Planta (Berl.)130, 257-263 (1976)
P l ~ J H ~ J 9 by Springer-Verlag 1976
The Adenosine-5'-Phosphosulfate Sulfotransferase from Spinach (Spinacea oleracea L.). Stabilization, Partial Purification, and Properties* Ahlert Schmidt Botanisches Institut der Universit/it Mfinchen, Menzinger Str. 67, D-8000 Miinchen 19, Federal Republic of Germany
Summary. Adenosine-5'-phosphosulfate (APS) sulfotransferase was purified 25-fold from spinach (Spinacea oleracea L.) leaves by Sephadex-G-200 gel filtration and chromatography on DEAE-cellulose. Enzyme activity was stabilized with 0.05 M Tris-HC1 pH 8.0 containing 10 mM mercaptoethanol (ME), 10raM MgCI2, and 30% glycerol. The molecular weight of the APS-sulfotransferase was estimated by gel filtration to be about 110,000 daltons. The enzyme is specific for the sulfonucleotide APS; PAPS is not a sulfur donor for this reaction. The apparent Km for APS was found to be 13 gM. The enzyme activity was determined with dithioerythritol (DTE) as acceptor, which has an apparent Km of 0.6 raM. Glutathione can substitute for DTE; other thiols such as mercaptoethanol and cysteine are less effective. The APS-sulfotransferase activity is inhibited by 5'-AMP, which increases the Km for APS but does not change Vmax, suggesting a competetive inhibition. Reduced methylviologen cannot substitute for a thiol in the spinach enzyme system. Thus it seems that assimilatory APS-sulfotransferase from spinach is different from the dissimilatory APS-reductase from Desulfovibrio or Thiobacillus, where methylviologen can be used as the electron donor.
Introduction It has been implied that APS is the sulfur donor for assimilatory sulfate reduction in higher plants (Ellis, 1969; Schmidt, 1972 a, b; 1975 a, b). However, different statements have been made recently (Burnell and Anderson, 1973; Schwenn and Hennies, 1974), * Abbreviations: APS : Adenosine-5'-phosphosulfate; PAPS: 3'Phosphoadenosine-5'-phosphosulfate; DTE: 1,4-Dithioerythritol; BAL: 2,3-Dimercaptopropanol; ME: Mercaptoethanol
suggesting PAPS to be the sulfur donor needed for reduction. Thus it became necessary to purify the enzyme systems involved. Extraction of active sulfotransferase from plant material has recently been achieved (Schmidt, 1975a, b, c). In this publication we will describe the purification of the APS-sulfotransferase from spinach and demonstrate the specificity of this enzyme preparation towards the sulfonucleotide APS. Furthermore, some properties of the enzyme will be presented.
Materials and Methods a) Protein Preparations Spinacea oleracea L. variety "Vital R " was grown in the botanical garden of this university. Leaves were homogenized in a "Braun Starmix" for 30 s at speed III using a buffer system containing 0.1 M Tris-HC1 pH 8.0; 0.1 M KC1; 0.02 M MgC12; and 0.01 M mercaptoethanol. 2 ml of buffer were used for 1 g of plant material (Schmidt, 1975a). The crude extract was squeezed through cheesecloth and centrifuged for 20 min at 10,000 x g. The supernatant was brought to 70% ammonium sulfate and stored at - 1 8 ~ until use. 20 ml of concentrated extract (about 50 mg of protein in 1 ml) were applied to a Sephadex-G-200 column (5 x 25 cm) equilibrated with a buffer containing 30% glycerol (v/v) in 0.02 M Tris-HC1 pH 8.0 with 10 mM MgClz and 10 mM ME. Fig. 1 shows the elution profile. The active fractions were pooled and adsorbed on a DEAE-cellulose column (2 x 10 cm) equilibrated with the glycerol containing buffer mentioned above. The protein was eluted with a linear gradient of increasing NaC1 dissolved in the buffer mentioned above ranging from 0 to 0.5 M NaC1; 200 ml were used for each reservoir. Fig. 2 shows the protein profile and the activity of the APS-sulfotransferase. The active fractions were pooled and stored in 60% ammonium sulfate. During these two steps a 25-fold purification over the ammonium sulfate cut was achieved with a specific activity of 933 nmol/mg prot. h. Further purification steps have not been successful so far since enzyme activity was rapidly lost when the enzyme was not stored in 70% ammonium sulfate. Chloroplast extract was prepared according to Schmidt and Trebst (1969). Isolated chloroplasts were prepared using the method of Jensen and Bassham (1966). The chloroplasts were broken
258
A. Schmidt: APS-Sulfotransferase from Spinach Fig. 1. Separation of the APS-sulfotransferase activity on a Sephadex-G-200 column. Fractions of 6 ml were collected. 0.2 ml of each second tube were used for the APS-sulfotransferase assay. Incubation conditions were as in Table 1. e - - - 9 = APS-sulfotransferase activity; = optical density at 280 nm
t~,
100
:
k
i
~:8o
" f
E o60 >.
t
g
i
§
i
i
Q.
.,....~' ~b
20
3'o
;o
6'o
so
7'0
Fraction No.
d) Preparation of Sulfonucleotides
/'~'
20.000i
APS and PAPS were synthesized enzymatically from sulfate and ATP (Hodson and Schiff, 1969; Schmidt etal., 1974). By this method labelled and non-labelled PAPS was prepared. PAPS was dephosphorylated to APS with a 3'-Phosphatase from potato (Boehringer, 15438).
l l
~ 15.000-
]
>. ~ I0.000-
e) Protein Determination
3
The method of Lowry (Lowry et al., 1951) was used. For protein with a high thiol content a turbidometric method (precipitation with tricloroacetic acid and measurement of the density at 420 nm) with reference to bovine serum albumin was used (Schmidt, 1975e).
r
-o
g
r ~v ~-~
,,
- i
~.
I ....
/,0
.,'
?
0
20
60 80 Fraction No
f) Chemicals I00
120
Fig. 2. Separation of the APS-sulfotransferase activity on a DEAEcellulose column. Fractions of 2.5 ml were collected. Incubation conditions as in Table I, however the specific activity of APS used was 126 cpm/nmol. 0.2 ml of each second fraction was used for determination of the sulfotransferase activity, o - - - e = A P S - s u l f o transferase activity; A---A=activity with APS-sulfotransferase added from PAPS; - - = o p t i c a l density at 280 mm
(35S) sulfate was obtained from Buchler (Ammersham). Nonlabelled APS, AMP, ADP, 3'-AMP, T-AMP, 3'-5'-ADP and cA M P as well as protein standards for molecular weight determinations were purchased from Boehringer (Mannheim). Methylviologen and DEAE-cellulose were obtained from Serva (Heidelberg); Sephadex-G-200 was obtained from Pharmacia (Sweden) and all other chemicals from Merck (Darmstadt).
Results in 0.02M Tris-HCl 10 m M MgC12.
pH8.0
containing
10raM
DTE
and
b) Determination of Sulfotransferase Activity This activity was determined by measuring acid-volatile radioactivity derived fiom either [35S]APS or [3sS]PAPS (Schmidt, 1975a) using dithioerythritol (DTE) as a thiol reagent.
c) Determination of Radioactivity Radioactivity was determined in a liquid scintillation counter, Beckmann LS-100 model, using the scintillation cocktail according to Patterson and Greene (1965).
