Upsala J Med Sci 83: 129-134, 1978 Separation of Rat-Liver Phosphoprotein Phosphatases Active on Phosphorylated Pyruvate Kinase (Type L) VINCENT P. K . TITANJI From the Institute of Medical and Physiological Chemistry, Biomedical Centre, University of Uppsula, Uppsala, Sweden ABSTRACT The substrate specificity of rat liver phosphoprotein phos- phatases has been investigated. The enzymes were resolved into three fractions, termed A , B and C, on elution from DEAE-cellulose with apparent molecular weights, as deter- mined by Sephadex G-200 chromatography, of approxi- mately 250 000, 250000 and 140 000, respectively. All frac- tions catalyzed the dephosphorylation of calf-thymus phos- phohistones, salmon phosphoprotamine and rabbit skeletal muscle phosphorylase a. The major portion of the activity towards these substrates was found in fraction B. The activity towards rat liver phosphopyruvate kinase (type L) resided almost exclusively in fractions B and C. It is con- cluded that rat liver contains multiple forms of phospho- protein phosphatases and that phosphatases of fraction B and C are the major activities towards phosphopyruvate kinase. INTRODUCTION The mechanism of hormonal regulation of carbo- hydrate metabolism in the liver has been studied extensively at the level of pyruvate kinase (type L) (for recent review see ref. 1). Experiments from this (1-3) and other laboratories (4-6) have estab- lished that pyruvate kinase (type L) is inhibited as a result of cyclic-AMP dependent phosphoryla- tion. Whereas the cyclic-AMP dependent protein kinases which catalyse the phosphorylation of pyru- vate kinase (type L) and other proteins have been investigated in great detail (for recent review and further references see 6), the phosphoprotein phos- phatases which reverse the phosphorylation are still under intensive research (see 7 for further ref- erences). After the discovery that a histone phosphatase preparation counterbalances the cyclic-AMP de- pendent phosphorylation of pyruvate kinase (type L) (8), attempts were made to purify the enzyme(s) and although an extensively purified preparation was obtained, severe losses in enzymatic activity were noted at the initial stages of the procedure (9). Since this inactivation was incompatible with the known stability of the final phosphatase prep- aration, it was postulated that labile enzyme forms were removed by the procedure. The aims of this investigation were to separate the phosphoprotein phosphatases of rat liver cyto- sol and to compare the specificity displayed to- wards phosphopyruvate kinase with that observed towards selected phosphoproteins from other organs. EXPERIMENTAL Materials. Recrystallized bovine serum albumin and phe- nylmethylsulfonyl fluoride were bought from Sigma. Sephadex G-200 was from Pharmacia Fine Chemicals, Uppsala. “Buffer A” containing 10 mM imidazoI/HCl, pH 7.5, 15 mM mercaptoethanol and 2.5 mM MgCI, was used as indicated. (32P)Phosphoproteins. Rat-liver pyruvate kinase was isolated and phosphorylated a s described earlier (8). Sedimentation equilibrium experiments with pyruvate kinase were performed by the meniscus-depletion tech- nique of Yphantis (10, 1 I ) . A Beckman Model E analytical ultracentrifuge equipped with an RTIC temperature control unit and an electronic control was used. All measurements were conducted in standard 12 mm double sector cells with sapphire windows. The centrifuge was run at 20°C for 18 h at 10000 rpm. Pyruvate kinase was dialysed against 20 mM potassium phosphate, pH 7.0, containing 0.1 mM fructose 1,6-diphosphate and 0.1 mM dithiothreitol. Assuming a partial specific volume, V , of 0.72 the molecular weight of pyruvate kinase was cal- culated to be 250000. Assuming E;:G=0.68 at 280 nm the maximal degree of phosphorylation was found to lie between 3.2 and 3.6 mol (32P)phosphate per mol pyru- vate kinase tetrarner. Analysis of cold phosphate on the unlabelled enzyme was not performed, although the dif- ferent maximal levels of phosphorylation obtained with different enzyme preparations suggests the presence of 9-782853 130 V . P . K . Titanji some phosphate on the unlabelled enzyme. Prior to use, phosphopyruvate kinase was chromatographed on a Sephadex (3-50 column