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Further characterization of mitochondrial protein phosphatase
ARCHIVES
OF BIOCHEMISTRY
Further
AND
Characterization G. CLARI,
Istituto
166, 318-322 (19%)
BIOPHYSICS
di Chimica
of Mitochondrial
A. DONELLA,
Biologica
dell’llniuersith Mitocondri”
L. A. PINNA,
Protein AND
Phosphatase
V. MORET
di Padoua and “Centro per lo Studio della Fisiologia de1 C.N.R., Padoua, Italy
dei
Received June 10, 1974 Mitochondrial protein phosphatase from rat liver exhibits rather wide substrate specificity, since it readily dephosphorylates, besides phosvitin, casein, and cytosol phosphoproteins, also ATP, ADP, inorganic pyrophosphate, p-nitrophenylphosphate. Aliphatic phosphate esters (&glycerophosphate, glucose&P, serine-phosphate) are not dephosphorylated to any detectable extent. Evidence for the participation of a single enzyme in the dephosphorylation of phosvitin and ATP is provided. However, the different affinity toward the two substrates and other evidence suggest that the enzyme has in uiuo the biological role of dephosphorylating, at least preferentially, the phosphoproteins.
In a previous paper (1) the partial purification from rat liver mitochondria of a protein phosphatase, active on both phosvitin and casein, has been described. In the present paper, an improved purification and characterization of this enzyme are reported in order to get more information about its role in the living cells. It has been found that mitochondrial protein phosphatase (whose molecular weight is about 32,000) dephosphorylates, besides phosvitin, casein and cytosol phosphoproteins, also a few nonprotein metabolites of physiological relevance, namely, ATP, ADP, and inorganic pyrophosphate. Evidence is reported, however, suggesting that the biological role of this enzyme in the mitochondria involves the dephosphorylation of phosphoproteins rather than that of nonprotein substrates. MATERIALS Purification
AND
of Mitochondrial
METHODS Protein
matographed on a carboxymethyl cellulose (CM cellulose) column (1.6 x 14 cm) equilibrated with the same buffer. Stepwise elution was carried out with 2 mM acetate buffer, pH 5.4, containing increasing concentrations of NaCl. Gel filtration. The fraction eluted from CM cellulose with 0.3 M NaCl fraction, once concentrated to a volume of 3 ml by ultrafiltration through Diaflo UM ‘2 membranes, was submitted to gel filtration through a Sephadex G 100 column (1.8 x 62 cm) equilibrated with 2 mM acetate plus 0.60 M NaC1, pH 5.4. Gel electrophoresis. Small aliquots of concentrated protein phosphatase eluted from CM cellulose by 0.3 M NaCl were submitted to 7.5% polyacrylamide gel electrophoresis, pH 4.3, according to the procedure described in Ref. 2. Before running the sample, ammonium persulfate was completely removed from the gel by preelectrophoresis for 15 hr at 2 mA per gel tube. At the end of the electrophoretic run the gels were sliced into 3-mm segments from which the enzyme was extracted by homogenization in 3 ml of cold 0.1 M succinate buffer, pH 5.9. All above operations were carried out in a cold room at 0-4°C.