The APS-sulfotransferase fraction purified as described in "Materials and Methods" was used for all experiments. An aliquot of the protein material stored in 60% ammonium sulfate was pelleted by centrifugation and dissolved in an appropriate amount of the original buffer used for leaf homogenization. This partially purified APS-sulfotransferase preparation still needed the addition of sodium sulfate with optimal concentrations at 0.5 mM, as was found with crude plant extracts (Schmidt, 1975a, b, c). The APS-sulfotransferase activity of this enzyme fraction is linear over the one-hour period normally used for incubation; the pH-optimum was found to be around
A. Schmidt: APS-Sulfotransferase from Spinach
259 Table 1. Substrate specificitytoward APS and PAPS
1OB-
Acid-vo/atile radioactivity formed cpm
2" A l f l ~'o
10s
APS Sulfot . . . . fe....
~
.
AlburninA ([b~ bumin(
} g9
24
nmol
Catolase
0.4 ml of fraction a + APS +MgClz + PAPS+MgC12
114 50
0.4 ml of fraction b + APS+MgC12 + PAPS + MgC12
34,135 860
26.5 0.7
0.4 ml a+0.4 ml b + APS+MgC12 + PAPS + MgC12
15,050 4,895
11.7 3.8
a) 0.1 ml enzyme + APS -MgC12 + APS + MgC12 + PAPS- MgCI~ + PAPS +MgCI~
101,330 96,130 6,415 5,865
78.6 74.5 5.0 04.6
b) 0.2 ml enzyme + APS+MgCIz + PAPS+MgC12
202,430 6,920
156.9 5.4
0.09 0.04
Concentrated APS-sulfotransferase
10~
2~6 ' 2'B ' 3'0 3'2 3~4 ' 3~6 Fraction No~
3'8
Fig. 3. Molecular weight determination of the APS-sulfotransferase from spinach on a calibrated Sephadex-G-200 column. The APSsulfotransferase activity was determined according to the corlditions of Table 1; the column was calibrated according to the method of Andrews (1964). Fractions of 3.1 ml were collected
p H 9.0; a n d the activity was f o u n d to be linear with protein concentration. Since this behaviour o f the enzyme did not change during the purification procedure, the data will n o t be given here again for the purified enzyme fraction (see Schmidt, 1975 a).
a) Molecular Weight Determination
Conditions (in gmoles) : Tris-HC1 pH 9.0: 100; MgC12 : 10; DTE : 20; Na2SO4: 500; APS: 0.2 (1 nmol = 1,290 cpm), when indicated; PAPS: 0.225 (l nmol=l,290cpm) when indicated; APSsulfotransferase: 0.9 mg/ml; fraction a=pooled fractions 49 and 50 from the DEAE-cellulose column; fraction b=pooled fractions 71 to 75 from the DEAE-celtulose column; total volume 1.5 ml; incubation for 1 h under N2; 37~ C.
A Sephadex-G-200 c o l u m n (2 x 55 cm) was equilibrated with the buffer containing glycerol and was run under these conditions with the protein standards egg albumin ( M W = 45,000), bovine albumin ( M W = 67,000), aldolase f r o m rabbit ( M W = 1 5 8 , 0 0 0 ) , and catalase f r o m bovine liver ( M W = 2 4 0 , 0 0 0 ) . F r o m these data (see Fig. 3) a molecular weight o f the spin a c h APS-sulfotransferase o f a b o u t 110,000 daltons is obtained, which is a b o u t 1/3 the molecular weight o f the APS-sulfotransferase f r o m Chlorella (fraction " S " , H o d s o n and Schiff, 1971).
fur donor. Only when protein fraction (a) is added, which has the capacity to dephosphorylate P A P S to APS, can P A P S replace A P S in the enzyme assay, Protein fraction (a) alone has no sulfotransferase activity, neither with A P S nor with PAPS. T h a t the APS-sulfotransferase f r o m spinach is specific for APS can be d e m o n s t r a t e d m o r e clearly with the concentrated APS-sulfotransferase. F r o m these data (Table 1) it is concluded that this enzyme has practically no activity with PAPS. This has already been suggested f r o m w o r k with chloroplast extract (Schmidt, 1972b) and f r o m data obtained with crude spinach extracts (Schmidt, 1975 a).
b) Substrate Specificity
c) Thiol Specificity
Purified APS-sulfotransferase activity and fractions (a) and (b) were obtained as fractions f r o m the DEAE-cellulose c o l u m n run as described in " M a t e rials and M e t h o d s " . These fractions were tested for their ability to use A P S and P A P S as sulfate donors. F r o m the data o f Table 1 it is obvious that the A P S sulfotransferase fraction (b) c a n n o t use P A P S as sul-
It has already been f o u n d with the chloroplast extract that different rates were obtained in the reaction f r o m sulfate to acid-volatile radioactivity when various thiols were tested (Schmidt, 1968; Schmidt and Trebst, 1969). As can be seen f r o m the data in Table 2, dithioerythritol and glutathione have practically the same activity towards the APS-sulfotransferase, while
260
A. Schmidt: APS-Sulfotransferase from Spinach nmotes
Table 2. Comparison of different thiols as acceptor for the APS-sulfotransferase from spinach Additions dithioerythritol (DTE)
10 m M 30 m M
nmol
% of DTE
21.1 20.9
100 99
cysteine
10 m M
4.8
22
mercaptoethanol
10 m M 30 m M
2.8 3.4
13 16
BAL
10 m M 30 m M
13.6 18.4
64 87
10 m M
20.6
98
glutathione
3' 2"
ME o"~ " ~'''~
I"
,
i
zo
'.'o
nmoles
5 "L ~o 4.]]="
6'o
do
--:
I~o ~mot~ ,,.e DTE
21"
z'o
;o
do
do
~;o ~r~ol,,
nmoles
dithionite dithionite + methylviologen
1.4 1.3
7 6
without addition
0.3
1
Conditions (in gmol): Tris-HC1 pH 9.0: 100; MgC12: 10; Na2SO4: 500; APS: 0.16 (1 n m o l = 5 0 9 c p m ) ; thiols: as indicated; methylviologen: 2; NazS204: 14; protein: 0.06rag; in a total volume of 1 ml. Incubation for 1 h under N2; 37 ~ C.
9 3" s'Lf 21o
,
i 20
i
4'0
BAL 9
6nO
810
4-
Table 3. Influence of nucleotides containing adenine on the APS-sulfotransferase activity Additions
5 n moles
,
nmol
la m o l e s
ystein
3" 2" 1-
Acid-volatile radioactivity formed
100
nmoles
i
20
i
i
/~0
,
i
60
,
i
80
% of control
1(~0 g moles
o--
Glutathione
none adenosine T-AMP 3'-AMP Y-AMP c-AMP 3'-5'-ADP ADP ATP
124.8 107.5 122.5 115.1 62.2 124.6 120.9 111.9 107.5
100 86 98 92 50 100 96 90 86
Conditions (in gmol): Tris-HCl pH 9.0: 100; MgC12: 10; Na2SO4: 500; APS: 0.2 (1 n m o l = 126 cpm); protein: 0.13 mg; nucleotide as indicated: 1; in a total volume of 1 ml. Incubation for 1 h under N 2 : 37 ~ C.
