Regulation of protein synthesis in rabbit reticulocyte lysates by guanosine triphosphate

Vol. 109, No. 3, 1982 December 15, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages

872-880

REGULATION OF PROTEIN SYNTHESIS IN RABBIT RETICULOCYTE LYSATES BY GUANOSINE TRIPHOSPHATE Rajinder

Singh

Ranu

Department of Microbiology and the Graduate Program in Cellular and Molecular Biology Colorado State University Fort Collins, Colorado 80523 Received

November

2, 1982

Summary: GTP *(2 mM) promotes protein synthesis in rabbit reticulocyte lysates in which protein chain initiation is inhibited bv the activation of specific adenosine 3':5' cyclic monophosphate independent protein kinases in: 1) heme deficiency; or 2) in hemin-supplemented lysates by the addition of the purified heme-regulated protein kinase (HRI); or 3) oxidized glutathione; or 4) by low levels of double stranded RNA. The molecular basis for the promotion of protein synthesis by GTP under these various conditions was investigated by examining the --in situ state The results show that GTP (2 mM) blocks eIF-2 of eIF-2 phosphorylation. phosphorylation and also promotes the dephosphorylation of phosphorylated eIF-2. These findings suggest that GTP restores protein synthesis by a common mechanism that involves the relief of eIF-2 from phosphorylation. The nonphosphorylated eIF-2 is, therefore, available for the maintenance and the restoration of protein chain initiation cycle. Protein dynamic

synthesis

process

in rabbit

that

involves

of the 38,000-dalton l-4).

the presence stranded

inhibit

protein

site(s) thione This

of initiation

level

of double

RNA activated chain

of eIF-2

initiation

The addition

(5-7). is

also

38,000-dalton

subunit

of a ternary

formation

also

associated

the

of eIF-2

with (2,3).

complex

the

cycle

The HRI and dR1 catalyzed

(4).

tion

complex

of eIF-2

with

In previous 2 mM) promotes supplemented

the

eIF-2

studies protein

lysates

it

protein

chain

factor in protein

of eIF-2

stabilization was shown that

synthesis

factor the

a that

same glutainitiation.

of the eIF-2

promotes

with

Met-tRNAf

chain

initiation

of eIF-2

by blocking

inhibits

the interac-

(1,8,9). addition

in heme deficient

872

rig/ml)

of the

phosphorylation

capacity

and in

activated

the phosphorylation

even on the addition

0 1982 by Academic Press, Inc. of reproduction in any form reserved.

are

in

HRI or oxidized

blocks

reaction

0006/291X/82/230872-09$01.00/0 Copyright All rights

RNA (0.1-100 (dRI)

Initiation

limiting

formation

@RI)

(eIF-2.Met-tRNAf.GTP)

and GTP (3) the ternary

first-rate

kinase

of purified

lysates

by a

(reviewed

by the phosphorylation

to hemin-supplemented inhibition

kinase

regulated

eIF-2

protein

stranded

protein

is

and dephosphorylation

factor

a heme-regulated

of a low

double

lysates

the phosphorylation

subunit

In heme deficiency,

reticulocyte

of purified

of GTP (about

lysates

and in hemin-

HRI or double

BlOCHEMlCAL

Vol. 109, No. 3, 1982 stranded

RNA or oxidized

GTP not

only

synthesis

in

also

prevents

promotes

glutathione the onset

lysates

in which

synthesis

protein

kinases

These

observations

that

in the presence suggestive

presented

lation

of eIF-2

eIF-2.

Thus,

under

in this

and also of protein

also

conditions,

restores

has been

inhibited

of other

translational of action

various

conditions

is

Materials

of GTP in

is

GTP, blocks

dephosphorylation available

initiation

(12-14). Never-

initiation cycle. and restorative

show that

eIF-2

GTP

inhibitory

of a common site

the

protein

(11,12).

of eIF-2

report

chain

these

the same site(s)

these

promotes

nonphosphorylated

and maintenance

but

of protein chain for the preventative

synthesis

RESEARCH COMMUNKATIONS

Under

of inhibition synthesis

the promotion and restoration the molecular basis theless, The results

(10-12).

phosphorylate are

of GTP on protein

AND BIOPHYSICAL

effects

not known.

the phosphory-

of phosphorylated for

the

resumption

cycle.

