Planta (Berl.) 124, 267--275 (1975) 9 by Springer-Verlag 1975
A Sulfotransferase from Spinach Leaves Using Adenosine-5'-Phosphosulfate Ahlert Schmidt Botanisehes Institut der Universit~t Mfinchen, Menzinger Stral3e 67, D-8 Miinchen, Federal Republic of Germany l~eceived 19 February; accepted 30 April, 1975
Summary. Active sulfotransferase can be extracted from spinach (Spinaeea oleracea L.) leaves (and other higher plants) using a buffer system containing 0.1 M KC1 and thiol reagents. This sulfotransferase is labile, it can, however, be stabilized by storage in 70 % ammonium sulfate containing 10 mM mereaptoethanol. This extract will reduce labelled adenosine-5'phosphosulfate (APS) and 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to acid-volatile radioactivity when dithioerythrol is added. The reduction from PAPS requires magnesium chloride and is inhibited by calcium chloride and sodium fluoride, whereas these chemicals have little effect on the APS-sulfotransferase activity. The reduction rates from both nucleotides are stimulated by increasing ionic strength and are inhibited by phosphate and cyanide. In the presence of non-labelled APS the acid-volatile radioactivity distilled from [~5S] PAPS is drastically reduced, whereas the opposite experiment using [8~S] APS in the presence of non-labelled PAPS has little effect. This indicates that APS is an obligatory intermediate in the conversion of [35S] PAPS to acid-volatile radioactivity. It is therefore concluded that the sulfotransferase from sp]nach is specific for APS. Activity with APS as sulfur-donor was found in 5 other plants in addition to spinach: Peunisetum, Zea (Gramineae); Brassiea (Crueiferae); Helianthus (Compositae) ; and Vicia (Papilionaceae). These experiments demonstrate the use of APS for assimilatory sulfate reduction in higher plants. This has been shown previously for the green alga Chlorella.
Introduction I t has been t h o u g h t for a long tinle t h a t a s s i m i l a t o r y sulfate r e d u c t i o n is d i s t i n g u i s h e d from d i s s i m i l a t o r y sulfate r e d u c t i o n b y t h e specific use of P A P S as sulfate donor. However, in recent y e a r s a specificity t o w a r d A P S for assimilat o r y sulfate r e d u c t i o n in t h e green alga Chlorella has been d e m o n s t r a t e d (Schmidt, 1972a; Schiff a n d H o d s o n , 1973). This was d e m o n s t r a t e d with purified enzymes (Schmidt, 1972 a) a n d b y w o r k w i t h Chlorella m u t a n t s (Schiff a n d Hodson, 1973 ; S c h m i d t et al., 1974). F o r higher p l a n t s knowledge is still f r a g m e n t a r y . I t has been shown t h a t P A P S is f o r m e d in spinach (Schmidt, 1968, 1972b) a n d this o b s e r v a t i o n has been confirmed b y t h e group of A n d e r s o n (Burnell a n d Anderson, 1973). I t h a d been suggested t h a t P A P S should be t h e sulfur donor n e e d e d for r e d u c t i o n in spinach (Asahi, 1964) a n d S c h m i d t a n d T r e b s t (1969) came to t h e same conclusion based on c o m p a r a t i v e b i o c h e m i s t r y a n d t h e finding t h a t P A P S was indeed f o r m e d in spinach. However, e x p e r i m e n t s w i t h chloroplast e x t r a c t showed t h a t A P S a n d P A P S were m e t a b o l i z e d (Schmidt, 1972b). I t was therefore concluded in a n a l o g y to t h e Chlorella s y s t e m t h a t A P S should be the sulfur donor
Abbreviations: APS: Adenosine-5'-Phosphosulfate; PAPS: 31-Phosphoadenosine-5'-phosphosulfate; DTE : Dithioerythrol. 18
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268
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n e e d e d for r e d u c t i o n (Schmidt, 1972a, b). However, Burnell a n d A n d e r s o n (1973) f a v o u r P A P S as i n t e r m e d i a t e in sulfate r e d u c t i o n in spinach. This p u b l i c a t i o n shows t h a t an active transferase can be e x t r a c t e d from spinach leaves. The experim e n t s w i t h A P S a n d P A P S a n d i n h i b i t o r studies i n d i c a t e t h a t A P S is t h e sulfur donor r e q u i r e d for f u r t h e r r e d u c t i o n in spinach. Materials and Methods Extract Preparation. Spinacea oleracea L. variety "Vital R " was grown in the botanical garden of this university. 50 g of leaves were homogenized in 100 m] of buffer in a "Braun Starmix" for 30 s at speed III. The buffer contained 0.1 M Tris-HC1 pH 8.0; 0.1 M KC1; 0.02 5~ MgC12; and 0.01 M mercaptoethanol. The crude extract was squeezed through cheesecloth and centrifuged afterwards for 20rain at 10000• The supernatant was brought to 70% ammonium sulfate and stored at --18 ~ C. An aliquot was thawed, centrifuged, and the pellet suspended in 1/2 of the original volume of fresh buffer. This extract was used for the experiments described. The extraction method has also been found suitable for other higher plants to yield active APS-sulfotransferase. Determination o/ Sul/otrans/erase Activity. This activity was determined by measuring acid-volatile radioactivity derived from either [35S] APS or [3~S] PAPS, using DTE as acceptor (Schmidt, 1972a). The acid-volatile radioactivity was distilled by adding nonlabelled sulfite and absorbed in 1 M triethanolamin and counted in a liquid scintillation counter Beckman Model LS-100 according to Patterson and Green (1965). Preparation o] Sul/onucleotides. APS and PAPS were synthesized enzymatically from sulfate and ATP (Hodson and Schiff, 1969; Schmidt et al., 1974). By this method labelled and non-labelled PAPS were prepared. PAPS was dephosphorylated to APS with a 3'-phosphatase from potato (Boehringer, No. 15438). Protein Determination. The method of Lowry was used (Lowry et al., 1951). Chemicals. [~5S] sulfate was ordered from Buchler, Braunschweig; ATP, 3'-AMP and APS were purchased from Boehringer, Mannheim; all other chemicals were obtained from ~r Darmstadt.
