Planta
Planta 144, 419-425 (1979)
9 by Springer-Verlag 1979
On the Role of S: Sulfotransferases in Assimilatory Sulfate Reduction by Plant Cell Suspension Cultures J.D. Schwenn, H. E1-Shagi, A. Kemena, and E. Petrak Abteilung ftir Biologie, Ruhr-Universit/it Bochum, Postfach 102148, D-4630 Bochum i, Federal Republic of Germany
Abstract. Cell suspension cultures of Catharanthus roseus (L.) G. Don were grown under S-auxotrophic (1.8 mM sulfate) and under S-heterotrophic (0.5 and 1.0 mM cysteine or methionine) conditions. The development of activity of the thiol sulfotransferases was followed during the complete growth period. Under auxotrophic growth, an NADPH-dependent S: sulfotransferase and a GSH-dependent S: sulfotransferase developed identically, whereas under heterotrophic growth, differences in the amount of enzymes and in the time course of their development occurred. The NADPH-dependent sulfotransferase appeared "repressed" by the S-amino acids but the GSH-dependent enzyme was "derepressed." In that phenomenon, the development of the GSH sulfotransferase paralleled the development of the ATP-sulfurylase (EC 2774) activity of the cells. Key words: Assimilatory sulfate reduction - Catharanthus Cell suspension culture - S-auxo/S-heterotrophic growth - Thiol sulfotransferase activity.
This reaction would be catalyzed by the enzyme PAPS: thiol sulfotransferase. The enzyme has not yet been isolated and characterized, but there is some evidence that the transfer requires the reduced acceptor XSH and PAPS (Asahi, 1964; Schwenn et al., 1976) to form the intermediate bound sulfite. In recent investigations, Schiff and co-workers (Schiffand Hodson, 1973 review) and Schmidt (1972, 1975; Schmidt et al., 1974) questioned the participation of PAPS in the assimilatory sulfate reduction as the direct donor of the sulfonyl group. They suggested that APS serves as the source of active sulfate in the reduction whereas PAPS is used for the formation of sulfate esters and sulfonates or even may serve as a reservoir of sulfate in algae and plants (Schiff and Hodson, 1973). The transfer described above would then be catalyzed by the enzyme APS: thiol sulfotransferase. According to Schmidt (1972) reduced glutathione can be used as the acceptor of the sulfonyl group in vitro:
APS + GSH --+ GS : SO3H + AMP
(2)
Introduction Previously, it was assumed that PAPS is the universal donor of "active sulfate" for the reduction of sulfate in the biosynthesis of cysteine (Asahi, 1964; Thompson, 1967) as well as for the biosynthesis of O-sulfate esters and sulfonates (Davies et al., 1966) in the plant. For assimilatory reduction, it has been proposed that the sulfonyl group of PAPS is transferred onto an endogenous acceptor thiol (" XSH") with the formation of a "bound sulfite" PAPS +XSH ~ XS: SO3H + PAP
(1)
Abbreviations: APS = adenylylphosphosulfate; GSH = reduced glu-
tathione; PAPS = phosphoadenylylphosphosulfate
Presumably, glutathione is not the physiologic acceptor for the sulfonyl group. This reaction, therefore, is assumed to indicate only the action of the APS: thiol sulfotransferase. Abrams and Schiff (1973) and Schmidt et al. (1974) suggested that in vivo the enzyme catalyzes the transfer of the sulfonate from APS onto an endogenous cofactor of the APS: thiol transferase (designated "CarSH "). The different results concerning the enzymatic mechanism of the transfer make it desirable to complement the biochemical studies with in vivo experiments. Reuveny and Filner (1977) recently showed that the biosynthesis of ATP-sulfurylase in plant cell suspension cultures is regulated by cysteine.
0032-0935/79/0144/0419/$01.40
420
J.D. Schwenn et al. : Role of S: Sulfotransferases in Sulfate Reduction
This finding may indicate that the enzymes of assimilatory sulfate reduction in higher plants can be controlled by their end product, as in bacteria or fungi (Thompson, 1967; Siegel, 1975). In this case one might expect that specific enzymes that are repressed during growth on cysteine or methionine, indeed, take part in the sulfate reduction. The development of sulfotransferase activity in higher plant tissue cultures has been investigated from this point of view. The specific activities of the NADPH-dependent and the GSH-dependent sulfotransferases have been followed under auxo- and heterotrophicgrowth. The ATP-sulfurylase has been monitored simultaneously as a reference enzyme for the capacity of the cells to activate inorganic sulfate.
ing amounts of APS (0.8 mM), PPi (0.8 mM), and Mg z+ (2 raM), the rate was strictly dependent on the amount of enzyme added. The assay was run in the presence of Tris-C1 (pH 8.0) 50 raM, glucose 10raM, NADP + 2raM, and 1I.U. each of hexokinase and glueose-6-phosphate dehydrogenase. The auxiliary enzymes were purchased "sulfate-free." APS as substrate was prepared according to Cherniak and Davidson (1964) and Horwitz et al. (1977). The concomitant AMP was removed by ion exchange chromatography on DEAE cellulose with a volatile NH~HCO3 buffer (Adams et al., 1971). The purity of the substrate was routinely controlled by HPLC on a reversed phase with tetrabutylammoniumnitrate as the complexing agent in a mixture of n-propanol/HzO (9.2%) as the mobile phase (Schwenn, 1978, manuscript in preparation).
Materials and Methods
I. GSH-Dependent Thiolsulfotransferase Activity. The assay contained per 1.0 ml of reaction mixture: Tris-C1 (pH 8.4) 50 raM, MgCI2 l0 raM, ATP (neutralized) 5 raM, 3sS-SO4 4 mM (specific activity: 10 gCi/gmol), reduced glutathione (neutralized) 2 raM, and 0.6 ml of crude cell extract.
1. Growth of Cell Suspension Cultures Cells of Catharanthus roseus (L.) have been cultured as described by Zenk et al. (1977) in a medium according to Linsmaier and Skoog (1965). The subcultures were started from cells that were at the logarithmic phase of growth. Five milliliters of packed cells were grown in Erlenmeyer flasks on a rotary shaker (100rpm at 23~ in dimmed light (5-10 Ix fluorescent tubes) or at 30~ in darkness. For S-auxotrophic growth, 1.8 mM SOs has been used as described by Linsmaier and Skoog or, for S-heterotrophic growth, 0.5 mM, or 1.0 mM cysteine or 0.5 mM methionine.
2. Extraction of Soluble Protein and Assay of the Enzymes
c) Assay of Sulfotransferase Activity Both enzyme activities were assayed from the crude extracts. Labeled sulfate and ATP served as the original substrates since the ATP-sulfurylase was always present in saturating amounts.
