Purification and characterization of the polycation-stimulated protein phosphatase catalytic subunit from porcine renal cortex

Biochimica et Biophysica Acta 872 (1986) 1-10 Elsevier

1

BBA 32470

P u r i f i c a t i o n and c h a r a c t e r i z a t i o n of t h e p o l y c a t i o n - s t i m u l a t e d p r o t e i n p h o s p h a t a s e catalytic subunit f r o m p o r c i n e renal c o r t e x K e i t h K. S c h l e n d e r *, S u s a n E. W i l s o n a n d R o n a l d L. M e l l g r e n Department of Pharmacology, Medical College of Ohio, C.S. 10008, Toledo, OH 43699 (U.S.A.) (Received March 4th, 1986)

Key words: Protein phosphatase; Polycation stimulation; Histone H1; Catalytic subunit purification; (Porcine kidney)

The wedominant form of phosphorylase phosphatase activity in porcine renal cortical extracts was a polycation-stimulated protein phosphatase. This activity was present in extracts in a high-molecular-weight form which could be converted to a free catalytic subunit by treatment with ethanol, urea, or freezing and thawing in the presence of jO-mercaptoethanol. The catalytic subunit of the polycation-stimulated phosphatase was purified by chromatography on DEAE-Sephacel, heparin-Sepharose, and Sephadex G-75. The phosphatase appeared to be homogeneous on SDS-polyacrylamide gel electrophoresis. The enzyme had an apparent M r of 3 5 0 0 0 on gel filtration and SDS-polyacrylamide gel electrophoresis. The purified phosphntase could be stimulated by histone H1, protamine, poly(D-lysine), poly(L-lysine) or polybrene utilizing phosphorylase a as the substrate. It preferentially dephosphorylated the a.subunit of phosphorylase kinase. The phosphatase was highly sensitive to inhibition by ATP. These results suggest that the renal polycationstimulated phosphatase catalytic subunit is very similar to or identical with the skeletal muscle phosphatase form which has been previously designated phusphatase-2Ac.

Introduction Protein phosphorylation-dephosphorylation is an important mechanism for the regulation of many cellular processes. Studies on the dephosphorylation of protein-bound phosphoserine and phosphothreonine have been complicated by the presence of multiple forms of protein phosphatases [1]. Ingebritsen and Cohen [2] have classifted mammalian protein phosphatases as type 1 and type 2. The type 1 enzyme, designated phosphatase-1, is inhibited by heat-stable inhibitor-1 or inhibitor-2 and it dephosphorylates the p-subunit of phosphorylase kinase. The type 2 phosphatases, designated 2A, 2B and 2C, are insensi-

* To whom correspondence should be addressed.

tive to inhibitor-1 or inhibitor-2 and preferentially dephosphorylate the a-subunit of phosphorylase kinase. Protein phosphatase-2B is calcium-dependent and protein phosphatase-2C is magnesiumdependent. Protein phosphatase-2A does not have a divalent metal ion requirement for activity. While studying the heat-stable phosphatase inhibitor proteins from kidney, we found a heat-stable protein that stimulated the phosphorylase phosphatase activity of a protein phosphatase preparation from rabl~t kidney cortex [3]. The activator protein was subsequently identified as histone H1 [4]. Histone HI-stimulated phosphatase was the major phosphorylase phosphatase isolated from the rabbit renal cortex [5]. Histone HIstimulated phosphatase activity was also found in extracts of a number of rabbit tissues (skeletal muscle, heart muscle, liver, and brain [6]) as well