monothiols like cysteine and mercaptoethanol are less effective. This behaviour is similar to activities found with the APS-sulfotransferase from Chlorella (Hodson and Schiff, 1971; Schmidt, 1972a). Methylviologen, reduced chemically by dithionite cannot substitute for the thiol requirement, although both chemicals are not inhibitory when tested with added dithioerythritol. Monothiols will, however, catalyze the APS-sulfotransferase reaction when applied in higher concentrations (Fig, 4). From these measurements an apparent Km for dithioerythritol of about 0.6 mM can be calculated. Surprisingly all thiols showed a good increases of the APS-sulfotransferase activity in the low concentration range. This leads to two different Km-regions for the monothiols mercapto-
# 2~0
4'0
'
6'0
8'0
100
~t m o t e s
Fig. 4. Dependence of the APS-sulfotransferase activity on thiol concentration. Conditions as in Table 1, however APS: 0.018 (1 nmol = 1,134 cpm) and protein: 0.08 mg was used; total volume 1.0 ml; incubation for 1 h: 37 ~ C.
ethanol and cysteine. One in the range around 1-2 mM and a second one in the range of about 20 mM. This was not found using dithiols like dithioerythritol of BAL and was not found with the monothiol glutathione.
d) Kin-determination for APS and Inhibition by 5'AMP
From data obtained with crude extracts from spinach, maize and Chlorella it was demonstrated that the APS-sulfotransferase is inhibited by Y-AMP (Schmidt, 1975b). The inhibition of the APS-sulfotransferase activity by Y-AMP was analyzed with our enzyme fraction. As can be seen from Table 3, different nucleotides containing adenine were tested for their ability to inhibit the APS-sulfotransferase activity. Among those nucleotides tested, only Y-AMP showed strong inhibition. This inhibition was ana-
A, Schmidt: APS-Sulfotransferase from Spinach
261
~oo~---~
%00-
J I I l
x
75-
+APS
-6?5E
1,
aE
o
._> "G 5 0 -
S0-
g
..o
o
o ~o
x
X 25-
25-
/ /
oi~ Fig, 5. Inhibition of the APS-sulfotransferase activity by Y-AMP. Conditions (in pmol): Tris-HC1 pH 9.0: 100; MgC12 : 10; DTE: 10; Na2SO,: 500; APS : 0.023 (1 nmol = 1,253 cpm); protein: 0.09 mg in a total volume of 1 ml. Incubation for 1 h under Nz; 37 ~ C. 100% = 13.1 nmoles acid-volatile radioactivity formed
E
z-
/
g o h5 O.S'
I
o > r
,--'-" o ~ ~
-80
o
;o
-I.'0
oh
ml chloroplast extroct
mMS/-AMP
2
*PAPS
~
Go
,~o
!
Fig. 7, Measurement of APS and PAPS sulfotransferase activity with spinach chloroplast extract. Conditions (in umoles) : Tris-HC1 p H 9 . 0 : 100; MgC12: 10; DTE: 10; Na2SO4: 500; APS: 0.16 (1 nmo1=856 cpm); PAPS: 0.2 (1 n m o l = 1,200 cpm); chloroplast extract: 0.8 mg/ml, amount added as indicated; total volume 1 ml. Incubation for 1 h under Nz, 37 ~ C
lyzed by keeping the APS concentration constant and varying the Y-AMP concentration. The results of this experiment are shown in Fig. 5. Inhibition by Y-AMP is found above 0.5 mM. Since AMP is the endproduct of the APS-sulfotransferase reaction and a product of APS degradation, the nature of this inhibition was investigated further. The Km for APS was determined in the absence and presence of Y-AMP (1 raM). The data of this experiment are shown in Fig. 6. The Km for APS was found to be 13 pM in the absence of Y-AMP. However, after the addition of 5'-AMP the Km increased to about 190 gM. Since Vmax did not change in the presence of Y-AMP it is concluded that the inhibition of Y-AMP is competetive to APS. It should be pointed out here that the low Km for APS was obtained only when less than 0.02 mg protein was used for one assay. If 0.1 mg of protein was used for the assay, the Km increased to about 80 taM for APS, which is probably due to APS-degrading activities still present in this preparation.
llM APS
Fig, 6. Lineweaver-Burk plot for the APS-sulfotransferase activity in the absence and presence o f 5"-AMP. Conditions (in gmo[): Tris-HCt p H 9.0: 100; MgCI2: 10; DTE: 10; NazSO4: 500; APS: varied as indicated, (1 n m o l = 1,330cpm); Y-AMP: 1, when indicated; protein: 0.018 mg; in a total volume of 1 ml. Incubation for 1 h at 37 ~ C; Nz. • x activity with 5'-AMP added; o - - o activity without 5"-AMP
e) Localization It was discovered some years ago that the entire sequence of enzymes needed for sulfate reduction is associated with chloroplasts from Spinach (Schmidt,
262 1968; Schmidt and Trebst, 1969; Trebst and Schmidt, t969; Schwenn, 1970; Schmidt and Schwenn, 1971). Chloroplast extract was therefore prepared (Schmidt and Trebst, 1969) and analyzed with APS and PAPS under optimal conditions for the APS-sulfotransferase assay (Schmidt, 1975a). The data are given in Fig. 7. APS is metabolized by this preparation with a high rate of 560 nmol/mg protein x h, which is about 18 times greater than the specific activity of the APS-sulfotransferase of crude leaf extract (31 nmol/mg protein x h; Schmidt, 1975c). PAPS is also metabolized, the rate, however, being about 56 nmol/mg protein x h, suggesting that the dephosphorylation of PAPS to APS is the limiting step. This would be in agreement with the observation of Burnell and Anderson (1973) that chloroplast extract has little 3'-phosphatase activity. From the fact that the specific activity of the APS-sulfotransferase measured with chloroplast extract was 18 times greater it is concluded that the APS-sulfotransferase in spinach leaves is localized within the chloroplast system.
Discussion
Higher plants contain an enzyme system catalyzing the transfer of activated sulfur onto suitable acceptors (Schmidt, 1972a, 1975a, b, c). This enzyme was found in the green alga Chlorella and was named APSsulfotransferase, since it was found to be specific for the sulfonucleotide APS (Schmidt, 1972 a; Schiff and Hodson, 1973). For higher plants we had suggested, on the basis of comparative biochemistry and on data obtained with spinach chloroplast extract, that APS is the sulfur donor needed for assimilatory sulfate reduction (Schmidt, 1972 a, b). However, this was not accepted by Burnell and Anderson (1973) and Schwenn and Hennies (1974) who suggested PAPS to be the sulfur donor in spinach. It therefore became necessary to purify this enzyme from plant material in order to determine the substrate specificity towards sulfonucleotides. From inhibitor studies with crude leaf extract it was suggested recently that the sulfotransferase from spinach should be specific for APS (see Schmidt, 1975 a) and the data of this paper clearly demonstrate with partially purified enzyme that the sulfotransferase from spinach is specific for APS and has little or no activity towards PAPS. The name APS-sulfotransferase is therefore suggested for this activity (see Schmidt, 1972a, b, 1975a). APS-sulfotransferase activity has been detected in over 50 plant families examined (Schmidt, 1975 c). Some properties of the APS-sulfotransferase from spinach will now be discussed.