equilibrated and eluted with 5 mM imidazol-HC1, pH 7.5, containing 10% glycerol, 50 mM KCI, 0.1 mM fructose-l,6-diphosphate and 0.1 mM dithio- threitol. (3‘)Phosphohistones (Sigma Type IIA) and (32P)phosphoprotamine were prepared and the alkali-labile phosphate content determined as described previously (9, 12). (32P)Phosphorylase a was prepared using 5-10 mg of rabbit skeletal muscle phosphorylase b (Boehringer) and 0.3 mg phosphorylase b kinase (Sigma) essentially ac- cording to the method of Fischer & Krebs (13). Before use, (32P)phosphorylase a , which contained about 4 nmol phosphate per mg protein, was dialyzed against 5 mM imidazoI/HCl, pH 7.5, containing 1.0 mM dithiothreitol. Enzyme assays and other methods. Phosphoprotein phosphatase activity was assayed according to a previous method (9). The final reaction mixture contained 50 mM trislHC1, pH 7.5, I mM dithiothreitol, 0.1 mg per rnl bovine serum albumin, 2 . 5 mM MnCI2 and the respective phosphoproteins: 5 p M phosphopyruvate kinase, 7 p M (32P)phosphorylase a , 20 p M (3’P)phosphohistones or 60 p M (32P)phosphoprotamine. The reaction mixture for the dephosphorylation of pyruvate kinase contained, in addi- tion, 5 % glycerol and 0.05 mM fructose-1,6-diphosphate to stabilize the enzyme. The volumes of the reaction mixtures were 40 p1 for the dephosphorylation of (3’P)- phosphopyruvate kinase and (3’P)phosphorylase a and 100 p1 for the other substrates. The reaction was started by addition of the protein phosphatase and allowed to continue at 30°C for 5-10 min. The release of (32P)- orthophosphate was linear for at least 15 min at the en- zyme concentrations used. One unit of protein phospha- tase is defined as the amount of enzyme which catalyses the release of I nmol of orthophosphate per min under these conditions. The substrate is expressed as the con- centration of the (3’P)phosphate moiety in the respective phosphoproteins. The specific radioactivity of the phos- phoproteins ranged from 20-100 cpm.pmol-’. Cyclic AMP-dependent protein kinase activity was determined in “cell-sap” prepared as described earlier. One unit of pro- tein kinase activity is defined as the amount of enzyme necessary to catalyze the incorporation of I pmol (”P))- phosphate into calf-thymus histones under the specified conditions (8). Protein in crude fractions was assayed according to the method of Lowry et al. (14) and in purified fractions from the absorbance at 280 nm assum- ing E:, ;;= I . Separation of protein phosphatases on DEAE-cellulose. Freshly excised livers (60 g) from male Sprague Dawley rats weighing 30g-350 g were homogenized in 3 vol of 250 mM sucrose, containing 15 rnM P-mercapteoethanol, 1 mM EDTA (pH 7.0, NaOH) and 0.1 mM phenyl- methylsulfonyl fluoride. A Potter-Elvehjem glass- homogenizer fitted with a teflon pestle was used and supernatant was applied to a DEAE-cellulose column (Whatman DE-52) (3.2X20 cm) equilibrated and eluted with buffer A containing 40 mM NaCI. The column was washed at a flow-rate of 2 0 0 ml/h for 5-6 h and the enzymes were eluted at a flow rate of 60 ml/h using a (500+500 ml) linear gradient of 40-350 mM NaCl in buffer A. Fractions of 10 ml were collected and assayed for pro- tein phosphatase activity (Fig. I ) Sephadex G-200 chromatography. T o each of t h e pooled fractions in Fig. 1 (about 50 mg protein) solid ammonium sulphate to 70% saturation was added with constant stirring. After 15 min each sample was centri- fuged at 16000Xg for 20 min and the precipitate dissolved in a minimal volume of buffer A and dialyzed against the same buffer containing 20% sucrose and 40 mM NaCI. The enzyme (about 2 ml) was then chromato- graphed on a Sephadex (3-200 column (2.4x57 cm) equili- brated and eluted with buffer A containing 0.1 mM NaCl at a flow rate of 10-12 ml/h; fractions of 2 . 6 2 . 