Mellgren, Ronald L.

Schlender, Keith K.

Wilson, Susan E.

Elsevier - Publisher Connector 

Isolation of a heat-stable protein activator of phosphorylase phosphatase 

Volume 146, number 2 FEBS LETTERS September 1982 
Isolation of a heat-stable protein activator of phosphorylase phosphatase 
Susan E, Wilson, Ronald L. Mellgren and Keith K. Schlender 
Department ofPharmacology and Therapeutics, Medical College of Ohio, CS 10008, Toledo, OH 43699, USA 
Received 6August 1982 
Phosphorylase Protein phosphatase Heat-stable protein 
(Kidney) 
Protein activator Protein inhibitor 
1. INTRODUCTION 
Phosphorylase phosphatase activity of protein 
phosphatase-1 [1]is believed to be regulated by: 
(i) Specific interaction of ligands with phosphor- 
ylase a; i.e.i by substrate directed-effects; 
(ii) Competition between phosphorylase and vari- 
ous other phosphoprotein substrates; 
(iii) Interaction of the phosphatase with heat- 
stable inhibitors [2]. 
Two heat-stable inhibitors have been isolated from 
skeletal muscle, inhibitor-1 and inhibitor-2 [3-5]. 
Inhibitor-1 exists in an active phosphorylated form 
and an inactive dephosphorylated form [3,4]. The 
degree of phosphorylation f inhibitor-1 is under 
hormonal control [1 ]. 
While studying the heat-stable protein inhibitors 
of phosphorylase phosphatase from kidney, we 
have found an activator of phosphorylase phos- 
phatase. To our knowledge such an activator has 
not been described. We have purified the heat- 
stable protein activator from swine renal cortex 
300-fold and have studied some of its properties. 
The activator stimulates phosphorylase phospha- 
tase activity primarily by decreasing the Km of the 
enzyme for phosphorylase a. 
2. MATERIALS AND METHODS 
2.1. Preparation of enzymes 
An enzyme, termed protein phosphatase C, was 
prepared from rabbit renal cortex as in [6]. The 
phosphatase preparation was >90% inhibited by 
renal heat-stable inhibitors and thus appears to be 
the putative catalytic subunit or active fragment of 
protein phosphatase-1 [ ]. Rabbit muscle phos- 
phatase C was purified through the first DEAE- 
Sephadex step [7] and further purified by chro- 
matofocusing on a Pharmacia PBE 94 column. The 
phosphatase eluted as a single peak at pH 4.7. A 
high-Mr and presumably native form of bovine 
heart phosphorylase phosphatase was partially pu- 
rified from a 10000 x g extract by DEAE- 
Sepharose chromatography followed by Bio-Gel 
A-1.5m chromatography. [32p]Phosphorylase  was 
prepared as in [7]. TPCK-treated crystalline tryp- 
sin and soybean trypsin inhibitor were purchased 
from Worthington. Catalytic subunit of cyclic 
AMP-dependent protein kinase [8] was a gift from 
Dr E. Reimann. Swine kidneys were obtained 
from a local slaughterhouse and transported tothe 
laboratory on ice. The cortex was removed and 
stored at-70"C. 
2.2. Phosphorylase phosphatase activator assay 
Phosphorylase phosphatase was assayed by 
measuring the release of 32p from [32p]phosphory- 
lase a [7]. A unit of phosphorylase phosphatase 
converts 1 nmol phosphorylase a dimer to phos- 
phorylase b/min. Phosphorylase phosphatase ac- 
tivator was assayed in a 100 p,l incubation mixture 
containing 10 munits/ml phosphatase C and 
1.03 /tM [32p]phosphorylase a (all phosphorylase 
concentrations a sume a dimer). A unit of activa- 
tor doubles the phosphatase activity under stan- 
dard reaction conditions. All activator assays and 
dilutions were carried out in glass test tubes treated 
with Prosil-28 (an organosilane surface-treating 
agent). 
Published by Elsevier Biomedical Press 
00145793/82/0000-0000/$2.75 ® 1982 Federation f European Biochemical Societies 331 
Volume 146, number 2 FEBS LETTERS September 1982 