Results S p i n a c h e x t r a c t s p r e p a r e d as d e s c r i b e d in " m a t e r i a l s a n d m e t h o d s " will t r a n s f e r t h e sulfo-group of A P S onto suitable acceptors in t h e presence of D T E . This can be d e t e r m i n e d as acid-volatile r a d i o a c t i v i t y from [asS] A P S after t h e a d d i t i o n of non-labelled sulfite as described earlier (Schmidt, 1972a, b). Fig. 1 shows t h a t t h e A P S - s u l f o t r a n s f e r a s e r e a c t i o n is linear over a c e r t a i n range w i t h p r o t e i n c o n c e n t r a t i o n a n d over t h e one h o u r p e r i o d n o r m a l l y used for i n c u b a t i o n (Fig. 2). This h a d a l r e a d y been d e m o n s t r a t e d for t h e r e d u c t i o n of sulfate t o G S - S O 3- using chloroplast e x t r a c t from spinach ( S c h m i d t a n d Trebst, 1969; S c h m i d t , 1972b). O p t i m a l thiol c o n c e n t r a t i o n for these r e a c t i o n s h a d been d e t e r m i n e d b y t h e spinach s y s t e m too, therefore 10 mM D T E were used as a c c e p t o r during these investigations. The p H - o p t i m n m of t h e A P S - s u l f o t r a n s f e r a s e r e a c t i o n was f o u n d to be a r o u n d 8.5 (Fig. 3) which is close to t h e p H - o p t i m u m d e t e r m i n e d for t h e A P S - s u l f o t r a n s f c r a s e of Chlorella (Schmidt, 1972a). I t was decided to i n c u b a t e always a t p H 9.0 to o b t a i n conditions c o m p a r a b l e to t h o s e of t h e ChlorelIa-system used b y t t o d s o n a n d Schiff (1971). The transferase a c t i v i t y was s t i m u l a t e d with increasing ionic strength. T a b l e 1 d e m o n s t r a t e s t h a t best rates were o b t a i n e d w i t h N%SOa, while NHaCI, KC1, or (NHd)2SO 4 were less effective. The r a t e of t h e A P S - s u l f o t r a n s f e r a s e r e a c t i o n is increased w i t h a d d e d Na2SOd; o p t i m a l conditions were f o u n d a r o u n d 0.5 M Na2SO 4 (Fig. 4). H i g h ionic s t r e n g t h was suitable for stabilizing t h e e n z y m e to some extent. The sulfotransferase
APS-Sulfotransferase Activity from Spinach Leaves
269
3sS dist n mot
80-
s,
i
I .z /
70-
/
jJ
60-
/
50/.0]
/
]
/
/
l/
]
30x/ /
20xl I /
10-
I f
o;2
o;,
0'.3
o;
o:s ~, oozym~
Fig. 1. Protein-dependence of the APS-sulfotransferase reaction. Conditions (in ~xmol): TrisHCI pI-I 9.0: 100; MgCle: 10; DTE: 10; KCI: 200; APS: 0.25 (851 cpm/nmol) and enzyme as indicated (10.7 mg/ml) in a total volume of 1.0 ml. Incubation time 1 h at 37 ~ C under N2 35Sdist nmol
50,/
50-
/
40/
/
/
x
f
• z"
/
30
x ,/
20
f
/ •
10. /
/ z"
/
15
3'0
4'5
6'0
min
Fig. 2. Time-dependence of the APS-sulfotransferase reaction. Conditions as in Fig. 1, however KC1 was replaced by Na2SO 4. Na2SO~: 250; protein: 1.07 mg
activity is inhibited by phosphate, which was already noted for the reduction of sulfate to GS-SO a- with the chloroplast extract system (Schmidt, 1968). Cyanide was found to be inhibitory for the APS-sulfotransferase reaction, which might explain w h y ferrieyanide and ferroeyanide had no effect on this reaction sequence (Table 2, 3). Fluoride (10 raM), calcium ions (10 raM), and 3'-AMP (6 mM) have no signifieant inhibitory effects and magnesium ions m a y be ommitted without loss of activity, although an approximate 30% decrease of activity was noted when the reaction was run in 0.2 M KC1 without the addition of Na~SO 4. The addition of eysteine had no effect on the APS-sulfotransferase reaction (Tables 2, 3). This etude extract also catalyzes the sulfotransferase reaction from P A P S under the conditions described, as ean be seen in Table 2 and 3. I-Iowever, some differences to the APS-sulfotransferase reaction were noted. The conversion of lS*
270
A. Schmidt 3sSdi~ n m(
30-
/
20
/
X
~
x
~
X
•
10
7'5
8:0
8:5
9:0
9:5 pH
Fig. 3. pit-optimum for the APS-sulfotransferase reaction. Conditions (in ~zmol): Tris-HC1 pH 7.5 to 9.5: 100; MgC12: 10; DTE: 10; APS: 0.25 (851 cpm/nmol); KCI: 200; protein: 1.07 rag; total volume 1.0 ml. 1 h incubation at 37 ~ C under N 2 Table 1. Stimulation of the APS-sulfotransferase reaction with ionic strength
Without addition With 0.2 M KC1 With 0.7 1 KC1 With 0.7 ~ NaC1 With 0.7 1V[NHaC1 With 0.7 M (NHa)2S04 With 0.7 M Na~SO4
cpm
nmol
6935 28405 38350 47950 46315 39655 64785
8.14 33.3 45.1 56.3 54.4 46.6 76.1
Controls with 0.2 M KC1 : Added enzyme heated Enzyme heated, S0a2- added 10 Enzyme heated, S2 added 5 Conditions (in ~zmol): Tris-HC1 pH 9: 100; MgC12: 10; DTE: 10; APS: 0.25 (851 epm/nmol); protein: 1.07 mg; KC1, NaC1, (NI-Ia)2SO4, NH~CI, and Na2SO4 as indicated; soae-: 100; S2 : 100; in a total volume of 1.0 ml. Incubation time 1 h at 37~ C under N 2.