and Methods

The following procedures have been described: the preparation of rabbit reticulocyte lysates ; protein synthesis mixture; assay of protein synthesis, preparation of purified RRI; preparation of purified eIF-2; protein kinase assay; sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and the autoradiography of polyacrylamide gels (15). The [\d32P]ATP (specific activity 3000 Ci/mmol) specifically purified by high performance liquid chromatography was obtained from New England Nuclear. Assay of in situ phosphorylation of eIF-2. Rabbit reticulocyte lysate protein synthesis reaction mixtures (20 ~1) were incubated with 50 pC of [y3*P]ATP at 30° either in absence of hemin or in the presence of optimal Other additions to hemin-supplemented hemin concentration (15 FM). lysates were: purified RR1 (0.4 ug) or double stranded RNA (17 rig/ml) or oxidized glutathione (0.5 mM). The mixtures contained energy generating system (creatine phospho-kinase (2 pg) and creatine phosphate (5 mM)) but no additional ATP or GTP unless otherwise indicated was added. At intervals, aliquots (3 ~1) were removed and transferred to 15 1.11 of denaturing solution containing 10% SDS and 5% fi-mercaptoethanol. The samples were heated at 100° for several minutes and were then subjected to electrophoresis in a 12% SDS-polyacrylamide gel at 100 volts for 6 hr. The polypeptides in gel were stained with Coomassie brilliant blue. The gel was dried and was then autoradiographed (15). The 38,000-dalton polypeptide of eIF-2 was excised from the gel. The radioactivity associated with the polypeptide was determined. Results The addition which

protein

3':5'

cyclic

deficiency;

and Discussion

of GTP (2 mM) promotes

chain

initiation

monophosphate

is

inhibited

independent

RRI;

3) by the

addition

synthesis

by the

protein

or 2) in hemin-supplemented

purified

protein

lysates

of oxidized

in lysates

activation

kinases by the glutathione

in:

in

of adenosine 1) heme

addition (GSSG);

of and 4)

of low levels of double stranded RNA (10-11). These findings raise the possibility that a common mechanism may be responsible for the promotion and the

restoration

by examining 38,000-dalton

the

of protein state

subunit

and the of eIF-2

synthesis.

We investigated the mechanism of -__ in situ phosphorylation of the in lysates in the presence of high specific level

873

Vol. 109, No. 3, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

123456766 Fig 1. -__ IN SITU PHOSPHORYLATION OF eIF-2 (38,000-DALTON SUBUNIT) IN HEMIN-SUPPLEMENTED LYSATES INCUBATED WITH PURIFIED HRI. Hemin-supplemented (15 PM) lysate protein synthesis mixtures were incubated at 30' with [Y32P]ATP. At intervals, aliquots (3 pl) were removed and analyzed by A; lanes 1-3, samples incubated electrophoresis in SDS-polyacrylamide gel. without BRI for 2, 4 and 6 minutes, respectively; lanes 4-6, samples incubated with HRI (0.4 pg) for 2, 4 and 6 minutes, respectively; lanes 7-9, samples incubated with HRI (0.4 ug) and GTP (2 mM) for 2, 4 and 6 respectively. The autoradiogram in this experiment was exposed minutes, for 22 hr. B; lanes 3-7, the samples were incubated without HRI for 2, 5, 7, 9 and 13 minutes, respectively; lanes 8-13, samples incubated with HRI (0.4 ug) for 2, 5, 7, 9, 11 and 13 minutes, respectively; lanes 14-19, samples incubated with HRI (0.4 ug) and GTP (2 mM) for 2, 5, 7, 9, 11 and 13 minutes, respectively; lanes 20-23, samples were incubated with After HRI (0.4 ug) alone for 5 minutes and then GTP (2 mM) was added. 7, 9, 11 and 13 minutes of incubation, respectively, aliquots (3 ~1) were The autoremoved for assay by SDS-polyacrylamide gel electrophoresis. radiogram in this experiment was exposed for 80 hr. Lane 1 in B shows position of stained 38,000-dalton subunit of eIF-2 and lane 2 is the autoradiogram of lane 1 showing phosphorylated 38,000-dalton subunit of eIF-2 as an internal control.