3. RESULTS 
3.1. Isolation and purificatio n of a heat-stable ac- 
tivator of phosphorylase phosphatase 
An extract of rabbit renal cortex [6] was heated 
to 95°C for 5 min, the denatured protein removed 
by centrifugation, and the supernatant assayed for 
heat-stable inhibitor. An aliquot of this prepara- 
tion could completely inhibit phosphorylase phos- 
phatase activity of phosphatase C (not shown). In 
an attempt to separate inhibitor-1 and inhibitor-2 
we fractionated rabbit renal cortical extracts by 
precipitation between 0-2% and 2-15% (w/v) tri- 
chloroacetic acid [9]. The 0-2% trichloroacetic 
acid fraction which was expected to contain inhibi- 
tor-2 [9], did have a large amount of inhibitory ac- 
tivity. However, the 2-15% trichloroacetic acid 
fraction which was expected to contain inhibitor-1 
actually stimulated phosphorylase phosphatase ac- 
tivity by >2-fold (not shown). Even after treat- 
ment with protein kinase to phosphorylate inhibi- 
tor-1 [4], the fraction still activated phosphorylase 
phosphatase activity. 
A preliminary study of the 2-15% trichloroace- 
tic acid fraction from swine renal cortex revealed a
heat-stable, non-dialyzable and trypsin-labile 
phosphorylase phosphatase activator. For purifica- 
tion of the activator, the 2-15% trichloroacetic 
acid precipitate was prepared from 390 g cortical 
tissue as in [9]. The precipitate was suspended in 
45 ml of 0.5 M potassium phosphate, 1.0 mM 
EDTA (pH 7.1). The pH of the suspension was ad- 
justed to 7.1 by the addition of 6 N NH4OH and 
Table 1 
Properties of heat-stable activator of phosphorylase 
phosphatase 
Trypsin-labile a + 
Stokes' radius (rs) 4.0 nm 
App. Mr (gel filtration) 90 000 
s20,w 1,5 
Frictional ratio 2.0 
M r (calc. [11 ]) 26 000 
aActivator was incubated with trypsin (activator/trypsin 
ratio, 40:1) in 50 mM imidazole, 5 mM EGTA, 2 mM 
DTT, pH 7.4 for 30 min at 30°C. The reaction was ter- 
minated with soybean trypsin inhibitor. The trypsin- 
trypsin inhibitor complex did not affect phosphatase 
activity 
extensively dialyzed against 10 mM potassium 
phosphate, 1 mM EDTA, pH 7.1 (PE buffer). The 
insoluble material was removed by centrifugation 
at 20 000 × g for 10 min and the supernatant 
(53 ml) was placed in a flask and heated in a boil- 
ing water bath. After the temperature reached 
90°C ( -5  min) heating was continued for 5 min. 
The suspension was chilled and centrifuged at 
8000 × g for 10 min to remove the denatured pro- 
tein. The heat-treated supernatant was mixed with 
3 ml phosphocellulose equilibrated with PE buffer. 
After gently stirring for 45 min, the gel was poured 
into a column (0.9 cm) and washed with PE buffer. 
Phosphatase inhibiting activity was present in the 
effluent. The column was washed with four bed 
volumes of PE buffer containing 0.5 M NaCI and 
then the activator was eluted with PE buffer con- 
taining 1.0 M NaCI. The fractions were dialyzed 
against PE buffer to remove salt before assay. 
The active fractions which contained 187000 
units of activator were pooled (2 ml) and could be 
¢1 
o~ 
09 
e- 
E 
0 
¢0 
.C  
r~ 
0 
0 
L 
I I I I r~ l  i I 
0.02  0 .04  0.1  
Phosphoce l lu lose  F ract ion  ( I J I )  
Fig.l. Effect of the activator on the activity of phos- 
phatase C. The assay was performed as described in the 
text. The protein concentration of the activator was 
2/tg//tl. Activity in the absence of activator (control) is 
shown as a solid horizontal line. 
332 
Volume 146, number 2 FEBS LETTERS September 1982 
>,  
~' ;>, 
a .E  
¢o 
o 
0 12 
J 
I I I I I I I 
3 6 9 
Phosphorylase a (jJM) 
Fig.2. Kinetics of the activation of phosphatase C by the 
activator. The assay conditions were the same as de- 
