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Fructose-1,6-diphosphatase from spinach leaf chloroplasts: Molecular weight transitions of the purified enzyme
Plant Science Letters, 5 (1975) 49--55 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
49
FRUCTOSE-1,6-DIPHOSPHATASE FROM SPINACH LEAF CHLOROPLASTS: MOLECULAR WEIGHT TRANSITIONS OF THE PURIFIED ENZYME
JUAN JOSE LAZARO, ANA CHUECA, JULIO LOPEZ GORGE and FEDERICO MAYOR*
Department o f Biochemistry, Estaeibn Experimental del Zaidin (S.C.I.C. ), Granada (Spain) (Received April 7, 1975) (Accepted April 23rd, 1975)
SUMMARY
Fructose-l,6-diphosphatase (FDPase) active fractions I and II earlier purified from spinach leaves show similar molecular weights, in the range 92 000--115 000, when calculated by Sephadex filtration and acrylamide gel electrophoresis. Both fractions are stable at acidic and neutral pH, but at pH 8.8 are partly splitted in similar subunits. Acrylamide electrophoresis at different gel concentration (Ferguson plots) show these subunits as monomers of native dimers I and II, with molecular weights between 54 000 and 60 000. INTRODUCTION
FDPase (EC 3.1.3.11) is a key enzyme in the regulation of the photosynthetic cycle [1]. This enzyme has been occasionally studied in navy-bean [2] and castor-bean [3] leaves, the algae Euglena gracilis [4], and the photosynthetic bacteria Rhodopseudomonas palustris [5] and Rhodospirillum rubrum [ 6]. Spinach leaf FDPase was analysed in some more detail, and has been purified from whole leaves [7] and chloroplastic lysates [8, 9]. In a previous work [10] we found that the spinach leaves' photosynthetic FDPase could be resolved in four active fractions, which were purified and isolated. An enzymatic diversity in connection with the existence of interconversible monomeric and dimeric forms has been demonstrated earlier in Rhodopseudomonas palustris photosynthetic enzyme [ 11], and in the gluconeogenic one from rabbit liver [12] and from the mold Candida utilis [13]. As a similar feature is possible for the spinach FDPase, we studied the structural relationship between these four fractions. "Present address: Department of Biochemistry, Faculty of Sciences, Universidad Autbnoma, Madrid (Spain). Abbreviation: FDPase, fructose-l,6-diphosphatase.
50 MATERIAL AND METHODS
Enzyme preparation All the experiments were performed with pure fractions I and H isolated with full FDPase activity as we described previously [ 10], and then lyophilized. This material was dissolved in the proper buffer before use, and its protein content determined according to Lowry et al. [ 14].
Assay for FDPase activity The reaction mixture was as follows: 0.1 M Tris HCI buffer pH 8.8, 4 mM famctose-l,6-diphosphate, 5 mM MgCI2, 1.6 ~,M EDTA, 5 mM cysteine and the enzyme preparation, in a final volume of 2 ml. After 30 min incubation at 28 ° the reaction was stopped with I ml of a 5% trichloroacetic acid solution, and the inorganic phosphate released determined according to Fiske and Subbarow [15]. One enzyme unit is expressed as the qua=~.tity of enzyme which releases I pit/of phosphorus per min in the alcove experimental conditions.