P A P S was s t i m u l a t e d to the same e x t e n t as was f o u n d for A P S b y high ionic s t r e n g t h ; a n d the i n h i b i t i o n b y K C N a n d phosphate was in the same order of m a g n i t u d e . However, in contrast to the APS-sulfotransferase reaction, a strict r e q u i r e m e n t for m a g n e s i u m ions using P A P S as sulfur donor (Table 2) was noted. F u r t h e r m o r e , the PAPS-sulfotransferase reaction is severely i n h i b i t e d b y fluoride a n d calcium ions, which h a d no effect on the APS-sulfotransferase reaction. This observation suggested t h a t P A P S m i g h t be dephosphorylated first to A P S a n d t h e n used as sulfur donor for reduction. If A P S is a n obligatory i n t e r m e d i a t e in the reduction of [~5S] P A P S , it should be possible to dilute the specific a c t i v i t y of [35S] A P S formed from [35S] P A P S b y the a d d i t i o n of non-labelled APS. This should be noticed i n a decrease of r a d i o a c t i v i t y transferred from [~5S] P A P S in the presence of non-labelled APS. As can be seen in Table 3, exactly this predicted
APS-Sulfotransferase Activity from Spinach Leaves
271
3SSdist nmol 70 60 50 40 30 20
/
I0' x
o.'25
0:s
o:75
i M N%SO,
Fig. 4. Dependence of the APS-sulfotransferase reaction from the sodium sulfate concentration. Conditions as in Fig. 3, however the reaction was run at pH 9.0 and KC1 was replaced by Na.2SQ as indicated
Table 2. Inhibitor studies using
APS and PAPS as sulfur donor for the sulfotransferase reaction cpm
nmol
% of control
28405 18 900 28 060 4415 140 1495 0 19 600 4 070 4 580 700
33.2 22.1 32.9 5.2 0.16 1.75 -52.4 10.9 12.2 1.9
100 67 99 16 -5 -100 21 23 4
57 915 57 690 57 640 28 445 3 610
68.1 67.8 67.7 76.1 9.7
100 99 99 100 13
0.2 M KC1
APS 4- MgC12 APS MgC12 AP S + MgC12-t-NaF APS -}- MgC12-}-KCN APS + MgC12- DTE APS @ MgCl~-- DTE -~ Fe(II)-cyanid APS ~ MgC12- DTE ~ Fe(III)-eyanid PAPS @ MgC1e PAPS -- MgCl 2 PAPS -~ MgC12@ NaF PAPS + MgCl~-l- KCN -
-
0.5 M Na2SO 4
APS ~ MgCI2 APS MgC12 APS -F MgC12-~ cysteine P A P S -}- MgCI~ PAPS -- MgCl~ -
-
Conditions (in Fmol) : Tris-HCl p H 9.0: 100; MgCl 2: 10; D T E : 10; APS : 0.25 (851 cpm/nmol) ; PAPS: 0.5 (374 cpm/nmol); eysteine: 5; N a F : 10; KCN: 10; Fe(II)-cyanid: 10; :Fe(III)cyanid: 10; KCI: 200; Na2SO4: 500; protein: 1.07 rag; total volume 1.0 ml. Incubation for 1 h at 37 ~ C under Ne.
d e c r e a s e in a c i d - v o l a t i l e r a d i o a c t i v i t y was f o u n d in s u c h a n e x p e r i m e n t . H o w e v e r , if P A P S is a n o b l i g a t o r y i n t e r m e d i a t e in t h e r e d u c t i o n s e q u e n c e of A P S , t h e a d d i t i o n of n o n - l a b e l l e d P A P S t o [35S] A P S s h o u l d d e c r e a s e t h e r a d i o a c t i v i t y
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Table 3. Inhibitor studies using APS and PAPS as sulfur donor for the sulfotransferase reaction cpm APS ~ APS § APS + APS ~ APS ~
nmol
% of control 100 99 100 21 90 100 33 83 18 21
MgCl2 MgCl2+ CaC12 MgCl2+ 3'-AMP MgC12-~phosphate MgCl2~-non-labelled PAPS
5460 5400 5450 5060
25.2 25.0 25.2 5.2 22.7
PAPS ~ MgCl~ PAPS ~ MgCl~ CaCl~ PAPS ~ MgCle-~3'-AMP PAPS ~ MgCI2~phosphate PAPS ~- MgC12~non-labelled APS
13325 4320 10990 2405 2860
35.6 11.6 29.4 6.4 7.6
1120
Conditions (in ~mol) : Tris-tIC1 pH 9.0: 100; MgCI2: 10; DTE : 10; APS : 0.25 (216 cpm/nmol) ; PAPS: 0.5 (374epm/nmol); Na2SOa: 500; CaC12: 10; 3'-AMP: 6; phosphate: 100; nonlabelled APS and PAPS: 1; protein: 0.53 mg; total volume 1.0 ml. Incubation time 1 h at 37~ C under N~.
transferred according to the same reasons stated above. The experiment, however, shows no significant decrease of radioactivity transferred! This suggests that APS is indeed an intermediate in the "PAPS-sulfotransferase reaction" and that the transferase is specific for APS. This has already been demonstrated for the APS-sulfotransferase from Chlorella (Schmidt, 1972a). Discussion
The formation of PAPS does not necessarily predict that this nucleotide is the sulfur donor needed for reduction (Schmidt, 1972a; Schiff and ttodson, 1973). For yeast it was shown by Hilz et al. (1959) that PAPS is needed for reduction, however Wilson et al. (1961) demonstrated that APS could be used as well, although with less activity. For Escherichia coli a specificity towards PAPS was found (Pasternak et al., 1965) and this has been confirmed by Tsang and Schiff (1974). Chlorella was shown originally to need PAPS for sulfate reduction (Hodson et al., 1968, 1971); however recent developments clearly demonstrate that the transferase is specific for APS and that the PAPS-confusion was due to a 3'-phosphatase activity converting PAPS to APS (Schmidt, 1972 ; Schiff and Hodson, 1973). The same situation has to be considered for spinach. Asahi (1964) concluded from his experiments that PAPS is the sulfur donor needed by spinach chloroplasts. On the other hand, Ellis (1969) suggested that APS might be used, since he could not demonstrate PAPS formation. I t was suggested that PAPS might be the sulfur donor in spinach, based on comparative biochemistry for assimilatory sulfate reduction and the fact that PAPS was indeed formed in spinach (Schmidt and Trebst, 1969). PAPS-formation in spinach was confirmed by Burnell and Anderson (1973). However, this group had to add 3'-AMP in order to find PAPS, whereas Schmidt (1972b) reported that thiol groups were needed for the spinach system and for the Chlorella-system for PAPS-formation. This suggests that two different mechanisms might be involved. I t seems possible that a
APS-Sulfotransfcrase Activity from Spinach Leaves
273
phosphatase exists, which transfers the phosphate group from 3'-AMP to APS forming PAPS. This would mimic the APS-kinase. Phosphotransferases have been found in microorganisms and plants (Brunngraber and Chargaff, 1967, 1973a, b). It was noted that for extraction and optimal assay conditions high ionic strength was needed. There could be various explanations for this: a) The sulfotransferase might be bound to membranes (for instance to chloroplast membranes (Sehmidt, 1968; Sehmidt and Trebst, 1969) and KC1 solubilizes the protein. b) Possible stabilization of the enzyme activity by high ionic strength. e) Perhaps the active site is unfolded under high ionic strength so that artificial thiols may reach the centre of the transferase. d) Possible inhibition of sulfatases. This would protect the sulfonueleotide from degradation, so that more sulfonueleotide is present for the transferase. Perhaps more than one argument discussed above might attribute to the good rates of the APS-sulfotransferase activity