Phosphatase Determination
Extraction and preliminary purification of mitochondrial protein phosphatase up to the (NH,),SO, precipitation were accomplished as already described (1). CM cellulose column chromarography. The precipitate, once dissolved in a small volume of 2 mM acetate buffer, pH 5.4, containing 0.5 mM NaCl, was dialyzed overnight against the same buffer and chro318 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
of Phosphatase
Activities
Protein phosphatase activity was assayed with phosvitin as substrate. The incubation medium contained in a final volume of 2 ml: 200 Mmoles succinate buffer, pH 5.9, 6 pmoles cysteine, 1 mg of phosvitin (containing 3 pmoles of alkali-labile phosphate). The reaction was started by addition of the enzyme. Incubation at 37°C lasted 30 min, unless different
MITOCHONDRIAL
PROTEIN
PHOSPHATASE
319
FIG. 1. CM cellulose chromatography of the mitochondrial protein phosphatase preparation partially purified as previously described (1). The stepwise elution was carried out as described in Materials and Methods. Four-milliliter fractions were collected and tested for the various enzyme activities. Absorbance at 280 nm (O---O); phosvitin phosphatase activity (arbitrary units)/O.Z ml (a---O); ATPase activity (arbitrary units)/O.l ml (A- --A). times are indicated in the legend. The reaction was stopped by adding 1.5 ml of cold 50% trichloroacetic acid. Inorganic phosphate was determined in the clear supernatant by the Gomori procedure (3). The same procedure was followed to test all the other phosphatase activities, except that phosvitin was replaced by 2 pmoles of suitable phosphorylated substrates. Phosphatase activity toward [32P]salmine and [32P]cytosol phosphoproteins was tested by incubating, under the above-described conditions, amounts of the labeled proteins equivalent to 50,000 cpm as protein-bound 3ZP. a*P1 released during incubation was determined by the same procedure used for the determination of the 32P, released from [y-“P]ATP (see below). Whenever a discrimination between protein phosphatase and ATPase activities in the same sample was required, unlabeled phosvitin was incubated together with [Y-~~P]ATP having a known specific radioactivity, under the same conditions described above. The reaction was stopped by addition of 0.8 ml of 50% trichloroacetic acid and 0.9 ml of Silico-tungstic acid solution (4) followed by centrifugation. Total P, released from both substrates was determined in an aliquot of the supernatant according to the Martin and Doty procedure (5). 3ZP1 released from [y32P]ATP was measured by counting a parallel aliquot of the isobutanol-benzene phase in a Packard TriCarb liquid scintillator model 3375. Pi released from phosvitin was calculated as the difference between the two previous values.
Proteins were determined according to the Lowry et al. procedure (6). Phosvitin was prepared from egg yolk according to Mecham and Olcott (7). [y-32P]ATP was from Radiochemical Centre. All other substrates were obtained from Sigma Chemical Company. 32P-labeled cytosol phosphoproteins were prepared as already described (8). S2P-labeled salmine was prepared following in its general lines the procedure described by Meisler and Langan (9) by using a cytosol protamine kinase preparation obtained by P cellulose chromatography (10).
RESULTS
AND
DISCUSSION
Figure 1 shows the pattern of phosphatase activities obtained when a preparation of phosvitin phosphatase, partially purified, as previously described (l), was submitted to chromatography on CM cellulose column. It can be seen that most of phosvitin phosphatase activity is present in the fraction eluted by 0.3 M NaCll while ATPase activity is resolved in at least three fractions, eluted, respectively, by the equilibrium buffer, by 0.1 M NaCl, and by 0.3 M NaCl, the last one being by far the minor among the three. The results reported in 1Sometimes a minor but significant peak of phosvitin phosphatase activity is also eluted by 0.4 M NaCl.
320
CLARI ET AL.
Table I show that the fraction eluted by 0.3 M NaCl, in contrast to those eluted at lower ionic strength, does not dephosphorylate AMP, glucose-6-PO ,, ,&glycerophosphate, phosphoserine (Table I). However, such a fraction is able to dephosphorylate, besides phosvitin, casein and cytosol phosalso nonprotein substrates phoproteins, such as ATP, ADP, pyrophosphate, and p-nitrophenylphosphate. All these phosphatase activities exhibited by such an enzyme fraction are inhibited by EDTA. On the contrary, Mg2+, under our conditions, has been found to decrease the ATPase, ADPase and pyrophosphatase activities at concentrations which have no effect on phosvitin phosphatase. Such a finding, probably due to different stabilities of Mg-substrate complexes, accounts for the apparent discrepancy from previously reported data (l), obtained in the presence of Mg2+, according to which no appreciable ATPase activity was detectable in the protein phosphatase fraction purified on CM cellulose. In order to establish whether or not the phosphatase activities exhibited by the 0.3 M NaCl fraction are due to a single multifunctional enzyme, such a fraction from
CM cellulose was submitted to further purification procedure and it was again tested for its activity toward protein substrate (phosvitin) and nonprotein substrate (ATP). Gel filtration through Sephadex G 100 gives rise to a single peak with protein phosphatase and ATPase activities overlapping as shown in Fig. 2. The molecular TABLE
I
SUBSTRATE SPECIFICITY OF THE ENZYME FRACTION ELUTED FROM CM CELLULOSE WITH 0.3 M NaCl”
Substrate
Phosphatase activity, P, released P&!
