Mg2+ counteracts the inhibitory effect of spermine on liver phosphorylase kinase

Vol. 121, No. 1, 1984 May 31, 1984

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 271-276

BIOCHEMICAL

Mg2+ COUNTERACTS THE INHIBITORY EFFECT OF SPERMINE ON LIVER PHOSPHORYLASE KINASE* Eikichi

Hashimoto,

Department Received April

Tomoko Kobayashi

of Biochemistry, Matsuoka, Fukui

and Hirohei

Fukui 910-11,

MedicaZ Japan

Yamamura School,

17, 1984

SUMMARY: When the effect of polyamines on pig liver phosphorylase kinase was examined, spermine was found to be the most inhibitory. Although putrescine stimulated the 'reaction slightly, the spermidine The effect was dependent on the phosphorylase b concentration. inhibitory effect of spermine was counteracted by increasing the Mg2+ concentration. At 0.3 mM Mg2+ , the apparent Km for phosphorylase & was increased g-fold by the addition of 5 mM spermine. However, increasing Mg2+ to 3 mM decreased the value to the initial level obtained at 0.3 mM Mg2' alone. These results suggest that of liver phosphorylase a possible role of Mg*+ in the regulation kinase is to protect the enzyme from the inhibitory action of a polyamine such as spermine.

There of

free

rylase

have been several

Mg2+ on the activities kinases

skeletal

muscle Recent

polyamines kinase

inhibitory.

involved

kinase

(8-11).

the Mg2+ concentration a role

for

Mg

.

several

direct

in the

this to

2-3 mM.

regulation

the

role phosphothat

of the

substrate,

ATP-

have shown that

various

When the

effect

properties

laboratories

effects

and liver

(7) indicated

to providing

were examined,

However,

muscle

Ca2+-binding

in controlling

through

2+

skeletal

a regulatory

and Heilmeyer

regulate

from

properties

phosphorylase

indicating

enzyme in addition

activities

substrate

to

reports

are

of

Rilimann

(l-6).

Mg2+ has the ability

Mg2+.

reports

types

of protein

on the enzymes or by modifying effect

of polyamines

spermine

was shown to be most

was counteracted These results of liver

on liver

by increasing may

suggest

phosphorylase

that

kinase

* This work was supported in part by research grants from the Scientific Research Fund of the Ministry of Education, Science and Culture of Japan, and the Yamanouchi Foundation for Research on Metabolic Disorders. 0006-291X/84 $1.50 271

All

Copyright 0 1984 rights of reproduction

by Academic Press, Inc. in any form reserved.