A. Schmidt: APS-Sulfotransferasefrom Spinach
a) Use of APS PAPS had been implied for assimilatory sulfate reduction since the unfavourable equilibrium constant for APS formation seemed to need a further step to pull the reaction to the product side and this was the phosphorylation of APS to PAPS. However, dissimilatory sulfate reduction proceeds without PAPS formation; thus the phosphorylation of APS to PAPS does not seem to be necessary. If another reaction with a similar low Km as reported for the APS-kinase could be found which leads to about the same standard free energy change as observed during the phosphorylation of APS to PAPS, one would have another explainable mechanism for the use of APS. The APSsulfotransferase seems to fulfil the requirements needed for such a reaction. The apparent Km for APS determined to be 13 gM and the standard energy change in this reaction should be in the order of AG~ - 12 kcal mol 1, assuming a standard free energy change for APS hydrolysis AG~ 18 kcal mol-1 (see Roy and Trudinger, 1970) and for S-sulfoglutathione hydrolysis (sulfate ester, organic thiosulfate) A G O = - 6 kcal mol-i, which, however, might be too high. This change of standard free energy is better for the APS-sulfotransferase reaction in this case, explaining why PAPS-formation is not a necessity for assimilatory sulfate reduction, since the standard free energy change for APS-phosphorylation is in the order of A G ~ 5 kcal mol- 1
b) Regulation The APS-sulfotransferase from spinach has a pH optimum of about 9. From work with whole cells it is known that sulfate in plants is reduced mainly during the light period (see Willenbrink, 1964). This can be explained by the rise of pH within the chloroplast during the light periods which in turn increases the activity of the APS-sulfotransferase. Furthermore, the ratio of APS to AMP will change due to photophosphorylation in the light period, which leads to a decrease of the AMP concentration. Since the APS-pool is small, a competetive inhibition of the APS-sulfotransferase activity might well have regulatory significance for light and dark periods with the change of pH.
c) Differences to Dissimilatory APS-reduetases Assimilatory APS-sulfotransferases from Chlorella (Schmidt, 1972a) or spinach (Schmidt, 1975a; and this publication) have a strict requirement for thiol
A. Schmidt: APS-Sulfotransferase from Spinach
groups to catalyze this reaction. Methylviologen is not an electron donor for the APS-sulfotransferase from spinach or Chlorella. Furthermore, assimilatory APS-sulfotransferase from Chlorella has been shown to catalyze the formation of bound and free sulfite depending on the conditions used (Schmidt, 1972 a, b; Schmidt, 1973; Schiff and Hodson, 1973; Abrams and Schiff, 1973). However, the formation of APS fiom bound or free sulfite has not been demonstrated so far with assimilatory APS-sulfotransferases. Another difference to dissimilatory APS-reductase is the failure to catalyze isotopic exchange reactions between sulfite and APS. We have not been able to demonstrate this exchange reaction with assimilatory APS-sulfotransferase from spinach or Chlorella (Schmidt, 1972 a, 1975 a; and this publication). Dissimilatory APS-reductase catalyzes the formation of APS from sulfite and AMP and the reverse reaction depending on the reaction conditions (Peck, 1974). The formation of acid-volatile radioactivity from [35S]APS with this enzyme can also be demonstrated using methylviologen as reductant (Peck et al., 1965). Thus dissimilatory APS-reductases catalyze the reaction in both directions where as assimilatory APS-sulfotransferases are "one way" catalysts. The expert technical assistance of Mrs. Christen is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft.
References Abrams, W.R., Schiff, J.A. : Studies of sulfate utilization by algae. 11. An enzyme-bound intermediate in the reduction of adenosine-5'-phosphosulfate (APS) by cell-free extracts of wild type Chlorella and mutants blocked for sulfate reduction. Arch. Mikrobiol. 94, 1-10 (1973) Andrews, P.: Estimation of the molecular weight of proteins by Sephadex-gel filtration. Biochem. J. 91, 222233 (1964) 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) Hodson, R.C., Schiff, J.A. : Preparation of adenosinc-3'-phosphate5'-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 adenosine-Y-phosphate5'-phosphosulfate to acidvolatile radioactivity. Plant Physiol. 47, 300-305 (1971) Jensen, R.G., Bassham, J.K.: Photosynthesis by isolated chloroplasts. Proc. nat. Acad. Sci. USA 56, 1095-1101 (1966)
263 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) Patterson, M.S., Greene, R.C. : Measurement of low energy betaemitters in aqueous solution by liquid scintillation counting of emulsions. Anal. Chem. 37, 854-857 (1956) Peck, H.D., Jr., Deacon, T.E., Davidson, J.T.: Studies on adenosine-5'-phosphosulfate reductase from Desulfovibrio desulfuricans and Thiobacillus thioparus. Biochim. Biophys. Acta 96, 429-446 (1965) Peck, H.D. Jr. : The evolutionary significance of inorganic sulfur metabolism. Symp. Soc. Gen. Microbiol. XXIV, 241262 (1974) Roy, A.B., Trudinger, P.A.: The biochemistry of inorganic compounds of sulphur. Cambridge: University press 1970 Schiff, J.A., Hodson, R.C. : The metabolism of sulfate. Ann. Rev. Plant Physiol. 24, 381414 (1973) Schmidt, A. : Ober Teilreaktionen der photosynthetischen Sulfatreduktion isolierter Chloroplasten. Thesis. G6ttingen (1968) Schmidt, A. : On the mechanism of photosynthetic sulfate reduction. An APS-sulfotransferase from Chlorella. Arch. Mikrobiol. 84, 77 86 (1972a) Schmidt, A. : Ober Teilreaktionen der photosynthetischen Sulfatreduktion in zellfreien Systemen aus Spinatchloroplasten und Chlorella. Z. Naturforsch. 27b, 183-192 (1972b) Schmidt, A. : Sulfate reduction in a cell-free system of Chlorella. The ferredoxin-dependent reduction of a protein-bound intermediate by a thiosulfonate reductase. Arch. Microbiol. 93, 2952 (1973) Schmidt, A. : A sulfotransferase from spinach leaves using adenosine-5'-phosphosulfate. Planta (Berl.) 124, 267-275 (1975) Schmidt, A. : Inhibition of the adenosine.5'-phosphosulfate-sulfotransferase activity from spinach, maize, and Chlorella by adenosine-5'-monophosphate. Planta (Berl.) 127, 93-95 (1975) Schmidt, A.: Distribution of the APS-sulfotransferase activity among higher plants. Plant Sci. Lett. 5, 407-415 (1975) Schmidt, A., Schwenn, J.D. : On the mechanism of photosynthetic sulfate reduction. Second. Int. Congr. Photosynthesis, Stresa, 507-513 (1971) Schmidt, A., Trebst, A. : The mechanism of photosynthetic sulfate reduction by isolated chloroplasts. Biochim. Biophys. Acta (Amst.) 180, 529-535 (1969) 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, 423434 (1974) Schwenn, J.D.: Untersuchungen zur Kinetik der photosynthetischen Sulfatreduktion isolierter Chloroplasten. Thesis. Bochum (1970) Schwenn, J.D., Hennies, H.H. : Enzymes and bound intermediate involved in photosynthetic sulfate reduction of spinach chloroplasts and Chlorella. In: M. Avron: Proceedings of the third Internat. Congr. on Photosynthesis. pp. 629-635. Amsterdam: Elsevier 1974 Trebst, A., Schmidt, A.: Photosynthetic sulfate and sulfite reduction by isolated chloroplasts. In: Progress in photosynthesis research, Vol. III, pp. 1510-1516. Ed.: Metzner, H. : Tfibingen 1969 Willenbrink, J.: Lichtabhfingiger 35S-Einbau in organische Bindung in Tomatenpflanzen. Z. Naturforsch. 19b, 356-357 (1964)
Received 15 January," accepted 9 February 1976
P l ~ J H ~ J 9 by Springer-Verlag 1976
The Adenosine-5'-Phosphosulfate Sulfotransferase from Spinach (Spinacea oleracea L.). Stabilization, Partial Purification, and Properties* Ahlert Schmidt Botanisches Institut der Universit/it Mfinchen, Menzinger Str. 67, D-8000 Miinchen 19, Federal Republic of Germany
Summary. Adenosine-5'-phosphosulfate (APS) sulfotransferase was purified 25-fold from spinach (Spinacea oleracea L.) leaves by Sephadex-G-200 gel filtration and chromatography on DEAE-cellulose. Enzyme activity was stabilized with 0.05 M Tris-HC1 pH 8.0 containing 10 mM mercaptoethanol (ME), 10raM MgCI2, and 30% glycerol. The molecular weight of the APS-sulfotransferase was estimated by gel filtration to be about 110,000 daltons. The enzyme is specific for the sulfonucleotide APS; PAPS is not a sulfur donor for this reaction. The apparent Km for APS was found to be 13 gM. The enzyme activity was determined with dithioerythritol (DTE) as acceptor, which has an apparent Km of 0.6 raM. Glutathione can substitute for DTE; other thiols such as mercaptoethanol and cysteine are less effective. The APS-sulfotransferase activity is inhibited by 5'-AMP, which increases the Km for APS but does not change Vmax, suggesting a competetive inhibition. Reduced methylviologen cannot substitute for a thiol in the spinach enzyme system. Thus it seems that assimilatory APS-sulfotransferase from spinach is different from the dissimilatory APS-reductase from Desulfovibrio or Thiobacillus, where methylviologen can be used as the electron donor.
