Starvation results in decreased initiation factor activity in rat skeletal muscle

VoI. 72, No. 4, 1976

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STARVATION RESULTS IN DECREASED INITIATION FACTOR ACTIVITY IN RAT SKELETAL MUSCLE BY D. Eugene Rannels, Anthony E. Pegg and Stephen R. Rannels Department of Physiology, The Milton S. Hershey Medical Center The Pennsylvania State University, Hershey, PennSylvania 17033

Received August27,1976 SUMMARY Activity of a factor forming a nitrocellulose filter-bound complex with [35S]met-tRNA~et and GTP was estimated in the postribosomal supernatant of psoas and heart muscle. Complex formation required initiator tRNA and GTP or GMP-P(CH2)P; it was inhibited by Mg 2+, GDP, aurintricarboxylic acid, spermine and spermidine. Starvation reduced complex-forming activity of supernatants from psoas but not heart muscle. Reduced activity was not due to increased dilution or deacylation of met-tRNA~ et or to increased hydrolysis of GTP by extracts from starved animals. INTRODUCTION

When rats were made alloxan-diabetic or starved for

48 hours, ribosomal subunits accumulated and polysomes were depleted in psoas muscle

(1,2).

No changes in ribosomal aggregation

were observed in hearts from these animals

(2).

Protein synthesis,

estimated in vitro, was inhibited in skeletal muscle from starved rats

(3).

These studies suggested that starvation restricted syn-

thesis of skeletal muscle proteins at the level of peptide-chain initiation. Reactions leading to formation of an initiation complex consisting of met-tRNA~ et and a 40S ribosomal subunit appeared to become rate limiting to protein synthesis in heme-deficient reticulocytes (4,5) and in the presence of low levels of double-stranded RNA or oxidized glutathione

(7).

(6)

Addition of the initiation factors

which mediate formation of this complex prevented or reversed these inhibitions

(8).

Formation of the complex may also be subject to

control by physiological factors such as substrate hormone

(Ii) availability or energy charge

Copyrtght© 1976 by A c a ~ m i c Press, Inc. All rights o] r~roduction in any [orm reserve.

1481

(12,13).

(9,10), and These ob-

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servations suggested the possible importance of initiation reactions as a site of regulation of protein synthesis. In ascites tumor cells, starvation for amino acids led to a loss of polysomes and a reduction in a met-tRNA~et-40S subunit complex,

the formation of which was mediated by an IF-E2-1ike

initiation factor

(9).

Thus, it appeared that the effects of

starvation on ribosomal aggregation in rat psoas could have been mediated by changes in activity of a similar factor.

This paper

describes formation of an initiation complex by post-ribosomal supernatants from heart and skeletal muscle of fed and starved rats. METHODS Heart or psoas muscle was removed from anesthetized rats, blotted, weighed and homogenized in a solution (2° ) containing 0.25 M KCI, 10 mM Tris, pH 7.4, and 3 ~ dithiothreitol. Following centrifugation of the homogenate for 70 minutes in a Beckman SW56 rotor (56,000 rpm), 50 ~i of the post-ribosomal supernatant was incubated (30 ° ) in a total volume of 300 ~i containing 90 mM KCI, 20 ~M Tris, pH 7.5, 3 r~4 dithiothreitol and 1.33 mM GTP ("complete" mixture) Reactions were started bv addition of a solution containing 4 pmoles of [35S]met-tRNA~ et (Specific Activity= 83.3 x 106 dpm/nmole). Reactions were stopped by addition of 5 ml of ice-cold buffer containing 90 ~M KCI, 20 mM Tris, pH 7.5, and 5 mM MgCI 2. This solution was poured over Millipore nitrocellulose filters (HAWP; pore size 0.45 ~m) which were then washed three times with 5 ml of the same cold buffer, dried, and counted in a toluene-based scintillator. Binding activity at 30 ° was too rapid for estimates of initial rates to be made. Values reported in this paper represent the steady state. When no supernatant was present, non-specific binding represented about 2% of that observed with the complete mixture. Rat liver tRNA, prepared by the method of Rogg et al. (14), was charged using E. Coli aminoacyl-tRNA synthetases, which charge only the initiator form of methionyl-tRNA (15). GTP and other nucleotides were estimated by high pressure liquid chromatography (16). Aminoacyl-tRNA synthetases were purchased from Miles Laboratories; [35S]-L-methionine from Amersham-Searle.