Vol. 121, No. 1, 1984

is

to protect

the

BIOCHEMICAL

enzyme from

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

inhibitory

action

by polyamine

such

as spermine. EXPERIMENTAL PROCEDURES Putrescine dihydrochloride, spermidine trihydrochloride, spermine tetrahydrochloride and polyethylene glycol (# 6,000) were obtained from Nakarai Chemicals, Kyoto. Phosphocreatine and creatine phosphokinase were purchased from Sigma. (U-14C)Glucose l-phosphate was purchased from New England Nuclear. Other experimental materials were obtained from commercial sources. Glycogen phosphorylase b was prepared as described by Fischer and Krebs (12). For the deteriination of activity, phosphorylase b was diluted in the buffer containing 20 mM Tris-HCl (pH 7.0), 30 mM 2-mercaptoethanol and 1 mg/ml bovine serum albumin. The suitably diluted enzyme solution (50 ~1) containing 2 mM 5'-AMP was mixed with the phosphorylase assay mixture (50 pl), which contained 100 mM glycerophosphate (pH 6.1), 20 mg/ml oyster glycogen and 49 mM (14C)glucose l-phosphate (24,000-28,000 cpm). After incubation at 30" for an appropriate time, the reaction was stopped by the addition of 75 % ethanol and the insoluble materials were treated as described previously (13). One unit of phosphorylase & was the amount of enzyme that transferred 1 pmol of glucose into glycogen per min under the conditions mentioned above. Enzyme with a specific activity of 55-95 units/mg was employed. Pig liver phosphorylase kinase was partially purified as described before (6) with slight modifications as indicated below. To the supernatant obtained by ultracentrifugation of the crude extract, 50 % (w/v) polyethylene glycol, dissolved in the homogenizing buffer (14) (0.25 M sucrose was replaced to 20 % (w/v) glycerol), was added to the final concentration of 5 % and the precipitated materials were dissolved in the homogenizing buffer. The insoluble materials were removed by ultracentrifugation at 105,000 g for 1 h. The supernatant solution (8.3 ml, 457 mg protein) was applied to two Sepharose CL-4B columns equilibrated with 25 mM Tris-HCl (pH 7.4), 10 mM 2-mercaptoethanol, 0.1 mM PMSFl/ and 20 % (w/v) glycerol. The gel filtration was performed with the same buffer. Phosphorylase kinase activity was determined as follows except otherwise noted. The first incubation mixture (50 ~1) contained 25 mM Tris-glycerophosphate (pH 8.5), 20 mu 2-mercaptoethanol, 4 mg/ml phosphorylase b, 10 mM MgC12, 1 mM ATP, 24 mM phosphocreatine, 16 units/ml creatrne phosphokinase and enzyme preparation. After incubation at 30' for an appropriate time, 5 1-11of reaction mixture was transferred to the 50 ~1 of the stopping solution containing 10 mu glycerophosphate (pH 6.1), 20 mM NaF, 40 mM 2-mercaptoethanol and 2 mM EDTA. To this solution, phosphorylase assay mixture (50 ~1) was added and phosphorylase a activity was determined as described above without 5'-AMP. In parallel with the complete system, the reaction was also performed in the absence of phosphorylase kinase and this activity was subtracted. After the purification by Sepharose CL-4B, phosphocreatine and creatine phosphokinase were omitted from the assay mixture. Definition of one unit of phosphorylase kinase and phosphorylase a was the same as described previously (13). The specific activity of the enzyme employed in this study was 75-94 units/mg. Protein was determined using Protein Assay (Bio-Rad) with bovine serum albumin as a standard. As shown in Fig. rylase I/

kinase

1, the

was variable,

The abbreviation

used is:

RESULTS effect of polyamines depending

on the

PMSF, phenyl 212

on liver

species

methylsulfonyl

phospho-

of amine and the fluoride.

8lOCHEMlCAL

Vol. 121, No. 1, 1984

AND BlOPHYStCAL RESEARCH COMMUNICATIONS

2

0246610 Polyamhe

(mM)

4

6

l%tpmim

81

hM)

Fig. 1. Effect of polyamines on liver phosphorylase kinase activity. The first incubation mixture (50 ~1) contained 25 mM Tris-glycerophosphate (pH 6.8), 20 mM 2-mercaptoethanol, 80 units/ml (A) or 320 units/ml (B) phosphorylase b, 0.1 mM ATP, 0.3 mM MgC12, phosphorylase Other kinase preparation and various concentrations of polyamines. assay conditions were as described in "EXPERIMENTALPROCEDURES". Phosphorylase kinase activities taken as 100 % were 0.42, 0.45 and 1.3 and 1.3 units/ml (B), respectively, 0.46 units/ml (A) or 1.2,

for the series of experiments to which putrescine (4) and spermine (0) were included.

phosphorylase reaction this

irrespective

effect

of the

was only

centrations

above

phorylase to

spermidine.

Among the

inhibitory.

At the

a biphasic

80 % at

in the

of

phosphorylase

from

of

was completely

counteracted

reactions

were performed

b and 5 mM spermine, units/ml

phosphorylase

In order kinetic

mM to

in the presence

this

2+

Mg

effect

(Fig.