Phosvitin Casein [s2P]Salmine [32P]Cytoso1 phosphoproteins Phosphorylserine Glucose-6-phosphate a-Glycerophosphate fl-Glycerophosphate AMP ADP ATP Na inorganic pyrophosphate p-Nitrophenylphosphate
wm
13.5 7.3 0.000 10.000 0.00 0.00 0.00 0.00 0.00 15.03 17.00 10.00 17.5
“The enzymatic activity was determined scribed in the Methods and Materials.
FIG. 2. Molecular filtration on Sephadex G 100 of the enzyme fraction eluted from CM and Methods. cellulose with 0.3 M NaCl. Genera1 conditions are described in the Materials Fractions of 3.7 ml were collected. The void volume of the column was 56 ml. Calibration curve was obtained with (1) horse heart cytochrome c (12, 400), (2) ovoalbumin (45,000), and (3) bovine serum albumin (67,000). Absorbance at 220 nm (0-O); phosvitin phosphatase activity (arbitrary units)/0.4 ml (O-O); ATPase activity (arbitrary units)/0.2 ml (A- --A). The recovery of both enzymatic activities approached 95%.
as de-
MITOCHONDRIAL r-
-
PROTEIN
7
L---? IL
c-----____------.
0 . .
.
.-
t Star’
3. Polyacrylamide gel electrophoresis. r;xperimental conditions as described in Materials and Methods. Solid line: phosvitin phosphatase activity; dashed line: ATPase activity. FIG.
TABLE
II
PRUTEIN PHOSPHATASE AND ATPASE ACTIVITIES EXHIBITED IN THE PRESENCE OF THE RELATED SUBSTRATES ADDED SEPARATELY AND TOGETHER~
Substrate Phosvitin (0.3 mg) [-y-32P]ATP (1 rmole) Phosvitin + [r-32P]ATP
fiPl released from phosvitin [r-“‘P]ATP 6.7 4.6
7.1 (12.047 cpm) 3.7 (6.270 cpm)
PHOSPHATASE
321
substrates are added together is less than that expected if two different enzymes acting independently on the two substrates were present. On the contrary, a mutual inhibition is observed as it would be expected if the two substrates compete for the same enzyme. Such a point of view is strengthened by other kinetic studies. Figure 4 shows the Lineweaver-Burk double-reciprocal plot for ATPase activity obtained by using [y-3ZP]ATP of known specific radioactivity in the absence and in the presence of unlabeled phosvitin. It can be seen that the inhibition by phosvitin results in a higher K,, with unchanged V, as expected if the two substrates ( [y-32P]ATP and phosvitin) are competing for the same catalytic site. However, the affinity of the enzyme for the two substrates is very different. The phosvitin phosphatase activity (Fig. 5), unlike ATPase activity, does not follow normal Michaelis-Menten kinetics, being inhibited by high concentrations of substrate. Nevertheless a fairly accurate evaluation of K, was possible by extrapolating the linear part of the graph in the doublereciprocal plot (inset for Fig. 5). Assuming
a The enzyme activity was determined as described in the Methods and Materials. The specific radioactivity of [T-~‘P]ATP was 52,500 counts/min per @mole.