Molecular weight determination Three different techniques were used, by interpolating against standard proteins: Column Sephadex G-150 filtration. 90 x 2.5 cm columns (Pharmacia, Uppsala) were used, with 0.05 M acetate pH 5.5 or 0.05 M Tris-HCl pH 8.2 as developing buffers, at a flow rate of 0.5 ml/min. Fractionation was monitored by absorption at 280 nm. Chymotrypsinogen A (MW 25 000), ovalbumin (MW 45 000), bovine serum albumin (MW 67 000), human transferrin (MW 74 000) and 7-globulin (MW 160 000), supplied by Serva Feinbiochemica, were used as standards and the elution volumes plotted ver,.us the corresponding log MW values. Thin-layer Sephadex G-150 filtration. 20 x 30 cm plates of Sephadex G-150 superfine (Pharmacia, Uppsala), 0.6--0.7 mm thickness, were developed with 0.05 M acetate pH 5.5, 0.05 M Tris--HCl pH 7.1 or 0.038 M Tris--glycine pH 8.8 buffers. In addition to the above standards hexokinase (MW 99 000, Sigma) was used. Replica papers in Whatmann 3MM were stained with amidoblack 10B, relative mobilities referred to the front calculated, and these, values plotted versus the corresponding log MW. Acrylamide gel electrophoresis. Seven acrylamide concentrations between 6 and 12% were used, with a constant acrylamide/bis-acrylamide ratio of 30. Gels of 0.5 x 7.5 cm were prepared in Tris--HCl buffer pH 8.9 in accordance with Hedrick and Smith [16], using hexokinase, rabbit muscle aldolase (MW 160 000, Serva Feinbiochemica) and bovine serum albumin as standards; after shows four fractions cor;tic (MW 134 000), trimeric as. The detection in situ of by incubating the gels for I h ination. The inorganic phos-
51
phate released was visualized by dipping the gels immediately in the solution: 5% ammonium molybdate, 1% hydroquinone, 20% Na2 SO3 and concentrated H2 SO4 (6:1:6:6). Relative mobilities referred to the front were calculated for each protein and acrylamide concentration, and their log values plotted versus the acrylamideconcentration. In accordance with Ferguson's equation [17] log M = -- KR C + log M0 (M = relative mobility at acrylamide concentration C; Mo = limit mobility at acrylamide concentration 0) this results in a straight line with a slope KR. The "size isomeric" proteins, having identical charge but different molecular size, yield nonparallel lines that can be extrapolated to a common point corresponding to the maximum acrylamide concentration without sieving effect. Molecular weights were calculated plotting the slopes versus thb corresponding standard molecular weights. RESULTS
Fig. I shows the Sephadex G-150 elution pattern at PH 5.5 of active fractions I and II; the molecular weights obtained were 92 000 and 100 000, respectively. When an alkaline buffer pH 8.2 was used as developing solution,
0.20-
0valbunpin Serumalbumin(~ChymotrypsDnogen ~ "
E 0.15- ~-giobulin
dJ
t-
E
O ¢O
N e"
-0.3 GI (J C RI
0.10
~J
-0.2
i__
O u) JO
<[
v
®
A= e~ to o
0.05
-0.1
12.
to o x
0
T
360
350
400
Effluent volume (ml} Fig. 1. Chromatography on Sephadex G-150 column (90 x 2.5 era) of standard proteins and active fractions I and If. Elution with 0.05 M acetate buffer pH 5.5. Standard proteins monitored in a continuous flow cell at 280 nm ( - - ) . FDPase activity o f fractions I (~---1) and II (~---o) were assayed in 5 ml fractions as described in the text.
52
+
i
x
x
X
X
I I
X
1[
i I
~[
I
X
)l
V
Chymotryp. I~ Ovalbum. Chymotryp. 0valbum.
Transl. 5erurnalb.(di )
Transl.
lla Serumalb. (di)
t
Hexok.
Hexok.
lib $erurnalb. ~ y-glob.
A
~
B i
Fig. 2. Thin-layer chromatography of active fraction II on Sephadex G-150 superfine. Elution with 0.05 M acetate buffer pH 5.5 (A) and 0.038 M Tris, glycine buffer pH 8.8 (B). Staining for proteins with amidoblack 10B.