measured. The data of this paper too show that PAPS is metabolized to acid-volatile radioactivity. It is suggested on the basis of the experiments described in this paper that APS is the sulfur donor needed for reduction. The activity with PAPS seems to be due to a 3'-phosphatase, which converts PAPS to APS. It is possible to demonstrate such enzyme activities in the crude spinach extract by determination of the phosphate released from PAPS and by chromatographic determination of the APS formed (data not shown). Furthermore, the inhibition data which NaF, CaCI~, and 3'-AMP and the magnesium requirement are additional evidence of phosphatase activities (Burnell and Anderson, 1973 ; Wells and IKageman, 1974). If APS is the sulfur donor needed for reduction and PAPS firstly has to be dephosphorylated, the addition of non-labelled APS to [35S] PAPS should reduce the specific activity of [35S] APS formed by isotopic dilution; this reduces the amonnt of radioactivity distilled. If, on the other hand, PAPS is the sulfur donor required for reduction, the addition of non-labelled PAPS to [aS] APS should, in a similar manner, reduce the amount of radioactivity distilled. This pair of experiments should elucidate which sulfonueleotide is used for further reduction. The present data show that the addition of non-labelled APS to [35S] PAPS drastically reduces the radioactivity distilled, whereas the opposite experiment had little effect. This clearly indicates that APS is an intermediate in the "PAPS-sulfotransferase reaction". This, of course, is evidence that the sulfotransferase is specific for APS and the name APS-sulfotransferasc is suggested for this enzymatic activity (Sehmidt, 1972a). The older observation of Asahi (1964) who postulated that PAPS is needed for the spinach system is explained by the fact that he had to add the sulfate activation system from yeast to get active extracts. It is suggested here that the yeast extract was contaminated with the yeast transferase, since APS was found by spinach chloroplasts without the addition of yeast extract and--as is shown in this publication--the spinach enzyme is already active with APS. Thus the experiment of Asahi shows a coupling of yeast transferase to spinach reducible thiol groups, but it is not
274
A. Schmidt
Table 4. Distribution of the APS-sulfotransferase activity among photosynthetic organisms a) Among algae a Cyanophyta
Oscillatoria wononichii Synechococcus sp.
Chrysophyta
Cyclotella Chaetocerus pseudocrinitis
Chlorophyta
Chlorella pyrenoidosa
Pyrrophyta
Peridinium trochoidium Gymnodinium sp.
Phaeophyta
Fucus vesiculosus
Rhodophyta
Chondrus crispus Porphyridium acrugineum
b) Among higher plants b Gramineae
Pennisetum purpureum Zea maya
Chenopodiaceae
Spinacea oleracea
Compositae
Helianthus annuus
Papilionaceae
Vicia /aba
a According to Tsang and Schiff (1974). b Schmidt, unpublished.
conclusive to d e m o n s t r a t e a specific sulfonucleotide r e q u i r e m e n t , n e i t h e r for spinach nor for yeast. E v i d e n c e for t h e a b i l i t y to use A P S as a sulfor d o n o r for a s s i m i l a t o r y sulfate r e d u c t i o n has been o b t a i n e d so far for r e d algae, b r o w n algae, blue green algae, a n d higher p l a n t s (see Table 4). This indicates t h a t a s s i m i l a t o r y sulfate r e d u c t i o n with A P S seems to be c o r r e l a t e d with organisms containing chloroplasts. The expert technical assistance of Mrs. Christen is gratefully acknowledged.
References Asahi, T.: Sulfur metabolism in higher plants. IV. Mechanism of sulfate reduction in chloroplasts. Biochim. biophys. Acta (Amst.) 82, 58-66 (1964) Brunngraber, E . F . , Chargaff, E. : Purification and properties of a nucleotide phosphotransferase from carrot. J. biol. Chem. 242, 4834-4840 (1967) Brunngraber, E. F., Chargaff, E.: A nucleotide phosphotransferase from Escherichia coli. Biochemistry 12, 3005-3012 (1973a) Brunngraber, E. F., Chargaff, E. : Nicotine adenine dinucleotide as substrate of the nucleotide phosphotransferase from Eschsrichia coll. Biochemistry 12, 3012-3016 (1973b) Burnell, J. N., Anderson, J.W. : Adenosine-5'-sulphatokinase activity in spinach leaf tissue. Biochem. J. 134, 565-579 (1973) Ellis, R. J. : Sulphate activation in higher plants. Planta (Berl.) 88, 34-42 (1969) Hilz, H., Kittler, M., Knappe, G.: Die Reduktion yon Sulfat in der Hefe. Biochem. Z. 332, 151-166 (1959)
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Hodson, R. C., Schiff, J. A., Searsella, A.J., Levinthal, M. : Studies of sulfate utilization by algae. 6. Adenosine-3'-phosphate-5'-phosphosulfate (PAPS) as an intermediate in thiosulfate formation from sulfate by cell-free extracts of Chlorella. Plant Physiol. 43, 563-569 (1968) Hodson, R. C., Schiff, J.A. : Preparation of adenosine-3'-phosphate-5'-phosphosulfate(PAPS) : An improved enzymatic method using Chlorella pyrenoidosa. Arch. Biochem. Biophys. 132, 151-156 (1969) Hodson, R. C., Schiff, J. A.: Studies of sulfate utilization by algae. 9. Fractionation of a cell-free system from Chlorella into two activities necessary for the reduction of adenosine3'-phosphate-5'-phosphosulfate to acid-volatile radioactivity. Plant Physiol. 47, 300 305 (1971) Lowry, O. H., Rosebrough, N.J., Farr, A.J., Randall, R. J. : Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 265-275 (1951) Pasternak, C. A., Ellis, R.J., Jones-Mortimer, M. C., Crichton, C. E. : The control of sulphate reduction in bacteria. Bioehem. J. 96, 270-275 (1965) Patterson, M. S., Greene, R.C.: Measurement of low energy beta-emitters in aqueous solution by liquid scintillation counting of emulsions. Anal. Chem. 117, 854-857 (1965) Schiff, J.A., Hodson, R. C.: The metabolism of sulfate. Ann. l~ev. Plant Physiol. 24, 381~114 (1973) Schmidt, A.: Untersuchungen zum Meehanismus der photosynthetisehen Sulfatreduktion isolierter Chloroplasten. Thesis, GSttingen 1968 Sehmidt, A.: An APS-sulfotransferase from Chlorella. Arch. mikrobiol. 84, 77 86 (1972a) Sehmidt, A.: Uber Teilreaktionen der photosynthetisehen Sulfatreduktion in zellfreien Systemen aus Spinatchloroplasten und Chlorella. Z. Naturforsch. 27b, 183-192 (1972b) Sehmidt, A., Abrams, W. R., Sehiff, J. A. : Studies of sulfate utilization by algae. Reduction of adcnosinc-5'-phosphosulfate (APS) to cysteine in extracts from Chlorella and mutants blocked for sulfate reduction. Enrop. J. Biochem. 47, 423-434 (1974) Schmidt, A., Schwenn, J. D. : On the mechanism of photosynthetic sulfate reduction. Second. Internat. Contr. on Photosynthesis, Stresa, 507-513 (1971) Schmidt, A., Trebst, A.: The mechanism of photosynthetic sulfate reduction by isolated chloroplasts. Biochim. biophys. Acta (Amst.) 189, 529 535 (1969) Tsang, M. L., Schiff, J. A. : A comparison of the enzymology of sulfate reduction in Chlorella and E. coll. Plant Physiol. 511, S 66 (1974) Tsang, M. L., Sehiff, J. A. : Distribution of adcnosine-5'-phosphosulfate (APS) and adenosine3'-phosphate-5'-phosphosulfate (PAPS) sulfotransferases in assimilatory sulfate reducers. Biol. Bull. 147, 502 (1974) Trebst, A., Schmidt, A.: Photosynthetic sulfate and sulfite reduction by isolated chloroplasts. In: Progress in photosynthesis research, vol. III, p. 1510-1516. Ed. H. Metzner: Tfibingen 1969 Wells, G. N., Hagemann, R. H. : Specificity for nicotinamide adenine dinueleatide by nitrate reductases from leaves. Plant Physiol. 54, 136-141 (1974) Wilson, L. G., Asahi, T., Bandurski, R. S. : Yeast sulfate-reducing system. I. Reduction of sulfate to sulfite. J. biol. Chem. 2116, 1822-1829 (1961)