2. NADPH-Dependent Thiolsulfotransferase Activity. The assay contained per 1.0 ml of reaction mixture: Tris-C1 (pH 8.4) 50 raM, MgCI2 10 raM, ATP (neutralized) 5 raM, 3sS-SO4 4 mM (specific activity: 10 gCi/gmol), NADP + l raM, glucose-6-phosphate 5 mM and crude extract as the source of enzyme and sulfonyl group acceptor (" X(SH)2 "). Glucose-6-phosphate dehydrogenase may be omitted because the cell extract contains nonlimiting amounts of the enzyme. The activity of both enzymes was determined after 30 min of incubation under N2 at 25~ C. The labeled bound sulfite was quantitated by isotope exchange with unlabeled carrier sulfite (20 ~tmol/ml of assay) and ion exchange chromatography as published earlier (Schwenn, et al., 1976).
a) Protein Determination The ceils were separated from the nutrient solution by gentle suction and subsequent washing with cold Tris-C1 buffer (pH 8.4) 50 raM, containing 2 mM MgC12 and 0.5 mM /3-mercaptoethanol. An aliquot of the cell material was taken and first homogenized in a Potter homogenizer (1 g fresh weight/2 ml buffer). The resultant slurry was sonicated with a Branson sonifier for 4 x 15 s with intervals of 15 s. The temperature was kept below 4~ C during sonification and all further treatment. A clear crude protein extract was obtained after 15 min of centrifugation at 18,000g. This extract was used to assay enzymatic activities. To determine the soluble protein, it was dialyzed overnight against Tris-C1 buffer. The protein content was measured by following basically the method of Lowry et al. (1951).
b) Assay of ATP-sulfurylase The enzymatic activity was determined spectrophotometrically in a split-beam photometer as the APS dependent generation of ATP in the presence of inorganic pyrophosphate ("reverse reaction"). The reverse reaction gives a more precise estimate of Vmax and thereby allows a more accurate calculation of the amount of enzyme than does the forward reaction. Other methods previously used for the isolation of the enzyme (Robbins, 1962; Shaw and Anderson, 1972, Reuveny and Filner, 1976) were less suitable for crude extracts or were time consuming. In the presence of saturat-
Results 1. Growth of the Cell Suspension Cultures
Plant cells from Catharanthus roseus were grown Sauxotrophically with 2 mM sulfate and S-heterotrophically with 0.5 mM cysteine, 1 mM cysteine, or 0.5 mM methionine as described in "Materials and Methods". The growth of the cell culture with these sulfur sources was found to differ only within small margins (Table 1). S-auxotrophic growth yielded a dry weight of 22 g/1 of culture. Approximately the same values were found for S-heterotrophic growth with cysteine or methionine. The amount of soluble protein varied from 2.20 g/1 to 1.56 g/1 of cell culture; the lowest concentration of soluble protein was observed with methionine as source of sulfur. The growth rate (as duplication of the dry weight/time) was 24 h for the S-auxotrophs at 30~ C. The heterotrophic cultures showed rates between 28 h and 35 h, It is noteworthy that cysteine as the sole source of
J.D. Schwenn et al. : Role of S: Sulfotransferases in Sulfate Reduction
421
"fable 1. Growth of C. roseus cell suspension cultures with different sources of sulfur Source of sulfur
Sulfate Cysteine Cysteine Methionine
zl/2 (h)
1.78 m M 0.5 m M 1.0 m M 0.5 m M
Soi. protein (g/l) ~
Dry weight (g/l) ~
Light
Dark
2.2 (9) 1.76 (16)
i7.6 22.0 24.0 20.0
Dark ~
Light b
Dark
24 35 35 28
31 28 -
1.96 1.8 2.12 1.56
(8) (7) (9) (10)
-
Light (6) (6) (7) (9)
22.0 (7) 22.0 (9) -
G r o w n at 30 ~ C G r o w n at 23 ~ C under 10 Ix=fluorescent light Maximal values of protein or dry weight observed after days of growth (as indicated in brackets). Logarithmic growth was maintained within five days after inoculation
b
2. Enzyme Activity '50 c
40~
13.
E -6 E
x c
10
30~
12I ~B
//v \v .o
CI n
rID AZ U X
el_ O)
E
20 E m
10 m { 0-
A~ ,
,
9
,
i
5
,
'
1'0
days
1~5
Fig. 1. Development of S : sulfotransferase activity and ATP-sulfurylase in cell suspension cultures of Catharanthus roseus. S-anxotrophic growth in the presence of 1.8 m M sulfate, fluorescent light 10 lx, 23 ~ C on a rotary shaker, v - v spec. activity of GSH-dependent transferase; A - a spec. activity of N A D P H - d e p e n d e n t transferase; o - o spec. activity of ATP-sulfurylase. For assay conditions see Materials and Methods
sulfur lead to the slowest growth rates as compared to the S-auxotrophic cells or cells growing on methionine. Assimilatory sulfate reduction by the higher plants is known to be associated with the chloroplasts (Schwenn and Trebst, 1976), which will only differentiate under appropriate illumination. As shown here, there was only a slight stimulation by dim fluorescent light (10 lx) of the growth under S-auxotrophic conditions, but the effect of this illumination upon enzyme activity was far more pronounced, as will be shown in the section below. However, it is important to note that this illumination did not allow the greening of the cells.
The sulfotransferases of the cells grown with sulfate approached their maximal specific activity after 4 to 5 days, which coincided with the period of maximal cell growth. The specific activity of the GSH-dependent sulfotransferase (Fig. 1) was up to 20% higher than the activity of the NADPH-dependent sulfotransferase. As growth proceeded, the specific activities of both enzymes declined, and after 10 days of growth their level was as low as at the beginning of the subculture. The time course of the development of the ATP-sulfurylase also reached its maximal specific activity after 4 to 5 days and afterward declined to the original lower specific activity. From this time course it appeared that during rapid growth the amount of enzyme was increased for a short period only. However, with further growth - approaching the stationary phase - the amount of enzyme was either kept at a constant level or decreased by degradation. The kinetics clearly indicates that the sulfotransferases and the ATP-sulfurylase are subject to a regulatory mechanism. If the cells were kept in absolute darkness (Fig. 2), the specific activity of the sulfotransferases remained extremely low and did not change during the complete period of growth. The ATP-sulfurylase reached approximately the same level of specific activity as in the "light-grown" culture, but now the decline started earlier. If under this condition the sulfate was replaced by cysteine or methionine, only the NADPH-dependent sulfotransferase did not alter its specific activity. As expected, the specific activity remained as low as under S-auxotrophic conditions during the complete period of growth in the presence of the end product of assimilatory sulfate reduction. In contrast to the apparent "repression" of the NADPHdependent sulfotransferase, the specific activity of the GSH-dependent sulfotransferase drastically increased (Fig. 3). The maximal activities ranged from 17 nmol-
422
LD. Schwenn et al, : Role of S: Sulfotransferases in Sulfate Reduction
50.
/
c'-
213.
"40~ E
E 10-
~0
E
9
9
9g ~0 x c-
{ o_ 30
30~
bm
o
9
O~
03
E
m
o
o ~a
3 20
o
O~ m ~J
U~ e~
x2
10~ ol
m I
I
n
, ~ A- ~_I ~ _
0
.
.
.
.
0
[-i
I-- I77--- l
,
/
ok m
,
,
5
0)
I
'
~b
days
n
~0
,
15
. . . .
Fig. 2. Development of S : sulfotransferase activity and ATP-sulfurylase in cell suspension cultures of Catharanthus roseus. S-auxotrophic growth as indicated in Fig. 1, dark, 30 ~ C. v - v spec. activity of GSH-dependent transferase; 9 1 4 9 spec. activity of N A D P H dependent transferase; o - 9 spec. activity of ATP-sulfurylase. For assay conditions see Materials and Methods
g
. . . .
,
1'0
,
days
,
,
1'5
Fig. 4. Development of ATP-sulfurylase in Catharanthus roseus under heterotrophic growth conditions as indicated in Fig. 3. Concentration of sulfur amino acids: o =0.5 m M m e t h i o n i n e ; , =0.5 m M cysteine and 9 = 1.0 m M cysteine
"E
/-o\
"6
o
E
r~ lD3
o
E
o
cE 10-
10-
E • c
\
o
9
c-
qJ
X
O)
E "5 /
~o/
o
o -20~
-
Ol
(J oj 131 C
AZ
?0 P r
\o
o
s CQ
ol r-
o
o
/
>, tJ m m
40.c_
m
-10 b
Q;
,= u~
oo
0
m i
,4CO .
.
.
.
]
5
.
.
.
.
u
10
.
.
.
.
days
i
15
13_
0
.
0
.