both fractions gave rise to two active peaks, named fractions Ia-Ib and lla-IIb. This is more remarkable in thin-layer Sephadex experiments. Fig. 2 shows thinlayer profiles of fraction lI with acetate pH 5.5 and Tris-glycine pH 8.8 as developing buffers. In the former, as also occurs at 7.1, only one protein spot is detected, whereas in the latter a second one with a slower mobility appears. The same happens with active fraction I. Polyacrylamide gel electrophoresis at pH 8.9 also gave rise to the splitting of fractions I and II. The plots of log M versus acrylamide concentration (Fig. 3) showed that both fully active pairs Ia-Ib and IIa-IIb have identical charge and different molecular weight; these were graphically determined in Fig. 4. Table I summarizes the molecular weight of active fractions, indicating the technique used for calculation. DISCUSSION
I spinach [7], pea [18] and tapioca Lkaline, acidic and neutral. The the latter the gluconeogenic one. tcidic FDPase, perhaps connected when a low CO2 fixation rate, be-
53
2.00
x C~
O
1.75
slope=- 6.09
o
1.50" slope =- 10.20
9 Acrylamide
10
11
12
concentration (%)
2.00)¢ ¢::)
•-
1.75
O
slope : - 6.57 1.50
A slope --- 9.80
/
|
|
!
'7
8
9
!
10
w
11
12
A c r y l a m i d e concentration {%}
Fig. 3. "Ferguson plots" of FDPase components. Fraction I a (~---o); fraction I b (~--~ ); fraction H a (J~--A); fraction II b (~--~). Conditions of acrylamide electrophoresis in the text.
TABLE I MOLECULAR WEIGHT OF FDPase ACTIVE FRACTIONS Fraction
MW
I
92 000
Hia
100 000 54 000 115 000
Ib Ha IIb
60 000
109 000
Method Sephadex G..150 filtration 8ephadex G-150 filtration Acrylamide gel electrophoresis Acrylamide gel electrophoresis Acrylamide gel electrophoresis Acrylamide gel electrophoresis
54
20.0"
umalbumm|tetra} 26 8000 $erumalbumin f i n ) 201000 •
15.0"
_f
Ser umaibumin ( d i ) 13/,000 ¢b t's
o t/}
......
•
Aldolase 160000
. . . . . . . . . . . . .
10.0" Se •umaibumin imono) 67000 •
•
cokinase 99000
•
-_:._----.
5.0J
lal 11a
II~ Ib
! ! ! S B
i
! I ! ¢
,
50000
•
I
l
100000
1
150000
|
i
•
200000
I
250000
•
300000
Molecular we,ght
Fig. 4. Molecular weight determination of FDPase fractions l¢IbJI8-11 b. Acrylamide eleetrophoresis conditions in the text. Slope values were taken from "Ferguson plots" of Fig. 3.
cause of its ability to hydrolyse this sugar-phosphate [20]. But the existence of fractions I and II is quite different: both are alkaline enzymes with narrow specificity for fructose-l,6-diphosphate and, as we have found in further experiments, with the same kinetic properties and very similar molecular characteristics [21]. R e molecular weights 92 000 and 100 000 found for fractions I and H are lower than that 1 9 5 0 0 0 zeported by Preiss and Kosuge [22], and those of 1 4 5 0 0 0 and 1 3 0 0 0 0 determined by Buchanan et al. [23] using ultracentrifugation and Sephadex G-200 filtration. Scala et al. [3] reported molecular weights in the range of 120000--135 000 for the three FDPases of castor-bean leaves, one of them being the photosynthetic enzyme, and Springgate and Stachow [5] 130 000 for the enzyme from Rhodopseudomonas palustris. The molecular weight of gluconeogenic FDPases are also higher: 130 000 in Candida erent organs of mammals [25--27 ]. i IIa-IIb obtained by polyacrylamide slow migration ones Ib and IIb have and H. The fast-moving ones Ia and IIa snow molecular we~jtts ot ~40UU and tJ0 000, and the "Ferguson plots" corroborate that they are subunits of half molecular weight of the corresponding Ib and Hb.
