Regulation of protein synthesis in rabbit reticulocyte lysates by 2,3-bisphosphoglycerate

Vol.

86,

No.

February

BIOCHEMICAL

3, 1979

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages 755-761

14, 1979

REGULATION OF PROTEIN SYNTHESZS IN RABBIT RETICULOCYTE LYSATES BY 2,3-BISPHOSPHOGLYCERATE Hiroshi

Narita,

Department Agriculture, Received

January

Koji

Ikura,

Ryuzo Sasaki

and Hideo

of Food Science and Technology, Kyoto University, Kyoto 606,

Chiba

Faculty Japan

of

2, 1979

Summary 2,3-Bisphosphoglycerate was the most potent effector of glycolytic intermediates tested for their effects on protein synthesis in gel-filtered lysates from rabbit reticulocytes. 2,3-Bisphosphoglycerate at low levels was stimulatory but became inhibitory at high levels. Both effects were dependent on Mgzf concentrations. The higher the concentration of Mq" , the higher the concentration of 2,3-bisphosphoglycerate required for maximal activation. 2,3-Bisphosphoglycerate concentrations required to exhibit an inhibitory effect increased as Mg'+ concentration increased. Both effects of 2,3-bisphosphoglycerate are discussed in terms of regulation of hemoglobin synthesis during maturation of erythroid cells. Introduction 2,3-Bisphosphoqlycerate

is

phosphate

in many mammalian

functions

as an effector

(reviewed

in Ref.1).

erythroid

cells,

micromolar this

synthase

respect

We have found

in basophilic

activity'.

The increase closely

to maturation

stages

be involved

presented

glycerate filtered 1 H. Narita

cells

here

and other lysates

et al.,

were

glycolytic

from

rabbit

manuscript

relate

acid-soluble

organic

and erythrocytes. of biological that

during

to about

This

events

of rabbit

5 ~$4 in reticulocytes,

levels

to the accumulation

done to examine intermediates

cells.

and that

of 2,3-bisphosphoqlycerate

in 2,3-bisphosphoqlycerate

in the regulation

cells

from the hundred-

to elevation

of erythroid

compound

in these

maturation

increases

attributable

activity

may, therefore, Studies

reticulocytes

in a number

is primarily

synthesizing

abundant

most

2,3-bisphosphoglycerate

level

increase

the

and its

of hemoglobin

with

2,3-Bisphosphoglycerate of hemoglobin

the effects on protein

synthesis.

of 2,3-bisphosphosynthesis

in gel-

reticulocytes.

in preparation 0006-291X/79/030755-07$01.00/0 755

Copyright All rights

@ 1979

by Academic Press, Inc. in anyform reserved.

of reproduction

Vol. 86, No. 3, 1979

Materials

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and Methods

Rabbit reticulocyte lysates were prepared according to the method of Housman et al.(2). The lysates were gel-filtered on a column of Sephadex G-25 previously equilibrated with 0.1 M K(OAc), 2 mM Mg(OAc)z, 20 mM HepesKOH (pH 7.6), and 2 mM dithiothreitol. The fractions corresponding to the excluded volume were combined and stored at -8O'C. The standard incubation mixture of cell-free protein synthesis assay contained the following components in 100 yl : 30 mM Hepes-KOH (pH 7.6), 80 mM K(OAc), 2 mM Mq(OAc)z, 2 n-M dithiothreitol, 2 mM ATP, 0.5 mkl GTP, 0.1 mM spermine, 3Oul/ml hemin, 3.75 uCi of L-[4,5-3H]leucine (105 Ci/mmole), 50 yM each of the other nonradioactive amino acids, and 37.5 ~1 gel-filtered lysates. In all instances, concentrations of Hepes-KOH, K(OAc), Mg(OAc) =, and dithiothreitol in reaction mixtures were corrected with those derived from the buffer solution used for gel-filtration of the lysates. Incubations were carried out at 37'C and 20 1.11 aliquots at intervals were applied to Whatman No.3 MM filter paper discs, previously dipped in 10 % trichloroacetic acid in acetone and then dried. The discs were further processed by a slightly modified method of Giloh (Freudenberq) and Maqer (3). 2,3-Bisphosphoqlycerate was measuredas described previously (4). Results Fig.1

shows the time

from rabbit

reticulocytes

glycerate.