A Sulfotransferase from Spinach Leaves Using Adenosine-5'-Phosphosulfate Ahlert Schmidt Botanisehes Institut der Universit~t Mfinchen, Menzinger Stral3e 67, D-8 Miinchen, Federal Republic of Germany l~eceived 19 February; accepted 30 April, 1975
Summary. Active sulfotransferase can be extracted from spinach (Spinaeea oleracea L.) leaves (and other higher plants) using a buffer system containing 0.1 M KC1 and thiol reagents. This sulfotransferase is labile, it can, however, be stabilized by storage in 70 % ammonium sulfate containing 10 mM mereaptoethanol. This extract will reduce labelled adenosine-5'phosphosulfate (APS) and 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to acid-volatile radioactivity when dithioerythrol is added. The reduction from PAPS requires magnesium chloride and is inhibited by calcium chloride and sodium fluoride, whereas these chemicals have little effect on the APS-sulfotransferase activity. The reduction rates from both nucleotides are stimulated by increasing ionic strength and are inhibited by phosphate and cyanide. In the presence of non-labelled APS the acid-volatile radioactivity distilled from [~5S] PAPS is drastically reduced, whereas the opposite experiment using [8~S] APS in the presence of non-labelled PAPS has little effect. This indicates that APS is an obligatory intermediate in the conversion of [35S] PAPS to acid-volatile radioactivity. It is therefore concluded that the sulfotransferase from sp]nach is specific for APS. Activity with APS as sulfur-donor was found in 5 other plants in addition to spinach: Peunisetum, Zea (Gramineae); Brassiea (Crueiferae); Helianthus (Compositae) ; and Vicia (Papilionaceae). These experiments demonstrate the use of APS for assimilatory sulfate reduction in higher plants. This has been shown previously for the green alga Chlorella.
Introduction I t has been t h o u g h t for a long tinle t h a t a s s i m i l a t o r y sulfate r e d u c t i o n is d i s t i n g u i s h e d from d i s s i m i l a t o r y sulfate r e d u c t i o n b y t h e specific use of P A P S as sulfate donor. However, in recent y e a r s a specificity t o w a r d A P S for assimilat o r y sulfate r e d u c t i o n in t h e green alga Chlorella has been d e m o n s t r a t e d (Schmidt, 1972a; Schiff a n d H o d s o n , 1973). This was d e m o n s t r a t e d with purified enzymes (Schmidt, 1972 a) a n d b y w o r k w i t h Chlorella m u t a n t s (Schiff a n d Hodson, 1973 ; S c h m i d t et al., 1974). F o r higher p l a n t s knowledge is still f r a g m e n t a r y . I t has been shown t h a t P A P S is f o r m e d in spinach (Schmidt, 1968, 1972b) a n d this o b s e r v a t i o n has been confirmed b y t h e group of A n d e r s o n (Burnell a n d Anderson, 1973). I t h a d been suggested t h a t P A P S should be t h e sulfur donor n e e d e d for r e d u c t i o n in spinach (Asahi, 1964) a n d S c h m i d t a n d T r e b s t (1969) came to t h e same conclusion based on c o m p a r a t i v e b i o c h e m i s t r y a n d t h e finding t h a t P A P S was indeed f o r m e d in spinach. However, e x p e r i m e n t s w i t h chloroplast e x t r a c t showed t h a t A P S a n d P A P S were m e t a b o l i z e d (Schmidt, 1972b). I t was therefore concluded in a n a l o g y to t h e Chlorella s y s t e m t h a t A P S should be the sulfur donor
Abbreviations: APS: Adenosine-5'-Phosphosulfate; PAPS: 31-Phosphoadenosine-5'-phosphosulfate; DTE : Dithioerythrol. 18
P l a n t a (BerI.), ~,rol. 124
268
A. Schmidt
n e e d e d for r e d u c t i o n (Schmidt, 1972a, b). However, Burnell a n d A n d e r s o n (1973) f a v o u r P A P S as i n t e r m e d i a t e in sulfate r e d u c t i o n in spinach. This p u b l i c a t i o n shows t h a t an active transferase can be e x t r a c t e d from spinach leaves. The experim e n t s w i t h A P S a n d P A P S a n d i n h i b i t o r studies i n d i c a t e t h a t A P S is t h e sulfur donor r e q u i r e d for f u r t h e r r e d u c t i o n in spinach. Materials and Methods Extract Preparation. Spinacea oleracea L. variety "Vital R " was grown in the botanical garden of this university. 50 g of leaves were homogenized in 100 m] of buffer in a "Braun Starmix" for 30 s at speed III. The buffer contained 0.1 M Tris-HC1 pH 8.0; 0.1 M KC1; 0.02 5~ MgC12; and 0.01 M mercaptoethanol. The crude extract was squeezed through cheesecloth and centrifuged afterwards for 20rain at 10000• The supernatant was brought to 70% ammonium sulfate and stored at --18 ~ C. An aliquot was thawed, centrifuged, and the pellet suspended in 1/2 of the original volume of fresh buffer. This extract was used for the experiments described. The extraction method has also been found suitable for other higher plants to yield active APS-sulfotransferase. Determination o/ Sul/otrans/erase Activity. This activity was determined by measuring acid-volatile radioactivity derived from either [35S] APS or [3~S] PAPS, using DTE as acceptor (Schmidt, 1972a). The acid-volatile radioactivity was distilled by adding nonlabelled sulfite and absorbed in 1 M triethanolamin and counted in a liquid scintillation counter Beckman Model LS-100 according to Patterson and Green (1965). Preparation o] Sul/onucleotides. APS and PAPS were synthesized enzymatically from sulfate and ATP (Hodson and Schiff, 1969; Schmidt et al., 1974). By this method labelled and non-labelled PAPS were prepared. PAPS was dephosphorylated to APS with a 3'-phosphatase from potato (Boehringer, No. 15438). Protein Determination. The method of Lowry was used (Lowry et al., 1951). Chemicals. [~5S] sulfate was ordered from Buchler, Braunschweig; ATP, 3'-AMP and APS were purchased from Boehringer, Mannheim; all other chemicals were obtained from ~r Darmstadt.