55
This different behaviour with regard to Sephadex filtration at acidic pH strongly suggest that at alkaline pH the native proteins give rise to two similar subunits, as it has been corroborated by the thin-layer filtration experiments. Both the monomers and dimers exhibit FDPase activity; Similar results have been reported for an active dimer of photosynthetic FDPase from Rhodopseudomonas palustris [ 11 ], with a molecular weight of 130 000 at pH 7.4 that at pH 8.5 is converted into a monomer of half molecular weight and a specific activity three times higher. ACKNOWLEDGMENTS
We are very grateful ~ Miss Matilde Garrido for skilful technical assistance. REFERENCES
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
J.A. Bassham, Proc. Natl. Acad. SCi. (U.S.), 68 (1971) 2877. J. Scala, G. Ketner and W.H. Jyung, Arch. Biochem. Biophys., 131 (1969) 111. J. Scala, C. Patrick and G. Macbeth, Arch. Biochem. Biophys., 127 (1968) 576. A.A. App and A.T. Jagendorf, Biochim. Biophys. Acta, 85 (1964) 427. C.P. Springgate and C.S. 8tachow, Arch. Biochem. Biophys., 152 (1972) 1. I.R. Joint, I. Morris and R.C. Puller, J. Biol. Chem., 247 (1972) 4833. E. Racker and E.A.R. Schroeder, Arch. Biochem. Biophys., 74 (1958) 326. H. Springer-Lederer, A.M. EI-Badry, H.C.J. Ottenheym and J.A. Bassham, Biochim. Biophys. Acta, 189 (1969) 464. J. Preiss, M.L. Biggs and E. Greenberg, J. Biol. Chem., 242 (1967) 2292. J.J. L~izaro, A. Chueca, J. L6pez Gorg6 and F. Mayor, Phytochemistry, 13 (1974) 2455. C.F. Springgate and C.8. Stachow, Bioehem. Biophys. Res. Commun., 49 (1972) 522. 8. Pontremoli, B. Luppis, 8. Traniello and A. Bargallesi, Arch. Biochem. Biophys., 114 (1966) 24. O.M. Rosen, P.L. Copeland and S.M. Rosen, J. Biol. Chem., 242 (1967) 2760. O.H. Lowry, N.J. Rosebrough, A.L. Parr and R.J. Randall, J. Biol. Chem., 193 (1951) 265. C.H. Fiske and Y. 8ubbarow, J. Biol. Chem., 66 (1925) 375. J.L. Hedrick and A.J. Smith, Arch. Biochem. Biophys., 126 (1968) 155. K.A. Ferguson, Metabolism, 13 (1964) 985. R.M. Smillie, Plant Physiol., suppl. 35 (Proc. Plant Physiol. Meetings) (1960). P.N. Viswanathan and P.S. Krishnan, Nature, 193 (1962) 166. M. Chakravorty, H.C. Chakrabortty and D.P. Burma, Arch. Biochem. Biophys., 82 (1959) 21. J.J. IAzaro, A. Chueca, J. Lbpez Gorg~ and F. Mayor, Phytochemistry, (1975) (in press). J. Preiss and T. Kosuge, Ann. Rev. Plant Physiol., 21 (1970) 433. B.B. Buchanan, P. Schfirmann and P.P. Kaiberer, J. Biol. Chem., 246 (1971) 5952. S. Traniello, M. Calcagno and S. Pontremoli, Arch. Biochem. Biophys., 146 (1971) 603. S. Tmniello, E. Melloni, S. Pontremoli, C.L. Sia and B.L. Horecker, Arch. Biochem. Biophys., 149 (1972) 222. W.L. Byrne, G.T. Rajagopalan, L.D. Griffin, E. Ellis, T.M. Harris, P. Hochachka, L. Reid and A. Geller, Arch. Biochem. Biophys., 146 (1971) 118. Y. Tashima, G. Tholey, G. Drummond, H. Bertrand, J.S. Rosenberg and B.L. Horecker, Arch. Biochem. Biophys., 149 (1972) 118.