0167-4838/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

as bovine aortic smooth muscle [7]. Core histones did not stimulate the phosphorylase phosphatase activity [4,6,8], but a number of other polycations including protamine [8], polylysine [7] and polybrene [6,9] were effective activators. The protein phosphatase which is stimulated by histone H1 and other polycations has been designated polycation-stimulated protein phosphatase [6,10]. Using partially purified phosphatase preparations, we found that polycation-stimulated protein phosphatase was not inhibited by inhibitor-1 or inhibitor-2 [5,6,8] and that the enzyme preferentially dephosphorylated the a-subunit of phosphorylase kinase [11]. Based on these properties, we suggested that polycation-stimulated protein phosphatase was a type 2 protein phosphatase [8,11]. In this report, we describe the purification and characterization of the catalytic subunit of the polycation-stimulated protein phosphatase from porcine renal cortex. The polycation-stimulated protein phosphatase, which represents the major phosphorylase phosphatase in renal cortical tissue, was identified as the type 2 enzyme designated phosphatase-2A by Ingebritsen and Cohen [2].

Protein phosphatase assays Phosphorylase phosphatase was assayed by measuring the release of [32p]p i from [32p]phosphorylase a as previously described [18]. For the standard polycation-stimulated protein phosphatase assay the concentration of [32p]phosphorylase was 1 #M (dimer), and histone H1 was included at 8 /~g/ml. If phosphatase-1 activity was present, it was inhibited with an excess of inhibitor-1 [11]. When polycation activation was investigated, phosphatase was included at a concentration which gave basal phosphatase activity of 5-10 munits/ml. One unit of phosphorylase phosphatase activity was defined as the amount of enzyme which dephosphorylates 1 nmol phosphorylase a dimer per rain. The phosphatase reaction was initiated by the addition of enzyme. Phosphorylase kinase phosphatase activity was determined as previously described [11]. The release of [32p]pi from the a- and fl-subunits of phosphorylase kinase was determined after separation of the subunits by SDS-polyacrylamide gel electrophoresis [20].

Other procedures Materials and Methods

Enzymes and proteins Phosphorylase b [12], phosphorylase kinase [13], histone H1 [14], core histones [4], heat-stable inhibitor-1 [15], and heat-stable inhibitor-2 [16], were prepared by published procedures. The catalytic subunit of bovine heart cAMP-dependent protein kinase [17] was a generous gift of Dr. Erwin Reimann. [32p]Phospharylase a was prepared as previously described [18]. Phosphorylase kinase was phosphorylated with the catalytic subunit of cAMP-dependent protein kinase [11]. The distribution of the 32p incorporated into the subunits of phosphorylase kinase was 34% in the a-subunit and 66% in the fl-subunit.

Chemicals Polybrene ( M r 40 000) was obtained from Serva Fine Biochemicals. Poly(L-lysine) ( M r 14000), poly(D-lysine) ( M r 12 000) and poly(DL-lysine) ( M r 5000) were purchased from Sigma Chemical Company. [y-32p]ATP was prepared according to the method of Walseth and Johnson [19].

Proteins were determined by the method of Bradford using bovine y-globulin as the standard [211. SDS-polyacrylamide gel electrophoresis was performed according to the procedure of Laemmli [22]. Proteins were located by silver staining [23]. Sucrose density-gradient centrifugation was done as described by Brandt et al. [24].

Purification of polycation-stimulated protein phosphatase catalytic subunit Swine kidneys obtained from a local slaughter house were placed on ice, transported to the laboratory, and dissected to remove the cortex. All further operations were done at 0 ° - 4 ° C unless otherwise stated. For a typical preparation, 500 g of fresh cortex were ground in a meat grinder and homogenized in a Waring blender with 1250 ml 25 mM imidazole-HC1/1 mM EDTA/1 mM dithiothreitol (pH 7.0 at I°C) (buffer A) containing 250 mM sucrose and a proteinase inhibitor cocktail (5 mM benzamidine/5 mM EGTA/0.5 mM phenylmethylsulfonyl fluoride/0.1 mM Na-ptosyl-L-lysine chloromethyl ketone/1 #g pepstatin A per ml/5 /~g leupeptin per ml). The homo-