Results S p i n a c h e x t r a c t s p r e p a r e d as d e s c r i b e d in " m a t e r i a l s a n d m e t h o d s " will t r a n s f e r t h e sulfo-group of A P S onto suitable acceptors in t h e presence of D T E . This can be d e t e r m i n e d as acid-volatile r a d i o a c t i v i t y from [asS] A P S after t h e a d d i t i o n of non-labelled sulfite as described earlier (Schmidt, 1972a, b). Fig. 1 shows t h a t t h e A P S - s u l f o t r a n s f e r a s e r e a c t i o n is linear over a c e r t a i n range w i t h p r o t e i n c o n c e n t r a t i o n a n d over t h e one h o u r p e r i o d n o r m a l l y used for i n c u b a t i o n (Fig. 2). This h a d a l r e a d y been d e m o n s t r a t e d for t h e r e d u c t i o n of sulfate t o G S - S O 3- using chloroplast e x t r a c t from spinach ( S c h m i d t a n d Trebst, 1969; S c h m i d t , 1972b). O p t i m a l thiol c o n c e n t r a t i o n for these r e a c t i o n s h a d been d e t e r m i n e d b y t h e spinach s y s t e m too, therefore 10 mM D T E were used as a c c e p t o r during these investigations. The p H - o p t i m n m of t h e A P S - s u l f o t r a n s f e r a s e r e a c t i o n was f o u n d to be a r o u n d 8.5 (Fig. 3) which is close to t h e p H - o p t i m u m d e t e r m i n e d for t h e A P S - s u l f o t r a n s f c r a s e of Chlorella (Schmidt, 1972a). I t was decided to i n c u b a t e always a t p H 9.0 to o b t a i n conditions c o m p a r a b l e to t h o s e of t h e ChlorelIa-system used b y t t o d s o n a n d Schiff (1971). The transferase a c t i v i t y was s t i m u l a t e d with increasing ionic strength. T a b l e 1 d e m o n s t r a t e s t h a t best rates were o b t a i n e d w i t h N%SOa, while NHaCI, KC1, or (NHd)2SO 4 were less effective. The r a t e of t h e A P S - s u l f o t r a n s f e r a s e r e a c t i o n is increased w i t h a d d e d Na2SOd; o p t i m a l conditions were f o u n d a r o u n d 0.5 M Na2SO 4 (Fig. 4). H i g h ionic s t r e n g t h was suitable for stabilizing t h e e n z y m e to some extent. The sulfotransferase
APS-Sulfotransferase Activity from Spinach Leaves
269
3sS dist n mot
80-
s,
i
I .z /
70-
/
jJ
60-
/
50/.0]
/
]
/
/
l/
]
30x/ /
20xl I /
10-
I f
o;2
o;,
0'.3
o;
o:s ~, oozym~
Fig. 1. Protein-dependence of the APS-sulfotransferase reaction. Conditions (in ~xmol): TrisHCI pI-I 9.0: 100; MgCle: 10; DTE: 10; KCI: 200; APS: 0.25 (851 cpm/nmol) and enzyme as indicated (10.7 mg/ml) in a total volume of 1.0 ml. Incubation time 1 h at 37 ~ C under N2 35Sdist nmol
50,/
50-
/
40/
/
/
x
f
• z"
/
30
x ,/
20
f
/ •
10. /
/ z"
/
15
3'0
4'5
6'0
min
Fig. 2. Time-dependence of the APS-sulfotransferase reaction. Conditions as in Fig. 1, however KC1 was replaced by Na2SO 4. Na2SO~: 250; protein: 1.07 mg
activity is inhibited by phosphate, which was already noted for the reduction of sulfate to GS-SO a- with the chloroplast extract system (Schmidt, 1968). Cyanide was found to be inhibitory for the APS-sulfotransferase reaction, which might explain w h y ferrieyanide and ferroeyanide had no effect on this reaction sequence (Table 2, 3). Fluoride (10 raM), calcium ions (10 raM), and 3'-AMP (6 mM) have no signifieant inhibitory effects and magnesium ions m a y be ommitted without loss of activity, although an approximate 30% decrease of activity was noted when the reaction was run in 0.2 M KC1 without the addition of Na~SO 4. The addition of eysteine had no effect on the APS-sulfotransferase reaction (Tables 2, 3). This etude extract also catalyzes the sulfotransferase reaction from P A P S under the conditions described, as ean be seen in Table 2 and 3. I-Iowever, some differences to the APS-sulfotransferase reaction were noted. The conversion of lS*
270
A. Schmidt 3sSdi~ n m(
30-
/
20
/
X
~
x
~
X
•
10
7'5
8:0
8:5
9:0
9:5 pH
Fig. 3. pit-optimum for the APS-sulfotransferase reaction. Conditions (in ~zmol): Tris-HC1 pH 7.5 to 9.5: 100; MgC12: 10; DTE: 10; APS: 0.25 (851 cpm/nmol); KCI: 200; protein: 1.07 rag; total volume 1.0 ml. 1 h incubation at 37 ~ C under N 2 Table 1. Stimulation of the APS-sulfotransferase reaction with ionic strength
Without addition With 0.2 M KC1 With 0.7 1 KC1 With 0.7 ~ NaC1 With 0.7 1V[NHaC1 With 0.7 M (NHa)2S04 With 0.7 M Na~SO4
cpm
nmol
6935 28405 38350 47950 46315 39655 64785
8.14 33.3 45.1 56.3 54.4 46.6 76.1
Controls with 0.2 M KC1 : Added enzyme heated Enzyme heated, S0a2- added 10 Enzyme heated, S2 added 5 Conditions (in ~zmol): Tris-HC1 pH 9: 100; MgC12: 10; DTE: 10; APS: 0.25 (851 epm/nmol); protein: 1.07 mg; KC1, NaC1, (NI-Ia)2SO4, NH~CI, and Na2SO4 as indicated; soae-: 100; S2 : 100; in a total volume of 1.0 ml. Incubation time 1 h at 37~ C under N 2.