2).

inhibitory Although

effect the

phosphorylase

observed

when 320

were present.

as summarized 273

concentra-

1 was obtained

of 80 units/ml

was also

showed

When the MgC12 con-

mM, the

the mode of action

were performed

polyamine

the higher

3

b and 10 mM spermine

to understand

analyses

0.3

with

the activity

in Fig.

0.1 mM ATP and 0.3 mM MgC12.

were increased

spermine

at

described

centrations

was in-

was shown to be most

this

effect

The results

of phos-

was observed

However,

of

at con-

concentration

b - concentration,

and inhibitory) &.

the extent

spermine

tested,

the

concentration

stimulation

10 mM spermine.

(stimulatory

presence

substrate

slight

polyamines

stimulated

spermidine

at lower

When the

lower

of phosphorylase

In contrast,

fold.

only

putrescine

concentrations,

mM was inhibitory

2.5

320 units/ml,

was inhibited

substrate

1.1-1.3

b (80 units/ml).

creased

tion

Although

& concentrations.

( q ), spermidine

of

spermine

in Table

I.

2+

and Mg In the

,

Vol. 121, No. 1, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

2 c 1.0
%i.7 00

2 0.5ly7 3 0

2 MgClt

3

1 1

(mM)

Fig. 2. Counteraction by MqL+ of spermine effect on liver phosphorylase kinase. The enzyme activity was determined as described in the legend to Fig. 1 except that the reaction mixture was modified as indicated below. (1) phosphorylase 9 concentration was 80 units/ml. (2) MqC12 concentrations were varied as indicated. (3) The reactions were performed in the absence and presence of 5 mM spermine. Phosphorylase kinase activities changed from 0.220.80 unit/ml and 0.43-0.82 unit/ml in the presence and absence of 5 mM spermine, respectively, by increasing the Mq2+ concentration.

presence

of

phorylase fold.

0.3 mM Mg*+,

b g-fold

However,

returned

the

although

the

increased apparent

of both

Km value

were changed,

Effector

to the both

Phosphorylase

Spermine

mf)

(mm

0.3

0

0.3

5

3

5

Km

the apparent

Vmax was also

Km for

phos-

increased

3.4-

3 mM Mg2+ and 5 mM spermine initial the

TABLE I of spermine and Mg 2+ on kinetic phosphorylase kinase.

Effect

Mq2+

the

presence

elevated

ATP concentrations

spermine

low level. apparent

properties

b

Vmax

When the

Km and Vmax were

of

liver

ATP Km

Vmax

(U*/ml)

(U**/mg)

(uM)

W**/mg)

11924

135+6

93+5

15927

465r21

6923

153?13

207+4

49k7

290t30

I,055278 109?8

*

unit of phosphorylase b ** unit of phosphorylase kinase The enzyme activity was determined as described in the legend to Fig. 1 except that the reaction mixture was modified as indicated (1) MqC12 and spermine were added as indicated. (2) When below. phosphorylase b concentration was changed, the concentrations of this substrate were varied from 22 units/ml to 440 units/ml and bovine serum albumin (4 mg/ml) was included for maintaining the total protein (3) When the ATP concentration was changed, the conconcentration. centrations of this substrate were varied from 30 DM to 150 PM and phosphorylase was 220 units/ml. -b concentration

274

Vol. 121, No. 1, 1984

not markedly

BIOCHEMICAL

influenced

by the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

addition

of

2+ concentration. Mg

These results

suggest

of spermine

phosphorylase

kinase

the

on liver

affinity

of

this

enzyme for

spermine

or increasing

that

inhibitory

the

is mainly

phosphorylase

effect

due to modifying

-b.

