/. Biochem., 81, 93-97 (1977)
Separation of Four Forms of the Rabbit Skeletal Muscle Enzyme Masanao KOBAYASHI* and Kanefusa KATO** •Department of Biochemistry, School of Medicine, Nagoya University, Showa-ku, Nagoya, Aichi 466, and "Department of Biochemistry, Medical College of Miyazaki, Kiyotake-cho, Miyazaki-gun, Miyazaki 889-16 Received for publication, June 16, 1976
Phosphoprotein phosphatase [phosphoprotein phosphohydrolase EC 3.1.3.16] in the soluble fraction of rabbit skeletal muscle, when assayed with phosphorylase a [EC 2.4.1.1] from rabbit skeletal muscle and phosphohistone as substrates, was resolved into three active fractions (Fractions I, II, and HI in order of elution) by DEAE-cellulose column chromatography. Sucrose density gradient centrifugation showed that these fractions were composed of subfractions of different molecular size (1: 7.3Sand4S; II: 8Sand4S; HI; 6.7S). Components with larger molecular size in the major fractions, II and III, were dissociated to a molecular size similar to that of the smallest component on freezing in the presence of mercaptoethanol. These results indicate that phosphoprotein phosphatase from skeletal muscle occurs in multiple forms very similar to those of the liver enzyme reported previously (Kobayashi, Kato and Sato (1975) Biochim. Biophys. Ada. 373, 343-355).
Phosphoprotein phosphatases [EC 3. 1.3. 16] from several sources have been reported to have a broad substrate specificity, catalyzing the dephosphorylation of several phosphoproteins, including phosphohistone (P-histone), glycogen synthetase D [UDPglucose: glycogen 4-a-glucosyltransferase, EC 2. 4. 1. 11], the phospho-form of phosphorylase b kinase [ATP: phosphorylase-6 phosphotransferase, EC 2. 7. 1. 38] and phosphorylase a [1, 4-crD-glucan: orthophosphate a-glucosyltransferase, EC 2.4. 1. 1] {1-8), and are thought to play an important role, in collaboration with protein kinases catalyzing phosphorylation, in the regulation of the activities of these and other functional proteins. Using P-histone, phosphorylase a and glycogen synthetase D as substrates, phosphoptotein phosphatase from rabbit skeletal muscle was shown to Vol. 81, No. 1, 1977
exist in two forms differing in some properties and in molecular size (s^y. 6.7 and 3.8) (3, 4). The liver phosphatase, on the other hand, could be separated by DEAE-cellulose chromatography into three fractions which consisted of at least four active subfractions of different molecular sizes (ji0)W: 7.8, 7.0, 5.8, and 4.0) as determined by sucrose density gradient centrifugation (5). Although some similarity between the enzymes from the two sources, prepared in somewhat different ways, was noted (3-5), no direct comparison was made. This communication reports that the muscle phosphatase, when prepared in the same way as the liver enzyme, shows multiple forms very similar to those of the liver phosphatase. A possible model for the multiple forms of the phosphatase from both sources is suggested. 93
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Multiple Molecular Forms of Phosphoprotein Phosphatase
M. KOBAYOSHI and K. KATO
94
RESULTS AND DISCUSSION
Rabbit muscle phosphorylase a labeled with "P was prepared by the method described previously (4). "P-Labeled P-histone was prepared by the method of Meisler and Langan (9), as modified by Kato and Bishop (7). Phosphorylase phosphatase and P-histone phosphatase activities were measured as described previously (5). One unit of phosphatase activity was difined as the amount catalyzing the hydrolysis of 1 nmole of "P-orthophosphate from the appropriate substarte.1 Previously, alcohol precipitation was included at an early stage of purification of muscle phosphoprotein phosphatase, and the enzyme was immediately eluted from a DEAE-cellulose column with a high concentration of NaCl (0.4 M) (5, 4). In this study, muscle phosphatase was prepared by a procedure similar to that used for the liver enzyme (5), employing a linear gradient of NaCl for elution of the enzyme from the DEAE-cellulose column. Freshly excised muscle was homogenized in 2.5 volumes of 20 mM Tris-HCl buffer (pH 7.5) containing 5 mM EDTA and 10 ITIM mercaptoethanol for 2 min using a blender (Hitachi VA850). This and all the following procedures were carried out at 4°. The homogenate was centnfuged at 12,000 x g for 30 mm and the supernatant, after filtration through two layers of gauze, was centrifuged at 78,000 x g for 60 min. The supernatant was fractionated by the addition of solid (NH4),SO4 (0-50% satn.). The precipitated fraction was dissolved in 20 mM Tris-HCl buffer (pH 7.5) containing 1 mM EDTA and 10 mM mercaptoethanol, dialyzed against the same buffer and applied to a DEAE-cellulose (Brown, Selectacel type 20) column. After elution of the phosphatase with increasing concentrations of NaCl, the peak fractions were pooled, concentrated by (NH^SQ, precipitation (55% satn), redissolved in a small volume of the above buffer, dialyzed against the same buffer and analyzed by sucrose density gradient centrifugation as described previously (5).