in the presence

In contrast,

stimulated

protein

at the physiological was strongly

of 0.5 mM 2,3-bisphosphoqlycerate incubation

for

2 min and

respectively.

of which

protein that

the

effects

sensitively

available

synthesizing

bisphosphoglycerate. by 2,3-bisphosphoglycerate

with

Mq*+, It are

protein

activity

was closely

protein

synthesis

As shown in Fiq,3, was observed

756

Hence the effect

at all

Mq'+

(Fiq.2). synthesis

shows that

2.1 mM in the

stimulation

was

the Mq'+

absence

of 2,3-

of protein

synthesis

concentrations

tested.

varied

be

by the change

investigated

Fig.2 is

of 2,3-bisphosphoglycerate

caused

on protein

(Fiq.3).

concentration

may, therefore,

synthesis.

of 2,3-bisphosphoglycerate

for

on the

dependent.

for

Mq2+ concentrations

optimal

concentrations

a complex

of 2,3-bisphosphoglycerate

the effect

by varying

concentration

is

of Mq'+

of Mg2+ on protein Subsequently

forms

synthesis

in concentrations

Optimal

lysates

of 2,3-bisphospho-

synthesis

by 253 % and 178% after

2,3-Bisphosphoqlycerate

tested

in gel-filtered

and absence

protein

the presence

synthesis

synthesis

was added

(5 mM) in reticulocytes,

inhibited.

argued

of protein

When 2,3-bisphosphoglycerate

concentration

30 min,

courses

depending

on Mq2+

Vol.

86,

No.

BIOCHEMICAL

3, 1979

AND

TIME

(min

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1

Effects of 2,3-bisphosphoglycerate on protein synthesis by a Figure 1. gel-filtered reticulocyte lysate. The lysate contained 30 ug/ml hemin and at 37'C (see Materials and Methods for 2 ml4 Mg(OAc)z , and was incubated details of preparation of lysate and assay system of protein synthesis). The t3H]leucine incorporation into protein in a 20 j.11 aliquot was measured with no addition (ti), 0.5 mM 2,3-bisphosphoglycerate (M), and 5.0 mM 2,3-bisphosphoglycerate (U).

10 I--

m b

X5E a ”

Figure 2. Mgzf -response of protein synthesis Experiments were done as described in Materials Mg=+ concentration. The final concentration the abscissa. The 13Hlleucine incorporation on the ordinate.

757

in a gel-filtered lysate. and Methods except for the of Mg2+ added is indicated on at 37°C for 2 min is expressed

Vol. 86, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

2,3-BISPHOSPHOGLYCERATE

CONCENTRATION

(mM)

Figure 3. Effects of 2,3-bisphosphoglycerate on protein synthesis at The lysate was incubated with 2,3-bisvarious concentrations of Mg*+ . phosphoglycerate under standard conditions (see Materials and Methods) except for the Mg'+ concentration. The ['Hlleucine incorporation at 37'C for 2 min (on the ordinate) was measured at the following concentrations of Mg2+ , 1.5 mM (a), 2.0 mM (U), 3.0 mM (w), and 4.0 mM (a--o) -

concentrations. give

a maximal

The extent Maximal 3.0,

The concentration enhancement

increased

of stimulation

activation

of 2,3-bisphosphoglycerate

also

was dependent

248,

addition

of 2,3-bisphosphoglycerate

ing Mg'+

at 1.5 mu and 2.0 mM (lower inhibit there

synthesis, results

by reducing

that

is

in fact

not by varying

activator

for

cluded and thus

that

the machinary

the concentration

of Mg"

protein

stimulation. exerts

its

synthesis. was metabolized

synthesis,

is

but

by functioning

to generate

as 2,3-bisphosphoglycerate

758

for

effect

The possibility

containof 2.1 mM)

evident

stimulatory

the Mg2+ concentrations of protein

systems

available

2.0, Although

concentration

It

increased.

of 1.0,

respectively.

synthesizing

the optimal

2,3-bisphosphoglycerate

stimulated

to protein than

2,3-bisphospoglycerate

synthesis

and 3010%,

to

concentration.