weight, estimated by comparison with standard proteins, is about 32,000 (inset of Fig. 2). By gel electrophoresis in polyacrylamide 7.5% at pH 4.3 protein phosphatase and ATPase are again superimposed in a single band (Fig. 3). Keeping in mind the high resolution of gel electrophoresis, one should conclude that the activities toward phosvitin and ATP are due to a single enzyme, rather than to different enzymes. Such a conclusion is also supported by experiments in which unlabeled phosvitin and [y-32P]ATP were simultaneously added. As shown in Table II, under these conditions, which allow the discrimination between the two activities, the Pi released when the two
FIG. 4. Lineweaver-Burk double-reciprocal plot for ATPase in the absence and in the presence of phosvitin. The incubation medium contained different concentration of [-y-S2P]ATP having a known specific radioactivity (50,000 cpm//lmole ATP) in the presence or in the absence of a fixed amount of unlabeled phosvitin (0.40 mg). Incubation time 10 min. The s2Pi released from [-r-S2P]ATP was determined as described in the Materials and Methods. Phosvitin absent (A- - -A); 0.40 mg phosvitin present in the incubation medium (0- - -0).
322
CLARI ET AL.
FIG. 5. Phosvitin phosphatase activity as a function of phosvitin concentration General conditions as described in Materials and Methods. Incubation time 15 min. In the inset the Lineweaver-Burk double-reciprocal plot for phosvitin phosphatase is reported.
34,000 as M, of phosvitin (ll), the Km for this substrate proved to be 5.0 x 10m6 M, while that for ATP is 5.0 x 10e4 M (Fig. 4). It must be underlined that: (1) the reported K, values were obtained in the absence of added Mg2+, i.e., under the optimal conditions for evidencing the ATPase activity of the enzyme; (2) the ATPase activity of the purified enzyme is quite negligible in comparison with the whole ATPase activity present in the crude mitochondrial extract and recovered in the fractions eluted from CM cellulose by lower ionic strength. Moreover the ATPase activity of the enzyme is well differentiated from other ATPases characterized up to now, since beside being inhibited by Mg2+ it is not stimulated either by Na+ plus I$+ or DNP, and it is inhibited neither by oligomycin nor by ouabain. In conclusion, the above results suggest that mitochondrial protein phosphatase, although displaying some ATPase activity in vitro, has in viuo the biological role of dephosphorylating, at least preferentially, the endogenous phosphoproteins. On this matter, it is worthwhile to emphasize that the enzyme is able to dephosphorylate the cytosol phosphoproteins (Table I) which, like phosvitin, are phosphorylated by a cytosol protein kinase, while they are not dephosphorylated by cytosol itself (8). Moreover, it should be mentioned that, upon purification, such cytosol phospho-
proteins display a very low average molecular weight (2000-3000) (12) which is consistent with the possibility of their penetration inside the outer mitochondrial membrane. ACKNOWLEDGMENT We thank Miss Carla Munari cal assistance.
for valuable
techni-
REFERENCES 1. MAGNI, G., CAULINI, G., AND MORET, V. (1971) Biochim. Biophys. Acto 242, 123-128. 2. MAURER, H. R. (1971) in Disc Electrophoresis (De Gruyter, ed.), p. 46, Berlin. 3. GOMORI, G. (1942) J. Lab. Clin. Med. 27,955-958. 4. LINDBERG, O., AND ERNSTER, L. (1956) in Methods of Biochemical Analysis (Glick, D., ed.), Vol. 3, p. 8, Znterscience, New York. 5. MARTIN, J. B., AND DOTY, D. M. (1949) Anal. Chem. 21, 965-967. 6. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 7. MECHAM, D. K., AND OLCOTT, H. S. (1949) J. Amer. Chem. Sot. 71, 3670-3677. 8. PINNA, L. A., CLARI, G., AND MORET, V. (1971) Biochim. Biophys. Acta 236, 270-278. 9. MEISLER, M. H., AND LANGAN, T. A. (1969) J. Biol. Chem. 244, 4961-4968. 10. BAGGIO, B., PINNA, L. A., MORET, V., AND SILIPRANDI, N. (1970) Biochim. Biophys. Acta 212, 515-517. 11. CLARK, R. C. (1970) Biochem. J. 118, 537-542. 12. PINNA, L. A., DONELLA, A., AND MORET, V. (1973) Fed. Eur. Biochem. Sot. Lett. 37, 183-187.