When the (NH«),SO4 fraction of the 78,000 xg supernatant of muscle extract was chromatographed on a DEAE-cellulose column as described for the liver preparation (5), both phosphorylase phosphatase and P-histone phosphatase activities were eluted in three peaks at NaCl concentration ranges similar to those in which "Fractions I, II, and III" of liver phosphatase were eluted (5); they
1
One unit defined here corresponds to one munit of the international unit recommended by IUB (/imole/min).
u* n
20
40 Fraction
B
60 number
80
Fig. I. DEAE-cellulose chromatography of phosphoprotein phosphatase ((NHO.SO* fraction). The (NH,),SO4 fraction corresponding to 290 g of skeletal muscle prepared as described in "EXPERIMENTAL" was placed on a DEAE-cellulose column (2.3x18 cm) equilibrated with 20 mM Tris-HCl buffer (pH 7.5) containing 1 mM EDTA, 10mM mercaptoethanol, and 0.07 M NaCl. After washing with the same buffer, elution was performed with 900 ml of a linear gradient of NaCl (0.07-0 35 M) in the same buffer and phosphorylase phosphatase (upper) and P-histone phosphatase (lower) activities were assayed as described in "EXPERIMENTAL," using 10 ft\ aliquots of the 10 ml fractions collected Fractions indicated in the figure as I, IIA, IIB, and III were pooled, concentrated, and used for sucrose density gradient centrifugation studies (Figs. 3 and 4). • and O indicate activity in the presence and absence of 5 mM MnCli, respectively.
/. Biochem.
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EXPERIMENTAL
MULTIPLE FORMS OF PHOSPHOPROTEIN PHOSPHATASE
When a crude extract (78,000 x g supernatant) was chromatographed in a similar manner, a similar elution pattern was observed (Fig. 2), suggesting that these fractions are not artifacts formed during (NH 4 ),SO 4 fractionation, but may exist in vivo.
The four fractions separated by DEAE-cellulose chromatography, Fractions I, IIA, 11B, and III in Fig. 1, were analyzed by sucrose density gradient centnfugation. As shown in Fig. 3, phosphoprotein phosphatase in skeletal muscle also exists in at least four forms of different molecular sizes. Fraction I was found to be a mixture of two components with 5MjW values of about 7.3 and 4. Fraction IIA was composed mainly of a small molecular size component with smjW of about 4, contaminated by a larger component with s^w of about 8. Fraction 11B appeared to contain the same components as Fraction IIA, but the proportion of the 8 S component was much greater. Fraction III consisted mainly of a medium-sized component with SjO,w of about 6.7 F ig. 4 shows the sedimentation profiles of Fractions IIA and III, which are taken as representative fractions of the muscle phosphatase, after treatment with mercaptoethanol as reported previously (3-5). After this treatment the forms of larger molecular size (8 S in Fraction IIA and 6.7 S in Fraction HI) disappeared and in both fractions only one species with a sedimentation coefficient similar to that of the smallest form was observed. In Fraction III, at the same time, the phosphorylase phosphatase activity was considerably increased, as reported with the " larger form " (4) and with the liver enzyme (5). The above results indicate that phosphoprotein phosphatase from skeletal muscle shows multiple forms very similar to those of the liver enzyme, except for some differences in sn>v values and the relative proportions of activity of each 20 40 60 F r a c t i o n number form. It exists in at least four forms differing in Fig. 2. DEAE-cellulose chromatography of phosphc- molecular size, with 5!0,w values of about 8, 7.3, protein phosphatase (crude extract) 