND L. P. HAGER, J. Biol. Chem., 236 (1961) lOl 3. E. FRIEDEMANN AND G. E. HAUGEN, J, Biol. Chem., 147 (1943) 415 • A. BRAY, Anal. Biochem., I (196o) 279. APlRION, Genet. Res., 6 (1965) 317 .
Received April 26th, 1968 Biochim Biophys Acta, 158 (1968) 491-493
BBA 21 221
S-(2-Hydroxy-2-carboxyethylthio)cysteine and in human urine
S-(carboxymethylthio)cysteine
During the studies on urinary cysteine conjugates, we have found a small amount of two unknown disulfides in the normal human urine. A patient who excretes a large amount of the above unknown disulfides after ingestion of cysteine has been found in the United States*. These unknown disulfides have now been identified as S-(2-hydroxy-2-carboxyethylthio)cysteine (HCETC, cysteine-fl-mercaptolactic mixed disulfide) and S(carboxymethylthio)cysteine (CMTC, cysteine-thioglycolic mixed disulfide). Although CMTC is known to be formed in vitro when monoiodoacetic acid is added to the reaction mixture of cystine and cystathionase 1, our present paper is the first report indicating the natural occurrence of CMTC and HCETC. Several papers have appeared on the synthesis of CMTC2, a, but the pure substance has not been obtained because of its instability in an aqueous solution. We synthesized about 80 % pure CMTC b y the following reaction: S-Sulfocysteine + thioglycolic acid --~ CMTC + SO 2
HCETC was synthesized from L-cysteine and fl-mercaptolactic acid, the latter prepared by the method of KOELSCH4. Details on the syntheses of these disulfides will be reported later. The isolation of these disulfides from the normal human urine was carried out as follows. The N-free ampholyte fraction, which was collected b y using a sulfonated Abbreviations: HCETC, S-(2-hydroxy-2-carboxyethylthio)cysteine; CMTC, S-(carboxymethylthio)cysteine; CMC, S - ( c a r b o x y m e t h y l ) c y s t e i n e ; DCEC, S-(i,2-dicarboxyethyl)cysteine. * M. L. EFRON of t h e Massachusetts General H o s p i t a l sent us a dried urine sample of the new p a t i e n t in Nov. 1966.
Biochim. Biophys. Acta, 158 (1968) 493-495
PRELIMINARY NOTES BBA 2 1 2 2 2
Regulation of sugar utilization by Aspergillus nidulans Mutants of micro-organisms which lack pyruvate dehydrogenase activity (pdh; pyruvate:lipoate oxidoreductase (acceptor-acetylating), EC 1.2.4.1) require acetate for growth; when glucose is added to cultures of such mutants, pyruvate is excreted into the growth medium 1. When this experiment was performed with a pdh- mutant of Aspergillus nidulans R46, it was unexpectedly observed that little or no pyruvate was produced from added glucose as long as acetate remained in the growth medium, although pyruvate excretion began immediately if such cultures were incubated with glucose in the absence of acetate. The same effect of acetate on pyruvate formation was noted when fructose was used instead of glucose. However, acetate did not retard the onset of pyruvate formation from sucrose (Fig. I). These findings suggest that acetate, or a product of acetate metabolism, inhibits the utilisation of some sugars; since this effect is observed with hexoses but not with sucrose, the inhibition appears to be exerted on the uptake of the sugars rather than on the activity of a glycolytic enzyme. It is the purpose of this communication to present evidence in support of this view. Fg. "B
P,O I/-
14
/
12
d 8
~4 0
0
I 5
I 10
I 15
I 20
5
10
15 20 Tirne(~)
Fig. I. Effect of a c e t a t e on p y r u v a t e f o r m a t i o n from v a r i o u s s u g a r s b y a pdh- m u t a n t of A. nidulans. Cells, g r o w n for 44 h on m e d i a c o n t a i n i n g IOO mM a c e t a t e as sole c a r b o n source, w e re h a r v e s t e d , w a s h e d a n d r e s u s p e n d e d in b a s a l m e d i u m plus t h e c a r b o n sources i n d i c a t e d . A. N o a c e t a t e w a s a d d e d . B. i o o mM a c e t a t e w a s a d d e d . P y r u v a t e f o r m a t i o n from t h e c a t a b o l i s m of 5 ° mM glucose (O), 5 ° mM fructose ( A ) or 25 mM sucrose ( × ) w a s m e a s u r e d on s a m p l e s of t h e i n c u b a t i o n med ia, b y t h e m e t h o d of FRIEDEMANN AND HAUGEN 2.