.
.
.
.
.
.
i
10
.
.
.
.
days
i
0~
15
Fig. 3. Comparison of GSH-dependent and N A D P H - d e p e n d e n t S : sulfotransferase activity under heterotrophic growth conditions. GSH-dependent transferase in the presence of 9 = 0.5 m M methionine; ~ =0.5 m M cysteine and 9 = 1.0 m M cysteine, N A D P H - d e pendent transferase in the presence of v = 0 . 5 m M methionine, =0.5 m M cysteine and 9 =1.0 m M cysteine - as sole source of sulfur. Dark, 30 ~ C
Fig. 5. S: sulfotransferase and ATP-sulfurylase activity in cell suspension cultures of Catharanthus roseus S-heterotrophic growth with 0.5 m M cysteine as sulfur supply, fluorescent light 10 Ix, 23 ~ C. v - v GSH-dependent transferase; A-zx N A D P H - d e p e n d e n t transferase activity; o - o ATP-sulfurylase
SO3/mg protein per assay and 6.8 nmol-SO3/mg protein per assay with 0.5 mM or 1.0 mM cysteine to 11.2 nmol-SO3/mg protein per assay with 0.5 mM methionine as the source of sulfur. With the low concentration of cysteine (0.5 mM) the maximal activity of the enzyme was found after 6 days of growth,
whereas the higher concentration lead to a slower increase (" derepression") of the enzyme activity with a maximum after 12 days of growth. In the presence of 0.5 mM methionine the GSH-dependent sulfotransferase activity reached a maximal level after 10 days. After these high values a rapid decline occurred,
J.D. Schwenn et al. : Role of S: Sulfotransferases in Sulfate Reduction
although the remaining specific activity was still higher at the end of growth than at the start of the subculture. This high specific activity differed markedly from the decreased activity of S-auxotrophically grown cells at the end of the stationary phase. The specific activity of the ATP-sulfurylase was affected under S-heterotrophic growth conditions in a similar way. With the low concentration of cysteine (0.5 raM) and methionine, the enzyme activity increased immediately after the start of the subculture, approaching values of 47 ~mol ATP/mg protein x rain (after 10 days of growth with cysteine) and 54 l~mol ATP/ mg x man (after 8 days of growth with methionine). The higher concentration of cysteine (1 mM) resulted in a long period of rather low specific activity which, after 9 days of growth, rapidly increased to 50 ~tmol ATP/mg protein • min (Fig. 4). S-heterotrophic growth on cysteine (0.5 raM) at the dimmed light lead to a fast rise of the specific activity of the ATP-sulfurylase (Fig. 5). Unlike the enzyme of the dark-grown cells, it attained maximal activity after 4 days of growth. Thereafter the specific activity decreased at a slower rate (from 53 I~mol ATP/mg protein x min to 18 ~tmol ATP/mg protein • rain) than in the dark-grown cells. The GSH-dependent sulfotransferase activity increased as before, and the NADPH-dependent sulfotransferase activity remained at the low specific activity throughout the complete growth period. Discussion
In the higher plants and green algae the metabolism of sulfate is branched into the assimilatory sulfate reduction with sulfur containing amino acids as end products and into the biosynthesis of sulfonates and sulfate ester compounds (Richmond, 1973; Schiff and Hodson, 1973). Both branches share the enzymes of sulfate activation in which the ATP-sulfurylase and APS-kinase sequentially activate the inorganic sulfate to the energetically favored PAPS. The reductive branch is entered by the action of the PAPS: thiol sulfotransferase. Hilz et al. (1959) proposed a mechanism for the enzyme involving the formation of a thiosulfate ester: SH / PAPS
+
Lip(SH)2
~
Lip.
+
PAP
(4)
"x
S : SO3H
In the higher plants, the transfer is assumed to require a reduced dithiol "X(SH)2", a small protein with a molecular weight of approximately 5,0006,000 (Schmidt and Schwenn, 1971), instead of the lipoamide: PAPS
+
/ X \
SH ~ SH
/ X \
SH
423
The bound sulfite thus formed will be reduced by a thiolsulfonate reductase to the corresponding bound sulfide: / X \
S : SOzH
PAP
(5)
> S " SO3H
/ X \
SH (6) S : SH
a dithiolpersulfide, which will provide its outer sulfan sulfur for the biosynthesis of cysteine. The PAPS: thiol sulfotransferase, according to this hypothesis, fulfills the same function in the plants as does the PAPS-reductase system in yeast, originally described by Bandurski and co-workers (Bandurski, 1965; Roy and Trudinger, 1970; Siegel, 1975). Porgu6 etal. (1970) suggested that thioredoxin serves as the physiologic dithiol of the enzyme PAPS-reductase in yeast. The mechanism of the PAPS-reductase system in many microorganisms has been supported by results obtained with auxotrophic mutants (Thompson, 1967). These investigations confirmed the sequence of enzymatic steps and, moreover, showed that the enzymes of the assimilatory sulfate reduction are repressed by cysteine. Since this control may also apply to higher plant ceils grown in suspension culture, we investigated the development of the thiol sulfotransferase activities under S-auxo- and S-heterotrophic growth conditions.
Sulfotransferase Activity Under S-Auxotrophic Growth Conditions The specific transferase activity of both assays (NADPH-dependent and GSH-dependent thiol sulfotransferase) increased with an identical time course shortly after the start of the subculture in fresh sulfate-containing nutrient solution. The maximum of specific activity coincided with the rapid phase of cell growth (4th day). Before the stationary phase of growth had been reached, the specific activity of the transferases had already begun to decline, reaching the initial low level found at the start. Plant cells grown in absolute darkness maintained the low specific activity throughout the complete growth period. This low level of activity may indicate that in the dark the demand for bound sulfite is satisfied by a smaller amount of enzyme than in the light.
Sulfotransferase Activity Under, S-Heterotrophic Growth Conditions The replacement of sulfate by one of the end products either cysteine or methionine - resulted in a sustained low specific activity of the NADPH-dependent sulfotransferase, as would be expected from an en-
-
+
SH
424 zyme that is "repressed" by an end product. Surprisingly, the GSH-dependent sulfotransferase activity increased rapidly during growth on cysteine or methionine. In the presence of 0.5 m M Cysteine the specific activity was up to five times higher than under auxotrophic growth. Moreover, the highest values of specific activity now were obtained after the stationary phase of growth had been reached. With the higher concentration of cysteine (1.0 raM), the increase of specific activity was retarded and the maximal values were only 30% to 50% of that observed with 0.5 m M cysteine. F r o m the results obtained in the presence of cysteine or methionine (the latter shows the same type of regulation of both sulfotransferase activities), it appears that the N A D P H - d e p e n d e n t enzyme remains repressed during S-heterotrophic growth. This apparent repression may also indicate that the N A D P H dependent thiol sulfotransferase takes part in the process of assimilatory sulfate reduction. The G S H dependent thiol sulfotransferase seemed derepressed during S-heterotrophic growth. The increase of specific activity under this condition is not consistent with participation of the enzyme as a true thiol sulfotransferase. The time course of its activity during Sheterotrophic growth closely resembled that of ATPsulfurylase.