OF BIOCHEMISTRY
Further
AND
Characterization G. CLARI,
Istituto
166, 318-322 (19%)
BIOPHYSICS
di Chimica
of Mitochondrial
A. DONELLA,
Biologica
dell’llniuersith Mitocondri”
L. A. PINNA,
Protein AND
Phosphatase
V. MORET
di Padoua and “Centro per lo Studio della Fisiologia de1 C.N.R., Padoua, Italy
dei
Received June 10, 1974 Mitochondrial protein phosphatase from rat liver exhibits rather wide substrate specificity, since it readily dephosphorylates, besides phosvitin, casein, and cytosol phosphoproteins, also ATP, ADP, inorganic pyrophosphate, p-nitrophenylphosphate. Aliphatic phosphate esters (&glycerophosphate, glucose&P, serine-phosphate) are not dephosphorylated to any detectable extent. Evidence for the participation of a single enzyme in the dephosphorylation of phosvitin and ATP is provided. However, the different affinity toward the two substrates and other evidence suggest that the enzyme has in uiuo the biological role of dephosphorylating, at least preferentially, the phosphoproteins.
In a previous paper (1) the partial purification from rat liver mitochondria of a protein phosphatase, active on both phosvitin and casein, has been described. In the present paper, an improved purification and characterization of this enzyme are reported in order to get more information about its role in the living cells. It has been found that mitochondrial protein phosphatase (whose molecular weight is about 32,000) dephosphorylates, besides phosvitin, casein and cytosol phosphoproteins, also a few nonprotein metabolites of physiological relevance, namely, ATP, ADP, and inorganic pyrophosphate. Evidence is reported, however, suggesting that the biological role of this enzyme in the mitochondria involves the dephosphorylation of phosphoproteins rather than that of nonprotein substrates. MATERIALS Purification
AND
of Mitochondrial
METHODS Protein
matographed on a carboxymethyl cellulose (CM cellulose) column (1.6 x 14 cm) equilibrated with the same buffer. Stepwise elution was carried out with 2 mM acetate buffer, pH 5.4, containing increasing concentrations of NaCl. Gel filtration. The fraction eluted from CM cellulose with 0.3 M NaCl fraction, once concentrated to a volume of 3 ml by ultrafiltration through Diaflo UM ‘2 membranes, was submitted to gel filtration through a Sephadex G 100 column (1.8 x 62 cm) equilibrated with 2 mM acetate plus 0.60 M NaC1, pH 5.4. Gel electrophoresis. Small aliquots of concentrated protein phosphatase eluted from CM cellulose by 0.3 M NaCl were submitted to 7.5% polyacrylamide gel electrophoresis, pH 4.3, according to the procedure described in Ref. 2. Before running the sample, ammonium persulfate was completely removed from the gel by preelectrophoresis for 15 hr at 2 mA per gel tube. At the end of the electrophoretic run the gels were sliced into 3-mm segments from which the enzyme was extracted by homogenization in 3 ml of cold 0.1 M succinate buffer, pH 5.9. All above operations were carried out in a cold room at 0-4°C.