7 ml were collected and assayed for phosphoprotein phosphatase activity. In order to estimate apparent molecular weights of the phosphatase fractions, the column was calibrated with catalase (240000), aldolase (158 OW), bovine serum albumin (67 000) and hen’s egg albumin (45 000) (results not illustrated). After the chromatography on Sephadex (3-200, the enzymes were concentrated by ammonium sulphate pre- cipitation (fractions A and B) or by ultrafiltration in a collodion bag (fraction C), and dialyzed at 1-2°C against buffer A containing 20% sucrose and 40 mM NaCI. The fractions were stored frozen in 0.1 ml portions and thawed only once prior to use. Little loss of activity occurred after 1 month. Ethanol precipitation at 20°C of phos- phatase fractions was performed according to the method of Brandt et al. (15), except that 2 4 mg protein per ml was added to 5 ml 95% ethanol. The precipitate was collected at 10000xg for 5 niin at 4”C, extracted with buffer A and dialysed against 1 I of the same buffer for 8-12 h prior to assay. RESULTS AND DISCUSSION Separation of rat-liver phosphoprotein phospharuses active on phosphorylated pyruvate kinase (type L ) Experiments were carried out in order to compare the elution profiles of phosphoprotein phosphatases active towards (32P)phosphopyruvate kinase with those active towards other phosphoproteins. Three fractions, termed A, B and C, were distinguished in order of elution from DEAE-cellulose (Fig. 1 ) . Fractions A , B and C were eluted between 0.1- 0.15, 0.17-0.25 and 0.27-0.3 M NaCl respectively. homogenization was performed in 30 sec with six strokes 4°C. The homogenate was centrifuged at 16000xg for 20 min and the supernatant obtained was further spun at 46000xg for 2 h. The postmicrosomal supernatant was filtered through glass wool to remove floating fat. The f l / l \ ~ I / n J M e d .s< I 83 The total recovery of (32P)phosphoprotamine phos- activity (Table I). Almost all the activity on ( 3 2 P ) ~ h o s ~ h o r y l a t e d pyruvate kinase was recovered in fractions B and at 940 rpm. This and other steps were Performed at 0- phatase was usually about 5@70% of the initial , Liver phosphopyruvute kinase phosphatases 13 1 n A B FRACTION NUMBER - E \ 2 2 0 > t ? + 9 1 5 w v) 3 I $ 10 a W z I- $ 05 a a m 0 - a % 20 LO 60 80 F R A C T I O N NUMBER Figs. I. Separation of phosphoprotein phosphatase on DEAE-cellulose. The two panels to the same experiment. Details are given in the text and in the legend to Table I . The horizontal bars indicate the fractions that were used for further experiments. C (Fig. 1). Fraction A showed only trace activity on this substrate. (32P)Phosphoprotamine, ("'P)- phosphohistones and (32P)phosphorylase a of rabbit skeletal muscle were the better substrates for phos- phatase fraction A. The relative ratio of (32P)phosphoprotamine/ (32P)phosphopyruvate kinase phosphatase activities was found t o be constant in different preparations of the postmicrosomal supernatant, but different for the separated fractions A , B and C (Fig. 1 , Table I). Fraction C showed the lowest ratio of phospho- protaminelphosphopyruvate kinase phosphatase activity, indicating increased preference for the latter substrate. In order to further compare the phosphatases, their physical properties were examined by chro- matography on a calibrated Sephadex (3-200 column. Phosphatase fractions A and B had a similar apparent molecular weight, approximately 250000 (based on the mean of two determinations for each fraction). Fraction C had an apparent molecular weight of 140000. Analysis of samples from the different regions of the Sephadex G-200 chromatograms revealed no further resolution of phosphoprotamine phosphatase from phosphopyru- vate kinase phosphatase activity (results not il- lustrated). The apparent molecular weights of the fractions are higher than those reported by Kobayashi et al. (see footnote in ref. 16, p. 353). The reason for this difference is not known. The conditions used for the preparation and chromatography of the ex- tracts in the present studies are different from those used by Kobayashi et al. I t has been suggested that multiple forms of liver phosphoprotein phos- phatases might be produced by the action of pro- teases, released from lysosomes during vigourous homogenisation of the tissue (17). To minimize the effects of proteolysis, homogenisation was per- formed under mild conditions and in the presence of phenylmethylsofonyl fluoride. Although com- plete inhibition of proteolysis could not be guaran- teed, its effects were considered to be small, be- cause fractions with apparent molecular weights lower than