[Y~~P]ATP

activity

this approach is preserved which

are

under

being: and there

associated

aliquots

are

in Fig

38,000-dalton lA,

lanes

lanes

l-3

is

various

little

of

is

is

system. stopped

1A and B show the

subunit

of

and Fig

lB,

4-6 and Fig

lB,

eIF-2

lanes

in

lanes 8-13)

kinetics

other

or in

situ

intervals,

are subjected

the 38,000-dalton

polypeptides.

of phosphorylation lysates

the presence

or in the presence 874

short

samples

hemin-supplemented

3-7)

in -~

artifacts

At very

in which

from

of

kinases

and the

gel

resolved

protein

of introducing

a fractionated

completely

the

likelihood

the reaction

The advantage

conditions.

specificity

in SDS-polyacrylamide

of eIF-2 Data

the

with

removed,

to electrophoresis subunit

these

that

of the alone

of HRI (Fig

(Fig

lA,

of HRI and GTP (Fig

BIOCHEMICAL

Vol. 109, No. 3, 1982

AND BIOPHYSICAL

12

RESEARCH COMMUNICATIONS

14

8

19

Fig 2. IN SITU PHOSPHORYLATIONOF eIF-2 (38,000-DALTON SUBUNIT) IN HEMIN DEFICIENT LYSATES. Hemin deficient lysate protein synthesis reaction mixtures were incubated with [Y3%']ATP at 30°. At intervals, and subjected to electrophoresis in SDSaliquots (3 pl) were removed polyacrylamide gel as described in Materials and Methods. The autoradiogram shows: lanes 1, hemin-supplemented lysates containing eIF-2 and HRI as an internal control for eIF-2 phosphorylation; lanes 2-7, hemin deficient lysates incubated for 2, 5, 7, 9, 11 and 13 minutes, respectively; lanes 8-13, hemin deficient lysates to which 2 mM GTP was added at the start of incubation and samples were removed after 2, 5, 7, 9, 11 and 13 minutes of incubation, respectively; and lanes 14-19, hemin deficient lysates were incubated with [Y3*P]ATP for 5 minutes and GTP (2 mM) was then added. The samples were removed after 7, 9, 11, 13 and 15 minutes of incubation, respectively.

lA,

lanes

exposed

7-9 and Fig for

a short

lB, time,

in hemin-supplemented the

autoradiogram

lation this

in control point

time.

This

samples

deliberate

is

over

in the state of hemin

and HRI (Fig under

exposed

1A and B; notice

presence

little

lysates is

14-19). if (Fig

for

otherwise

lA,

alone lanes identical

any eIF-2 lA,

of fully

the dramatic (Fig

l-3).

period lB,

the

is

If

is

the

lanes

and partially difference

expense is

observed

1-3)

and in the presence

compared

with

Fig

lanes

In the presence 875

hand phosphory-

a long

of reducing

A, lanes

lB,

other

To emphasize for

phosphorylated that

observed

eIF-2

3-7).

autoradiograms

at the

conditions).

is

on the

of time,

(Fig

exposed

autoradiogram

phosphorylation

lanes

a long

exposure

4-6);

When the

manifested

we have deliberately

differences (Fig

lanes

the

eIF-2 in the of hemin

3-7 and 8-13, of BRI,

eIF-2

Vol. 109, No. 3, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

1

16

Fig OF eIF-2 (38,000-DALTON SUBUNIT) IN HEMIN--IN SITU PHOSPHORYLATION SUPPLEMENTED LYSATES IN THE PRESENCE OF OXIDIZED GLUTATHIONE. Heminsupplemented (15 pM) lysate protein synthesis reaction mixtures containing 0.5 mM GSSG were incubated with [Y3*P]ATP; lanes l-6, samples incubated for 2, 5, 7, 9, 11 and 13 minutes, respectively; lanes 7-12, samples containing GSSG and 2 mM GTP (added at the start of incubation) were incubated for 2, 5, 7, 9, 11 and 13 minutes, respectively and; lanes 13-16, GSSG containing reaction mixture incubated for 5 minutes and then GTP (2 mM) was added and samples were removed after 7, 9, 11 and 13 minutes of incubation, respectively.

phosphorylation

is

(Fig

minutes

incubation tion

lB,

rapid,

8-13).

in the presence

during

first

progressive reaches

very

lanes

but

of HRI,

2 minutes slow

a plateau

(Fig

rate

in about

phosphorylation

in hemin-supplemented

incubation

in

and progressive is

assayed

the

presence

of

of eIF-2

during

in

lane

14),

in this (Fig

results hemin

(Fig

in this

for

little

eIF-2

thereafter,

lB,

lanes

lB,

lanes is

5 minutes,

first

there

2 minutes,

observed

deficiency

(Fig

876

(Fig

2);

or

in

to that When GTP (2

during is

a rapid

lB, eIF-2

a

The rate

similar

followed

when --in situ

is

that

14-19). 3-7).