ATP-Sulfurylase Activity Under S-Auxo- and S-Heterotrophic Growth Conditions Auxotrophic growth of the cells led to a short initial period of increased specific activity of the enzyme. Even before the stationary phase of growth had been reached, the specific activity declined to the original level. When the sulfate was replaced by cysteine or methionine as the single source of sulfur, the enzyme activity was first decreased. In the later phase of growth the specific activity was substantially increased. The low activity at the beginning presumably indicates a repression, signaled by cysteine. As growth proceeds, cysteine is removed by incorporation into the proteins, and the repressive signal disappears. The increased activity in the late phase of growth, on the other hand, could be initiated through the sulfate deficiency 1. Thus ATP-sulfurylase seems to be regulated by two different signals: Sulfate deficiency acts as a trigger for a de novo synthesis of the enzyme, whereas cysteine has an inhibitory effect. Reuveny and Filner (1977) obtained similar results with to1 The activities of ATP-sulfurylase, GSH-dependent thiol sulfotransferase, and O-acetyl serine sulfhydrylaserapidly increase after the sulfate from the nutrient solution has been used up (Bergmann, L., Schwenn, J.D., Urlaub, H., 1978 to be published)
J.D. Schwenn et al. : Role of S : Sulfotransferases in Sulfate Reduction bacco cells grown on cysteine or djenkolate. They also showed that the derepression is inhibited by cycloheximide, which strongly suggests de novo synthesis of the ATP-sulfurylase. The apparent repression of the ATP-sulfurylase by cysteine appeared to be completely overruled when the cells were grown in the light instead of in absolute darkness. The immediate increase of specific activity of the enzyme in this case may well be driven by the demand of the cell for sulfate-ester compounds, which, in the light, may originate from the biosynthesis of the chloroplast m e m b r a n e system (Thomas et al,, 1970).
Conclusions
The development of N A D P H - d e p e n d e n t thiol sulfotransferase activity differed from that of GSH-dependent thiol sulfotransferase in that the latter rapidly increased under S-heterotrophic conditions. This increase of specific activity, however, disagrees with the proposal that this enzyme furnishes the assimilatory sulfate reduction with bound sulfite. The apparent repression of the N A D P H - d e p e n d e n t thiol sulfotransferase in heterotrophically grown cells rather suggests that it is this enzyme that takes part in the assimilatory reduction. This work is supported by the Deutsche Forschungsgemeinschaft. The authors are indebted to Prof. Zenk for providing the facilities to grow the cell cultures and to Mrs. Sichler for expert technical assistance.
References
Asahi, T. : Sulfur metabolism in higher plants: IV Mechanism of sulfate reduction in chloroplasts. Bioehim. Biophys. Acta 82, 5846 (1964) Abrams, W.R., Schiff, J.A. : Studies of sulfate utilization by algae: XI An enzyme-bound intermediate in the reduction of adenosine-5'-phosphosulfate (APS) by cell-free extracts of wild-type Chlorellaand mutants blocked for sulfate reduction. Arch. Mikrobiol. 94, 1-10 (1973) Adams, C.A., Warnes, G.M., Nicholas, D.J.D.: Preparation of labelled adenosine-5'-phosphosulfateusing APS-reductase from Thiobacillus denitrificans. Anal. Biochem. 42, 207 213 (1971) Bandurski, R.S.: Biological reduction of sulfate and nitrate in: Plant Biochemistry, pp. 467-490, Bonner, J., Varner, J.E., eds. New York: Academic Press 1965 Cherniak, P., Davidson, E.A. : Synthesis of adenylyl sulfate and adenylylsulfate-3-phosphate. J. Biol. Chem. 239, 2986-2990 (1964) Davies, W.H., Mercer, E.I., Goodwin, T.W.: Some observations on the biosynthesis of the plant sulfolipid by Euglena gracilis Biochim. J. 98, 369-373 (1966) Hilz, H., Kittler, M., Knape, G. : Die Reduktion yon Sutfat in der Hefe. Biochem. Z. 332, 15t-166 (1959)
J.D. Schwenn et al. : Role of S: Sulfotransferases in Sulfate Reduction Horwitz, J.P., Neenan, J.P., Misra, R.S., Rozhin, J., Huo, A., Philips, K.D. : Studies on bovine adrenal estrogen sulfotransferase: lII Facile synthesis of 3'-phospho- and T-phospho-adenosine-5'-phosphosulfate. Biochim. Biophys. Acta 4811, 376-38i (1977) Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. : Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275 (1951) Linsmaier, E.M., Skoog, F. : Organic growth factor requirements of tobacco tissue cultures. Physiol. Plant. 18, 100 127 (1965) Porqu6, P.D., Baldsten, A., Reiehard, P.: The involvement of the thioredoxin system in the reduction of methionine sulfoxide and sulfate. J. Biol. Chem. 245, 2371 2374 (1970) Reuveny, Z., Filner, P. : A new assay for ATP-sulfnrylase based on differential solubility of the sodium salts of adenosine-5'phosphosulfate and sulfate. Anal. Biochem. 75, 410-428 (1976) Reuveny, Z., Filner, P. : Regulation of adenosine triphosphate sulfurylase in cultured tobacco cells. J. Biol. Chem. 252, 1858 1864 (1977) Richmond, D.V.: Sulfur compounds, in Phytochemistry Vol. 3, pp. 42-73, Miller, L.P. ed. New York, V.N. Reinold 1973 Robbins, P.W.: Sulfate activating enzymes, in Methods of Enzymology, vol. 5, pp. 964 977, Colowick, S.P., Kaplan, N.O., eds. New York: Academic Press 1962 Roy, A.B., Trudinger, P.A.: The biochemistry of inorganic compounds of sulphur, pp 269-272. Cambridge: The University 1970 Schmidt, A., Schwenn, J.D. : On the mechanism of photosynthetic sulfate reduction, in Proc. IInd Int. Congr. Photosynthesis Stresa 1971, pp. 507 514, Forti, G., Avron, M., Melandri, eds. The Hague: W. Junk 1971 Schmidt, A. : On the mechanism of photosynthetic sulfate reduction An APS-sulfotransferase from Chlorella. Arch. Mikrobiol. 84, 77-86 (1972)
425 Schmidt, A., Abrams, W.R., Schiff; J.A. : Reduction of adenosine5'-phosphosulfate to cysteine in extracts from Chlorella and mutants blocked for sulfate reduction. Eur. J. Biochem. 47, 423-434 (1974) Schmidt, A. : A sulfotransferase from spinach leaves using adenosine-5!-phosphosulfate. Planta 124, 267 275 (1975) Schiff, J.A., Hodson, R.C. : The metabolism of sulfate. Ann. Rev. Plant Physiol. 24, 382-414 (1973) Schwenn, J.D., Depka, B., Hennies, H.H.: Assimilatory sulfate reduction in chloroplasts: Evidence for the participation of both stromal and membrane-bound enzymes. Plant Cell Physiol. 17, 165 176 (1976) Schwenn, J.D., Trebst, A.: Photosynthetic sulfate reduction by chloroplasts, in The intact chloroplast, pp. 315-334, Barber, J., ed. Amsterdam: Elsevier/NH 1976 Shaw, W.H., Anderson, J.W.: Purification, properties and substrate specificity of adenosine triphosphate sulfurylase from spinach leaf tissue. Biochem. J. 127, 237-247 (1972) Siegel, L.M.: Biochemistry of the sulfur cycle in Metabolic Pathways, vol. 7, pp. 228~30, Greenberg, H. ed. New York: Academic Press 1975 Thompson, J.F.: Sulfur metabolism in plants. Ann. Rev. Plant Physiol. 18, 59-84 (1967) Thomas, D.R., Stobart, A.K.: Lipids of tissue cultures of Kalanchoe crenata. J. Exp. Bot. 21,274-285 (1970) Zenk, M.H., E1-Shagi, H., Arens, H., St6ckigt, J., Weiler, E.W., Deus, B.: Formation of the indole alkaloids serpentine and ajmalicine in ceil suspension cultures of Catharanthus roseus, in Plant tissue culture and its bio-technological application, pp. 27-43. Barz. W., Reinhard, E., Zenk, M.H., eds. Berlin, Heidelberg, New York: Springer 1977
Received 13 July; accepted 7 December 1978
Planta 144, 419-425 (1979)
9 by Springer-Verlag 1979
On the Role of S: Sulfotransferases in Assimilatory Sulfate Reduction by Plant Cell Suspension Cultures J.D. Schwenn, H. E1-Shagi, A. Kemena, and E. Petrak Abteilung ftir Biologie, Ruhr-Universit/it Bochum, Postfach 102148, D-4630 Bochum i, Federal Republic of Germany
Abstract. Cell suspension cultures of Catharanthus roseus (L.) G. Don were grown under S-auxotrophic (1.8 mM sulfate) and under S-heterotrophic (0.5 and 1.0 mM cysteine or methionine) conditions. The development of activity of the thiol sulfotransferases was followed during the complete growth period. Under auxotrophic growth, an NADPH-dependent S: sulfotransferase and a GSH-dependent S: sulfotransferase developed identically, whereas under heterotrophic growth, differences in the amount of enzymes and in the time course of their development occurred. The NADPH-dependent sulfotransferase appeared "repressed" by the S-amino acids but the GSH-dependent enzyme was "derepressed." In that phenomenon, the development of the GSH sulfotransferase paralleled the development of the ATP-sulfurylase (EC 2774) activity of the cells. Key words: Assimilatory sulfate reduction - Catharanthus Cell suspension culture - S-auxo/S-heterotrophic growth - Thiol sulfotransferase activity.