When wild-type A. nidulans grew on glucose as sole carbon source, in media containing the hexose at initial concentrations of 4.5, 9 or 18 mg/ml, the glucose content of the media fell linearly with increase in cell mass. At these concentrations of the hexose, the formation of i mg dry weight of cells was accompanied by the utilisation of 2. 9, 3.2 and 3.0 mg of glucose, respectively. Growth of similar cultures, in media of the same glucose content but also containing ioo mM sodium acetate, proceeded at similar rates and to a similar extent; however, under these conditions, 0.7, i . i and I . i mg of glucose, respectively, were removed from the media for each increase of I m g dry weight of cells, The presence of acetate thus decreased the utilisation of glucose for cellular syntheses by nearly 70 %. That this effect was due largely to an exclusion of glucose from the cells was shown by studies with [14C6]glucose. When [14C6]glucose was added (at I . i ffC/ml of medium; 0.04 izC/ffmole) to a culture of A. nidulans growing on 25 mM glucose, 14C Biochim. Biophys. Acta, I58 (I968) 4 9 1 - 4 9 3
492
PRELIMINARY NOTES
was rapidly incorporated into cellular components: in IO h, the specific activity of the organisms rose to about 16ooo counts/rain per mg of dried cells. In contrast, a similar culture growing on 25 mM glucose and IOO mM acetate took up only a small amount of [~4CG]glucose: the specific activity rose to only 2ooo counts/rain per mg of dried cells in the first 2 h, and was maintained at this low level throughout the duration of the experiment (Fig. 2). These effects were noted also with washed cells of A. ,nidula~s, which had been grown on media containing either glucose or glucose and acetate. In these experiments, the cells were incubated for only 15 rain with I mM [14C61glucose (o.II ffC/ffnaole), F,q. 3
O
O
"! 5C --
/
Fig.2
c~ (3
O~O--O--O I I I 2
4
4C --
zx
I
e 8 1O T i m e (h)
[irrl<~ ' rrl;r~
Fig. 2. I n c o r p o r a t i o n of I~C from [14Cejglucose (I.I ttC/ml) by A. nidulans growing on 2 5 mM glucose (O) or 25 mM glucose and ioo mM acetate ( 0 ) - Samples of the growing cultures were filtered on Millipore filters, which were washed with cold basal m e d i u m and were assayed for 14C, whilst suspended in BRAY'Sa fluid, with a P a c k a r d scintillation counter. Separate samples of the cells were dried to c o n s t a n t weight and weighed. Fig. 3- I n c o r p o r a t i o n of 14C from [laCe]glucose (O,I I ffC/inl) by washed suspension of A. nidulans, Cells were g r o w n for 18 h on basal m e d i u m containing 5 ° mM glucose, with or w i t h o u t ioo mM acetate as indicated. T h e y were harvested, washed w i t h 5 ° mM s o d i u n l - p o t a s s i u l n p h o s p h a t e (pH 7.0) and resuspended in this buffer. The incorporation of tiC from ~14Celglucose was m e a s u r e d as described in tile legend of Fig. 2, except t h a t the glucose c o n t e n t of all media was only I mM, O, glucose-grown cells, no added acetate; O , glucose-grown cells incubated with IOO m3I acetate; 2x, cells grown on glucose and acetate, no acetate added to incubation m e d i u m ; Jk, cells grown on glucose and acetate, incubated with ioo mM acetate.