genate was then centrifuged at 10000 × g for 45 rain and the resulting supernatant was filtered through glass wool. The pH of the extract was adjusted to 7.2 by the addition of 6 M NH4OH and then brought to 70% saturation by the addition of 0.45 g ammonium sulfate/ml. The mixture was stirred until all of the ammonium sulfate was in solution. After standing without stirring for 1 h, the precipitate was collected by centrifuging at 10000 × g for 20 rain. The pellet was suspended in 150 ml buffer A containing the proteinase inhibitor cocktail and dissolved with the aid of a glass-Teflon tissue homogenizer. The catalytic subunit was generated by the rapid addition, with mixing, of 5 vol. room-temperature ethanol. The mixture was immediately centrifuged at 5000 × g for 5 rain and the pellet was extracted in a Waring blender with 125 ml buffer A containing the proteinase inhibitor cocktail. The suspension was centrifuged at 10000 × g for 15 rain and the supematant was dialyzed for 1-2 h against 2 1 buffer A and then overnight against 2 1 buffer A containing 10% glycerol. The insoluble material was removed by centrifugation at 20000 × g for 15 min. The solution was applied to a DEAE-

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Sephacel column (2.6 x 10 cm) equilibrated with buffer A containing 50 mM N a C I / 1 0 ~ glycerol. The column was washed with approx. 25 ml equilibration buffer and then with buffer A containing 100 mM NaC1/10% glycerol until the absorbance at 280 nm returned to near baseline. Protein phosphatase activity was then eluted with buffer A containing 300 mM NaCI/10% glycerol. The active fractions were pooled and precipitated by the addition of 0.45 g ammonium sulfate/ml. After standing for 1 h, the precipitate was collected by centrifuging at 10000 x g for 20 min. The pellet was dissolved with 1 ml 10 mM imidazole-HC1/0.4 mM E D T A / 1 mM dithiothreitol/10% glycerol (pH 7.0 at I°C) (buffer B). The solution was extensively dialyzed against buffer B until the conductivity of the dialysis buffer remained constant. The dialyzed sample was applied to a heparin-Sepharose column (2.5 × 7.5 cm) equilibrated with buffer B. It was important that the sample was well dialyzed and applied to the column at a slow flow-rate (less than 0.6 ml/min). The column was washed with buffer B until the absorbance at 280 nm returned to near baseline. Polycation-sti/nulated protein phosphatase activ-

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Fraction Number Fig. 1. Heparin-Sepharose chromatography. The extensively dialyzed sample from the DEAE-Sephacel step (Table I) was applied to a heparin-Sepharose column (1.6 x9.3 cm) as described in Materials and Methods. Polycation-c~timulated phosphatase activity was eluted by increasing the ionic strength with NaCI to 0.05 p. Inhibitor-sensitive phosphatase activity could be eluted by increasing the ionic strength with NaCI to 0.5/~. Fractions (5 ml) were assayed for phosphorylase phosphatase activity with no additions (O), with 7.2 pg histone H1/ml present (O), or with inhibitor-1 present (z0.

ity was then elutea with buffer B containing 40 m M NaCI (Fig. 1). The active fractions were pooled and concentrated to approx. 2 ml with an immersible Millipore Cx ultrafilter. The concentrated sample was applied to a Sephadex G-75 superfine column (1.6 × 75 cm) equilibrated with 25 m M imidazole-HCl/1 m M E D T A (pH 7.0 at I ° C ) containing 100 m M N a C 1 / 1 m M dithiothreitol/10% glycerol. The column was eluted at a flow rate of approx. 8 m l / h . 2-ml fractions were collected and assayed for polycation-stimulated phosphatase activity. The active fractions were combined and dialyzed against 25 m M imidazoleH C I / 1 m M E D T A / ( p H 7.0 at I ° C ) containing 1 m M d i t h i o t h r e i t o l / 5 0 % glycerol. The concentrated enzyme was stored at - 2 0 ° C . Under these conditions, the enzyme could be stored for several months with little loss in activity.