reported above could not be detected even after storage of the cytosol fraction for 6 h before further purification. This observation, taken together with the differences in kinetic prop- erties elaborated below, supports the possibility that fraction A , B and C probably serve different functions. Stability of the phosphatases Under the conditions chosen for chromatography and upon storage at 2°C phosphoprotein phospha- tase, fraction B seemed to be the most stable, 132 V . P . K . Titanji Table I. Purification of phospkoprotein phosphatases f r o m rat-liver cell-sap Phosphoprotamine and phosphopyruvate kinase phosphatase activities varied widely between 0.12-0.4 and 0.01- 0.03 pmol (3ZP)phosphate released per g wet weight of liver and min respectively (based on recoveries from cell-sap). The recovery of protein in the same fractions was about 100 mg per g of liver. In this particular experiment 14400 units of phosphoprotamine phosphatase and 1 200 units of phosphopyruvate kinase phosphatase were re- covered in the cell-sap from 60 g of liver. The calculation of the recovery of enzyme activity after the Sephadex column is based on the values obtained after chromatography of a portion of the activity from the DEAE-cellulose step. The slight activation of phosphatase B was not always obtained. The activity ratio is defined as the rate of dephosphorylation of (3zP)phosphoprotamine (60 pM) divided by the rate of the dephosphorylation of (32P)phospho- pyruvate kinase (5 pM) Phosphopyruvate kinase phosphatase Phospho- Phosphatase Activity ratioa protarnine yield u/mg P - p r o t a m in e Step (ulmg protein) (%) protein % yield 32P-pyruvate kinase Cell-sap 3.0 100 0.3 100 11.6k0.6 DEAE-cellulose A 11.9 17.0 b 0.8 b B 25.2 44.6 0.9 19.7 16.1k2.6 C 7.4 7.1 1.7 19.2 4 . 3 5 1 . 5 A 43.5 17.8 b b Sephadex G-200 b B C 114.0 59.6 3.5 22.7 31.2 13.0 2.8 2.2 7.5 5.9 Activity ratio: mean+S.D. of four preparations are given for cell-sap and DEAE-cellulose fractions; mean values In most cases the rates of the dephosphorylation of (32P)phosphopyruvate kinase were too low for reliable estimates of two preparations are given for the Sephadex G-200 fractions. to be made. followed by fraction A. Phosphatase fraction C was the least stable; enzymatic activity decreased dur- ing freezing and thawing in the absence of sucrose. Under similar conditions, phosphatases A and B maintained full enzymatic activity. As can be seen in Table I , only about 3 0 % of the phosphatase C activity applied to the Sephadex (3-200 column was recovered, whereas the other two fractions were almost quantitatively recovered. Earlier, a phosphoprotein phosphatase (M. W. 32 000) active towards phosphorylated pyruvate kinase was purified through an unusual ethanol precipitation step at 20°C as described by Brandt et al. (9, 15). The stability of the phosphatase frac- tions A , B and C , was also investigated using the ethanol treatment. We found that the enzymatic activity of fractions A and B towards (32P)phospho- rylase a were increased by 50-100% after ethanol treatment (15). However, the activity of the same fraction A towards (32P)phosphoprotamine and (32P)phosphopyruvate kinase decreased to about 3 0 % of the initial values. With phosphatase frac- tion B, the decrease was to about 70% of the initial activity. Regardless of the substrate used, ethanol treatment either completely inactivated phos- phatase C o r reduced the activity to less than 30- Upsulu J M e d Sci 83 50% of the initial values. Whereas the precipitation of crude extracts with ethanol at 20°C facilitates the further purification of a phosphoprotein phos- phatase, the same treatment reduces the activity of the DEAE-cellulose fractions towards all the other substrates studied except phosphorylase a. These observations might serve to explain the losses in enzymatic activity observed during our earlier attempts (9) t o purify a phosphoprotamine plus phosphopyruvate kinase phosphatase through the unusual ethanol precipitation step of Brandt et al. (15). The available data do not permit us to identify the fraction yielding the low molecular weight phosphoprotein phosphatase which was purified earlier in this laboratory (9). In order to resolve this question, further