78,000 x p super- 6 7, and 4. The component of larger molecular natant corresponding to 38 g of skeletal muscle was size, reported as " Phosphatase 1" (5) or the chromatographed on a DEAE-cellulose column (1.6x7 " larger form " (4) in previous papers, appears to cm) as described in Fig. 1, using a 400 ml NaCI linear have been a mixture of the first three forms degradient of the same concentration range. Aliquots of scribed above. Previously, the phosphatase was 10 n\ and 30 /iI of the 5 ml fractions were used for eluted from the DEAE-cellulose column simply phosphorylase phosphatase (upper) and P-histone phosphatase (lower) assay, respectively. • , plus with a high concentration of NaCI. Therefore, the relatively small differences in molecular size MnCl,, O, minus MnCl,. Vol. 81, No. 1, 1977
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were also referred to as Fractions I, II, and III in order of elution (Fig. 1). The phosphorylase phosphatase activity of Fraction III, like that of Fraction III of liver, was stimulated by inclusion of 5 mM MnCl, in the assay mixture, while the effects of MnCl, on the activity of the other two fractions were variable. With regard to P-histone phosphatase activity, the activity of all the fractions was more or less stimulated by MnG,. The activity, especially that of phosphorylase phosphatase, of Fraction II always showed a shoulder or splitting into two peaks (referred to as Fraction IIA and TIB in Fig. 1) with different sensitivities to MnQ,, showing that this fraction contains more than one component. Both phosphatase activities were highest in Fraction II, wheras in liver the highest phosphorylase phosphatase activity was always observed in Fraction III when assayed in the presence of MnQ, (5). The effect of metal ions has already been discussed (5) and studied extensively (70).
95
M. KOBAYASHI and K. KATO
96 II B
III
1
o >,
II B U)
in en o
•-
10
20
10 20 10 20 Fraction number(from the bottom)
10
20
Fig. 3. Sedimentation profiles of phosphoprotein phosphatase and P-histone phosphatase activities of the phosphoprotein phosphatase fractions in a sucrose density gradient. Appropriately diluted 150 ft\ aliquots of the concentrated Fractions I, HA, ILB, and HI (protein content 1.6, 0.62, 2.0, and 0.73 mg, respectively), as indicated in Fig. 1, were subjected to sucrose density gradient centrifugation. Assay conditions were as described in Fig. 2. Arrows indicate the position of bovine serum albumin, used as a reference protein may not have permitted separation of these three forms by gel filtration or sucrose density gradient centrifugation, unless preceded by gross fractionation with a linear NaCl gradient, as reported here. The results presented in this report support the hypothesis previously proposed for the multiple forms of the liver phosphatase, that a common catalytic subunit is bound to different regulatory (or inhibitory) proteins (5). The smallest form, with 5 M|W of about 4, may represent free catalytic unit unbound to any inhibitory protein. If such a model is correct, the muscle phosphatase appears to exist much more as unbound " active " form
10 20 10 20 Fraction number(from the bottom)
Fig. 4. Sedimentation profiles of phosphorylase phosphatase and P-histone phosphatase activities of the mercaptoethanol-treated phosphoprotein phosphatase fractions. The same procedure as in Fig. 3 was carried out with Fractions IIA and ID after mercaptoethanol treatment as described previously (5). The state of dilution of each fraction is equivalent to that in Fig. 3, and other conditions were as described in Fig. 3. J. Biochem.