DISCUSSION Recently,

experimental

results

the effects

of Mg2+ are closely

of

muscle

skeletal

also

shown that

affinity

cogen phosphorylase

b is

kinase

of

the

increased

indicating

to the Ca2+ -binding

related

phosphorylase

the

have accumulated

(7).

property

In addition,it

skeletal greatly

that

muscle

was

enzyme for

in the presence

gly-

of

enough

r

amount of related

Ca2+ (15).

effects

kinase

on the

case of liver

from Exton's

and our

in the control results

of

obtained

tector

of the

though

the

effective

nity

that

skeletal

YgLf

muscle

of

and Ca2+ have phosphorylase

and proper

respect

phosphorylase

spermine

through

especially

at

b. -

occupation

low Mg2'

Acknowledgementfor her secretarial

to both

for

(5,6). 2+

Mg

to the

The

as a pro-

of spermine. -b was greatly

initial

-binding

low level

binding

site of

Alenhanced

2+

the Mg

was in spite

may be compatible

may exist

this

Ca 2+ -binding

cation

capacity

on liver may influence and the affi-

of Mg2+ may be blocked -binding

site

on this

are grateful 275

by

enzyme,

concentrations.

The authors assistance.

Mg2+

free

the Mg2+ concentration

These effects of

role

reports

of

conditions

These results

2+

a Mg

a role

action

increasing

the polyamine.

kinase

more recent

phosphorylase

the value

that

the

the inhibitory

spermine,

supposition

various

propose

glycogen

in decreasing

enzyme with for

study

for

kinase,

have indicated

enzyme under

in this

of

the presence the

laboratories

this

Km value

phosphorylase the

of

phosphorylase

enzyme from

in the presence

with

expression

suggest

activity. In the

of

These results

to Miss K. Mihara

Vol. 121, No. 1, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.

Chelala, C.A. and Torres, H.N. (1968) Biochem. Biophys. Res Commun. 32, 704-709 m-Palasi, C. and Wei, S.H. (1970) Proc. Natl. Acad. Sci. ---U.S.A. 67, 345-350 Clerch, L.B. and Huijing, F. (1972) Biochim. Biophys. Acta. 268, 654-662 Singh, T.J., Akatsuka, A. and Huang, K.-P. (1982) Arch. Biochem. Biophys. 218, 360-368 Chrisman, T.D., Sobo, G.E. and Exton, J.H. (1984) FEBS Lett. -167, 295-300 Hashimoto, E., Mizuta, K. and Yamamura, H. Submitted to -J. Biochem. Kilimann, M. and Heilmeyer, Jr.L.M.G. (1977) Eur. 2. Biochem. 2, 191-197 Takai, Y., Nakaya, S., Inoue, M., Kishimoto, A., Nishiyama, K., Yamamura, H. and Nishizuka, Y. (1976) 2. Biol. -Chem. 251, 1481-1487

9. 10. 11. 12. 13.

14. 15.

Criss, W.E., Yamamoto, M., Takai, Y., Nishizuka, Y. and Morris, H.P. (1978) Cancer Research 38, 3532-3539 Ahmed, K., WE M.J. and Goueli, S.A. (1978) Biochem. -J. 176, 739-750 D.-F., Schatzman, R.C., Mazzei, G.J., Turner, R.S., Raynor, Qi, R.L., Liao, S. and Kuo, J.F. (1983) Biochem. J. 213, 281-288 Fischer, E.H. and Krebs, E.G. (1962) Methods Enzymol. 2, 369-376 Taira, T., Kii, R., Sakai, K., Tabuchi, H., Takimoto, S., Nakamura, S., Takahashi, J., Hashimoto, E., Yamamura, H. and Nishizuka, Y. (1982) J. Biochem. 91, 883-888 Hashimoto, E., Mizuta; K., Tsutou, A., Nakamura, S. and Yamamura, H. (1983) J. Biochem. 93, 939-942 Heilmeyer,-Jr.L.M.G., Meyer, F., Haschke, R.H. and Fischer, E.H. (1970) 2. Biol. Chem. 245, 6649-6656 --

276