This reaction would be catalyzed by the enzyme PAPS: thiol sulfotransferase. The enzyme has not yet been isolated and characterized, but there is some evidence that the transfer requires the reduced acceptor XSH and PAPS (Asahi, 1964; Schwenn et al., 1976) to form the intermediate bound sulfite. In recent investigations, Schiff and co-workers (Schiffand Hodson, 1973 review) and Schmidt (1972, 1975; Schmidt et al., 1974) questioned the participation of PAPS in the assimilatory sulfate reduction as the direct donor of the sulfonyl group. They suggested that APS serves as the source of active sulfate in the reduction whereas PAPS is used for the formation of sulfate esters and sulfonates or even may serve as a reservoir of sulfate in algae and plants (Schiff and Hodson, 1973). The transfer described above would then be catalyzed by the enzyme APS: thiol sulfotransferase. According to Schmidt (1972) reduced glutathione can be used as the acceptor of the sulfonyl group in vitro:
APS + GSH --+ GS : SO3H + AMP
(2)
Introduction Previously, it was assumed that PAPS is the universal donor of "active sulfate" for the reduction of sulfate in the biosynthesis of cysteine (Asahi, 1964; Thompson, 1967) as well as for the biosynthesis of O-sulfate esters and sulfonates (Davies et al., 1966) in the plant. For assimilatory reduction, it has been proposed that the sulfonyl group of PAPS is transferred onto an endogenous acceptor thiol (" XSH") with the formation of a "bound sulfite" PAPS +XSH ~ XS: SO3H + PAP
(1)
Abbreviations: APS = adenylylphosphosulfate; GSH = reduced glu-
tathione; PAPS = phosphoadenylylphosphosulfate
Presumably, glutathione is not the physiologic acceptor for the sulfonyl group. This reaction, therefore, is assumed to indicate only the action of the APS: thiol sulfotransferase. Abrams and Schiff (1973) and Schmidt et al. (1974) suggested that in vivo the enzyme catalyzes the transfer of the sulfonate from APS onto an endogenous cofactor of the APS: thiol transferase (designated "CarSH "). The different results concerning the enzymatic mechanism of the transfer make it desirable to complement the biochemical studies with in vivo experiments. Reuveny and Filner (1977) recently showed that the biosynthesis of ATP-sulfurylase in plant cell suspension cultures is regulated by cysteine.
0032-0935/79/0144/0419/$01.40
420
J.D. Schwenn et al. : Role of S: Sulfotransferases in Sulfate Reduction
This finding may indicate that the enzymes of assimilatory sulfate reduction in higher plants can be controlled by their end product, as in bacteria or fungi (Thompson, 1967; Siegel, 1975). In this case one might expect that specific enzymes that are repressed during growth on cysteine or methionine, indeed, take part in the sulfate reduction. The development of sulfotransferase activity in higher plant tissue cultures has been investigated from this point of view. The specific activities of the NADPH-dependent and the GSH-dependent sulfotransferases have been followed under auxo- and heterotrophicgrowth. The ATP-sulfurylase has been monitored simultaneously as a reference enzyme for the capacity of the cells to activate inorganic sulfate.
ing amounts of APS (0.8 mM), PPi (0.8 mM), and Mg z+ (2 raM), the rate was strictly dependent on the amount of enzyme added. The assay was run in the presence of Tris-C1 (pH 8.0) 50 raM, glucose 10raM, NADP + 2raM, and 1I.U. each of hexokinase and glueose-6-phosphate dehydrogenase. The auxiliary enzymes were purchased "sulfate-free." APS as substrate was prepared according to Cherniak and Davidson (1964) and Horwitz et al. (1977). The concomitant AMP was removed by ion exchange chromatography on DEAE cellulose with a volatile NH~HCO3 buffer (Adams et al., 1971). The purity of the substrate was routinely controlled by HPLC on a reversed phase with tetrabutylammoniumnitrate as the complexing agent in a mixture of n-propanol/HzO (9.2%) as the mobile phase (Schwenn, 1978, manuscript in preparation).
Materials and Methods
I. GSH-Dependent Thiolsulfotransferase Activity. The assay contained per 1.0 ml of reaction mixture: Tris-C1 (pH 8.4) 50 raM, MgCI2 l0 raM, ATP (neutralized) 5 raM, 3sS-SO4 4 mM (specific activity: 10 gCi/gmol), reduced glutathione (neutralized) 2 raM, and 0.6 ml of crude cell extract.
1. Growth of Cell Suspension Cultures Cells of Catharanthus roseus (L.) have been cultured as described by Zenk et al. (1977) in a medium according to Linsmaier and Skoog (1965). The subcultures were started from cells that were at the logarithmic phase of growth. Five milliliters of packed cells were grown in Erlenmeyer flasks on a rotary shaker (100rpm at 23~ in dimmed light (5-10 Ix fluorescent tubes) or at 30~ in darkness. For S-auxotrophic growth, 1.8 mM SOs has been used as described by Linsmaier and Skoog or, for S-heterotrophic growth, 0.5 mM, or 1.0 mM cysteine or 0.5 mM methionine.
2. Extraction of Soluble Protein and Assay of the Enzymes
c) Assay of Sulfotransferase Activity Both enzyme activities were assayed from the crude extracts. Labeled sulfate and ATP served as the original substrates since the ATP-sulfurylase was always present in saturating amounts.