verted to free catalytic subunits with an apparent Mr of 30000-35000 by treating with 80~ roomtemperature ethanol, 4 M urea, or freeze-thawing in the presence of 0.2 M fl-mercaptoethanol [1]. When renal cortical extracts were freeze-thawed in the presence of fl-mercaptoethanol and then chromatographed on Superose 12, essentially all of the phosphorylase phosphatase activity appeared to 3.2"

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Protein phosphatase activity of extracts from porcine renal cortex Renal cortical extracts were prepared in the presence of proteinase inhibitors as described in Materials and Methods and assayed for phosphorylase phosphatase activity under three assay conditions. Spontaneously active phosphatase activity (assayed without modulators added) was 27.3 + 3.1 u n i t s / g tissue (mean + S.E., n - 6). Inhibitor-l-sensitive activity (activity with no additions minus activity in the presence of inhibitor-l) was only 3.2 + 1.0 u n i t s / g tissue (n = 6). Inhibitor-sensitive activity was not increased by preincubation of the extracts with inhibitor-1. When activity was determined in the presence of 8 #g histone H 1 / m l and inhibitor-l, the phosphatase activity was 120.3 + 19.3 u n i t s / g tissue (n = 4). Thus, nearly all of the phosphorylase phosphatase activity of renal cortical extracts was insensitive to inhibitor-1 and the activity was stimulated about 5-fold by histone H1. Gel filtration of renal cortical extracts on Superose 12 revealed a major histone HI-stimulated phosphatase peak eluting with an apparent Mf of approx. 150000 and a Stokes radius of 4.7 nm (Fig. 2A). There was a small peak of activity eluting in the M r 30000-35000 range. High-M r forms of several protein phosphatases can be con-

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Fig. 2. Superose 12 gel filtration chromatography.(A) Cortical extract prepared as described in Materials and Methods was diluted 15-fold with 25 mM imidazole-HC1/100 mM NaCI/1 mM dithiothreitol (pH 7.4) and 200 pi were applied to a Superose 12 column (1 ×30 cm) equilibrated with diluting buffer. The column was run at room temperature with a flow rate of 0.5 ml/min. Fractions (0.25 ml) were assayed for phosphorylase phosphatase activity in the absence (O) and in the presence of 8/~g histone H1/ml (e). The arrows indicate the elution positions of blue dextran (V0), thyroglobulin (T), catalase (C), aldolase (A), ovalbumin (O) and myogiobin (M). (B) A sample of the extract was adjusted to pH 7.3 by addition of 1 M Tris-bas¢ and freeze-thawed in the presence of 0.2 M #-mercaptoethanol. The treated sample was centrifuged at 15000x g for 10 min to remove insoluble protein and diluted 24-fold in diluting buffer. It was then chromatographed and assayed as described above.

TABLE I PURIFICATION OF POLYCATION-STIMULATED PROTEIN PHOSPHATASE CATALYTIC SUBUNIT The enzyme was purified from 500 g of porcine renal cortex. Details of the purification procedure are given under Materials and Methods. Phosphatase activity was determined in the presence of 8 ~tg histone H1/ml. Step

Protein (rag)

Activity (units)

Extract

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be converted to a low-Mr catalytic subunit with a Stokes radius of 2.5 nm (Fig. 2B). The activity of the low-Mr form was stimulated by addition of histone H1. Similar results were obtained when extracts were treated with urea (data not shown). 1

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Recovery (~)

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149

Purification of the catalytic subunit Polycation-stimulated protein phosphatase catalytic subunit was purified as described in Materials and Methods. The catalytic subunit was generated by treatment of a resuspended 70~ ammonium sulfate precipitate of the extract with 80~ room-temperature ethanol [25]. Polycationstimulated catalytic subunit was then purified by chromatography on DEAE-Sephacel, heparin-

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Fig. 3. SDS-polyacrylamide gel electrophoresis of polycationstimulated phosphatase catalytic subunit. Electrophoresis was carried out on a 105 gel"and the gel was silver-stained as described in the text. Enzyme (0.3/tg) was applied to lane 1. Marker proteins applied to lane 2 were ovalbumin (44 kDa), giyceraldehyde-3-phosphate dehydrogenase (36 kDa), carbonic anhydrase (30 kDa) and lysozyme (14 kDa).