49
FRUCTOSE-1,6-DIPHOSPHATASE FROM SPINACH LEAF CHLOROPLASTS: MOLECULAR WEIGHT TRANSITIONS OF THE PURIFIED ENZYME
JUAN JOSE LAZARO, ANA CHUECA, JULIO LOPEZ GORGE and FEDERICO MAYOR*
Department o f Biochemistry, Estaeibn Experimental del Zaidin (S.C.I.C. ), Granada (Spain) (Received April 7, 1975) (Accepted April 23rd, 1975)
SUMMARY
Fructose-l,6-diphosphatase (FDPase) active fractions I and II earlier purified from spinach leaves show similar molecular weights, in the range 92 000--115 000, when calculated by Sephadex filtration and acrylamide gel electrophoresis. Both fractions are stable at acidic and neutral pH, but at pH 8.8 are partly splitted in similar subunits. Acrylamide electrophoresis at different gel concentration (Ferguson plots) show these subunits as monomers of native dimers I and II, with molecular weights between 54 000 and 60 000. INTRODUCTION
FDPase (EC 3.1.3.11) is a key enzyme in the regulation of the photosynthetic cycle [1]. This enzyme has been occasionally studied in navy-bean [2] and castor-bean [3] leaves, the algae Euglena gracilis [4], and the photosynthetic bacteria Rhodopseudomonas palustris [5] and Rhodospirillum rubrum [ 6]. Spinach leaf FDPase was analysed in some more detail, and has been purified from whole leaves [7] and chloroplastic lysates [8, 9]. In a previous work [10] we found that the spinach leaves' photosynthetic FDPase could be resolved in four active fractions, which were purified and isolated. An enzymatic diversity in connection with the existence of interconversible monomeric and dimeric forms has been demonstrated earlier in Rhodopseudomonas palustris photosynthetic enzyme [ 11], and in the gluconeogenic one from rabbit liver [12] and from the mold Candida utilis [13]. As a similar feature is possible for the spinach FDPase, we studied the structural relationship between these four fractions. "Present address: Department of Biochemistry, Faculty of Sciences, Universidad Autbnoma, Madrid (Spain). Abbreviation: FDPase, fructose-l,6-diphosphatase.
50 MATERIAL AND METHODS
Enzyme preparation All the experiments were performed with pure fractions I and H isolated with full FDPase activity as we described previously [ 10], and then lyophilized. This material was dissolved in the proper buffer before use, and its protein content determined according to Lowry et al. [ 14].
Assay for FDPase activity The reaction mixture was as follows: 0.1 M Tris HCI buffer pH 8.8, 4 mM famctose-l,6-diphosphate, 5 mM MgCI2, 1.6 ~,M EDTA, 5 mM cysteine and the enzyme preparation, in a final volume of 2 ml. After 30 min incubation at 28 ° the reaction was stopped with I ml of a 5% trichloroacetic acid solution, and the inorganic phosphate released determined according to Fiske and Subbarow [15]. One enzyme unit is expressed as the qua=~.tity of enzyme which releases I pit/of phosphorus per min in the alcove experimental conditions.