2. NADPH-Dependent Thiolsulfotransferase Activity. The assay contained per 1.0 ml of reaction mixture: Tris-C1 (pH 8.4) 50 raM, MgCI2 10 raM, ATP (neutralized) 5 raM, 3sS-SO4 4 mM (specific activity: 10 gCi/gmol), NADP + l raM, glucose-6-phosphate 5 mM and crude extract as the source of enzyme and sulfonyl group acceptor (" X(SH)2 "). Glucose-6-phosphate dehydrogenase may be omitted because the cell extract contains nonlimiting amounts of the enzyme. The activity of both enzymes was determined after 30 min of incubation under N2 at 25~ C. The labeled bound sulfite was quantitated by isotope exchange with unlabeled carrier sulfite (20 ~tmol/ml of assay) and ion exchange chromatography as published earlier (Schwenn, et al., 1976).
a) Protein Determination The ceils were separated from the nutrient solution by gentle suction and subsequent washing with cold Tris-C1 buffer (pH 8.4) 50 raM, containing 2 mM MgC12 and 0.5 mM /3-mercaptoethanol. An aliquot of the cell material was taken and first homogenized in a Potter homogenizer (1 g fresh weight/2 ml buffer). The resultant slurry was sonicated with a Branson sonifier for 4 x 15 s with intervals of 15 s. The temperature was kept below 4~ C during sonification and all further treatment. A clear crude protein extract was obtained after 15 min of centrifugation at 18,000g. This extract was used to assay enzymatic activities. To determine the soluble protein, it was dialyzed overnight against Tris-C1 buffer. The protein content was measured by following basically the method of Lowry et al. (1951).
b) Assay of ATP-sulfurylase The enzymatic activity was determined spectrophotometrically in a split-beam photometer as the APS dependent generation of ATP in the presence of inorganic pyrophosphate ("reverse reaction"). The reverse reaction gives a more precise estimate of Vmax and thereby allows a more accurate calculation of the amount of enzyme than does the forward reaction. Other methods previously used for the isolation of the enzyme (Robbins, 1962; Shaw and Anderson, 1972, Reuveny and Filner, 1976) were less suitable for crude extracts or were time consuming. In the presence of saturat-
Results 1. Growth of the Cell Suspension Cultures
Plant cells from Catharanthus roseus were grown Sauxotrophically with 2 mM sulfate and S-heterotrophically with 0.5 mM cysteine, 1 mM cysteine, or 0.5 mM methionine as described in "Materials and Methods". The growth of the cell culture with these sulfur sources was found to differ only within small margins (Table 1). S-auxotrophic growth yielded a dry weight of 22 g/1 of culture. Approximately the same values were found for S-heterotrophic growth with cysteine or methionine. The amount of soluble protein varied from 2.20 g/1 to 1.56 g/1 of cell culture; the lowest concentration of soluble protein was observed with methionine as source of sulfur. The growth rate (as duplication of the dry weight/time) was 24 h for the S-auxotrophs at 30~ C. The heterotrophic cultures showed rates between 28 h and 35 h, It is noteworthy that cysteine as the sole source of
J.D. Schwenn et al. : Role of S: Sulfotransferases in Sulfate Reduction
421
"fable 1. Growth of C. roseus cell suspension cultures with different sources of sulfur Source of sulfur
Sulfate Cysteine Cysteine Methionine
zl/2 (h)
1.78 m M 0.5 m M 1.0 m M 0.5 m M
Soi. protein (g/l) ~
Dry weight (g/l) ~
Light
Dark
2.2 (9) 1.76 (16)
i7.6 22.0 24.0 20.0
Dark ~
Light b
Dark
24 35 35 28
31 28 -
1.96 1.8 2.12 1.56
(8) (7) (9) (10)
-
Light (6) (6) (7) (9)
22.0 (7) 22.0 (9) -
G r o w n at 30 ~ C G r o w n at 23 ~ C under 10 Ix=fluorescent light Maximal values of protein or dry weight observed after days of growth (as indicated in brackets). Logarithmic growth was maintained within five days after inoculation
b
2. Enzyme Activity '50 c
40~
13.
E -6 E
x c
10
30~
12I ~B
//v \v .o
CI n
rID AZ U X
el_ O)
E
20 E m
10 m { 0-
A~ ,
,
9
,
i
5
,
'
1'0
days
1~5
Fig. 1. Development of S : sulfotransferase activity and ATP-sulfurylase in cell suspension cultures of Catharanthus roseus. S-anxotrophic growth in the presence of 1.8 m M sulfate, fluorescent light 10 lx, 23 ~ C on a rotary shaker, v - v spec. activity of GSH-dependent transferase; A - a spec. activity of N A D P H - d e p e n d e n t transferase; o - o spec. activity of ATP-sulfurylase. For assay conditions see Materials and Methods
sulfur lead to the slowest growth rates as compared to the S-auxotrophic cells or cells growing on methionine. Assimilatory sulfate reduction by the higher plants is known to be associated with the chloroplasts (Schwenn and Trebst, 1976), which will only differentiate under appropriate illumination. As shown here, there was only a slight stimulation by dim fluorescent light (10 lx) of the growth under S-auxotrophic conditions, but the effect of this illumination upon enzyme activity was far more pronounced, as will be shown in the section below. However, it is important to note that this illumination did not allow the greening of the cells.
The sulfotransferases of the cells grown with sulfate approached their maximal specific activity after 4 to 5 days, which coincided with the period of maximal cell growth. The specific activity of the GSH-dependent sulfotransferase (Fig. 1) was up to 20% higher than the activity of the NADPH-dependent sulfotransferase. As growth proceeded, the specific activities of both enzymes declined, and after 10 days of growth their level was as low as at the beginning of the subculture. The time course of the development of the ATP-sulfurylase also reached its maximal specific activity after 4 to 5 days and afterward declined to the original lower specific activity. From this time course it appeared that during rapid growth the amount of enzyme was increased for a short period only. However, with further growth - approaching the stationary phase - the amount of enzyme was either kept at a constant level or decreased by degradation. The kinetics clearly indicates that the sulfotransferases and the ATP-sulfurylase are subject to a regulatory mechanism. If the cells were kept in absolute darkness (Fig. 2), the specific activity of the sulfotransferases remained extremely low and did not change during the complete period of growth. The ATP-sulfurylase reached approximately the same level of specific activity as in the "light-grown" culture, but now the decline started earlier. If under this condition the sulfate was replaced by cysteine or methionine, only the NADPH-dependent sulfotransferase did not alter its specific activity. As expected, the specific activity remained as low as under S-auxotrophic conditions during the complete period of growth in the presence of the end product of assimilatory sulfate reduction. In contrast to the apparent "repression" of the NADPHdependent sulfotransferase, the specific activity of the GSH-dependent sulfotransferase drastically increased (Fig. 3). The maximal activities ranged from 17 nmol-
422
LD. Schwenn et al, : Role of S: Sulfotransferases in Sulfate Reduction
50.
/
c'-
213.
"40~ E
E 10-
~0
E
9
9
9g ~0 x c-
{ o_ 30
30~
bm
o
9
O~
03
E
m
o
o ~a
3 20
o
O~ m ~J
U~ e~
x2
10~ ol
m I
I
n
, ~ A- ~_I ~ _
0
.
.
.
.
0
[-i
I-- I77--- l
,
/
ok m
,
,
5
0)
I
'
~b
days
n
~0
,
15
. . . .
Fig. 2. Development of S : sulfotransferase activity and ATP-sulfurylase in cell suspension cultures of Catharanthus roseus. S-auxotrophic growth as indicated in Fig. 1, dark, 30 ~ C. v - v spec. activity of GSH-dependent transferase; 9 1 4 9 spec. activity of N A D P H dependent transferase; o - 9 spec. activity of ATP-sulfurylase. For assay conditions see Materials and Methods
g
. . . .
,
1'0
,
days
,
,
1'5
Fig. 4. Development of ATP-sulfurylase in Catharanthus roseus under heterotrophic growth conditions as indicated in Fig. 3. Concentration of sulfur amino acids: o =0.5 m M m e t h i o n i n e ; , =0.5 m M cysteine and 9 = 1.0 m M cysteine
"E
/-o\
"6
o
E
r~ lD3
o
E
o
cE 10-
10-
E • c
\
o
9
c-
qJ
X
O)
E "5 /
~o/
o
o -20~
-
Ol
(J oj 131 C
AZ
?0 P r
\o
o
s CQ
ol r-
o
o
/
>, tJ m m
40.c_
m
-10 b
Q;
,= u~
oo
0
m i
,4CO .
.
.
.
]
5
.
.
.
.
u
10
.
.
.
.
days
i
15
13_
0
.
0
.
.
