Protein synthesis elongation factor 1 from rat liver: A zinc metalloenzyme

VoL67,No.

BIOCHEMICAL

3,1975

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

PROTEIN SYNTHESIS ELONGATION FACTOR 1 FROM RAT LIVER: A ZINC METALLOENZYME Philip

S. Kotsiopoulos

and Scott

Department Boston Boston, Received

October

C. Mohr*

of Chemistry University

Massachusetts

02215

3,1975

Highly purified elongation factor 1 (light form, EFlL) from rat liver contains zinc as determined by atomic absorption spectrophotometry. Analysis has been performed on the most active protein fraction from DEAE-Sephadex chromatography (estimated purity: 90%) and on the main band obtained from this fraction by polyacrylamide gel electrophoresis. The data are consistent with a stoichiometry of approximately one g-atom of zinc per 54,000 daltons of EFlL protein. A functional role for Zn2+ is suggested by the fact that 0.3 mM l,lO-phenanthroline completely abolishes GTP binding by EFlL (measured by the nitrocellulose filter retention assay), while the isomeric non-chelator 1,7-phenanthroline has no effect. This inhibition can be overcome by the addition of excess zinc ion. Tightly a number include (3),

aspartate all

(5).

from

In this

ribosome

next

of the newly

*to

protein,

Hydrolysis steps

(4),

factors

A site

e,

important

through

and murine,

and nucleic (l),

EF-T

(E. coli) the

lengthened

used:

synthesis,

should EFlL -EFlN --

feline, a study

binding

I (2)

homologous

liver)**.

formation from

the ribosomal

of the peptide the A site

factor factor

viruses

protein

synthefactor

to the mRNA-

site

ternary (6).

comThe

bond and translocation

to the P site

1 (light 1 (heavy

myelo-

Elongation

be addressed elongation elongation

examples

of avian

and RD-114 RNA tumor of the

in

and RNA polymerase

of aminoacyl-tRNA

from

roles

Notable

of an aminoacyl-tRNA*EFl*GTP

EFl*GDP

peptidyl-tRNA

metabolism.

transcriptases

and EFl (rat

formation

of GTP releases

in protein

acid

and functional

DNA polymerase

simian,

we have begun

the

structural

and the reverse

catalyzes

whom correspondence

**Abbreviations

play

transcarbamylase

laboratory

elongation

ions

of nucleotide

Escherichia

virus

1, a soluble

plex.

zinc

of enzymes

blastosis

sis

bound

form), form).

are cata-

Vol.67,No.3,

1975

lyzed

by peptidyl

soluble

protein,

factor

transferase EF2 (rat

EF-T consists

lized

(7).

partly

resulted

sucrose

because

reticulocytes

ranging

to be five

times

(l'),

suggesting

that

reports

of the

preparations

association

Using

the

daltons

the

former

of zinc

presence

is

(10).

EFlL

of this MATERIALS

the

functionally

in EF-TU

have been crystal-

(8,9)

has been difficult

(12)

prompted

Its

(EFlN)

EFl from and comprises

from calf ternary active

isolation

to the

electrophoresis

same criteria,

in forming the

gel

homogeneity

each

The prokaryotic

phospholipids.

by disc these

and another

which

M.W. >200,000

to apparent

more efficient

presence

for

from

subunit)

respectively.

sources with

as determined

of 62,000

ribosomal

and EF-Ts,

animal

in size

has been purified

shown

large

EF-Tu

various

centrifugation.

subunits

of the

or EF-G (E. coli),

of its

M.W. 50-60,000,

gradient

identical

liver)

EF? from

in aggregates

(EFlL),

(part

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of two entities,

Previously

to purify,

form

BIOCHEMICAL

brain

complex species.

us to examine

light and rabbit three has been

than

EFlN

Unpublished our

EFlL

metal. AND METHODS

The procedure used in the purification of EF? was that of Collins et al. A combined column of Sepharose 46 over(8) with the following modifications: layed with Sephadex G-200 (2:l vol/vol) was used instead of Sepharose 6B gel filtration. DEAE-Sephadex A-50 chromatorgraphy (1.5 x 40 cm column) followed the hydroxylapatite step and used a linear 0.1-0.5 M KC? gradient in 20 mM TrisHC? (pH 7.5), 1 mM DTT and 0.1 mM EDTA. The final column eluted EFlL at 0.4 M KC?. (See Table I.) Unfractionated according to (13).