RESULTS AND DISCUSSION met-tRNA~ et to ~ l l i p o r e muscle

Activity of heart supernatants in binding filters was three times that from psoas

(Table I) when extracts were prepared in 0.25 M KCI.

In

separate experiments, muscles were homogenized in the presence of 0.25, 0.50, 0.75 and 1.00 M KCI.

1482

After centrifugation,

the

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TABLE Characteristics of I n i t i a t o r

I

of S u p e r n a t a n t - d e p e n d e n t

Binding

tRNA to Filters.

[35S]met-tRNA~ et bound, CONDITION

OF

10 -3.

INCUBATION

EXPERIMENT

cpm/g m u s c l e

PSOAS

HEART

I

Complete

690

1316

-GTP

248

126

+GDP

270

384

88

64

602

708

273

ND

-GTP

29

ND

+ATA

6

ND

105

ND

-GTP + GDP -GTP + G M P - P ( C H 2 ) P

EXPERIMENT

II

Complete

+Bacterial

tRNA

H e a r t and psoas m u s c l e w e r e p r e p a r e d as d e s c r i b e d in methods. As indicated, GDP (1.33 mM), G M P - P ( C H 2 ) P (1.33 mM), a u r i n t r i c a r b o x y l i c acid, A T A (60 ~M), or a c y l a t e d b a c t e r i a l tRNA (0.5 mg) w e r e p r e s e n t from the start of incubation. A f t e r i0 (Experiment I) or 5 (Experiment II) m i n u t e s b i n d i n g was a s s a y e d as d e s c r i b e d in methods. ND = not determined.

post-ribosomal trifuged

supernatant

to r e m o v e

ing s u p e r n a t a n t prepared

over

was

was d i l u t e d

the a c t o m y o s i n unchanged

this range

to 0.25 M KCI and recen-

formed.

w h e n psoas

Activity or h e a r t

of KCI c o n c e n t r a t i o n s .

1483

of the r e s u l t

extracts Therefore,

were full

Vol. 72, No. 4, 1976

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Ioo

IOO

~ 6o ! ~ m

ff

4o

(z:

'

\\.,

20

i

[ P20i

~,sp~ ~

o.,

~

'l~Mg 2+

O--osp ]

I

0

I

I

j I

I

I

2 5 4 5 CONCENTRATION

| .

~;sp

ga+

,,o_osp I

I

0 I 2 5 4 ADDED, mM

I

I

I

5

Figure_!l. Effects of Magnesium or Polyamines on Binding of Met-tRNA~ et to Filters. Tissue preparation and binding assays were as described in methods. Psoas muscles were homogenized in 4 volumes of buffer; hearts, 5 volumes. Incubation time was i0 minutes; MgCI 2 (Mg2+), spermidine (Spd), or spermine (Sp) were added to the assay to give the concentration indicated. Under3~ontrol conditions (no additions), 6272 and 13,505 x 102 cpm [ S]met-tRNA met were bound per gram of psoas or heart, respectively, f

activity appeared to be released to the supernatant in the presence of 0.25 M KCI. In both tissues, binding activity required GTP and was inhibited by GDP.

Inhibition amounted to 60 to 70 percent when GTP

and GDP were present in equal-molar concentrations periment I).

(Table I, Ex-

The non-hydrolyzable GTP analog, GMP-P(CH2)P,

supported 55 to 85 percent of full activity.

Binding activity

in psoas supernatants was abolished by aurintricarboxylic acid (ATA), but not by a large excess of acylated b a c t e r i a l tRNA

(Table

I, Experiment II). Addition of increasing concentrations of MgCI 2, spermidine, or spermine to the incubation buffer reduced binding of tRNA~ et

(Figure i).