Fig. 4. Effect of histon~ on phosphorylase phosphatase activity. The activity of polycation-stimulated catalytic subunit was determined in the presence of different concentrations of histone H1 (O), core histones (v), or histone H1 in the presence of 15 mM NaCI (e). Activity in the absence of added histone was inhibited 25~$by 15 mM NaC1.

Sepharose, and Sephadex G-75. The contaminating phosphatase-1 catalytic subunit was removed by a modification of the heparin-Sepharose chromatographic procedure first used by Gergely et al. [26] to separate catalytic subunits of phosphatase-1 and phosphatase-2A. By reducing the concentration of the imidazole buffer to 10 raM, the polycation-stimulated catalytic subunit was adsorbed to the heparin-Sepharose column and the activity was eluted by the addition of 40 mM NaC1 (Fig. 1). All of the inhibitor-sensitive activity (phosphatase-1) was also adsorbed but a higher ionic strength buffer was required to elute it (Fig. 1). Purification data for a typical preparation are shown in Table I. The overall purification of the phosphatase was about 2000-fold. The enzyme was found to be essentially homogeneous on SDS-polyacrylamide gel electrophoresis and migrated with an apparent M r of 35 000 (Fig. 3). When the purified preparation was subjected to nondenaturing polyacrylamide gel electrophoresis, all of the phosphatase activity which could be eluted from the gel comigrated with a protein band which upon reelectrophoresis in the presence of SDS migrated with an apparent M r of 35 000 (data not shown).

Physical properties The polycation-stimulated catalytic subunit eluted from a calibrated Sephadex G-75 superfine column with an apparent M r of 35 000. Using the same column, the Stokes radius was determined to be 2.5 nm. On sucrose density-gradient centrifugation, the enzyme had a sedimentation coefficient of 3.6. The M, calculated by the method of Siegel and Monty [27] was 39000 and the frictional ratio was 1.1.

Polycation stimulation The phosphorylase phosphatase activity of the purified catalytic subunit was stimulated about 8-fold by histone H1 (Fig. 4). The concentration of histone H1 required for half-maximal activation was 0.1/~M. Core histories were without effect on phosphatase activity. The extent of histone H1 activation was affected by low concentrations of added salt. Histone H1 stimulated phosphatase activity 20-fold when 15 mM NaC1 was included in the assay (Fig. 4). Similar results were obtained when the assays were done in the presence of 15 mM KC1 (data not shown). Other polycations were tested as possible activators of phosphatase activity. Protamine stimulated activity at low concentrations (2-fold activation at 3.2 /~g/ml) but inhibited at higher

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Protarnine ( p g / m l ) Fig. 5. Effect of protamin¢ on phosphorylase phosphatase activity. The activity of purified polycation-stimulated catalytic subunit was determined in the presence of different concentrations of protamine.

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Fig. 7. Effect of polybrene on phosphorylase phosphatase activity. The activity of polycation-stimulated catalytic subunit was determined in the presence of different concentrations of polybrene. The structure of the repeating unit of polybrene is shown in the inset.

100

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t concentrations (Fig. 5). Poly(D-lysine) and poly(L-lysine) also stimulated the enzyme (Fig. 6). However, poly(DL-lysine) was without effect at low concentrations and inhibited at concentrations above 1/~g/ml. All of the above polycations which stimulated phosphatase activity were amino acid derivatives. The effect of polybrene, a synthetic poly quaternary ammonium cation, on phosphorylase phosphatase activity was determined. Although the activation curve was reproducibly multiphasic, polybrene was an effective activator of phosphorylase phosphatase activity (Fig. 7).