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MULTIPLE FORMS OF PHOSPHOPROTEIN PHOSPHATASE
97
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2. Zieve, F J. & Glinsmann, W.H (1973) Biochem. Biophys Res. Commun. 50, 872-878 3. Kato, K. & Sato, S. (1974) Biochim. Biophys. Ada 358, 299-307 4. Kato, K., Kobayashi, M., & Sato, S. (1974) Biochim. Biophys Ada 371, 89-101 5. Kobayashi, M., Kato, K., & Sato, S. (1975) Biochim. Biophys. Acia 377, 343-355 6 Nakai, C. & Thomas, J.A. (1974) /. Biol. Chem. 249, 6459-6467 7. KiUilea, S.D., Brandt, H., Lee, E.Y.C., & Whelan, W.J. (1976) J. Biol. Chem. 251, 2363-2368 8. Lee, E.Y C , Brandt, H., Capulong, Z.L., & Killilea, S.D. (1976) in Advances in Enzyme Regulation (Weber, G , ed.) Vol. 14, pp. 467^*90, Pergamon-Press, Oxford-New York 9. Meisler, M.H. & Langan, T.A. (1969) J. Biol. Chem. 244, 4961-4968 10. Kato, K., Kobayashi, M., & Sato, S. (1975) / . Biochem. 77, 811-815 11. Brandt, H., KiUilea, S D., & Lee, E.Y.C. (1974) Biochem. Biophys. Res. Commun. 61, 598-604 12. Brandt, H., Lee, E.Y.C , & Killilea, S.D. (1975) Biochem. Biophys. Res. Commun. 63, 950-956 13 Abe, N. & Tsuiki, S. (1974) Biochim. Biophys. Ada 350, 383-391 14. Tan, E.L , Levine, M.A., Yang, C.S., & Li, H.-C. (1976) Int. J. Biochem. 7, 21-26 15. Kalala, L.R., Goris, J., & Merlevede, W. (1973) FEBS Lett. 32, 284-288 16. Thomas, J.A., Young, T., & Mellgren, R. (1974) Federation Proc. 33, 1315, Abstr. No. 526 17. Li, H-C (191'5) FEBS Lett 55,134-137 18. Maeno, H. & Greengard, P. (1972) / . Biol. Chem. 247, 3269-3277 19. Miyamoto, E. & Kakiuchi, S. (1975) Biochim. Biophys. Ada 384, 458-465 20. Brandt, H., Capulong, Z.L., & Lee, E.Y.C. (1975) /. Biol. Chem. 250, 8038-8044 21. Gratecos, D., Detwiler, T., & Fischer, E.H. (1974) in Metabolic Interconversion of Enzymes 1973 (Fischer, The authors are grateful to Ms. T. Baba for valuable E.H., Krebs, E.G., Neurath, H., & Stadtman, E.R., assistance. eds.) pp. 43-52, Springer-Verlag, New York 22. Huang, F.L. & Glinsmann, W.H. (1975) Proc. Natl. REFERENCES Acad. Sci. U.S. 72, 3004-3008 1. Kato, K. & Bishop, J.S. (1972) /. Biol. Chem 247, 23. Huang, F.L. & Glinsmann, W. (1976) FEBS Lett. 62, 326-329 7420-7429
than that of liver. Moreover, besides liver (9,1115) and skeletal muscle, more or less similar multiple forms of the enzyme have been reported for phosphatases from heart (16,17), brain {18,19), and adrenal cortex (15), and such a model may be expected to be applicable to phosphoprotein phosphatases from other tissues Activation and dissociation of phosphorylase phosphatase, which acts on several phosphoprotein substrates, by a high concentration of ethanol (c.f. our mercaptoethanol treatment) has been reported by Lee and co-workers (8,11,12), and the dissociated form was highly purified from rabbit liver and skeletal muscle (20). They also proposed essentially the same model as described above, although they attribute the observed multiple forms to artifacts of the homogenization procedure (8). Gratecos et al. reported extensive purification of a specific phosphorylase phosphatase, probably dissociated by the use of 8 M urea, from glycogenprotein complex of rabbit muscle; this tended to aggregate to yield apparent multiple forms (21). The relationship between the phosphatase from this source and that from the soluble fraction is unclear as yet. The exact nature of the inhibitory proteins in the above models is not clear at present, although the existence of some heat-stable inhibitory proteins has been reported in liver (12) and muscle (12, 22, 23). Purification of undissociated forms of the phosphatase appears to be essential for unequivocal elucidation of the nature of the multiple forms of this enzyme, and the discovery of physiological conditions causing dissociation and/or reassociation would greatly assist an understanding of their significance in metabolic regulation in vivo.