[35S]met-

Spermine and spermidine were both effective

at concentrations within the physiological range

1484

(17), with sper-

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE Effect

of S t a r v a t i o n

Supernatant

II

on F i l t e r

from Heart

Binding

or P s o a s

[35S]Met-tRNA~et

of M e t - t R N A ~ et by

Muscle.

BOUND

CONDITION OF A N I M A L

10 -3. c p m / g Muscle

TISSUE

EXPERIMENT

l0 -2- c p m / m g Protein

RNA mg/g Muscle

I Psoas

738 + 32

(5)

2 days

626 + 20

(5) a

3 days

570

+ 16

4 days

Fed

i14

+ 5

1.74

+

.07

(6)

98 + 3 a

1.34

+

.05

(7) b

(5) b

89 + 3b

1.30

+

.05

(7) b

511 + 21

(5) b

77 + 3 b

1.31

+

.05

(7) b

544 + 32

(6)

81 + 4 273 + 18

.05

(6)

.01

(7) b

Starved:

EXPERIMENT

II Psoas

Fed

ND

Heart

1508

+ 87

(3)

Psoas

419

+ 25

(6) a

51 + 5 b

Heart

1541

+ 196

(3)

255 + 32

2.50

+

Starved: 2 days

ND 2.26

+

Rats w e r e f a s t e d as i n d i c a t e d a n d b i n d i n g a c t i v i t y d e t e r m i n e d in s u p e r n a t a n t s f r o m h e a r t or p s o a s as d e s c r i b e d in m e t h o d s in 15 m i n u t e i n c u b a tions. R N A was e s t i m a t e d by a l k a l i n e h y d r o l y s i s as d e s c r i b e d p r e v i o u s l y (22). V a l u e s r e p r e s e n t the m e a n + S.E.M. of the n u m b e r of d e t e r m i n a t i o n s s h o w n in p a r e n t h e s i s . ND = not determined a = p< .05 vs f e d b = p< .01 vs fed

mine

being

about

twice

a molar

basis.

These

similar

to t h a t

of

as p o t e n t

as s p e r m i d i n e

experiments

IF-E 2

(18),

indicated

IF-I

1485

(19,20)

when

compared

on

t h a t an a c t i v i t y or I F - M P

(21), w h i c h

Vol. 72, No. 4, 1976

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

form a ternary initiation complex between factor,

initiator tRNA,

and GTP, could be estimated in the post ribosomal supernatant of psoas and heart muscle.

These factors have been reported to bind

to ribosomes and to be released by high salt concentrations. The data in Table II show that 48 hours of starvation reduced binding activity of psoas supernatants whether expressed per gram muscle or per mg supernatant protein

(Experiment I).

Activity of

psoas extracts declined as starvation continued while activity of heart supernatants was unchanged after two days

(Experiment II).

Psoas RNA content decreased after two days of starvation, but did not decline thereafter; a similar period.

heart RNA was reduced somewhat after

Details of the relationship between changes in

binding activity and content of specific RNA species remain to be investigated. Similar effects of starvation were observed when assays contained 5 mM MgCI 2.

Activity was greatly reduced under these con-

ditions, but the complex formed in the presence of Mg 2+ may be more directly related to the physiological precursor of the ribosomal initiation complex

(23).

Decreased activity in supernatants from psoas of starved rats did not appear to result from increased dilution of

[35S]met-tRNA~ et

by endogenous initiator tRNA or from accelerated rates of deacylation of substrate tRNA. fold range of

Binding activity was investigated over a 10-

[35S]met-tRNA~et concentration.

Activity was re-

duced in psoas of starved rats at all tRNA concentrations panel A).