scribed in the text except hat the phosphatase was in- 
cluded at 5 munits/ml and the [32p]phosphorylase con- 
centration was varied. Assays were performed in the 
absence (0) and in the presence (,) of 0.045/~g activator 
preparation. Kinetics constants were determined by fit- 
ting the data to the Michaelis-Menten quation by non- 
linear regression analysis [18]. Vmax-values were: with- 
out activator, 2.28 munits/assay; with activator, 
2.71 munits/assay. Kin-values were: without activator, 
4.13/~M; with activator, 0.19/~M. 
stored for several months at 4 ° C without loss of ac- 
tivity. Making the assumption that the assay of the 
2-15% trichloroacetic acid fraction (the first step 
which could be assayed) measures all of the activa- 
tor present in the extract, the activator was purified 
320-fold by the procedure outlined above. On 
SDSgel electrophoresis [10], the preparation had 2 
major protein bands (Mr 30 000-34 000) and a 
minor protein band (M r ~28 000). Activator ac- 
tivity was destroyed by SDS treatment and could 
not be detected after elution of the gels. A small 
amount of inhibitory activity was stable during 
SDS electrophoresis and was eluted from the re- 
gion of the gel containing the minor protein band. 
Activator purified through the phosphocellulose 
step was used for the studies reported in this paper. 
3.2. Properties of the phosphorylase phosphatase ac- 
tivator 
Some of the physical properties of the activator 
are summarized in table 1. Incubation of activator 
with trypsin completely abolished stimulation of 
phosphatase activity indicating that the activator is 
probably a polypeptide. When the activator was 
chromatographed on Sephadex G-200 both the ac- 
tivator and the protein emerged as a single peak 
with rs 4.0 nm and app. Mr 90 000. The s20,w de- 
termined by sucrose density gradient centrifuga- 
tion was 1.5. The activator appeared to be highly 
asymmetric with a frictional ratio of 2.0. The cal- 
culated Mr [11] was 26 000. 
3.3. Effect of activator on phosphorylase phos- 
phatase activity 
Phosphorylase phosphatase activity of renal 
phosphatase C in the presence of activator was 3 -  
4-times greater than in the absence of activator, 
under standard assay conditions (fig.l). Phos- 
phatase C purified from rabbit skeletal muscle and 
a high Mr phosphorylase phosphatase isolated 
from bovine heart were stimulated to a similar ex- 
tent by the activator (not shown). At high concen- 
trations of activator, activition was decreased 
(fig.l). This may have been due to the slight con- 
tamination of the activator with heat-stable inhibi- 
tor. The activator itself had no phosphorylase 
phosphatase activity. 
When the concentration f [32p]phosphorylase  
was varied, the activator increased the Vmax by 
only -20% (fig.2). In the absence of activator, the 
Km for phosphorylase a was 4.13 /tM. This was 
comparable to the value of 2.4 #M reported in [12] 
for skeletal muscle phosphatase. In the presence of 
activator, there was a marked decrease in the Km 
for phosphorylase a to 0.19/LM. 
4. DISCUSSION 
To our knowledge, this is the first description of 
a heat-stable protein activator of phosphorylase 
phosphatase. Khandelwal and Zinman [13] re- 
ported on a heat-stable protein isolated from rab- 
bit liver which stimulated the dephosphorylation 
of [32p]histone catalyzed by a rabbit liver phos- 
phoprotein phosphatase. However, their protein 
factor had no effect on phosphatase activity when 
[32p]phosphorylase a was the substrate [13]. The 
properties of the purified phosphorylase phos- 
phatase activator were similar to other heat-stable 
protein modulators which are believed to have 