.
.
.
.
.
i
10
.
.
.
.
days
i
0~
15
Fig. 3. Comparison of GSH-dependent and N A D P H - d e p e n d e n t S : sulfotransferase activity under heterotrophic growth conditions. GSH-dependent transferase in the presence of 9 = 0.5 m M methionine; ~ =0.5 m M cysteine and 9 = 1.0 m M cysteine, N A D P H - d e pendent transferase in the presence of v = 0 . 5 m M methionine, =0.5 m M cysteine and 9 =1.0 m M cysteine - as sole source of sulfur. Dark, 30 ~ C
Fig. 5. S: sulfotransferase and ATP-sulfurylase activity in cell suspension cultures of Catharanthus roseus S-heterotrophic growth with 0.5 m M cysteine as sulfur supply, fluorescent light 10 Ix, 23 ~ C. v - v GSH-dependent transferase; A-zx N A D P H - d e p e n d e n t transferase activity; o - o ATP-sulfurylase
SO3/mg protein per assay and 6.8 nmol-SO3/mg protein per assay with 0.5 mM or 1.0 mM cysteine to 11.2 nmol-SO3/mg protein per assay with 0.5 mM methionine as the source of sulfur. With the low concentration of cysteine (0.5 mM) the maximal activity of the enzyme was found after 6 days of growth,
whereas the higher concentration lead to a slower increase (" derepression") of the enzyme activity with a maximum after 12 days of growth. In the presence of 0.5 mM methionine the GSH-dependent sulfotransferase activity reached a maximal level after 10 days. After these high values a rapid decline occurred,
J.D. Schwenn et al. : Role of S: Sulfotransferases in Sulfate Reduction
although the remaining specific activity was still higher at the end of growth than at the start of the subculture. This high specific activity differed markedly from the decreased activity of S-auxotrophically grown cells at the end of the stationary phase. The specific activity of the ATP-sulfurylase was affected under S-heterotrophic growth conditions in a similar way. With the low concentration of cysteine (0.5 raM) and methionine, the enzyme activity increased immediately after the start of the subculture, approaching values of 47 ~mol ATP/mg protein x rain (after 10 days of growth with cysteine) and 54 l~mol ATP/ mg x man (after 8 days of growth with methionine). The higher concentration of cysteine (1 mM) resulted in a long period of rather low specific activity which, after 9 days of growth, rapidly increased to 50 ~tmol ATP/mg protein • min (Fig. 4). S-heterotrophic growth on cysteine (0.5 raM) at the dimmed light lead to a fast rise of the specific activity of the ATP-sulfurylase (Fig. 5). Unlike the enzyme of the dark-grown cells, it attained maximal activity after 4 days of growth. Thereafter the specific activity decreased at a slower rate (from 53 I~mol ATP/mg protein x min to 18 ~tmol ATP/mg protein • rain) than in the dark-grown cells. The GSH-dependent sulfotransferase activity increased as before, and the NADPH-dependent sulfotransferase activity remained at the low specific activity throughout the complete growth period. Discussion
In the higher plants and green algae the metabolism of sulfate is branched into the assimilatory sulfate reduction with sulfur containing amino acids as end products and into the biosynthesis of sulfonates and sulfate ester compounds (Richmond, 1973; Schiff and Hodson, 1973). Both branches share the enzymes of sulfate activation in which the ATP-sulfurylase and APS-kinase sequentially activate the inorganic sulfate to the energetically favored PAPS. The reductive branch is entered by the action of the PAPS: thiol sulfotransferase. Hilz et al. (1959) proposed a mechanism for the enzyme involving the formation of a thiosulfate ester: SH / PAPS
+
Lip(SH)2
~
Lip.
+
PAP
(4)
"x
S : SO3H
In the higher plants, the transfer is assumed to require a reduced dithiol "X(SH)2", a small protein with a molecular weight of approximately 5,0006,000 (Schmidt and Schwenn, 1971), instead of the lipoamide: PAPS
+
/ X \
SH ~ SH
/ X \
SH
423
The bound sulfite thus formed will be reduced by a thiolsulfonate reductase to the corresponding bound sulfide: / X \
S : SOzH
PAP
(5)
> S " SO3H
/ X \
SH (6) S : SH
a dithiolpersulfide, which will provide its outer sulfan sulfur for the biosynthesis of cysteine. The PAPS: thiol sulfotransferase, according to this hypothesis, fulfills the same function in the plants as does the PAPS-reductase system in yeast, originally described by Bandurski and co-workers (Bandurski, 1965; Roy and Trudinger, 1970; Siegel, 1975). Porgu6 etal. (1970) suggested that thioredoxin serves as the physiologic dithiol of the enzyme PAPS-reductase in yeast. The mechanism of the PAPS-reductase system in many microorganisms has been supported by results obtained with auxotrophic mutants (Thompson, 1967). These investigations confirmed the sequence of enzymatic steps and, moreover, showed that the enzymes of the assimilatory sulfate reduction are repressed by cysteine. Since this control may also apply to higher plant ceils grown in suspension culture, we investigated the development of the thiol sulfotransferase activities under S-auxo- and S-heterotrophic growth conditions.
Sulfotransferase Activity Under S-Auxotrophic Growth Conditions The specific transferase activity of both assays (NADPH-dependent and GSH-dependent thiol sulfotransferase) increased with an identical time course shortly after the start of the subculture in fresh sulfate-containing nutrient solution. The maximum of specific activity coincided with the rapid phase of cell growth (4th day). Before the stationary phase of growth had been reached, the specific activity of the transferases had already begun to decline, reaching the initial low level found at the start. Plant cells grown in absolute darkness maintained the low specific activity throughout the complete growth period. This low level of activity may indicate that in the dark the demand for bound sulfite is satisfied by a smaller amount of enzyme than in the light.
Sulfotransferase Activity Under, S-Heterotrophic Growth Conditions The replacement of sulfate by one of the end products either cysteine or methionine - resulted in a sustained low specific activity of the NADPH-dependent sulfotransferase, as would be expected from an en-
-
+
SH
424 zyme that is "repressed" by an end product. Surprisingly, the GSH-dependent sulfotransferase activity increased rapidly during growth on cysteine or methionine. In the presence of 0.5 m M Cysteine the specific activity was up to five times higher than under auxotrophic growth. Moreover, the highest values of specific activity now were obtained after the stationary phase of growth had been reached. With the higher concentration of cysteine (1.0 raM), the increase of specific activity was retarded and the maximal values were only 30% to 50% of that observed with 0.5 m M cysteine. F r o m the results obtained in the presence of cysteine or methionine (the latter shows the same type of regulation of both sulfotransferase activities), it appears that the N A D P H - d e p e n d e n t enzyme remains repressed during S-heterotrophic growth. This apparent repression may also indicate that the N A D P H dependent thiol sulfotransferase takes part in the process of assimilatory sulfate reduction. The G S H dependent thiol sulfotransferase seemed derepressed during S-heterotrophic growth. The increase of specific activity under this condition is not consistent with participation of the enzyme as a true thiol sulfotransferase. The time course of its activity during Sheterotrophic growth closely resembled that of ATPsulfurylase.