P A P S was s t i m u l a t e d to the same e x t e n t as was f o u n d for A P S b y high ionic s t r e n g t h ; a n d the i n h i b i t i o n b y K C N a n d phosphate was in the same order of m a g n i t u d e . However, in contrast to the APS-sulfotransferase reaction, a strict r e q u i r e m e n t for m a g n e s i u m ions using P A P S as sulfur donor (Table 2) was noted. F u r t h e r m o r e , the PAPS-sulfotransferase reaction is severely i n h i b i t e d b y fluoride a n d calcium ions, which h a d no effect on the APS-sulfotransferase reaction. This observation suggested t h a t P A P S m i g h t be dephosphorylated first to A P S a n d t h e n used as sulfur donor for reduction. If A P S is a n obligatory i n t e r m e d i a t e in the reduction of [~5S] P A P S , it should be possible to dilute the specific a c t i v i t y of [35S] A P S formed from [35S] P A P S b y the a d d i t i o n of non-labelled APS. This should be noticed i n a decrease of r a d i o a c t i v i t y transferred from [~5S] P A P S in the presence of non-labelled APS. As can be seen in Table 3, exactly this predicted
APS-Sulfotransferase Activity from Spinach Leaves
271
3SSdist nmol 70 60 50 40 30 20
/
I0' x
o.'25
0:s
o:75
i M N%SO,
Fig. 4. Dependence of the APS-sulfotransferase reaction from the sodium sulfate concentration. Conditions as in Fig. 3, however the reaction was run at pH 9.0 and KC1 was replaced by Na.2SQ as indicated
Table 2. Inhibitor studies using
APS and PAPS as sulfur donor for the sulfotransferase reaction cpm
nmol
% of control
28405 18 900 28 060 4415 140 1495 0 19 600 4 070 4 580 700
33.2 22.1 32.9 5.2 0.16 1.75 -52.4 10.9 12.2 1.9
100 67 99 16 -5 -100 21 23 4
57 915 57 690 57 640 28 445 3 610
68.1 67.8 67.7 76.1 9.7
100 99 99 100 13
0.2 M KC1
APS 4- MgC12 APS MgC12 AP S + MgC12-t-NaF APS -}- MgC12-}-KCN APS + MgC12- DTE APS @ MgCl~-- DTE -~ Fe(II)-cyanid APS ~ MgC12- DTE ~ Fe(III)-eyanid PAPS @ MgC1e PAPS -- MgCl 2 PAPS -~ MgC12@ NaF PAPS + MgCl~-l- KCN -
-
0.5 M Na2SO 4
APS ~ MgCI2 APS MgC12 APS -F MgC12-~ cysteine P A P S -}- MgCI~ PAPS -- MgCl~ -
-
Conditions (in Fmol) : Tris-HCl p H 9.0: 100; MgCl 2: 10; D T E : 10; APS : 0.25 (851 cpm/nmol) ; PAPS: 0.5 (374 cpm/nmol); eysteine: 5; N a F : 10; KCN: 10; Fe(II)-cyanid: 10; :Fe(III)cyanid: 10; KCI: 200; Na2SO4: 500; protein: 1.07 rag; total volume 1.0 ml. Incubation for 1 h at 37 ~ C under Ne.
d e c r e a s e in a c i d - v o l a t i l e r a d i o a c t i v i t y was f o u n d in s u c h a n e x p e r i m e n t . H o w e v e r , if P A P S is a n o b l i g a t o r y i n t e r m e d i a t e in t h e r e d u c t i o n s e q u e n c e of A P S , t h e a d d i t i o n of n o n - l a b e l l e d P A P S t o [35S] A P S s h o u l d d e c r e a s e t h e r a d i o a c t i v i t y
272
A. Sehmidt
Table 3. Inhibitor studies using APS and PAPS as sulfur donor for the sulfotransferase reaction cpm APS ~ APS § APS + APS ~ APS ~
nmol
% of control 100 99 100 21 90 100 33 83 18 21
MgCl2 MgCl2+ CaC12 MgCl2+ 3'-AMP MgC12-~phosphate MgCl2~-non-labelled PAPS
5460 5400 5450 5060
25.2 25.0 25.2 5.2 22.7
PAPS ~ MgCl~ PAPS ~ MgCl~ CaCl~ PAPS ~ MgCle-~3'-AMP PAPS ~ MgCI2~phosphate PAPS ~- MgC12~non-labelled APS
13325 4320 10990 2405 2860
35.6 11.6 29.4 6.4 7.6
1120
Conditions (in ~mol) : Tris-tIC1 pH 9.0: 100; MgCI2: 10; DTE : 10; APS : 0.25 (216 cpm/nmol) ; PAPS: 0.5 (374epm/nmol); Na2SOa: 500; CaC12: 10; 3'-AMP: 6; phosphate: 100; nonlabelled APS and PAPS: 1; protein: 0.53 mg; total volume 1.0 ml. Incubation time 1 h at 37~ C under N~.