in the presence

1430,

a marked

of Mg'+

on the Mg'+

by 2,3-bisphosphoglycerate

and 4.0 mM Mg2+ was 212,

should

as the concentration

required

protein from these on protein as an was ex-

ATP or GTP concen-

Vol. 86, No. 3, 1979

trations that

were

BIOCHEMICAL

almost

stimulation

Mg2+ 02.1

or its

glycerate

at high (1.5

complex

observed

caused

by lowering

the machinery

Mg'+

concentrations

with

for

high

on protein

concentrations concentrations

protein

synthesis

It

synthesis

Fructose

in extracts

protein

becomes

(0.5

appreciable

synthesis,

site(s)

or

at which complex

to compare

synthesis

but

6-phosphate

had no effect.

Of the compounds

potent

and the only

6-phosphate, tested,

compound

lysates

1,6-bisphosphate the inhibitory

effect

the effects

account

their lysates

(0.5 (0.01

mM), glucose

physiofrom

3-phosphoglycerate,

inhibitory

1,6-bis-

mM and 0.5 mM) showed

2,3-bisphosphoglycerate with

(5).

of 2,*3-bisphosphoglycerate.

1,6-bisphosphate

Fructose

(5,6).

We examined into

affect

6-phosphate

in gel-filtered

the effects

Glucose

mM), and fructose

fructose

synthesis,

and ICAD+, taking

with

cells

reticulocyte

of N?+D' (6) .

on protein

sugars

and glucose

lysates,

protein

sugars

stimulation.

activator

protein

of mammalian

rabbit

reticulocyte

I shows the results.

phosphate

stimulatory may be

phosphorylated

6-phosphate

in the presence

reticulocytes,

certain

in gel-filtered

inhibit

concentrations,

rabbit Table

6-phosphate

phosphorylated

logical

that

fructose

rabbit

stimulatory

various

for

have specific

from a variety

synthesis

In non-gel-filtered and glucose

when Mgzf concentrations

or a Mg.2,3-bisphosphoglycerate

reported

1,6-bisphosphate,

stimulate

2,3-Bisphospho-

of 2,3-bisphosphoglycerate

may

of

effect.

has been recently

protein

range

and the decreased

available

likely

of 2,3-bisphospho-

synthesis.

inhibition

is

due to the reduction

effect

became inhibitory

Mg'+

an inhibitory

stimulatory

This

It

cases.

effect

stimulatory

of 2,3-bisphosphoglycerate

gives

of

with

in all

seen in the inhibitory

as a direct

mM and 2.0 mM).

effect

binding

incubation

an indirect

Mgzf as well

glycerate

low

during

by 2,3-bisphosphoglycerate

mM) involves

of inhibitory

were

constant

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and WAD+ was the

effect

most

at physiological

concentrations. Discussion It has been proved phatase

activities

that

both

in erythrocytes

2,3-bisphosphoglycerate of rabbit

759

(7) and other

synthase

and phos-

mammals (B-13)

are

Vol. 86, No. 3, 1979

Table

BIOCHEMICAL

I.

Effects

of

Compounds added

glycolytic

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

intermediates on protein synthesis Leucine incorporated into protein after incubation for 2 min 30 min 100% 100%

Concn. (mM)

NOIN2 Glucose

6-P

0.1 0.5a

85 122

100 124

Glucose

1,6-diP

O.lb 0.5

93 130

94 138

Fructose

6-P

O-la 0.5

95 99

107 99

Fructose

1,6-diP

O.Ola 0.5

120 152

102 120

Glycerate

3-P

0.02a 0.5

103 92

99 95

Glycerate

2,3-diP

0.5 5.0a

253 14

178 15

b 0.1 96 113 NAD+ 0.5 108 114 Protein synthesis was measured after 2 min and 30 min incubation under standard conditions as described in Materials and Methods. zhysiological concentrations in rabbit reticulocytes. Physiological concentrations in rabbit erythrocytes.

manifested

by

inhibited

the

same

by the product,

vable

from these

facts

and the results accumulate

regulating

rates

2,3-bisphosphoglycerate. stimulate tion,

the

and conversely,

substrate

concentration.