Phosphatase Determination
Extraction and preliminary purification of mitochondrial protein phosphatase up to the (NH,),SO, precipitation were accomplished as already described (1). CM cellulose column chromarography. The precipitate, once dissolved in a small volume of 2 mM acetate buffer, pH 5.4, containing 0.5 mM NaCl, was dialyzed overnight against the same buffer and chro318 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
of Phosphatase
Activities
Protein phosphatase activity was assayed with phosvitin as substrate. The incubation medium contained in a final volume of 2 ml: 200 Mmoles succinate buffer, pH 5.9, 6 pmoles cysteine, 1 mg of phosvitin (containing 3 pmoles of alkali-labile phosphate). The reaction was started by addition of the enzyme. Incubation at 37°C lasted 30 min, unless different
MITOCHONDRIAL
PROTEIN
PHOSPHATASE
319
FIG. 1. CM cellulose chromatography of the mitochondrial protein phosphatase preparation partially purified as previously described (1). The stepwise elution was carried out as described in Materials and Methods. Four-milliliter fractions were collected and tested for the various enzyme activities. Absorbance at 280 nm (O---O); phosvitin phosphatase activity (arbitrary units)/O.Z ml (a---O); ATPase activity (arbitrary units)/O.l ml (A- --A). times are indicated in the legend. The reaction was stopped by adding 1.5 ml of cold 50% trichloroacetic acid. Inorganic phosphate was determined in the clear supernatant by the Gomori procedure (3). The same procedure was followed to test all the other phosphatase activities, except that phosvitin was replaced by 2 pmoles of suitable phosphorylated substrates. Phosphatase activity toward [32P]salmine and [32P]cytosol phosphoproteins was tested by incubating, under the above-described conditions, amounts of the labeled proteins equivalent to 50,000 cpm as protein-bound 3ZP. a*P1 released during incubation was determined by the same procedure used for the determination of the 32P, released from [y-“P]ATP (see below). Whenever a discrimination between protein phosphatase and ATPase activities in the same sample was required, unlabeled phosvitin was incubated together with [Y-~~P]ATP having a known specific radioactivity, under the same conditions described above. The reaction was stopped by addition of 0.8 ml of 50% trichloroacetic acid and 0.9 ml of Silico-tungstic acid solution (4) followed by centrifugation. Total P, released from both substrates was determined in an aliquot of the supernatant according to the Martin and Doty procedure (5). 3ZP1 released from [y32P]ATP was measured by counting a parallel aliquot of the isobutanol-benzene phase in a Packard TriCarb liquid scintillator model 3375. Pi released from phosvitin was calculated as the difference between the two previous values.
Proteins were determined according to the Lowry et al. procedure (6). Phosvitin was prepared from egg yolk according to Mecham and Olcott (7). [y-32P]ATP was from Radiochemical Centre. All other substrates were obtained from Sigma Chemical Company. 32P-labeled cytosol phosphoproteins were prepared as already described (8). S2P-labeled salmine was prepared following in its general lines the procedure described by Meisler and Langan (9) by using a cytosol protamine kinase preparation obtained by P cellulose chromatography (10).
RESULTS
AND
DISCUSSION
Figure 1 shows the pattern of phosphatase activities obtained when a preparation of phosvitin phosphatase, partially purified, as previously described (l), was submitted to chromatography on CM cellulose column. It can be seen that most of phosvitin phosphatase activity is present in the fraction eluted by 0.3 M NaCll while ATPase activity is resolved in at least three fractions, eluted, respectively, by the equilibrium buffer, by 0.1 M NaCl, and by 0.3 M NaCl, the last one being by far the minor among the three. The results reported in 1Sometimes a minor but significant peak of phosvitin phosphatase activity is also eluted by 0.4 M NaCl.
320
CLARI ET AL.
Table I show that the fraction eluted by 0.3 M NaCl, in contrast to those eluted at lower ionic strength, does not dephosphorylate AMP, glucose-6-PO ,, ,&glycerophosphate, phosphoserine (Table I). However, such a fraction is able to dephosphorylate, besides phosvitin, casein and cytosol phosalso nonprotein substrates phoproteins, such as ATP, ADP, pyrophosphate, and p-nitrophenylphosphate. All these phosphatase activities exhibited by such an enzyme fraction are inhibited by EDTA. On the contrary, Mg2+, under our conditions, has been found to decrease the ATPase, ADPase and pyrophosphatase activities at concentrations which have no effect on phosvitin phosphatase. Such a finding, probably due to different stabilities of Mg-substrate complexes, accounts for the apparent discrepancy from previously reported data (l), obtained in the presence of Mg2+, according to which no appreciable ATPase activity was detectable in the protein phosphatase fraction purified on CM cellulose. In order to establish whether or not the phosphatase activities exhibited by the 0.3 M NaCl fraction are due to a single multifunctional enzyme, such a fraction from
CM cellulose was submitted to further purification procedure and it was again tested for its activity toward protein substrate (phosvitin) and nonprotein substrate (ATP). Gel filtration through Sephadex G 100 gives rise to a single peak with protein phosphatase and ATPase activities overlapping as shown in Fig. 2. The molecular TABLE
I
SUBSTRATE SPECIFICITY OF THE ENZYME FRACTION ELUTED FROM CM CELLULOSE WITH 0.3 M NaCl”
Substrate
Phosphatase activity, P, released P&!