Dephosphorylation of phosphorylase kinase The specificity toward the a- and fl-subunits of phosphorylase kinase in the absence and presence of histone H1 was determined. In the absence of histone H1, the purified catalytic subunit dephosphorylated the a-subunit at a rate 3-4-fold greater than that for the B-subunit (Fig. 8A). Histone H1 increased the rate of dephosphorylation of both the a- and the fl-subunit (Fig. 8B). However, there was a preferential activation of a-subunit dephosphorylation. ,In the presence of histone H1, the

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Fig. 8. Dephosphorylation of phosphorylase kinase. The dephosphorylation of skeletal muscle phosphorylas¢ kinase by polycation-stimulated catalytic subunit was determined as noted in Materials and Method. [32P]Pi remaining in the a-subunit (O) and ~8-subunit (ll) was determined at various times. The reaction was run in the absence (A) or presence of 25 /~g histone H 1 / m l (B). Initial rates were determined by linear regression analysis.

rate of dephosphorylation of the a-subunit was about 8-fold greater than that of the fl-subunit.

A TP inhibition Ingebritsen and co-workers [2,28] have noted that the catalytic subunit of phosphatase-1 and phosphatase-2A can be distinguished by preincubation with ATP. They found that phosphatase1 was relatively insensitive (50% inactivated by 0.35 mM ATP), whereas phosphatase 2A was relatively sensitive (90% inactivated by 0.1 mM ATP). The purified polycation-stimulated catalytic subunit was tested for activity after preincubation

ATP (mM) 0.01

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Fig. 9. Effect of ATP preincubation on phosphorylase phosphatase activity. Polycation-stimulated .catalytic subunit was preincubated for 10 rain at 300C in 50 mM imidazole-HCl, 2.5 mM EGTA, 3.3 mM thoophyllin¢, 2 naM dithiothreitol, 0.1 nag bovine serum albumin/ml (pH 7.4) and the indicated concentration of ATE Phosphorylase phosphatase activity was then determined in the absence of histone H1.

with ATP (Fig. 9). Phosphorylase phosphatase activity was inactivated 80% by preincubation with 0.1 mM ATP. In a parallel experiment phosphatase-1, isolated from rat liver, was not inactivated by preincubation with 0.1 mM ATP (data not shown). Discussion

There are two notable properties of the phosphorylase phosphatase activity of fresh porcine renal extracts. The first is the small fraction of the activity which was sensitive to heat-stable inhibitor-1. Only about 12% of the spontaneously active phosphatase was inhibited by the addition of excess inhibitor-1. Treatment with either 4 M urea, or 80% ethanol, or free,e-thawing in the presence of fl-mercaptoethanol to generate catalytic subunits did not significantly change the fraction of phosphatase activity which was inhibited by addition of inhibitor-1 [6]. In some tissues, a significant amount of the inhibitor-sensitive phosphatase is present as an inactive form known as the ATP-Mg-dependent protein phosphatase [10]. The latter form, a complex of the catalytic subunit of phosphatase-1 and inhibitor-2, is activated by incubation with ATP-Mg, and a protein kinase designated FA or GSK-3 [29,30]. We were unable to detect significant amounts of ATP-Mg-dependent phosphatase activity in fresh porcine cortical ex-