little ordered structure [4,5,14]. It is possible that 
these proteins are active only after harsh treat- 
333 
Volume 146, number 2 FEBS LETTERS September 1982 
ment. However, there is evidence that several are 
active in their native state [4,14]. 
The activator described here may represent a
new mechanism for the regulation of phospho- 
protein phosphatase activity. The activator in- 
creases phosphatase activity primarily by decreas- 
ing the Km for phosphorylase a by -20-fold. The 
activator may be of particular importance in 
tissues uch as the kidney where the phosphorylase 
concentration is very low. The activity of phos- 
phorylase in swine renal cortex is < 1% of the 
activity in rabbit skeletal muscle [15,16]. If one as- 
sumes that the specific activity of renal phosphory- 
lase is similar to muscle phosphorylase [17], it can 
be calculated that the phosphorylase concentration 
of swine renal cortex is -0.1 ~M. Thus, the activa- 
tor could have a dramatic effect on phosphorylase 
phosphatase activity in vivo. The brain is another 
tissue in which the phosphorylase concentration is 
low [15]. A similar heat-stable activator has been 
found in a 2-15% trichloroacetic acid precipitate 
of rat brain (unpublished). 
It will also be of interest o see if other putative 
substrates of protein phosphatase-1 are affected by 
the activator. One of the major questions relating 
to a multifunctional phosphoprotein phosphatase 
is how the activity of an enzyme with several sub- 
strates is regulated [ 1,2,12]. An activator which can 
decrease the Km for one substrate provides a 
mechanism by which relative specificity, and thus 
control, can be conferred on a multisubstrate en- 
zyme. 
ACKNOWLEDGEMENTS 
This work was supported in part by USPHS 
grants AM 14723, AM 28277 and by USPHS. Re- 
search Career Development Award AM 00337 to 
K.K.S. 
REFERENCES 
[1] Cohen, P., Foulkes, J.G., Goris, J., Hemmings, 
B.A., Ingebritsen, T.S., Stewart, A.A. and Strada, 
S.T. (1981) in: Metabolic Interconversion of En- 
zymes 1980 (Holzer, H. ed) pp. 28-43, Springer- 
Verlag, Berlin, Heidelberg, New York. 
[2] Krebs, E.G. and Beavo, J.A. (1979) Annu. Rev. 
Biochem. 48, 923-959. 
[3] Huang, F.L. and Glinsmann, W.H. (1976) Eur. J. 
Biochem. 70, 419-426. 
[4] Nimmo, G.A. and Cohen, P. (1978) Eur. J. Bio- 
chem. 87, 341-351. 
[5] Foulkes, J.G. and Cohen, P. (1980) Eur. J. Bio- 
chem. 105, 195-203. 
[6] Mellgren, R.L. and Schlender, K.K. (1982) J. Cyclic 
Nucl. Res. in press. 
[7] Killilea, S.D., Aylward, J.H., Metlgren, R.L. and 
Lee, E.Y.C. (1978) Arch. Biochem. Biophys. 191, 
638-646. 
Reimann, E.M. and Beham, R.A. (1982) Methods 
Enzymol. in press. 
Foulkes, J.G., Jefferson, L.S. and Cohen, P. (1979) 
FEBS Lett. 112, 21-24. 
Laemmli, U.K. (1970) Nature 227, 680-685. 
Siegel, L.M. and Monty, K.J. (1966) Biochim. Bio- 
phys. Acta 112, 346-362. 
Lee, E.Y.C., Silberman, S.R., Ganapathi, M.K., 
Petrovic, S. and Paris, H. (1980) Adv. Cyclic Nucl. 
Res. 13, 95-131. 
Khandelwal, R.L. and Zinman, S.M. (1978) Bio- 
chem. Biophys. Res. Commun. 82, 1340-1345. 
Weber, H. and Rosen, O.M. (1977) J. Cyclic Nucl. 
Res. 3, 415-424. 
Villar-Palasi, C. and Lamer, J. (1960) Arch. Bio- 
chem. Biophys. 86, 270-273. 
Villar-Palasi, C. and Gazquez-Martinez, I. (1968) 
Biochim. Biophys. Acta 159, 479-489. 
Mendicino, J., Abou-Issa, H., Medicus, R. and Kra- 
towick, N. (1975) Methods Enzymol. 42, 375-397. 
Cleland, W.W. (1967) Adv. Enzymol. 29, 1-32. 
[81 
191 
II01 
[Ill 
[12] 
[13] 
[14] 
[15l 
[16] 
[171 
[181 
334 


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Isolation of a heat-stable protein activator of phosphorylase phosphatase

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