Introduction It has been implied that APS is the sulfur donor for assimilatory sulfate reduction in higher plants (Ellis, 1969; Schmidt, 1972 a, b; 1975 a, b). However, different statements have been made recently (Burnell and Anderson, 1973; Schwenn and Hennies, 1974), * Abbreviations: APS : Adenosine-5'-phosphosulfate; PAPS: 3'Phosphoadenosine-5'-phosphosulfate; DTE: 1,4-Dithioerythritol; BAL: 2,3-Dimercaptopropanol; ME: Mercaptoethanol
suggesting PAPS to be the sulfur donor needed for reduction. Thus it became necessary to purify the enzyme systems involved. Extraction of active sulfotransferase from plant material has recently been achieved (Schmidt, 1975a, b, c). In this publication we will describe the purification of the APS-sulfotransferase from spinach and demonstrate the specificity of this enzyme preparation towards the sulfonucleotide APS. Furthermore, some properties of the enzyme will be presented.
Materials and Methods a) Protein Preparations Spinacea oleracea L. variety "Vital R " was grown in the botanical garden of this university. Leaves were homogenized in a "Braun Starmix" for 30 s at speed III using a buffer system containing 0.1 M Tris-HC1 pH 8.0; 0.1 M KC1; 0.02 M MgC12; and 0.01 M mercaptoethanol. 2 ml of buffer were used for 1 g of plant material (Schmidt, 1975a). The crude extract was squeezed through cheesecloth and centrifuged for 20 min at 10,000 x g. The supernatant was brought to 70% ammonium sulfate and stored at - 1 8 ~ until use. 20 ml of concentrated extract (about 50 mg of protein in 1 ml) were applied to a Sephadex-G-200 column (5 x 25 cm) equilibrated with a buffer containing 30% glycerol (v/v) in 0.02 M Tris-HC1 pH 8.0 with 10 mM MgClz and 10 mM ME. Fig. 1 shows the elution profile. The active fractions were pooled and adsorbed on a DEAE-cellulose column (2 x 10 cm) equilibrated with the glycerol containing buffer mentioned above. The protein was eluted with a linear gradient of increasing NaC1 dissolved in the buffer mentioned above ranging from 0 to 0.5 M NaC1; 200 ml were used for each reservoir. Fig. 2 shows the protein profile and the activity of the APS-sulfotransferase. The active fractions were pooled and stored in 60% ammonium sulfate. During these two steps a 25-fold purification over the ammonium sulfate cut was achieved with a specific activity of 933 nmol/mg prot. h. Further purification steps have not been successful so far since enzyme activity was rapidly lost when the enzyme was not stored in 70% ammonium sulfate. Chloroplast extract was prepared according to Schmidt and Trebst (1969). Isolated chloroplasts were prepared using the method of Jensen and Bassham (1966). The chloroplasts were broken
258
A. Schmidt: APS-Sulfotransferase from Spinach Fig. 1. Separation of the APS-sulfotransferase activity on a Sephadex-G-200 column. Fractions of 6 ml were collected. 0.2 ml of each second tube were used for the APS-sulfotransferase assay. Incubation conditions were as in Table 1. e - - - 9 = APS-sulfotransferase activity; = optical density at 280 nm
t~,
100
:
k
i
~:8o
" f
E o60 >.
t
g
i
§
i
i
Q.
.,....~' ~b
20
3'o
;o
6'o
so
7'0
Fraction No.
d) Preparation of Sulfonucleotides
/'~'
20.000i
APS and PAPS were synthesized enzymatically from sulfate and ATP (Hodson and Schiff, 1969; Schmidt etal., 1974). By this method labelled and non-labelled PAPS was prepared. PAPS was dephosphorylated to APS with a 3'-Phosphatase from potato (Boehringer, 15438).
l l
~ 15.000-
]
>. ~ I0.000-
e) Protein Determination
3
The method of Lowry (Lowry et al., 1951) was used. For protein with a high thiol content a turbidometric method (precipitation with tricloroacetic acid and measurement of the density at 420 nm) with reference to bovine serum albumin was used (Schmidt, 1975e).
r
-o
g
r ~v ~-~
,,
- i
~.
I ....
/,0
.,'
?
0
20
60 80 Fraction No
f) Chemicals I00
120
Fig. 2. Separation of the APS-sulfotransferase activity on a DEAEcellulose column. Fractions of 2.5 ml were collected. Incubation conditions as in Table I, however the specific activity of APS used was 126 cpm/nmol. 0.2 ml of each second fraction was used for determination of the sulfotransferase activity, o - - - e = A P S - s u l f o transferase activity; A---A=activity with APS-sulfotransferase added from PAPS; - - = o p t i c a l density at 280 mm
(35S) sulfate was obtained from Buchler (Ammersham). Nonlabelled APS, AMP, ADP, 3'-AMP, T-AMP, 3'-5'-ADP and cA M P as well as protein standards for molecular weight determinations were purchased from Boehringer (Mannheim). Methylviologen and DEAE-cellulose were obtained from Serva (Heidelberg); Sephadex-G-200 was obtained from Pharmacia (Sweden) and all other chemicals from Merck (Darmstadt).
Results in 0.02M Tris-HCl 10 m M MgC12.
pH8.0
containing
10raM
DTE
and
b) Determination of Sulfotransferase Activity This activity was determined by measuring acid-volatile radioactivity derived fiom either [35S]APS or [3sS]PAPS (Schmidt, 1975a) using dithioerythritol (DTE) as a thiol reagent.
c) Determination of Radioactivity Radioactivity was determined in a liquid scintillation counter, Beckmann LS-100 model, using the scintillation cocktail according to Patterson and Greene (1965).