Separation of Four Forms of the Rabbit Skeletal Muscle Enzyme Masanao KOBAYASHI* and Kanefusa KATO** •Department of Biochemistry, School of Medicine, Nagoya University, Showa-ku, Nagoya, Aichi 466, and "Department of Biochemistry, Medical College of Miyazaki, Kiyotake-cho, Miyazaki-gun, Miyazaki 889-16 Received for publication, June 16, 1976
Phosphoprotein phosphatase [phosphoprotein phosphohydrolase EC 3.1.3.16] in the soluble fraction of rabbit skeletal muscle, when assayed with phosphorylase a [EC 2.4.1.1] from rabbit skeletal muscle and phosphohistone as substrates, was resolved into three active fractions (Fractions I, II, and HI in order of elution) by DEAE-cellulose column chromatography. Sucrose density gradient centrifugation showed that these fractions were composed of subfractions of different molecular size (1: 7.3Sand4S; II: 8Sand4S; HI; 6.7S). Components with larger molecular size in the major fractions, II and III, were dissociated to a molecular size similar to that of the smallest component on freezing in the presence of mercaptoethanol. These results indicate that phosphoprotein phosphatase from skeletal muscle occurs in multiple forms very similar to those of the liver enzyme reported previously (Kobayashi, Kato and Sato (1975) Biochim. Biophys. Ada. 373, 343-355).
Phosphoprotein phosphatases [EC 3. 1.3. 16] from several sources have been reported to have a broad substrate specificity, catalyzing the dephosphorylation of several phosphoproteins, including phosphohistone (P-histone), glycogen synthetase D [UDPglucose: glycogen 4-a-glucosyltransferase, EC 2. 4. 1. 11], the phospho-form of phosphorylase b kinase [ATP: phosphorylase-6 phosphotransferase, EC 2. 7. 1. 38] and phosphorylase a [1, 4-crD-glucan: orthophosphate a-glucosyltransferase, EC 2.4. 1. 1] {1-8), and are thought to play an important role, in collaboration with protein kinases catalyzing phosphorylation, in the regulation of the activities of these and other functional proteins. Using P-histone, phosphorylase a and glycogen synthetase D as substrates, phosphoptotein phosphatase from rabbit skeletal muscle was shown to Vol. 81, No. 1, 1977
exist in two forms differing in some properties and in molecular size (s^y. 6.7 and 3.8) (3, 4). The liver phosphatase, on the other hand, could be separated by DEAE-cellulose chromatography into three fractions which consisted of at least four active subfractions of different molecular sizes (ji0)W: 7.8, 7.0, 5.8, and 4.0) as determined by sucrose density gradient centrifugation (5). Although some similarity between the enzymes from the two sources, prepared in somewhat different ways, was noted (3-5), no direct comparison was made. This communication reports that the muscle phosphatase, when prepared in the same way as the liver enzyme, shows multiple forms very similar to those of the liver phosphatase. A possible model for the multiple forms of the phosphatase from both sources is suggested. 93
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Multiple Molecular Forms of Phosphoprotein Phosphatase
M. KOBAYOSHI and K. KATO
94
RESULTS AND DISCUSSION
Rabbit muscle phosphorylase a labeled with "P was prepared by the method described previously (4). "P-Labeled P-histone was prepared by the method of Meisler and Langan (9), as modified by Kato and Bishop (7). Phosphorylase phosphatase and P-histone phosphatase activities were measured as described previously (5). One unit of phosphatase activity was difined as the amount catalyzing the hydrolysis of 1 nmole of "P-orthophosphate from the appropriate substarte.1 Previously, alcohol precipitation was included at an early stage of purification of muscle phosphoprotein phosphatase, and the enzyme was immediately eluted from a DEAE-cellulose column with a high concentration of NaCl (0.4 M) (5, 4). In this study, muscle phosphatase was prepared by a procedure similar to that used for the liver enzyme (5), employing a linear gradient of NaCl for elution of the enzyme from the DEAE-cellulose column. Freshly excised muscle was homogenized in 2.5 volumes of 20 mM Tris-HCl buffer (pH 7.5) containing 5 mM EDTA and 10 ITIM mercaptoethanol for 2 min using a blender (Hitachi VA850). This and all the following procedures were carried out at 4°. The homogenate was centnfuged at 12,000 x g for 30 mm and the supernatant, after filtration through two layers of gauze, was centrifuged at 78,000 x g for 60 min. The supernatant was fractionated by the addition of solid (NH4),SO4 (0-50% satn.). The precipitated fraction was dissolved in 20 mM Tris-HCl buffer (pH 7.5) containing 1 mM EDTA and 10 mM mercaptoethanol, dialyzed against the same buffer and applied to a DEAE-cellulose (Brown, Selectacel type 20) column. After elution of the phosphatase with increasing concentrations of NaCl, the peak fractions were pooled, concentrated by (NH^SQ, precipitation (55% satn), redissolved in a small volume of the above buffer, dialyzed against the same buffer and analyzed by sucrose density gradient centrifugation as described previously (5).