Molecular weight determination Three different techniques were used, by interpolating against standard proteins: Column Sephadex G-150 filtration. 90 x 2.5 cm columns (Pharmacia, Uppsala) were used, with 0.05 M acetate pH 5.5 or 0.05 M Tris-HCl pH 8.2 as developing buffers, at a flow rate of 0.5 ml/min. Fractionation was monitored by absorption at 280 nm. Chymotrypsinogen A (MW 25 000), ovalbumin (MW 45 000), bovine serum albumin (MW 67 000), human transferrin (MW 74 000) and 7-globulin (MW 160 000), supplied by Serva Feinbiochemica, were used as standards and the elution volumes plotted ver,.us the corresponding log MW values. Thin-layer Sephadex G-150 filtration. 20 x 30 cm plates of Sephadex G-150 superfine (Pharmacia, Uppsala), 0.6--0.7 mm thickness, were developed with 0.05 M acetate pH 5.5, 0.05 M Tris--HCl pH 7.1 or 0.038 M Tris--glycine pH 8.8 buffers. In addition to the above standards hexokinase (MW 99 000, Sigma) was used. Replica papers in Whatmann 3MM were stained with amidoblack 10B, relative mobilities referred to the front calculated, and these, values plotted versus the corresponding log MW. Acrylamide gel electrophoresis. Seven acrylamide concentrations between 6 and 12% were used, with a constant acrylamide/bis-acrylamide ratio of 30. Gels of 0.5 x 7.5 cm were prepared in Tris--HCl buffer pH 8.9 in accordance with Hedrick and Smith [16], using hexokinase, rabbit muscle aldolase (MW 160 000, Serva Feinbiochemica) and bovine serum albumin as standards; after shows four fractions cor;tic (MW 134 000), trimeric as. The detection in situ of by incubating the gels for I h ination. The inorganic phos-
51
phate released was visualized by dipping the gels immediately in the solution: 5% ammonium molybdate, 1% hydroquinone, 20% Na2 SO3 and concentrated H2 SO4 (6:1:6:6). Relative mobilities referred to the front were calculated for each protein and acrylamide concentration, and their log values plotted versus the acrylamideconcentration. In accordance with Ferguson's equation [17] log M = -- KR C + log M0 (M = relative mobility at acrylamide concentration C; Mo = limit mobility at acrylamide concentration 0) this results in a straight line with a slope KR. The "size isomeric" proteins, having identical charge but different molecular size, yield nonparallel lines that can be extrapolated to a common point corresponding to the maximum acrylamide concentration without sieving effect. Molecular weights were calculated plotting the slopes versus thb corresponding standard molecular weights. RESULTS
Fig. I shows the Sephadex G-150 elution pattern at PH 5.5 of active fractions I and II; the molecular weights obtained were 92 000 and 100 000, respectively. When an alkaline buffer pH 8.2 was used as developing solution,
0.20-
0valbunpin Serumalbumin(~ChymotrypsDnogen ~ "
E 0.15- ~-giobulin
dJ
t-
E
O ¢O
N e"
-0.3 GI (J C RI
0.10
~J
-0.2
i__
O u) JO
<[
v
®
A= e~ to o
0.05
-0.1
12.
to o x
0
T
360
350
400
Effluent volume (ml} Fig. 1. Chromatography on Sephadex G-150 column (90 x 2.5 era) of standard proteins and active fractions I and If. Elution with 0.05 M acetate buffer pH 5.5. Standard proteins monitored in a continuous flow cell at 280 nm ( - - ) . FDPase activity o f fractions I (~---1) and II (~---o) were assayed in 5 ml fractions as described in the text.
52
+
i
x
x
X
X
I I
X
1[
i I
~[
I
X
)l
V
Chymotryp. I~ Ovalbum. Chymotryp. 0valbum.
Transl. 5erurnalb.(di )
Transl.
lla Serumalb. (di)
t
Hexok.
Hexok.
lib $erurnalb. ~ y-glob.
A
~
B i
Fig. 2. Thin-layer chromatography of active fraction II on Sephadex G-150 superfine. Elution with 0.05 M acetate buffer pH 5.5 (A) and 0.038 M Tris, glycine buffer pH 8.8 (B). Staining for proteins with amidoblack 10B.
both fractions gave rise to two active peaks, named fractions Ia-Ib and lla-IIb. This is more remarkable in thin-layer Sephadex experiments. Fig. 2 shows thinlayer profiles of fraction lI with acetate pH 5.5 and Tris-glycine pH 8.8 as developing buffers. In the former, as also occurs at 7.1, only one protein spot is detected, whereas in the latter a second one with a slower mobility appears. The same happens with active fraction I. Polyacrylamide gel electrophoresis at pH 8.9 also gave rise to the splitting of fractions I and II. The plots of log M versus acrylamide concentration (Fig. 3) showed that both fully active pairs Ia-Ib and IIa-IIb have identical charge and different molecular weight; these were graphically determined in Fig. 4. Table I summarizes the molecular weight of active fractions, indicating the technique used for calculation. DISCUSSION
I spinach [7], pea [18] and tapioca Lkaline, acidic and neutral. The the latter the gluconeogenic one. tcidic FDPase, perhaps connected when a low CO2 fixation rate, be-
53
2.00
x C~
O
1.75
slope=- 6.09
o
1.50" slope =- 10.20
9 Acrylamide
10
11
12
concentration (%)
2.00)¢ ¢::)
•-
1.75
O
slope : - 6.57 1.50
A slope --- 9.80
/
|
|
!