Phosvitin Casein [s2P]Salmine [32P]Cytoso1 phosphoproteins Phosphorylserine Glucose-6-phosphate a-Glycerophosphate fl-Glycerophosphate AMP ADP ATP Na inorganic pyrophosphate p-Nitrophenylphosphate
wm
13.5 7.3 0.000 10.000 0.00 0.00 0.00 0.00 0.00 15.03 17.00 10.00 17.5
“The enzymatic activity was determined scribed in the Methods and Materials.
FIG. 2. Molecular filtration on Sephadex G 100 of the enzyme fraction eluted from CM and Methods. cellulose with 0.3 M NaCl. Genera1 conditions are described in the Materials Fractions of 3.7 ml were collected. The void volume of the column was 56 ml. Calibration curve was obtained with (1) horse heart cytochrome c (12, 400), (2) ovoalbumin (45,000), and (3) bovine serum albumin (67,000). Absorbance at 220 nm (0-O); phosvitin phosphatase activity (arbitrary units)/0.4 ml (O-O); ATPase activity (arbitrary units)/0.2 ml (A- --A). The recovery of both enzymatic activities approached 95%.
as de-
MITOCHONDRIAL r-
-
PROTEIN
7
L---? IL
c-----____------.
0 . .
.
.-
t Star’
3. Polyacrylamide gel electrophoresis. r;xperimental conditions as described in Materials and Methods. Solid line: phosvitin phosphatase activity; dashed line: ATPase activity. FIG.
TABLE
II
PRUTEIN PHOSPHATASE AND ATPASE ACTIVITIES EXHIBITED IN THE PRESENCE OF THE RELATED SUBSTRATES ADDED SEPARATELY AND TOGETHER~
Substrate Phosvitin (0.3 mg) [-y-32P]ATP (1 rmole) Phosvitin + [r-32P]ATP
fiPl released from phosvitin [r-“‘P]ATP 6.7 4.6
7.1 (12.047 cpm) 3.7 (6.270 cpm)
PHOSPHATASE
321
substrates are added together is less than that expected if two different enzymes acting independently on the two substrates were present. On the contrary, a mutual inhibition is observed as it would be expected if the two substrates compete for the same enzyme. Such a point of view is strengthened by other kinetic studies. Figure 4 shows the Lineweaver-Burk double-reciprocal plot for ATPase activity obtained by using [y-3ZP]ATP of known specific radioactivity in the absence and in the presence of unlabeled phosvitin. It can be seen that the inhibition by phosvitin results in a higher K,, with unchanged V, as expected if the two substrates ( [y-32P]ATP and phosvitin) are competing for the same catalytic site. However, the affinity of the enzyme for the two substrates is very different. The phosvitin phosphatase activity (Fig. 5), unlike ATPase activity, does not follow normal Michaelis-Menten kinetics, being inhibited by high concentrations of substrate. Nevertheless a fairly accurate evaluation of K, was possible by extrapolating the linear part of the graph in the doublereciprocal plot (inset for Fig. 5). Assuming
a The enzyme activity was determined as described in the Methods and Materials. The specific radioactivity of [T-~‘P]ATP was 52,500 counts/min per @mole.