When the (NH«),SO4 fraction of the 78,000 xg supernatant of muscle extract was chromatographed on a DEAE-cellulose column as described for the liver preparation (5), both phosphorylase phosphatase and P-histone phosphatase activities were eluted in three peaks at NaCl concentration ranges similar to those in which "Fractions I, II, and III" of liver phosphatase were eluted (5); they
1
One unit defined here corresponds to one munit of the international unit recommended by IUB (/imole/min).
u* n
20
40 Fraction
B
60 number
80
Fig. I. DEAE-cellulose chromatography of phosphoprotein phosphatase ((NHO.SO* fraction). The (NH,),SO4 fraction corresponding to 290 g of skeletal muscle prepared as described in "EXPERIMENTAL" was placed on a DEAE-cellulose column (2.3x18 cm) equilibrated with 20 mM Tris-HCl buffer (pH 7.5) containing 1 mM EDTA, 10mM mercaptoethanol, and 0.07 M NaCl. After washing with the same buffer, elution was performed with 900 ml of a linear gradient of NaCl (0.07-0 35 M) in the same buffer and phosphorylase phosphatase (upper) and P-histone phosphatase (lower) activities were assayed as described in "EXPERIMENTAL," using 10 ft\ aliquots of the 10 ml fractions collected Fractions indicated in the figure as I, IIA, IIB, and III were pooled, concentrated, and used for sucrose density gradient centrifugation studies (Figs. 3 and 4). • and O indicate activity in the presence and absence of 5 mM MnCli, respectively.
/. Biochem.
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EXPERIMENTAL
MULTIPLE FORMS OF PHOSPHOPROTEIN PHOSPHATASE
When a crude extract (78,000 x g supernatant) was chromatographed in a similar manner, a similar elution pattern was observed (Fig. 2), suggesting that these fractions are not artifacts formed during (NH 4 ),SO 4 fractionation, but may exist in vivo.
The four fractions separated by DEAE-cellulose chromatography, Fractions I, IIA, 11B, and III in Fig. 1, were analyzed by sucrose density gradient centnfugation. As shown in Fig. 3, phosphoprotein phosphatase in skeletal muscle also exists in at least four forms of different molecular sizes. Fraction I was found to be a mixture of two components with 5MjW values of about 7.3 and 4. Fraction IIA was composed mainly of a small molecular size component with smjW of about 4, contaminated by a larger component with s^w of about 8. Fraction 11B appeared to contain the same components as Fraction IIA, but the proportion of the 8 S component was much greater. Fraction III consisted mainly of a medium-sized component with SjO,w of about 6.7 F ig. 4 shows the sedimentation profiles of Fractions IIA and III, which are taken as representative fractions of the muscle phosphatase, after treatment with mercaptoethanol as reported previously (3-5). After this treatment the forms of larger molecular size (8 S in Fraction IIA and 6.7 S in Fraction HI) disappeared and in both fractions only one species with a sedimentation coefficient similar to that of the smallest form was observed. In Fraction III, at the same time, the phosphorylase phosphatase activity was considerably increased, as reported with the " larger form " (4) and with the liver enzyme (5). The above results indicate that phosphoprotein phosphatase from skeletal muscle shows multiple forms very similar to those of the liver enzyme, except for some differences in sn>v values and the relative proportions of activity of each 20 40 60 F r a c t i o n number form. It exists in at least four forms differing in Fig. 2. DEAE-cellulose chromatography of phosphc- molecular size, with 5!0,w values of about 8, 7.3, protein phosphatase (crude extract) 78,000 x p super- 6 7, and 4. The component of larger molecular natant corresponding to 38 g of skeletal muscle was size, reported as " Phosphatase 1" (5) or the chromatographed on a DEAE-cellulose column (1.6x7 " larger form " (4) in previous papers, appears to cm) as described in Fig. 1, using a 400 ml NaCI linear have been a mixture of the first three forms degradient of the same concentration range. Aliquots of scribed above. Previously, the phosphatase was 10 n\ and 30 /iI of the 5 ml fractions were used for eluted from the DEAE-cellulose column simply phosphorylase phosphatase (upper) and P-histone phosphatase (lower) assay, respectively. • , plus with a high concentration of NaCI. Therefore, the relatively small differences in molecular size MnCl,, O, minus MnCl,. Vol. 81, No. 1, 1977
Downloaded from https://academic.oup.com/jb/article-abstract/81/1/93/836019 by Western Sydney University Library user on 14 January 2019
were also referred to as Fractions I, II, and III in order of elution (Fig. 1). The phosphorylase phosphatase activity of Fraction III, like that of Fraction III of liver, was stimulated by inclusion of 5 mM MnCl, in the assay mixture, while the effects of MnCl, on the activity of the other two fractions were variable. With regard to P-histone phosphatase activity, the activity of all the fractions was more or less stimulated by MnG,. The activity, especially that of phosphorylase phosphatase, of Fraction II always showed a shoulder or splitting into two peaks (referred to as Fraction IIA and TIB in Fig. 1) with different sensitivities to MnQ,, showing that this fraction contains more than one component. Both phosphatase activities were highest in Fraction II, wheras in liver the highest phosphorylase phosphatase activity was always observed in Fraction III when assayed in the presence of MnQ, (5). The effect of metal ions has already been discussed (5) and studied extensively (70).