E. coli

tRNA was prepared

and aminoacylated

with

[14Cl-phe

The assay for EF? activity was based on the ability of EFl to bind GTP, forming a complex which is retained on nitrocellulose filters (14). The reaction of the EFl*GTP complex with phe-tRNA to form a phe-tRNA*EFl*GTP ternary complex that is not retained on nitrocellulose filters was routinely performed as described (14) with the modification that phe-tRNA was incubated with EFl*GTP for only 30 sec. at 0". All radioactivity determinations were made using a Picker scintillation counter and a water-miscible scintillation fluid (15). Sodium-dodecyl-sulfate gel electrophoresis (16) and sucrose gradient centrifugation, 5-20%, (17) were used to determine the molecular weight of EFlL Both methods agreed closely with a using known standard proteins as markers. The EF? preparations following either hydroxylapatite M.W. of 54,000 + 4,000. or DEAE-Sephade? A-50 chromatography were analyzed using polyacrylamide gel electrophoresis (18) and determined to be approximately 60% and 90% pure EFlL, To assay for EF-1 activity, the gels respectively, by densitometric analysis. were sliced and treated as described in (8). GTP-binding activity was eluted from two major bands with approximately 60% recovery of EF? activity (Figure 1).

980

Vol.67,No.

BIOCHEMICAL

3,1975

EF IL

DYE

pmoles ’ GTP bound

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

2 I -

II-JIL migrotion

Figure

1.

Analysis

of EFl Preparation

+

by Polyacrylamide

Gel Electrophoresis.

Top frame: Photograph of gel stained with Coomassie Blue and densitometer tracing of the same sample (from hydroxylapatite). Bottom frame: EFl activity profile derived from slices of a gel identical to that depicted above. Protein was extracted from the slices and assayed as described in MATERIALS AND METHODS.

To assay for Zn2+ all glassware and standards were treated as described by Thiers (19) to avoid metal ion contamination. The gel slices corresponding to EFlL were digested with 2.0 ml concentrated HCl/HNO3 (3:l) and if necessary further oxidized with 1.0 ml hydrogen peroxide (BDH, ultra-pure). The residue In cases where analyses was dissolved in 1.0 ml 1% HN03 to give a clear solution. were performed directly on free enzyme in solution, the sample was digested with 8 N HN03 to evaporation and the residue dissolved as above. All assays were done using a Perkin-Elmer 305A atomic absorption,spectrophotometer with the flame attachment and the resonance line at 2138 A (20). In routine analyses protein concentration was determined by the method of Warburg and Christian (21). Absorbances at 280 and 260 nm were measured on a Zeiss PMQ II spectrophotometer. When highly pure enzyme was used protein concentration was also determined by the Lowry method (22) with crystalline bovine The two methods agreed within 10%. serum albumin as standard. [14C]-phenylalanine and [ 3H]-GTP were obtained from New England Nuclear; ATP, GTP, pyruvate kinase and phosphoenolpyruvate were from Sigma; hydroxylapatite (hypatite C) was from Clarkson Chemical Co.; Sephadex G-200, Sepharose

981

Vol.67,No.3,

1975

BIOCHEMICAL

Purification

TABLE I.

of EFlL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

from

Rat Liver

Figures in the table refer to a preparation starting from 340 g (wet weight) One unit of EFl activity binds one pmol GTP per minute per of frozen liver. milligram protein in the standard assay at 37". Yields given in the table reflect the selection of the EFlU fractions after gel filtration and hydroxylapatite chromatography and the EFlL fractions after DEAE-Sephadex chromatography. Fraction

Protein

(ml)

(mg)

SN-lOOa

500

28,000

56

4.8

100

Ammonium sulfate (35-7O%)b

150

9,750

65

11.4

83

Gel filtration

40

600

15

32.0

14

Hydroxylapatite

30

75

DEAE-Sephadex

10

apost-microsomal bdialyzed

Protein cont. (mg/ml)

Yield

Vol.