The APS-sulfotransferase fraction purified as described in "Materials and Methods" was used for all experiments. An aliquot of the protein material stored in 60% ammonium sulfate was pelleted by centrifugation and dissolved in an appropriate amount of the original buffer used for leaf homogenization. This partially purified APS-sulfotransferase preparation still needed the addition of sodium sulfate with optimal concentrations at 0.5 mM, as was found with crude plant extracts (Schmidt, 1975a, b, c). The APS-sulfotransferase activity of this enzyme fraction is linear over the one-hour period normally used for incubation; the pH-optimum was found to be around
A. Schmidt: APS-Sulfotransferase from Spinach
259 Table 1. Substrate specificitytoward APS and PAPS
1OB-
Acid-vo/atile radioactivity formed cpm
2" A l f l ~'o
10s
APS Sulfot . . . . fe....
~
.
AlburninA ([b~ bumin(
} g9
24
nmol
Catolase
0.4 ml of fraction a + APS +MgClz + PAPS+MgC12
114 50
0.4 ml of fraction b + APS+MgC12 + PAPS + MgC12
34,135 860
26.5 0.7
0.4 ml a+0.4 ml b + APS+MgC12 + PAPS + MgC12
15,050 4,895
11.7 3.8
a) 0.1 ml enzyme + APS -MgC12 + APS + MgC12 + PAPS- MgCI~ + PAPS +MgCI~
101,330 96,130 6,415 5,865
78.6 74.5 5.0 04.6
b) 0.2 ml enzyme + APS+MgCIz + PAPS+MgC12
202,430 6,920
156.9 5.4
0.09 0.04
Concentrated APS-sulfotransferase
10~
2~6 ' 2'B ' 3'0 3'2 3~4 ' 3~6 Fraction No~
3'8
Fig. 3. Molecular weight determination of the APS-sulfotransferase from spinach on a calibrated Sephadex-G-200 column. The APSsulfotransferase activity was determined according to the corlditions of Table 1; the column was calibrated according to the method of Andrews (1964). Fractions of 3.1 ml were collected
p H 9.0; a n d the activity was f o u n d to be linear with protein concentration. Since this behaviour o f the enzyme did not change during the purification procedure, the data will n o t be given here again for the purified enzyme fraction (see Schmidt, 1975 a).
a) Molecular Weight Determination
Conditions (in gmoles) : Tris-HC1 pH 9.0: 100; MgC12 : 10; DTE : 20; Na2SO4: 500; APS: 0.2 (1 nmol = 1,290 cpm), when indicated; PAPS: 0.225 (l nmol=l,290cpm) when indicated; APSsulfotransferase: 0.9 mg/ml; fraction a=pooled fractions 49 and 50 from the DEAE-cellulose column; fraction b=pooled fractions 71 to 75 from the DEAE-celtulose column; total volume 1.5 ml; incubation for 1 h under N2; 37~ C.
A Sephadex-G-200 c o l u m n (2 x 55 cm) was equilibrated with the buffer containing glycerol and was run under these conditions with the protein standards egg albumin ( M W = 45,000), bovine albumin ( M W = 67,000), aldolase f r o m rabbit ( M W = 1 5 8 , 0 0 0 ) , and catalase f r o m bovine liver ( M W = 2 4 0 , 0 0 0 ) . F r o m these data (see Fig. 3) a molecular weight o f the spin a c h APS-sulfotransferase o f a b o u t 110,000 daltons is obtained, which is a b o u t 1/3 the molecular weight o f the APS-sulfotransferase f r o m Chlorella (fraction " S " , H o d s o n and Schiff, 1971).
fur donor. Only when protein fraction (a) is added, which has the capacity to dephosphorylate P A P S to APS, can P A P S replace A P S in the enzyme assay, Protein fraction (a) alone has no sulfotransferase activity, neither with A P S nor with PAPS. T h a t the APS-sulfotransferase f r o m spinach is specific for APS can be d e m o n s t r a t e d m o r e clearly with the concentrated APS-sulfotransferase. F r o m these data (Table 1) it is concluded that this enzyme has practically no activity with PAPS. This has already been suggested f r o m w o r k with chloroplast extract (Schmidt, 1972b) and f r o m data obtained with crude spinach extracts (Schmidt, 1975 a).
b) Substrate Specificity
c) Thiol Specificity
Purified APS-sulfotransferase activity and fractions (a) and (b) were obtained as fractions f r o m the DEAE-cellulose c o l u m n run as described in " M a t e rials and M e t h o d s " . These fractions were tested for their ability to use A P S and P A P S as sulfate donors. F r o m the data o f Table 1 it is obvious that the A P S sulfotransferase fraction (b) c a n n o t use P A P S as sul-
It has already been f o u n d with the chloroplast extract that different rates were obtained in the reaction f r o m sulfate to acid-volatile radioactivity when various thiols were tested (Schmidt, 1968; Schmidt and Trebst, 1969). As can be seen f r o m the data in Table 2, dithioerythritol and glutathione have practically the same activity towards the APS-sulfotransferase, while
260
A. Schmidt: APS-Sulfotransferase from Spinach nmotes
Table 2. Comparison of different thiols as acceptor for the APS-sulfotransferase from spinach Additions dithioerythritol (DTE)
10 m M 30 m M
nmol
% of DTE
21.1 20.9
100 99
cysteine
10 m M
4.8
22
mercaptoethanol
10 m M 30 m M
2.8 3.4
13 16
BAL
10 m M 30 m M
13.6 18.4
64 87
10 m M
20.6
98
glutathione
3' 2"
ME o"~ " ~'''~
I"
,
i
zo
'.'o
nmoles
5 "L ~o 4.]]="
6'o
do
--:
I~o ~mot~ ,,.e DTE
21"
z'o
;o
do
do
~;o ~r~ol,,
nmoles
dithionite dithionite + methylviologen
1.4 1.3
7 6
without addition
0.3
1
Conditions (in gmol): Tris-HC1 pH 9.0: 100; MgC12: 10; Na2SO4: 500; APS: 0.16 (1 n m o l = 5 0 9 c p m ) ; thiols: as indicated; methylviologen: 2; NazS204: 14; protein: 0.06rag; in a total volume of 1 ml. Incubation for 1 h under N2; 37 ~ C.
9 3" s'Lf 21o
,
i 20
i
4'0
BAL 9
6nO
810
4-
Table 3. Influence of nucleotides containing adenine on the APS-sulfotransferase activity Additions
5 n moles
,
nmol
la m o l e s
ystein
3" 2" 1-
Acid-volatile radioactivity formed
100
nmoles
i
20
i
i
/~0
,
i
60
,
i
80
% of control
1(~0 g moles
o--
Glutathione
none adenosine T-AMP 3'-AMP Y-AMP c-AMP 3'-5'-ADP ADP ATP
124.8 107.5 122.5 115.1 62.2 124.6 120.9 111.9 107.5
100 86 98 92 50 100 96 90 86
Conditions (in gmol): Tris-HCl pH 9.0: 100; MgC12: 10; Na2SO4: 500; APS: 0.2 (1 n m o l = 126 cpm); protein: 0.13 mg; nucleotide as indicated: 1; in a total volume of 1 ml. Incubation for 1 h under N 2 : 37 ~ C.