stimulates

hemoglobin

cells

makes

during of their

activity inhibit

likely

that

this

would

glycerate

accumulates

to concentrations

effect. level

This

inhibitory

of hemoglobin

operate

Hemoglobin

increase

binds

with

would

may contribute

in

until

at which

effect

hemoglobin

the maturation

functions

synthesis

by lowering

inhibithe

of 2,3-bisphosphoglycerate that

continuously

760

cells,

therefore,

activity

during

erythrocytes.

and

the enzyme from product

on protein

in matured

is concei-

hemoglobin

syntheses.

regulation

stimulation

that

of erythroid

Our finding

operative

2,3-bisphosphoglycerate

here

strongly

It

maturation

the phosphatase

concomitantly

is

(8,10,14).

by relieving

synthesis.

activity

of hemoglobin,

The resulting

increase it

synthase

presented

Accumulation

synthase

phosphoglycerate

the

2,3-bisphosphoglycerate

2,3-bisphosphoglycerate co-operatively

and that

protein

the

of erythroid

ViVo.

Such

a co-

2,3-bisphosphostimulatory

is overcome for

and 2,3-bis-

by its

determining

An attempt

effect

of

inhibitory the upper

is in progress

to

Vol. 86, No. 3, 1979

reveal

BIOCHEMICAL

the mechanism

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the stimulatory

and inhibitory

effects

of

2,3-bis-

phosphoglycerate. The dependency tration

suggests

However, tration

there

of the effects

that is

Mgzf participates

no information

in the maturation Reticulocytes

to mention synthesis

that

phosphoglycerate in the presence

lysates

must be included

in the

results

synthase

synthesis. of Mg'+

It

are used for filtration,

which

concen-

concen-

cells.

to gel

lysates

protein

5 mM 2,3-bisphosphoglycerate.

of compounds

on Mg'+

on the variation

subjected

2,3-bisphosphoglycerate gel-filtered

available

when reticulocyte being

in modulating

of erythroid

contain

without

of 2,3-bisphosphoglycerate

experiments the effects

obtained.

are metabolized should

is worthwhile

be interpreted

of protein of 2,3-bis-

Results

obtained

to become substrates carefully

of

even when

are used.

ACKNOWLEDGEMENT : This work was supported Education, Science, and Culture of Japan.

by grants

from the Ministry

of

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Chiba, H., and Sasaki, R.(1978) in "Current Topics in Cellular Regulation" (B. L. Horecker, and E. R. Stadtman, eds) Vo1.14, pp.75-112. Academic Press, New York. M., RajBhandary, U. L., and Lodish, H. F. Housman, D., Jacobs-Lorene, (1970) Nature 227, 913-918. Biochim. Biophys. Acta, Giloh (Freudenberg), H., and Mager, J.(1975) 414, 293-308. Sasaki, R., Ikura, K., Sugimoto, E., and Chiba, H.(1974) Anal. Biochem., 61, 43-47. Lenz, J. R., Chatterjee, G. E., Maroney, P. A., and Baglioni, C-(1978) Biochemistry 17, 80-87. Wu, J. M., Cheung, C. P., and Suhadolinik, R. J.(1978) Biochim. Biophys. Res. Conunun. 82, 921-928. Narita, H., Utsumi, S., Ikura, K., Sasaki, R., and Chiba, H.(1979) Int. J. Biochem., 10, in the press. Sasaki, R., Ikura, K., Sugimoto, E., and Chiba, H.(1975) Eur. J. Biochem ., 50, 581-593. Ikura, K., Sasaki, R., Narita, H., Sugimoto, E., and Chiba, H.(1976) Eur. J. Biochem., 66, 515-522. Sasaki, R., Ikura, K., Narita, H., and Chiba, H.(1976) Agric. Biol. Chem ., 40, 2213-2221. Rosa, R., Audit, I., and Rosa, J-(1975) Biochimie, 57, 1059-1063. Hass, L. F., and Miller, K. B.(l975) Biochem. Biophys. Res. Commun., 66, 970-979. Rose, Z. B., and Dube, S-(1976) Arch. Biochem. Biophys., 117, 284-292. Rose, Z. B-(1968) J. Biol. Chem., 243, 4810-4820.

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