of

phosphoryla-

there

achieved

dephosphorylation

are

3-4 start

phosphorylation

phosphorylation HRI

about

at the

in

added

of GTP is very

control

eIF-2

increase

maximum

is very

in the presence

full

The same basic tion

lB,

after

dephosphorylation

there

7-9 minutes

of eIF-2

added

a near

of increase

observed mM) is

reaching

When GTP (2 mM) is

preinitial

by a slow lanes

20-23).

phosphoryla-

hemin-supplemented

8lOCHEMlCAL

Vol. 109, No. 3, 1982

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

19

12

Fig. 4: IN SITU PHOSPHORYLATIONOF eIF-2 (38,000-DALTON SUBUNIT) IN HEMIN-SUPPLEMENTED LYSATES IN THE PRESENCEOF DOUBLE STRANDEDRNA: Hemin-supplemented (15 PM) lysate protein synthesis reaction mixtures containing double stranded RNA (17 &ml) were incubated with [Y3'P]ATP: lanes 2 to 7, samples incubated for 2, 5, 7, 9, 11 and 13 minutes respectively; lanes 8 to 13, samples in addition contained 2 mM GTP and incubated for 2, 5, 7, 9, 11 and 13 minutes, respectively; lanes 14-19, the were incubated for 5 minutes double stranded RNA containing reaction mixture and then GTP (2 mM) was 17 minutes of incubation gel containing eIF-2 as

lysates

in the presence

RNA (Fig

4).

deficiency

A notable and these

which

eIF-2

hemin

deficiency

(Fig

lB,

(Fig

lysates

levels

lanes

level

3-7)

start

levels

other

during

of eIF-2

(Fig

1,2,3).

associated eIF-2

with is

13,

15,

of stained

stranded

in heme-

rapidity

50-75x

that

should

with

of incubation ideal

hemin

observed be noted

that

of eIF-2

obtained

phosphorylated,

(under

of optimum

with it

the 38,000-dalton fully

ation

two minutes

phosphorylation

even below (data

11,

in

2).

compared

Nearly form

9,

of double

is the

first

in the presence

of phosphorylation

7,

phosphoryl

modes of inhibition

2, lane

are

after

3) or low in eIF-2

of incubation,

in dephosphorylated after

of GSSG (Fig difference

conditions)

GTP at the the

an autoradiogram

is phosphorylated

When the synthesis

added, samples were removed Lane 1 is respectively. an internal maker of eIF-2.

by the that

observed under

subunit within 877

of eIF-2). 2 minutes

addition

GTP often

of reduces

in hemin-supplemented

this

by counting

protein

concentrations

condition

exists

the radioactivity When GTP is about

25-50x

added of

Vol. 109, No. 3, 1982 eIF-2

is

from

dephosphorylated

double

stranded

In these lysates

BIOCHEMICAL

fall

rapidly

during

first

with

lysates

do we see differences subunit

significantly

higher

eIF-2

phosphorylation

GTP serving not

increase

in eIF-2

that

these

(lo),

phosphorylation eIF-2

degree If

of dephosphorylation order:

dR1. is

also

These HRI-like

in these full

four

restoration samples

added

determined observations inhibitors fully

in the presence

if

this

is to

about

an

the block

in

to unlabelled

was the

of eIF-2

case one

phosphorylation

in

of dephosphory-

significant protein

the

of low

in view synthesis

is observed

> plus

of double

of the four results rescue

(11).

by GTP in double

levels

level

of dsRNA activate of rescue

by the

inhibitor

suggest

that

only

of synthesis concentration

there

in GSSG treated of GSSG such that

addition

often

difficult stranded

of double limited

amount

in the presence

stranded of of HRI

(unpublished

results).

amount

of HRI (or

may be limited lysates)

is

The

of protein

modes of inhibitions

controlled

It

and

dsRNA.

of protein

the degree

modes

show the

HRI > plus

in GTP induced

a carefully

in heme

of phosphorylation

by GTP, the

synthesis

of

of purified

levels

sensitivity

to prevention

of eIF-2

unless

and these

Similarly

or in the presence inhibition

ATP is

be attributed

promote

GSSG > heme-deficiency

of sensitivity

RNA treated RNA is

with

can be attributed

of GSSG, in the presence

synthesis

following synthesis

deficient

which

GTP (16),

by 2 mM GTP are

one considers

of protein

promotion

to get

in hemin

and the promotion

GTP concentrations

RNA (11).

of inhibition

same order

4-13

associated

Since

Moreover,

in the presence

and to a lesser

stranded

that

rate

the amount

of incubation

therefore,

in the slow

of phosphorylated fact

of label

of

equilibrium

during

(only

than

due to

samples.

deficiency HRI,

by GTP cannot,

see the

The block the

donor

donor.