monothiols like cysteine and mercaptoethanol are less effective. This behaviour is similar to activities found with the APS-sulfotransferase from Chlorella (Hodson and Schiff, 1971; Schmidt, 1972a). Methylviologen, reduced chemically by dithionite cannot substitute for the thiol requirement, although both chemicals are not inhibitory when tested with added dithioerythritol. Monothiols will, however, catalyze the APS-sulfotransferase reaction when applied in higher concentrations (Fig, 4). From these measurements an apparent Km for dithioerythritol of about 0.6 mM can be calculated. Surprisingly all thiols showed a good increases of the APS-sulfotransferase activity in the low concentration range. This leads to two different Km-regions for the monothiols mercapto-
# 2~0
4'0
'
6'0
8'0
100
~t m o t e s
Fig. 4. Dependence of the APS-sulfotransferase activity on thiol concentration. Conditions as in Table 1, however APS: 0.018 (1 nmol = 1,134 cpm) and protein: 0.08 mg was used; total volume 1.0 ml; incubation for 1 h: 37 ~ C.
ethanol and cysteine. One in the range around 1-2 mM and a second one in the range of about 20 mM. This was not found using dithiols like dithioerythritol of BAL and was not found with the monothiol glutathione.
d) Kin-determination for APS and Inhibition by 5'AMP
From data obtained with crude extracts from spinach, maize and Chlorella it was demonstrated that the APS-sulfotransferase is inhibited by Y-AMP (Schmidt, 1975b). The inhibition of the APS-sulfotransferase activity by Y-AMP was analyzed with our enzyme fraction. As can be seen from Table 3, different nucleotides containing adenine were tested for their ability to inhibit the APS-sulfotransferase activity. Among those nucleotides tested, only Y-AMP showed strong inhibition. This inhibition was ana-
A, Schmidt: APS-Sulfotransferase from Spinach
261
~oo~---~
%00-
J I I l
x
75-
+APS
-6?5E
1,
aE
o
._> "G 5 0 -
S0-
g
..o
o
o ~o
x
X 25-
25-
/ /
oi~ Fig, 5. Inhibition of the APS-sulfotransferase activity by Y-AMP. Conditions (in pmol): Tris-HC1 pH 9.0: 100; MgC12 : 10; DTE: 10; Na2SO,: 500; APS : 0.023 (1 nmol = 1,253 cpm); protein: 0.09 mg in a total volume of 1 ml. Incubation for 1 h under Nz; 37 ~ C. 100% = 13.1 nmoles acid-volatile radioactivity formed
E
z-
/
g o h5 O.S'
I
o > r
,--'-" o ~ ~
-80
o
;o
-I.'0
oh
ml chloroplast extroct
mMS/-AMP
2
*PAPS
~
Go
,~o
!
Fig. 7, Measurement of APS and PAPS sulfotransferase activity with spinach chloroplast extract. Conditions (in umoles) : Tris-HC1 p H 9 . 0 : 100; MgC12: 10; DTE: 10; Na2SO4: 500; APS: 0.16 (1 nmo1=856 cpm); PAPS: 0.2 (1 n m o l = 1,200 cpm); chloroplast extract: 0.8 mg/ml, amount added as indicated; total volume 1 ml. Incubation for 1 h under Nz, 37 ~ C
lyzed by keeping the APS concentration constant and varying the Y-AMP concentration. The results of this experiment are shown in Fig. 5. Inhibition by Y-AMP is found above 0.5 mM. Since AMP is the endproduct of the APS-sulfotransferase reaction and a product of APS degradation, the nature of this inhibition was investigated further. The Km for APS was determined in the absence and presence of Y-AMP (1 raM). The data of this experiment are shown in Fig. 6. The Km for APS was found to be 13 pM in the absence of Y-AMP. However, after the addition of 5'-AMP the Km increased to about 190 gM. Since Vmax did not change in the presence of Y-AMP it is concluded that the inhibition of Y-AMP is competetive to APS. It should be pointed out here that the low Km for APS was obtained only when less than 0.02 mg protein was used for one assay. If 0.1 mg of protein was used for the assay, the Km increased to about 80 taM for APS, which is probably due to APS-degrading activities still present in this preparation.
llM APS
Fig, 6. Lineweaver-Burk plot for the APS-sulfotransferase activity in the absence and presence o f 5"-AMP. Conditions (in gmo[): Tris-HCt p H 9.0: 100; MgCI2: 10; DTE: 10; NazSO4: 500; APS: varied as indicated, (1 n m o l = 1,330cpm); Y-AMP: 1, when indicated; protein: 0.018 mg; in a total volume of 1 ml. Incubation for 1 h at 37 ~ C; Nz. • x activity with 5'-AMP added; o - - o activity without 5"-AMP
e) Localization It was discovered some years ago that the entire sequence of enzymes needed for sulfate reduction is associated with chloroplasts from Spinach (Schmidt,
262 1968; Schmidt and Trebst, 1969; Trebst and Schmidt, t969; Schwenn, 1970; Schmidt and Schwenn, 1971). Chloroplast extract was therefore prepared (Schmidt and Trebst, 1969) and analyzed with APS and PAPS under optimal conditions for the APS-sulfotransferase assay (Schmidt, 1975a). The data are given in Fig. 7. APS is metabolized by this preparation with a high rate of 560 nmol/mg protein x h, which is about 18 times greater than the specific activity of the APS-sulfotransferase of crude leaf extract (31 nmol/mg protein x h; Schmidt, 1975c). PAPS is also metabolized, the rate, however, being about 56 nmol/mg protein x h, suggesting that the dephosphorylation of PAPS to APS is the limiting step. This would be in agreement with the observation of Burnell and Anderson (1973) that chloroplast extract has little 3'-phosphatase activity. From the fact that the specific activity of the APS-sulfotransferase measured with chloroplast extract was 18 times greater it is concluded that the APS-sulfotransferase in spinach leaves is localized within the chloroplast system.
Discussion
Higher plants contain an enzyme system catalyzing the transfer of activated sulfur onto suitable acceptors (Schmidt, 1972a, 1975a, b, c). This enzyme was found in the green alga Chlorella and was named APSsulfotransferase, since it was found to be specific for the sulfonucleotide APS (Schmidt, 1972 a; Schiff and Hodson, 1973). For higher plants we had suggested, on the basis of comparative biochemistry and on data obtained with spinach chloroplast extract, that APS is the sulfur donor needed for assimilatory sulfate reduction (Schmidt, 1972 a, b). However, this was not accepted by Burnell and Anderson (1973) and Schwenn and Hennies (1974) who suggested PAPS to be the sulfur donor in spinach. It therefore became necessary to purify this enzyme from plant material in order to determine the substrate specificity towards sulfonucleotides. From inhibitor studies with crude leaf extract it was suggested recently that the sulfotransferase from spinach should be specific for APS (see Schmidt, 1975 a) and the data of this paper clearly demonstrate with partially purified enzyme that the sulfotransferase from spinach is specific for APS and has little or no activity towards PAPS. The name APS-sulfotransferase is therefore suggested for this activity (see Schmidt, 1972a, b, 1975a). APS-sulfotransferase activity has been detected in over 50 plant families examined (Schmidt, 1975 c). Some properties of the APS-sulfotransferase from spinach will now be discussed.