'7
8
9
!
10
w
11
12
A c r y l a m i d e concentration {%}
Fig. 3. "Ferguson plots" of FDPase components. Fraction I a (~---o); fraction I b (~--~ ); fraction H a (J~--A); fraction II b (~--~). Conditions of acrylamide electrophoresis in the text.
TABLE I MOLECULAR WEIGHT OF FDPase ACTIVE FRACTIONS Fraction
MW
I
92 000
Hia
100 000 54 000 115 000
Ib Ha IIb
60 000
109 000
Method Sephadex G..150 filtration 8ephadex G-150 filtration Acrylamide gel electrophoresis Acrylamide gel electrophoresis Acrylamide gel electrophoresis Acrylamide gel electrophoresis
54
20.0"
umalbumm|tetra} 26 8000 $erumalbumin f i n ) 201000 •
15.0"
_f
Ser umaibumin ( d i ) 13/,000 ¢b t's
o t/}
......
•
Aldolase 160000
. . . . . . . . . . . . .
10.0" Se •umaibumin imono) 67000 •
•
cokinase 99000
•
-_:._----.
5.0J
lal 11a
II~ Ib
! ! ! S B
i
! I ! ¢
,
50000
•
I
l
100000
1
150000
|
i
•
200000
I
250000
•
300000
Molecular we,ght
Fig. 4. Molecular weight determination of FDPase fractions l¢IbJI8-11 b. Acrylamide eleetrophoresis conditions in the text. Slope values were taken from "Ferguson plots" of Fig. 3.
cause of its ability to hydrolyse this sugar-phosphate [20]. But the existence of fractions I and II is quite different: both are alkaline enzymes with narrow specificity for fructose-l,6-diphosphate and, as we have found in further experiments, with the same kinetic properties and very similar molecular characteristics [21]. R e molecular weights 92 000 and 100 000 found for fractions I and H are lower than that 1 9 5 0 0 0 zeported by Preiss and Kosuge [22], and those of 1 4 5 0 0 0 and 1 3 0 0 0 0 determined by Buchanan et al. [23] using ultracentrifugation and Sephadex G-200 filtration. Scala et al. [3] reported molecular weights in the range of 120000--135 000 for the three FDPases of castor-bean leaves, one of them being the photosynthetic enzyme, and Springgate and Stachow [5] 130 000 for the enzyme from Rhodopseudomonas palustris. The molecular weight of gluconeogenic FDPases are also higher: 130 000 in Candida erent organs of mammals [25--27 ]. i IIa-IIb obtained by polyacrylamide slow migration ones Ib and IIb have and H. The fast-moving ones Ia and IIa snow molecular we~jtts ot ~40UU and tJ0 000, and the "Ferguson plots" corroborate that they are subunits of half molecular weight of the corresponding Ib and Hb.
55
This different behaviour with regard to Sephadex filtration at acidic pH strongly suggest that at alkaline pH the native proteins give rise to two similar subunits, as it has been corroborated by the thin-layer filtration experiments. Both the monomers and dimers exhibit FDPase activity; Similar results have been reported for an active dimer of photosynthetic FDPase from Rhodopseudomonas palustris [ 11 ], with a molecular weight of 130 000 at pH 7.4 that at pH 8.5 is converted into a monomer of half molecular weight and a specific activity three times higher. ACKNOWLEDGMENTS
We are very grateful ~ Miss Matilde Garrido for skilful technical assistance. REFERENCES
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
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