95
M. KOBAYASHI and K. KATO
96 II B
III
1
o >,
II B U)
in en o
•-
10
20
10 20 10 20 Fraction number(from the bottom)
10
20
Fig. 3. Sedimentation profiles of phosphoprotein phosphatase and P-histone phosphatase activities of the phosphoprotein phosphatase fractions in a sucrose density gradient. Appropriately diluted 150 ft\ aliquots of the concentrated Fractions I, HA, ILB, and HI (protein content 1.6, 0.62, 2.0, and 0.73 mg, respectively), as indicated in Fig. 1, were subjected to sucrose density gradient centrifugation. Assay conditions were as described in Fig. 2. Arrows indicate the position of bovine serum albumin, used as a reference protein may not have permitted separation of these three forms by gel filtration or sucrose density gradient centrifugation, unless preceded by gross fractionation with a linear NaCl gradient, as reported here. The results presented in this report support the hypothesis previously proposed for the multiple forms of the liver phosphatase, that a common catalytic subunit is bound to different regulatory (or inhibitory) proteins (5). The smallest form, with 5 M|W of about 4, may represent free catalytic unit unbound to any inhibitory protein. If such a model is correct, the muscle phosphatase appears to exist much more as unbound " active " form
10 20 10 20 Fraction number(from the bottom)
Fig. 4. Sedimentation profiles of phosphorylase phosphatase and P-histone phosphatase activities of the mercaptoethanol-treated phosphoprotein phosphatase fractions. The same procedure as in Fig. 3 was carried out with Fractions IIA and ID after mercaptoethanol treatment as described previously (5). The state of dilution of each fraction is equivalent to that in Fig. 3, and other conditions were as described in Fig. 3. J. Biochem.
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97
Vol. 81, No. 1, 1977
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than that of liver. Moreover, besides liver (9,1115) and skeletal muscle, more or less similar multiple forms of the enzyme have been reported for phosphatases from heart (16,17), brain {18,19), and adrenal cortex (15), and such a model may be expected to be applicable to phosphoprotein phosphatases from other tissues Activation and dissociation of phosphorylase phosphatase, which acts on several phosphoprotein substrates, by a high concentration of ethanol (c.f. our mercaptoethanol treatment) has been reported by Lee and co-workers (8,11,12), and the dissociated form was highly purified from rabbit liver and skeletal muscle (20). They also proposed essentially the same model as described above, although they attribute the observed multiple forms to artifacts of the homogenization procedure (8). Gratecos et al. reported extensive purification of a specific phosphorylase phosphatase, probably dissociated by the use of 8 M urea, from glycogenprotein complex of rabbit muscle; this tended to aggregate to yield apparent multiple forms (21). The relationship between the phosphatase from this source and that from the soluble fraction is unclear as yet. The exact nature of the inhibitory proteins in the above models is not clear at present, although the existence of some heat-stable inhibitory proteins has been reported in liver (12) and muscle (12, 22, 23). Purification of undissociated forms of the phosphatase appears to be essential for unequivocal elucidation of the nature of the multiple forms of this enzyme, and the discovery of physiological conditions causing dissociation and/or reassociation would greatly assist an understanding of their significance in metabolic regulation in vivo.