ATP-Sulfurylase Activity Under S-Auxo- and S-Heterotrophic Growth Conditions Auxotrophic growth of the cells led to a short initial period of increased specific activity of the enzyme. Even before the stationary phase of growth had been reached, the specific activity declined to the original level. When the sulfate was replaced by cysteine or methionine as the single source of sulfur, the enzyme activity was first decreased. In the later phase of growth the specific activity was substantially increased. The low activity at the beginning presumably indicates a repression, signaled by cysteine. As growth proceeds, cysteine is removed by incorporation into the proteins, and the repressive signal disappears. The increased activity in the late phase of growth, on the other hand, could be initiated through the sulfate deficiency 1. Thus ATP-sulfurylase seems to be regulated by two different signals: Sulfate deficiency acts as a trigger for a de novo synthesis of the enzyme, whereas cysteine has an inhibitory effect. Reuveny and Filner (1977) obtained similar results with to1 The activities of ATP-sulfurylase, GSH-dependent thiol sulfotransferase, and O-acetyl serine sulfhydrylaserapidly increase after the sulfate from the nutrient solution has been used up (Bergmann, L., Schwenn, J.D., Urlaub, H., 1978 to be published)
J.D. Schwenn et al. : Role of S : Sulfotransferases in Sulfate Reduction bacco cells grown on cysteine or djenkolate. They also showed that the derepression is inhibited by cycloheximide, which strongly suggests de novo synthesis of the ATP-sulfurylase. The apparent repression of the ATP-sulfurylase by cysteine appeared to be completely overruled when the cells were grown in the light instead of in absolute darkness. The immediate increase of specific activity of the enzyme in this case may well be driven by the demand of the cell for sulfate-ester compounds, which, in the light, may originate from the biosynthesis of the chloroplast m e m b r a n e system (Thomas et al,, 1970).
Conclusions
The development of N A D P H - d e p e n d e n t thiol sulfotransferase activity differed from that of GSH-dependent thiol sulfotransferase in that the latter rapidly increased under S-heterotrophic conditions. This increase of specific activity, however, disagrees with the proposal that this enzyme furnishes the assimilatory sulfate reduction with bound sulfite. The apparent repression of the N A D P H - d e p e n d e n t thiol sulfotransferase in heterotrophically grown cells rather suggests that it is this enzyme that takes part in the assimilatory reduction. This work is supported by the Deutsche Forschungsgemeinschaft. The authors are indebted to Prof. Zenk for providing the facilities to grow the cell cultures and to Mrs. Sichler for expert technical assistance.
References
Asahi, T. : Sulfur metabolism in higher plants: IV Mechanism of sulfate reduction in chloroplasts. Bioehim. Biophys. Acta 82, 5846 (1964) Abrams, W.R., Schiff, J.A. : Studies of sulfate utilization by algae: XI An enzyme-bound intermediate in the reduction of adenosine-5'-phosphosulfate (APS) by cell-free extracts of wild-type Chlorellaand mutants blocked for sulfate reduction. Arch. Mikrobiol. 94, 1-10 (1973) Adams, C.A., Warnes, G.M., Nicholas, D.J.D.: Preparation of labelled adenosine-5'-phosphosulfateusing APS-reductase from Thiobacillus denitrificans. Anal. Biochem. 42, 207 213 (1971) Bandurski, R.S.: Biological reduction of sulfate and nitrate in: Plant Biochemistry, pp. 467-490, Bonner, J., Varner, J.E., eds. New York: Academic Press 1965 Cherniak, P., Davidson, E.A. : Synthesis of adenylyl sulfate and adenylylsulfate-3-phosphate. J. Biol. Chem. 239, 2986-2990 (1964) Davies, W.H., Mercer, E.I., Goodwin, T.W.: Some observations on the biosynthesis of the plant sulfolipid by Euglena gracilis Biochim. J. 98, 369-373 (1966) Hilz, H., Kittler, M., Knape, G. : Die Reduktion yon Sutfat in der Hefe. Biochem. Z. 332, 15t-166 (1959)
J.D. Schwenn et al. : Role of S: Sulfotransferases in Sulfate Reduction Horwitz, J.P., Neenan, J.P., Misra, R.S., Rozhin, J., Huo, A., Philips, K.D. : Studies on bovine adrenal estrogen sulfotransferase: lII Facile synthesis of 3'-phospho- and T-phospho-adenosine-5'-phosphosulfate. Biochim. Biophys. Acta 4811, 376-38i (1977) Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. : Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275 (1951) Linsmaier, E.M., Skoog, F. : Organic growth factor requirements of tobacco tissue cultures. Physiol. Plant. 18, 100 127 (1965) Porqu6, P.D., Baldsten, A., Reiehard, P.: The involvement of the thioredoxin system in the reduction of methionine sulfoxide and sulfate. J. Biol. Chem. 245, 2371 2374 (1970) Reuveny, Z., Filner, P. : A new assay for ATP-sulfnrylase based on differential solubility of the sodium salts of adenosine-5'phosphosulfate and sulfate. Anal. Biochem. 75, 410-428 (1976) Reuveny, Z., Filner, P. : Regulation of adenosine triphosphate sulfurylase in cultured tobacco cells. J. Biol. Chem. 252, 1858 1864 (1977) Richmond, D.V.: Sulfur compounds, in Phytochemistry Vol. 3, pp. 42-73, Miller, L.P. ed. New York, V.N. Reinold 1973 Robbins, P.W.: Sulfate activating enzymes, in Methods of Enzymology, vol. 5, pp. 964 977, Colowick, S.P., Kaplan, N.O., eds. New York: Academic Press 1962 Roy, A.B., Trudinger, P.A.: The biochemistry of inorganic compounds of sulphur, pp 269-272. Cambridge: The University 1970 Schmidt, A., Schwenn, J.D. : On the mechanism of photosynthetic sulfate reduction, in Proc. IInd Int. Congr. Photosynthesis Stresa 1971, pp. 507 514, Forti, G., Avron, M., Melandri, eds. The Hague: W. Junk 1971 Schmidt, A. : On the mechanism of photosynthetic sulfate reduction An APS-sulfotransferase from Chlorella. Arch. Mikrobiol. 84, 77-86 (1972)
425 Schmidt, A., Abrams, W.R., Schiff; J.A. : Reduction of adenosine5'-phosphosulfate to cysteine in extracts from Chlorella and mutants blocked for sulfate reduction. Eur. J. Biochem. 47, 423-434 (1974) Schmidt, A. : A sulfotransferase from spinach leaves using adenosine-5!-phosphosulfate. Planta 124, 267 275 (1975) Schiff, J.A., Hodson, R.C. : The metabolism of sulfate. Ann. Rev. Plant Physiol. 24, 382-414 (1973) Schwenn, J.D., Depka, B., Hennies, H.H.: Assimilatory sulfate reduction in chloroplasts: Evidence for the participation of both stromal and membrane-bound enzymes. Plant Cell Physiol. 17, 165 176 (1976) Schwenn, J.D., Trebst, A.: Photosynthetic sulfate reduction by chloroplasts, in The intact chloroplast, pp. 315-334, Barber, J., ed. Amsterdam: Elsevier/NH 1976 Shaw, W.H., Anderson, J.W.: Purification, properties and substrate specificity of adenosine triphosphate sulfurylase from spinach leaf tissue. Biochem. J. 127, 237-247 (1972) Siegel, L.M.: Biochemistry of the sulfur cycle in Metabolic Pathways, vol. 7, pp. 228~30, Greenberg, H. ed. New York: Academic Press 1975 Thompson, J.F.: Sulfur metabolism in plants. Ann. Rev. Plant Physiol. 18, 59-84 (1967) Thomas, D.R., Stobart, A.K.: Lipids of tissue cultures of Kalanchoe crenata. J. Exp. Bot. 21,274-285 (1970) Zenk, M.H., E1-Shagi, H., Arens, H., St6ckigt, J., Weiler, E.W., Deus, B.: Formation of the indole alkaloids serpentine and ajmalicine in ceil suspension cultures of Catharanthus roseus, in Plant tissue culture and its bio-technological application, pp. 27-43. Barz. W., Reinhard, E., Zenk, M.H., eds. Berlin, Heidelberg, New York: Springer 1977
Received 13 July; accepted 7 December 1978