weight, estimated by comparison with standard proteins, is about 32,000 (inset of Fig. 2). By gel electrophoresis in polyacrylamide 7.5% at pH 4.3 protein phosphatase and ATPase are again superimposed in a single band (Fig. 3). Keeping in mind the high resolution of gel electrophoresis, one should conclude that the activities toward phosvitin and ATP are due to a single enzyme, rather than to different enzymes. Such a conclusion is also supported by experiments in which unlabeled phosvitin and [y-32P]ATP were simultaneously added. As shown in Table II, under these conditions, which allow the discrimination between the two activities, the Pi released when the two
FIG. 4. Lineweaver-Burk double-reciprocal plot for ATPase in the absence and in the presence of phosvitin. The incubation medium contained different concentration of [-y-S2P]ATP having a known specific radioactivity (50,000 cpm//lmole ATP) in the presence or in the absence of a fixed amount of unlabeled phosvitin (0.40 mg). Incubation time 10 min. The s2Pi released from [-r-S2P]ATP was determined as described in the Materials and Methods. Phosvitin absent (A- - -A); 0.40 mg phosvitin present in the incubation medium (0- - -0).
322
CLARI ET AL.
FIG. 5. Phosvitin phosphatase activity as a function of phosvitin concentration General conditions as described in Materials and Methods. Incubation time 15 min. In the inset the Lineweaver-Burk double-reciprocal plot for phosvitin phosphatase is reported.
34,000 as M, of phosvitin (ll), the Km for this substrate proved to be 5.0 x 10m6 M, while that for ATP is 5.0 x 10e4 M (Fig. 4). It must be underlined that: (1) the reported K, values were obtained in the absence of added Mg2+, i.e., under the optimal conditions for evidencing the ATPase activity of the enzyme; (2) the ATPase activity of the purified enzyme is quite negligible in comparison with the whole ATPase activity present in the crude mitochondrial extract and recovered in the fractions eluted from CM cellulose by lower ionic strength. Moreover the ATPase activity of the enzyme is well differentiated from other ATPases characterized up to now, since beside being inhibited by Mg2+ it is not stimulated either by Na+ plus I$+ or DNP, and it is inhibited neither by oligomycin nor by ouabain. In conclusion, the above results suggest that mitochondrial protein phosphatase, although displaying some ATPase activity in vitro, has in viuo the biological role of dephosphorylating, at least preferentially, the endogenous phosphoproteins. On this matter, it is worthwhile to emphasize that the enzyme is able to dephosphorylate the cytosol phosphoproteins (Table I) which, like phosvitin, are phosphorylated by a cytosol protein kinase, while they are not dephosphorylated by cytosol itself (8). Moreover, it should be mentioned that, upon purification, such cytosol phospho-
proteins display a very low average molecular weight (2000-3000) (12) which is consistent with the possibility of their penetration inside the outer mitochondrial membrane. ACKNOWLEDGMENT We thank Miss Carla Munari cal assistance.
for valuable
techni-
REFERENCES 1. MAGNI, G., CAULINI, G., AND MORET, V. (1971) Biochim. Biophys. Acto 242, 123-128. 2. MAURER, H. R. (1971) in Disc Electrophoresis (De Gruyter, ed.), p. 46, Berlin. 3. GOMORI, G. (1942) J. Lab. Clin. Med. 27,955-958. 4. LINDBERG, O., AND ERNSTER, L. (1956) in Methods of Biochemical Analysis (Glick, D., ed.), Vol. 3, p. 8, Znterscience, New York. 5. MARTIN, J. B., AND DOTY, D. M. (1949) Anal. Chem. 21, 965-967. 6. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 7. MECHAM, D. K., AND OLCOTT, H. S. (1949) J. Amer. Chem. Sot. 71, 3670-3677. 8. PINNA, L. A., CLARI, G., AND MORET, V. (1971) Biochim. Biophys. Acta 236, 270-278. 9. MEISLER, M. H., AND LANGAN, T. A. (1969) J. Biol. Chem. 244, 4961-4968. 10. BAGGIO, B., PINNA, L. A., MORET, V., AND SILIPRANDI, N. (1970) Biochim. Biophys. Acta 212, 515-517. 11. CLARK, R. C. (1970) Biochem. J. 118, 537-542. 12. PINNA, L. A., DONELLA, A., AND MORET, V. (1973) Fed. Eur. Biochem. Sot. Lett. 37, 183-187.