in the presence or absence of i00 mM acetate. Again, the presence of acetate reduced the rate of isotope incorporation by glucose-grown cells to less than 2o % of that noted in the absence of acetate. Although cells which had been grown on medium containing glucose and acetate contained only about one-third of the glucokinase activity (EC 2.7.I.2) of cells grown on glucose as sole carbon source, such cells manifested but a relatively slight reduction in the rate of isotope incorporation from [14Ce]glucose, and the presence of acetate again caused a dramatic inhibition of this incorporation (Fig. 3)No effect of acetate on glucose utilisation was observed with a mutant 4 of A. nidulans devoid of acetyl-CoA synthetase activity (EC 6 . 2 . 1 . 1 ) , which shows that it is a metabolic product of acetate and not acetate itself that inhibits hexose utilisation. The results presented here suggest that, in A. nidulans, acetyl-CoA, which is 13iochim. Biophys. Acta, 158 (1968) 491-493
PRELIMINARY NOTES
493
the major end-product of glycolysis, m a y control the rate of glycolysis b y regulating the entry of some sugars into the cell. We thank Dr. C. F. ROBERTS for advice on the care and feeding of A. nidulans and of its mutants, and Miss JOAN FARR for technical assistance. This work was performed during the tenure by A. H . R . of a Senior Postdoctoral Research Fellowship of the National Science Foundation.
Department of Biochemistr% University of Leicester, Leicester (Great Britain) 1 2 3 4
A. T. G. D.
A. H. ROMANO H. L. KORNBERG
D. GOUNARIS AND L. P. HAGER, J. Biol. Chem., 236 (1961) lOl 3. E. FRIEDEMANN AND G. E. HAUGEN, J, Biol. Chem., 147 (1943) 415 • A. BRAY, Anal. Biochem., I (196o) 279. APlRION, Genet. Res., 6 (1965) 317 .
Received April 26th, 1968 Biochim Biophys Acta, 158 (1968) 491-493
BBA 21 221
S-(2-Hydroxy-2-carboxyethylthio)cysteine and in human urine
S-(carboxymethylthio)cysteine
During the studies on urinary cysteine conjugates, we have found a small amount of two unknown disulfides in the normal human urine. A patient who excretes a large amount of the above unknown disulfides after ingestion of cysteine has been found in the United States*. These unknown disulfides have now been identified as S-(2-hydroxy-2-carboxyethylthio)cysteine (HCETC, cysteine-fl-mercaptolactic mixed disulfide) and S(carboxymethylthio)cysteine (CMTC, cysteine-thioglycolic mixed disulfide). Although CMTC is known to be formed in vitro when monoiodoacetic acid is added to the reaction mixture of cystine and cystathionase 1, our present paper is the first report indicating the natural occurrence of CMTC and HCETC. Several papers have appeared on the synthesis of CMTC2, a, but the pure substance has not been obtained because of its instability in an aqueous solution. We synthesized about 80 % pure CMTC b y the following reaction: S-Sulfocysteine + thioglycolic acid --~ CMTC + SO 2
HCETC was synthesized from L-cysteine and fl-mercaptolactic acid, the latter prepared by the method of KOELSCH4. Details on the syntheses of these disulfides will be reported later. The isolation of these disulfides from the normal human urine was carried out as follows. The N-free ampholyte fraction, which was collected b y using a sulfonated Abbreviations: HCETC, S-(2-hydroxy-2-carboxyethylthio)cysteine; CMTC, S-(carboxymethylthio)cysteine; CMC, S - ( c a r b o x y m e t h y l ) c y s t e i n e ; DCEC, S-(i,2-dicarboxyethyl)cysteine. * M. L. EFRON of t h e Massachusetts General H o s p i t a l sent us a dried urine sample of the new p a t i e n t in Nov. 1966.
Biochim. Biophys. Acta, 158 (1968) 493-495