(Figure 2,

As also shown in Figure 2 (panel B), addition of tissue

extracts from psoas of fed or fasted rats reduced the amount of [35S]met-tRNA met which remained acylated during i n c u b a ~ o n . However, f the rate of deacylation was similar with each tissue source. Walton and Gill

(12,13) recently suggested that activity of

1486

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BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS

E

I

=

,J°

,~ I00

E

"- 8 0

/"

/

/

FED

/

i Bo o

./

b_ 6c

I0 0 !

i • 60

~4c

•

40

,20 ¸

B

L

I

I

I

020

B0

;0

Met-tRNAf ADDED, prnoles

;

;

;

J0

INCUBATION TIME, rain

Figure 2. Effect of Availability of Met-tRNA~ et on Binding Activity in Supernatants from Fed and Starved Rats. In the experiments shown in panel A, binding activity was assayed in supernatant from psoas muscle of fed (closed symbols) or starved (48 hours, open symbols) rats, as described in methods. Assays were incubated 5 minutes. In a second experiment (panel B), samples were withdrawn at the times indicated. The fraction of met-tRNA~ et which remained acylated at each time was estimated by spo%ting the sample on filter paper discs and counting the washed discs in toluene containing 0.6% omnifluor (15). Radioactivity bound to the discs at each point was expressed as a fraction of that bound at zero time. Assays contained either no tissue extract (open squares), or extract from psoas muscle of fed (closed circles) or starved (48 hours, open cirlces) animals.

a ternary complex-forming factor, partially purified from reticulocytes,

could be modified by changes in the GTP:GDP ratio and thus

by the adenylate energy charge through the nucleotide diphosphate kinase reaction.

In the present experiments,

these factors did

not appear to account for inhibition of protein synthesis in psoas muscle in vivo.

Whole tissue levels of GDP, GTP, AMP, ADP, and

ATP were unaltered by 48-72 hours of starvation; charge

(24) also was unchanged

servations). vation,

Furthermore,

(D. E. Rannels,

adenylate energy

unpublished ob-

reduced binding activity during star-

as assayed in vitro,

did not appear to result from loss

of GTP or accumulation of GDP during incubation.

1487

Approximately

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BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS

TABLE E f f e c t of C r e a t i n e on A c t i v i t y

III

Phosphate

from Psoas

and C r e a t i n e

Phosphokinase

of F e d and S t a r v e d

Rats.

CONDITION

CP+

[ 3 5 S ] M e t - t R N A ~ et bound,

Nucleotide Concentration ~moles/ml GTP GDP

OF A N I M A L

CPK

10 -2.

Fed

-

(5) 45 ± 2

.99 ± .04

.27 ± .05

Starved

-

(5) 32 ± 2 a

.86 ± .06

.34 ± .03

Fed

+

(5) 67 ± 2 b

1.09

± .07

.14 ± .03 c

Starved

+

(5) 45 ± i a'b

i.i0

± .03 b

.18 ± .04 c

cpm/mg protein

Rats were s t a r v e d for 96 hours and b i n d i n g a c t i v i t y of psoas extracts a s s a y e d in 5 m i n u t e i n c u b a t i o n s as d e s c r i b e d in methods. The c o n c e n t r a t i o n s of GTP and GDP in the assay m i x t u r e at zero time w e r e 1.16 ± .06 and 0.04 ± .01 ~ m o l e s / m l , r e s p e c t i v e l y . Creatine phosphate (CP) and c r e a t i n e p h o s p h o k i n a s e (CPK) w e r e a d d e d to the assay m i x t u r e at c o n c e n t r a t i o n s of 3.33 n ~ and 0.07 mg/ml, r e s p e c t i v e l y . Similar changes in n u c l e o t i d e c o n c e n t r a t i o n s w e r e o b t a i n e d f o l l o w i n g a 15 minute incubation. V a l u e s r e p r e s e n t the m e a n ± S.E.M. of the n u m b e r of d e t e r m i n a t i o n s s h o w n in p a r e n t h e s i s . a = p < .01 vs F e d b = p < .01 vs - (CP + CPK) c = p < .05 vs - (CP + CPK)

20% of the GTP i n i t i a l l y lyzed d u r i n g or s t a r v e d

a 5 minute

rats

phosphokinase

ferences

in b i n d i n g

from both

regenerated.

Reduced

maintained

of this

of s t a r v e d

mechanism

activity

rats.