tracts [6]. We conclude that there are relatively small amounts of protein phosphatase-1 in the cytosolic fraction of porcine renal cortex. The second notable property of the phosphorylase phosphatase activity of porcine renal cortical extracts was the marked stimulation by certain polycations. Histone H1 stimulated activity about 4-6-fold. Other polycations (poly(L-lysine), poly(D-lysine), protamine and polybrene) also activated [6]. The phosphorylase phosphatase activity determined in the presence of polycation was about 40-times greater than phosphatase-1 activity. Thus, when determined in the presence of activator, the polycation-stimulated activity is by far the major phosphorylase phosphatase activity of renal cortical extracts. On the basis of our previous studies using partially purified enzymes, we had suggested that the polycation-stimulated phosphatase from rabbit skeletal muscle [8,11] or renal cortex [5] was a type 2 phosphatase. To determine whether the type 2 renal phosphatase which was stimulated by polycations is a unique enzyme or whether it represents a newly described property of a known phosphatase, we have purified the catalytic subunit of polycation-stimulated phosphatase to near homogeneity. The purified enzyme, which was free of detectable phosphatase-1 catalytic subunit, has physical properties very similar to those reported for the catalytic subunit of skeletal muscle phosphatase-2A designated 2Ac by Cohen and coworkers [31]. The purified renal catalytic subunit was stimulated by the same polycations (Figs. 4-7) that stimulated the activity of renal cortical extracts [6]. With one enzyme preparation, a phosphatase fraction was eluted off the heparin-Sepharose column at a slightly higher ionic strength than that required to elute the polycation-stimulated catalytic subunit. The former fraction was only slightly stimulated by histone H1. Treatment with trypsin converted it to a histone HI-dependent form. The relationship between these two forms of catalytic sub,unit is currently under investigation. The purified renal enzyme preferentially dephosphorylated the a-subunit of phosphorylase kinase (Fig. 8) and its activity was very sensitive to preincubation with ATP (Fig. 9). Thus, it is coneluded that the renal polycation-stimulated protein phosphatase is identical or similar to the

enzyme classified as phosphatase-2A by Ingebritsen and Cohen [2]. Recently Cohen and coworkers, using partially purified enzymes from liver [32] and purified enzymes from skeletal muscle [31], have come to the same conclusion. The mechanism of polycation activation remains to be elucidated. We have reported that the kinetics for phosphorylase a determined in the presence of histone H1 were complex, but at low concentrations of phosphorylase a (i.e., below 1.2 #M) the data followed Michaelis-Menton kinetics [6]. The K m for phosph0rylase a was lowered from 25 laM in the absence to 0.09 #M in the presence of histone H1. The decrease in the K m in the presence of a polycation could be due to a complex between polycation and the substrate. Krebs [33] reported that protamine could complex with phosphorylase a and inhibit its activity. At concentrations of 15 #g histone H 1 / m l or less, we did not see any inhibition of phosphorylase a activity or turbidity [11]. However, high concentrations of histone H1 and other polycations, including polybrene, complexed with phosphorylase a to form turbid solutions. This may explain the multiphasic saturation plot with polybrene (Fig. 7). Maximal activation was achieved in the presence of a large molar excess of phosphorylase a relative to polycation activator [5,31,34]. In addition, polycations stimulated the dephosphorylation of a number of phosphoprotein substrates [32]. Thus it seems unlikely that the polycations increase protein phosphatase activity by interacting with the phosphoprotein substrates. It seems more likely that the polycations activate by a phosphatase-directed mechanism. Further studies will be required to determine the physiological significance of polycation modulation of phosphatase-2A activity. While it seems unlikely that the active polycations tested thus far would regulate a cytosolic protein phosphatase in vivo, studies with polycations have uncovered a possible mechanism for the regulation of protein phosphatase activity. As noted above, histone H1 increases phosphorylase phosphatase activity by decreasing the K m for phosphorylase a. A modulator which decreases the K m for some substrates but has no effect or increases the K m for other substrates could selectively regulate protein phosphatase activity.

Acknowledgments This work was supported by USPHS grant AM 14723. R.L.M. is an Established Investigator of the American Heart Association. We thank Scott Jakes for the sample of liver protein phosphatase-1 and Martha Heck and Debra LeBarr for the typescript.

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