purification of the enzymes would be required. The different stability properties sup- port the existence of different phosphoprotein phosphatases, perhaps with overlapping specifici- ties. Kinetic results In view of the differences in molecular size and stability properties described above, some kinetic experiments were made to further compare the frac- Liver phosphopyruvate kinase phosphatases 133 Table 11. Effectors of phosphoprotein phosphata- ses B and C The standard reaction medium containing 5 p M (32P)- phosphopyruvate kinase was used. Divalent cations were not included in the tests, except where specifically in- dicated. Phosphatase B (6.78 pg per test) and phosphatase C (4.22 p g per test) were from the DEAE-cellulose step. The meanfS.D. of data from four determinations are shown. Other conditions were described in the Experi- mental section pmol releasedb min Phos- Phos- Effector phatase B phatase C 1. No additions 2. 2.5 mM EDTA 3. 2.5 mM MgCIZ 4. 2.5 mM MnC1, 5 . 5 mM potassium 6. 25 mM NaF 7. 1 mM ATP 8. 1 mM ATP+2.5 mM phosphate pH 7.5 MgCl, 5.6f0.2 2.2k0.1 48.1 f 1.1 28.15-0.08 3.02 f 0.4 2.25f0.3 2.42f1.5 46.5?2.5 29.1k 1.3 20.9k 1.9 55.5fO 46.0f2.8 13.2k0.9 10.0f0.6 8.7f0.3 46.8+0 tions B and C which showed the highest recovery of enzymatic activity towards pyruvate kinase. Mn2+ and Mg2+ stabilized or stimulated the activity of both fractions. The extent of stimulation ob- served was greater with phosphatase B than with phosphatase C. Orthophosphate, ATP and N a F were inhibitory (Table 11). Partial inhibition of en- zymatic activity by EDTA indicates that divalent cations are not absolutely required. The amount of phosphoprotein phosphatase ac- tivity towards phosphopyruvate kinase found in the post-microsomal supernatant (Table 1) was esti- mated to be about 30 units per g wet weight of liver. This would be sufficient to completely de- phosphorylate in about 10 secs all the pyruvate kinase present in normally fed rats (about 1 pM subunit). In other experiments, the total recovery of cyclic AMP-dependent protein kinase in a post- microsomal fraction was found to be 30000 units per g wet weight of liver. Since histones were phos- phorylated at about 50% of the rate of pyruvate kinase (Ref. 8, Fig. 2 ) , it was estimated that com- plete phosphorylation of pyruvate kinase in the livers of normally fed rats would take about 1 sec. These findings, together with the identification of fractions B and C as the main phosphopyruvate kinase phosphatase in vitro give further support for the physiological relevance of the reversible dephosphorylation. ACKNOWLEDGEMENTS This investigation was supported by grants from the Swedish Medical Research Council 13X-4485 and the Medical Faculty, the University of Uppsala. Ultracentri- fugation analyses were kindly performed by Dr H. Per- toft at this Institute. REFERENCES 1. 2. 3. 4. 5 . 6. 7. 8. 9. 10. 11. 12. 13. Engstrom, L.: Regulation of liver pyruvate kinase by phosphorylation-dephosphorylation reactions. In Regulatory Mechanisms of Carbohydrate Metabo- lism (ed. V . Esmann), p. 53. Pergamon Press, New York, 1978. Ljungstrom, O., Hjelmqvist, G . & Engstrom, L.: Phosphorylation of purified rat liver pyruvate kinase by cyclic-AMP stimulated protein kinase. Biochim Biophys Acta358: 289, 1974. Engstrom, L., Berglund, L., Bergstrom, G., Hjelm- qvist, G . & Ljungstrom, 0.: Regulatory phosphoryla- tion of purified pig liver pyruvate kinase. In Lipmann Symposium: Energy, Biosynthesis and Regulation in Molecular Biology, p. 192. Walter de Gruyter, Berlin, 1974. 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C.: Activa- tion of phosphorylase phosphatase by a novel proce- dure. Biochern Biophys Res Comrn 6 1 : 598, 1974. 16. Kobayashi, M., Kato, K. & Sato, S . : Multiple molec- ular forms of phosphoprotein phosphatase. Biochim Biophys Acta377: 343, 1975. 17. Lee, E. Y . C., Brandt, H., Capulong, 2. L. & Killi- lea, S . D.: Properties and regulation of liver phospho- rylase phosphatase. Enzyme Regull4: 467, 1976. Received M a y 30, 1978 Address for reprints: Vincent P. K. Titanji Department of Medical and Physiological Chemistry Biomedical Center Box 575 University of Uppsala S-75123, Uppsala Sweden Upsula J Med Sci 83