Because

appreciably

of [Y~~P]ATP).

because

reaches

subunit

2 minutes,

phosphate

as a phosphate

GTP containing lation

activity better

also

to

on the kinetics

of incubation.

change

first

but

redistribution

2 and 4 minutes

during

of specific

this

added

not only

phosphate

in the amount

during

obtained

[Y~~P]ATP

Our results

38,000-dalton

does not

38,000-dalton

of magnitude

that

of

of incubation,

ATP.

2 minutes

of incubation

would

within

associated

order

period

suggest

are generally

activity

ATP and creatine

minutes

lowering

specific

of label

of radioactivity

percentages

RESEARCH COMMUNICATIONS

samples).

initial

of unlabelled

phosphorylation

very

the

during

some redistribution eIF-2

lower

RNA treated

experiments

will

of presence

(the

AND BIOPHYSICAL

activated

in heme deficiency

of GTP restores

or prevents

and rapidly.

A question must be asked and blocks eIF-2 phosphorylation?

as to how GTP promotes Several possibilities 878

dephosphorylation may be considered:

Vol. 109, No. 3, 1982 1.

BIOCHEMICAL

GTP may serve

In this

as a cofactor

respect,

above)

it

inhibit

should

(17,18).

the

eIF-2

be noted

that

high

binding

site

of eIF-2

on the protein of eIF-2

phosphoprotein

phosphatase,

would

GTP would,

result.

synthesis.

This

view

kinase;

observed

the

promote

the

reversion

(19) al., inactivates

in

have

shown that

the

form

results).

together,

promotion

of protein

promotion lation

(Fig

4).

It

state

level

phosphorylation

was indicated

for

a period

reported

synthesis

eIF-2

There Gross

of another

under

these

3-4 minutes

that

both

(20).

factors. in the less

eIF-2 serve

efficient

dephosphory-

to block

the

dRI.

A similar

phosphorylation

the

or in the

from

efficient

the activated

in a previous

that

as effective

evident less

GTP (2 mM)

of these

quite

rapid

study Recently

here

and assayed

at 30°,

reaching

rate

in which Hronis

of eIF-2 assays

were

and Traugh

in heme deficient

lysates

this phosphorylation was kinetically in 20 minutes. Inhibition of protein

conditions

would

of incubation

be expected

(16,22,23)

to take

and maximum eIF-2

place

slow during

phosphory-

will precede (by about 0.8-l minute) the onset of inhibition chain initiation. The reason for this extremely slow rate phosphorylation These

GTP promoted

results protein

is not described synthesis

is et factor

RR1 (unpublished suggest

GTP (2 mM) does not

of 2 minutes

--in situ eIF-2 incubated at 34". Curiously, and did not go to completion

lation protein

would

and

form.

We have noted

is

in 2-4 minutes.

concept.

Recently

of --in situ eIF-2 phosphorylation described of 15 minutes of incubation is very rapid

a period

performed

This

to inactivate

(10-13).

this

of phosphorylated

and also

may be that

and fails

a steady

first

(10,19).

observations

synthesis.

the

polypeptides for

by GTP in heme deficiency

phosphorylation

The rate

(21)

lysate

support to inactive

of dsRNA, GTP (2 mM) is not

of protein

in eIF-2

activation

over

synthesis

that

synthesis

of GSSG or RR1 may be due to a combination

In the presence block

these

eIF-2 protein

of RR1 in heme-deficiency

of RRI.

the dephosphorylation

presence

provide

the

eIF-2

finding

GTP and dGTP in the presence

reversible

Taken

of the other

contention

and dGTP enhance

to block active

to promote

on protein

inhibitior

of this

the ATP

of phosphorylated

the activation

of active

support

of a very

GTP effect

of most

in

ATP (1 mM). for

by the earlier

of GTP, also

3. GTP (2 mM) may prevent

of GDP (2 mM and phosphatase

be expected

supported

phosphatase.