transferred according to the same reasons stated above. The experiment, however, shows no significant decrease of radioactivity transferred! This suggests that APS is indeed an intermediate in the "PAPS-sulfotransferase reaction" and that the transferase is specific for APS. This has already been demonstrated for the APS-sulfotransferase from Chlorella (Schmidt, 1972a). Discussion
The formation of PAPS does not necessarily predict that this nucleotide is the sulfur donor needed for reduction (Schmidt, 1972a; Schiff and ttodson, 1973). For yeast it was shown by Hilz et al. (1959) that PAPS is needed for reduction, however Wilson et al. (1961) demonstrated that APS could be used as well, although with less activity. For Escherichia coli a specificity towards PAPS was found (Pasternak et al., 1965) and this has been confirmed by Tsang and Schiff (1974). Chlorella was shown originally to need PAPS for sulfate reduction (Hodson et al., 1968, 1971); however recent developments clearly demonstrate that the transferase is specific for APS and that the PAPS-confusion was due to a 3'-phosphatase activity converting PAPS to APS (Schmidt, 1972 ; Schiff and Hodson, 1973). The same situation has to be considered for spinach. Asahi (1964) concluded from his experiments that PAPS is the sulfur donor needed by spinach chloroplasts. On the other hand, Ellis (1969) suggested that APS might be used, since he could not demonstrate PAPS formation. I t was suggested that PAPS might be the sulfur donor in spinach, based on comparative biochemistry for assimilatory sulfate reduction and the fact that PAPS was indeed formed in spinach (Schmidt and Trebst, 1969). PAPS-formation in spinach was confirmed by Burnell and Anderson (1973). However, this group had to add 3'-AMP in order to find PAPS, whereas Schmidt (1972b) reported that thiol groups were needed for the spinach system and for the Chlorella-system for PAPS-formation. This suggests that two different mechanisms might be involved. I t seems possible that a
APS-Sulfotransfcrase Activity from Spinach Leaves
273
phosphatase exists, which transfers the phosphate group from 3'-AMP to APS forming PAPS. This would mimic the APS-kinase. Phosphotransferases have been found in microorganisms and plants (Brunngraber and Chargaff, 1967, 1973a, b). It was noted that for extraction and optimal assay conditions high ionic strength was needed. There could be various explanations for this: a) The sulfotransferase might be bound to membranes (for instance to chloroplast membranes (Sehmidt, 1968; Sehmidt and Trebst, 1969) and KC1 solubilizes the protein. b) Possible stabilization of the enzyme activity by high ionic strength. e) Perhaps the active site is unfolded under high ionic strength so that artificial thiols may reach the centre of the transferase. d) Possible inhibition of sulfatases. This would protect the sulfonueleotide from degradation, so that more sulfonueleotide is present for the transferase. Perhaps more than one argument discussed above might attribute to the good rates of the APS-sulfotransferase activity measured. The data of this paper too show that PAPS is metabolized to acid-volatile radioactivity. It is suggested on the basis of the experiments described in this paper that APS is the sulfur donor needed for reduction. The activity with PAPS seems to be due to a 3'-phosphatase, which converts PAPS to APS. It is possible to demonstrate such enzyme activities in the crude spinach extract by determination of the phosphate released from PAPS and by chromatographic determination of the APS formed (data not shown). Furthermore, the inhibition data which NaF, CaCI~, and 3'-AMP and the magnesium requirement are additional evidence of phosphatase activities (Burnell and Anderson, 1973 ; Wells and IKageman, 1974). If APS is the sulfur donor needed for reduction and PAPS firstly has to be dephosphorylated, the addition of non-labelled APS to [35S] PAPS should reduce the specific activity of [35S] APS formed by isotopic dilution; this reduces the amonnt of radioactivity distilled. If, on the other hand, PAPS is the sulfur donor required for reduction, the addition of non-labelled PAPS to [aS] APS should, in a similar manner, reduce the amount of radioactivity distilled. This pair of experiments should elucidate which sulfonueleotide is used for further reduction. The present data show that the addition of non-labelled APS to [35S] PAPS drastically reduces the radioactivity distilled, whereas the opposite experiment had little effect. This clearly indicates that APS is an intermediate in the "PAPS-sulfotransferase reaction". This, of course, is evidence that the sulfotransferase is specific for APS and the name APS-sulfotransferasc is suggested for this enzymatic activity (Sehmidt, 1972a). The older observation of Asahi (1964) who postulated that PAPS is needed for the spinach system is explained by the fact that he had to add the sulfate activation system from yeast to get active extracts. It is suggested here that the yeast extract was contaminated with the yeast transferase, since APS was found by spinach chloroplasts without the addition of yeast extract and--as is shown in this publication--the spinach enzyme is already active with APS. Thus the experiment of Asahi shows a coupling of yeast transferase to spinach reducible thiol groups, but it is not
274
A. Schmidt
Table 4. Distribution of the APS-sulfotransferase activity among photosynthetic organisms a) Among algae a Cyanophyta
Oscillatoria wononichii Synechococcus sp.
Chrysophyta
Cyclotella Chaetocerus pseudocrinitis
Chlorophyta
Chlorella pyrenoidosa
Pyrrophyta
Peridinium trochoidium Gymnodinium sp.
Phaeophyta
Fucus vesiculosus
Rhodophyta
Chondrus crispus Porphyridium acrugineum
b) Among higher plants b Gramineae
Pennisetum purpureum Zea maya
Chenopodiaceae
Spinacea oleracea
Compositae
Helianthus annuus
Papilionaceae
Vicia /aba
a According to Tsang and Schiff (1974). b Schmidt, unpublished.
conclusive to d e m o n s t r a t e a specific sulfonucleotide r e q u i r e m e n t , n e i t h e r for spinach nor for yeast. E v i d e n c e for t h e a b i l i t y to use A P S as a sulfor d o n o r for a s s i m i l a t o r y sulfate r e d u c t i o n has been o b t a i n e d so far for r e d algae, b r o w n algae, blue green algae, a n d higher p l a n t s (see Table 4). This indicates t h a t a s s i m i l a t o r y sulfate r e d u c t i o n with A P S seems to be c o r r e l a t e d with organisms containing chloroplasts. The expert technical assistance of Mrs. Christen is gratefully acknowledged.
References Asahi, T.: Sulfur metabolism in higher plants. IV. Mechanism of sulfate reduction in chloroplasts. Biochim. biophys. Acta (Amst.) 82, 58-66 (1964) Brunngraber, E . F . , Chargaff, E. : Purification and properties of a nucleotide phosphotransferase from carrot. J. biol. Chem. 242, 4834-4840 (1967) Brunngraber, E. F., Chargaff, E.: A nucleotide phosphotransferase from Escherichia coli. Biochemistry 12, 3005-3012 (1973a) Brunngraber, E. F., Chargaff, E. : Nicotine adenine dinucleotide as substrate of the nucleotide phosphotransferase from Eschsrichia coll. Biochemistry 12, 3012-3016 (1973b) Burnell, J. N., Anderson, J.W. : Adenosine-5'-sulphatokinase activity in spinach leaf tissue. Biochem. J. 134, 565-579 (1973) Ellis, R. J. : Sulphate activation in higher plants. Planta (Berl.) 88, 34-42 (1969) Hilz, H., Kittler, M., Knappe, G.: Die Reduktion yon Sulfat in der Hefe. Biochem. Z. 332, 151-166 (1959)
APS-Sulfotransferase Activity from Spinach Leaves
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