A. Schmidt: APS-Sulfotransferasefrom Spinach
a) Use of APS PAPS had been implied for assimilatory sulfate reduction since the unfavourable equilibrium constant for APS formation seemed to need a further step to pull the reaction to the product side and this was the phosphorylation of APS to PAPS. However, dissimilatory sulfate reduction proceeds without PAPS formation; thus the phosphorylation of APS to PAPS does not seem to be necessary. If another reaction with a similar low Km as reported for the APS-kinase could be found which leads to about the same standard free energy change as observed during the phosphorylation of APS to PAPS, one would have another explainable mechanism for the use of APS. The APSsulfotransferase seems to fulfil the requirements needed for such a reaction. The apparent Km for APS determined to be 13 gM and the standard energy change in this reaction should be in the order of AG~ - 12 kcal mol 1, assuming a standard free energy change for APS hydrolysis AG~ 18 kcal mol-1 (see Roy and Trudinger, 1970) and for S-sulfoglutathione hydrolysis (sulfate ester, organic thiosulfate) A G O = - 6 kcal mol-i, which, however, might be too high. This change of standard free energy is better for the APS-sulfotransferase reaction in this case, explaining why PAPS-formation is not a necessity for assimilatory sulfate reduction, since the standard free energy change for APS-phosphorylation is in the order of A G ~ 5 kcal mol- 1
b) Regulation The APS-sulfotransferase from spinach has a pH optimum of about 9. From work with whole cells it is known that sulfate in plants is reduced mainly during the light period (see Willenbrink, 1964). This can be explained by the rise of pH within the chloroplast during the light periods which in turn increases the activity of the APS-sulfotransferase. Furthermore, the ratio of APS to AMP will change due to photophosphorylation in the light period, which leads to a decrease of the AMP concentration. Since the APS-pool is small, a competetive inhibition of the APS-sulfotransferase activity might well have regulatory significance for light and dark periods with the change of pH.
c) Differences to Dissimilatory APS-reduetases Assimilatory APS-sulfotransferases from Chlorella (Schmidt, 1972a) or spinach (Schmidt, 1975a; and this publication) have a strict requirement for thiol
A. Schmidt: APS-Sulfotransferase from Spinach
groups to catalyze this reaction. Methylviologen is not an electron donor for the APS-sulfotransferase from spinach or Chlorella. Furthermore, assimilatory APS-sulfotransferase from Chlorella has been shown to catalyze the formation of bound and free sulfite depending on the conditions used (Schmidt, 1972 a, b; Schmidt, 1973; Schiff and Hodson, 1973; Abrams and Schiff, 1973). However, the formation of APS fiom bound or free sulfite has not been demonstrated so far with assimilatory APS-sulfotransferases. Another difference to dissimilatory APS-reductase is the failure to catalyze isotopic exchange reactions between sulfite and APS. We have not been able to demonstrate this exchange reaction with assimilatory APS-sulfotransferase from spinach or Chlorella (Schmidt, 1972 a, 1975 a; and this publication). Dissimilatory APS-reductase catalyzes the formation of APS from sulfite and AMP and the reverse reaction depending on the reaction conditions (Peck, 1974). The formation of acid-volatile radioactivity from [35S]APS with this enzyme can also be demonstrated using methylviologen as reductant (Peck et al., 1965). Thus dissimilatory APS-reductases catalyze the reaction in both directions where as assimilatory APS-sulfotransferases are "one way" catalysts. The expert technical assistance of Mrs. Christen is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft.
References Abrams, W.R., Schiff, J.A. : Studies of sulfate utilization by algae. 11. An enzyme-bound intermediate in the reduction of adenosine-5'-phosphosulfate (APS) by cell-free extracts of wild type Chlorella and mutants blocked for sulfate reduction. Arch. Mikrobiol. 94, 1-10 (1973) Andrews, P.: Estimation of the molecular weight of proteins by Sephadex-gel filtration. Biochem. J. 91, 222233 (1964) 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) Hodson, R.C., Schiff, J.A. : Preparation of adenosinc-3'-phosphate5'-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 adenosine-Y-phosphate5'-phosphosulfate to acidvolatile radioactivity. Plant Physiol. 47, 300-305 (1971) Jensen, R.G., Bassham, J.K.: Photosynthesis by isolated chloroplasts. Proc. nat. Acad. Sci. USA 56, 1095-1101 (1966)
263 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) Patterson, M.S., Greene, R.C. : Measurement of low energy betaemitters in aqueous solution by liquid scintillation counting of emulsions. Anal. Chem. 37, 854-857 (1956) Peck, H.D., Jr., Deacon, T.E., Davidson, J.T.: Studies on adenosine-5'-phosphosulfate reductase from Desulfovibrio desulfuricans and Thiobacillus thioparus. Biochim. Biophys. Acta 96, 429-446 (1965) Peck, H.D. Jr. : The evolutionary significance of inorganic sulfur metabolism. Symp. Soc. Gen. Microbiol. XXIV, 241262 (1974) Roy, A.B., Trudinger, P.A.: The biochemistry of inorganic compounds of sulphur. Cambridge: University press 1970 Schiff, J.A., Hodson, R.C. : The metabolism of sulfate. Ann. Rev. Plant Physiol. 24, 381414 (1973) Schmidt, A. : Ober Teilreaktionen der photosynthetischen Sulfatreduktion isolierter Chloroplasten. Thesis. G6ttingen (1968) Schmidt, A. : On the mechanism of photosynthetic sulfate reduction. An APS-sulfotransferase from Chlorella. Arch. Mikrobiol. 84, 77 86 (1972a) Schmidt, A. : Ober Teilreaktionen der photosynthetischen Sulfatreduktion in zellfreien Systemen aus Spinatchloroplasten und Chlorella. Z. Naturforsch. 27b, 183-192 (1972b) Schmidt, A. : Sulfate reduction in a cell-free system of Chlorella. The ferredoxin-dependent reduction of a protein-bound intermediate by a thiosulfonate reductase. Arch. Microbiol. 93, 2952 (1973) Schmidt, A. : A sulfotransferase from spinach leaves using adenosine-5'-phosphosulfate. Planta (Berl.) 124, 267-275 (1975) Schmidt, A. : Inhibition of the adenosine.5'-phosphosulfate-sulfotransferase activity from spinach, maize, and Chlorella by adenosine-5'-monophosphate. Planta (Berl.) 127, 93-95 (1975) Schmidt, A.: Distribution of the APS-sulfotransferase activity among higher plants. Plant Sci. Lett. 5, 407-415 (1975) Schmidt, A., Schwenn, J.D. : On the mechanism of photosynthetic sulfate reduction. Second. Int. Congr. Photosynthesis, Stresa, 507-513 (1971) Schmidt, A., Trebst, A. : The mechanism of photosynthetic sulfate reduction by isolated chloroplasts. Biochim. Biophys. Acta (Amst.) 180, 529-535 (1969) 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, 423434 (1974) Schwenn, J.D.: Untersuchungen zur Kinetik der photosynthetischen Sulfatreduktion isolierter Chloroplasten. Thesis. Bochum (1970) Schwenn, J.D., Hennies, H.H. : Enzymes and bound intermediate involved in photosynthetic sulfate reduction of spinach chloroplasts and Chlorella. In: M. Avron: Proceedings of the third Internat. Congr. on Photosynthesis. pp. 629-635. Amsterdam: Elsevier 1974 Trebst, A., Schmidt, A.: Photosynthetic sulfate and sulfite reduction by isolated chloroplasts. In: Progress in photosynthesis research, Vol. III, pp. 1510-1516. Ed.: Metzner, H. : Tfibingen 1969 Willenbrink, J.: Lichtabhfingiger 35S-Einbau in organische Bindung in Tomatenpflanzen. Z. Naturforsch. 19b, 356-357 (1964)
Received 15 January," accepted 9 February 1976