GTP may serve

dephosphorylation

phosphorylation

in the presence

good evidence

levels

with

hence,

therefore,

of ATP antagonizes

The reduced

phosphoprotein

and in the presence

is also

RESEARCH COMMUNICATIONS

phosphoprotein

2. GTP (2 mM) may compete

phosphorylation

addition

for

the activity

lysates

AND BIOPHYSICAL

of of

clear. here

provide

under

diverse 879

a unitary conditions

molecular

basis

and also

for

provide

Vol. 109, No. 3, 1982 further tion

support of eIF-2

dynamic shifts

optimum

of eIF-2

that

by blocking

the eIF-2

and dephosphoryla-

of protein

synthesis

dephosphorylation

kinase eIF-2

is

RESEARCH COMMUNICATIONS

the phosphorylation

in which

towards

initiation

AND BIOPHYSICAL

conditions

protein

equilibrium

synthesis

synthesis

the view

of equilibrium

level this

protein

for under

state

increased

BIOCHEMICAL

(following

its

phosphorylation.

inhibited protein

(18). kinase

are

is

in a

favored.

An

activation) As a consequence,

GTP promotes catalyzed

protein

phosphorylation

of eIF-2. ACKNOWLEDGEMENT. This Experiment Station/USDA

work was supported by a NSF Grant 80 21969 and Animal Health and Disease Research Program.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

Ochoa, S. and deHaro, S. (1979) Ann. -~ Rev. Biochem. 48, 549-580. Gross, M. (1980) Molecular CellularBiochem. 31, 25-36. Maitra, U., Stringer, E. A. and Chaudhuri, A. (1982) Ann. Rev. -Biochem. 51, 869-900. Biochem. Biophys. Res. Commun. 107, 828-833. Ranu, R. S. (1982) Ranu. R. S. (1979) Biochem. Biophys. &. Commun. 91, 1437-1444. -Ranu, R. S. (1980) Biochem. Biophys. Res. Commun. 97, 252-262. Ernst, V., Levin, D. H. and London, I. r (1980) Proc. Natl. Acad. --Sci. USA 77, 1286-1290. Proc. Natl. Acad. Sci. USA. ?&u,R. S. and London, I. M. (1979) -__--76, 1079-1083. Das, A., Ralston, R. O., Grace, M., Roy, R., Ghosh-Dastidas, P., Das, H. K., Yaghmai, B., Palmieri, S., and Gupta, N. K. (1979) Proc. Natl. Acad. Sci. USA. 76, 5076-5079. Balkow, K., Hunt, rand Jackson, R. J. (1975) Biochem. Biophys. Res. Commun. 67, 366-374. I. M. (1976) Ernst, V.. Levin, D. H., Ranu, R. S. and London, Acad: 73, 1112-1116. Proc.'Nd. ~ -Sci. USA -. Delaunav, J , Ranu, R. S., Levin, D. H., Ernst, V. and London, I.M. (i977 1 ----Proc. Natl. Acad. Sci. USA. 74, 2264-2268. Ranu, R. S. (1980) Biochem. Biophys. Rx. Commun. 97, 1124-1132. Ranu, R. S. (1980) FEBS Lett. 112, 211-215. Ranu, R. S. and London,.M. (1979) Methods Enzymol. 60, 459-484. R. J. and Trachsel, H. Farrell, P. Balkow, K., Hunt, T., Jackson, (1977) Cell 11, 187-200. Proc. Natl. Acad. Sci. USA. Safer, B. and Jagus, R. (1979) ----~ 76, 1094-1098. Res. Ranu, R. S. and Bhalla, K. K. (1981) Biochem. Biophys. Commun. 102, 30-37. Gross, M., Watt-Morse, P., and Kaplansky, D. A. (1981) Biochem et Biophys. Acta 654, 219-226. Acad. Ernst, V., Levin, D. H. and London, I. M. (1979) Proc. Natl. Sci. USA. 76, 2118-2122. &nis, T. and Traugh, .I. (1982) J. Biol. -Chem. 256, 11409-11412. Maxwell, C. R., Kamper, C. S. and Rabinovitz, M. (1971) --J. Mol. gi&. 58, 317-327. Hunt, T., Vanderhoff, G. A. and London, I. M. (1972) J . Ms. Biol. 66, 471-481.

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