IF-E2-1ike

Further

is m o d i f i e d

of the factor.

1488

and

b u t dif-

Activity

of

45% h i g h e r w h e n GTP was

for the i n h i b i t i o n

in s k e l e t a l m u s c l e

purification

(Table III).

from fed

phosphate

GTP in b o t h extracts,

remained

activity

supernatants

of c r e a t i n e

sources was a b o u t

at least in part,

by w h i c h

of psaos

Addition

activity

supernatants

in the assay m i x t u r e was h y d r o -

incubation

(Table III).

creatine

account,

present

factor may

of p r o t e i n definition

during

synthesis of the

starvation

requires

Vol. 72, No. 4, 1976

BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS

ACKNOWLEDGEMENTS The authors are grateful to Dr. L. S. Jefferson and Dr. H. E. Morgan for their encouragement and financial support. This work was supported by grants HLI1534, AM15658 and CA18138 from the National Institutes of Health. REFERENCES i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

Jefferson, L. S., Koehler, J. O. and Morgan, H. E. (1972) Proc. Nat. Acad. Sci. (U.S.) 69____C816-820. Rannels, D. E., Hjalmarson, and Morgan, H. E. (1974) Amer. J. Physiol., 226, 528-539. Rannels, S. R., Li, J. B. and Jefferson, L. S. (1976). Diabetes 25, Suppl. i, 333. Balkow, K., Mizuno, S., Fisher, J. M. and Rabinovitz, M. (1973) Biochem. Biophys. Acta., 324, 397-409. Legon, S., Jackson, R. J. and Hunt, T. (1973) Nature New Biol., 241, 150-152. Ehrenfeld, E. and Hunt, T. (1971) Proc. Nat. Acad. Sci. (U.S.), 68, 1075-1078. K-~sower, N.S., Vanderhoff, G. A. and Kosower, E. M. (1972) Biochim. Biophys. Acta., 272, 623-637. Beuzard, Y. and London, I. M. (1974) Proc. Nat. Acad. Sci., (U.S.) 71, 2863-2866. Pain, V. M. and Henshaw, E. C. (1975) Eur. J. Biochem., 57 335-342. Smith, K. E. and Henshaw, E. C. (1975) Biochemistry i_44, 10601067. Liang, T. and Liao, S. (1975) Proc. Nat. Acad. Sci. (U.S.) 72, 706-709. Walton, G. M. and Gill, G. N. (1975) Biochim. Biophys. Acta 390, 231-245. Walton, G. M. and Gill, G. N. (1976) Biochim. Biophys. Acta, 418, 195-203. Rogg, H., Wehrli, W. and Staehelin, M. (1969) Biochim. Biophys. Acta, 195, 13-15. Stanley, W. M., Jr. (1974) In: Methods in Enzymology, Vol. XXIX (L. Grossman and K. Moldave, eds.) pp. 530-546, Academic Press New York. Whitfield, C. F., and Morgan, H. E. (1973) Biochim. Biophys. Acta 307, 181-196. Raina, A., and J~nne, J. (1975) Medical Biology 5_33, 121-147. Schreier, M. H. and Staehelin, T. (1973) Nature New Biol., 242, 35-38. Dettman, G. L. and Stanley, W. M., Jr. (1973) Biochim. Biophys, Acta 299, 142-147. Gupta, N° K., Woodley, C. L., Chen, Y. C. and Bose, K. K. (1973) J. Biol. Chem. 248, 4500-4511. Safer, B., Anderson, W. F. and Merrick, W. C. (1975) J. Biol. Chem. 250, 9067-9075. Morgan, H. E., Jefferson, L. S., Wolpert, E. B. and Rannels, D. E. (1971) J. Biol. Chem. 246, 2163-2170. Gupta, N. K., Chatterjee, B., Chen, Y. C. and Majumdar, A. (1975) J. Biol. Chem. 250, 853-862. Atkinson, D. E. and Walton, G. M. (1967) J. Biol. Chem. 242, 3239-3241.

1489