1.5

Specific activity (units/mg)

(X)

2.5

133

7.4

0.15

780

0.9

fraction

vs. 50 mM Tris-HCl

(pH 7.2)

and 1 mM DTT

filters were from 4B and DEAE-Sephadex A-50 were from Pharmacia ; nitrocellulose was purchased from K & K Laboratories, the Millipore Co.; l,lO-phenanthroline and 1,7- and 4,7-phenanthroline were purchased from the G.F. Smith Co. The latter two compounds were once-recrystallized from ethanol/water mixtures before All other chemicals were reagent grade. use. RESULTS Metal

Content

of EFlL

We performed the EFlL well liver

protein

as the free EFlL

atomic

band from

54,000.

halved

there

the Zn2+/protein

which

1.3 + 0.3

As shown

ratio

after

increased

for

II,

Zn2+ on gel could

DEAE-Sephadex

(SD) g-atom

in Table

was good correlation

analyses enzyme activity

enzyme solution

contained

weight

absorption

Zn2+/mole

be eluted

from 0.4

982

net

(Figure

1) as The rat

of enzyme of molecular the

ppm Zn*+ observed.

ug Zn2+/mg

containing

chromatography.

in each case where

in the

slices

protein

enzyme sample The fact at the

was that

hydroxyla-

Vol.67,No.3,

1975

TABLE II.

Zinc

BIOCHEMICAL

Content

of EFll

from

The mean value for zinc content of (mol. wt. 54,000). Preparation A EFlL by densitometric analysis of DEAE-Sephadex A-50, 90% pure. All where noted. The blank consisted case of free enzyme analyses which albumin.

(0.050)

1.46

A-Z

60

0.083

(0.045)

1.14

30

0.040

(0.045)

1.10

36

0.068

(0.072)

1.56

18

0.036

(0.072)

1.65

A-4b

60

0.073

(0.042)

1.01

B-l

77

0.123

(0.052)

1.32

B-2b

36

0.042

(0.055)

0.96

in the tablearethe net are given in parentheses.

were

of purification

to nearly

1.0 pg Zn2+/mg

stage

Zn2+ is

indicates

on EFlL*GTP

complete

on nitrocellulose

the

mixture

concentrations (see

Table

10m4 M Zn2+.

throline,

of EFl*GTP filters)

had no effect

activity

equal

directly

in 8 N HN03

protein

at the final

of the enzyme.

(i.e.

no binary

to or greater were

The activity

on the

corresponding

when l,lO-phenanthroline

of l,lO-phenanthroline

The non-chelating

part

the

Activity

at a concentration

III).

hydrolyzed

an integral

Binding

inhibition

was retained reaction

observed;

solutions

of Chelators We found

ppm zinc

protein 60% pure is from except in the serum

Zinc Content (g-atom mole)

0.115

purification

with

Observed Zinca (Pm)

65

stage

activity

EFlL is 1.3 + 0.3 g-atom per mole is from hydroxylapatite, estimated polyacrylamide gels; preparation B analyses were done with gel slices of a protein-free gel slice except had a blank containing 50 pg bovine

A-l

bin these cases EFlL with gentle heating.

Lower

Rat Liver

EFl Present (us EF~L)

aValues blanks

Effect

RESEARCH COMMUNICATIONS

Preparation No.

A-3

patite

AND BIOPHYSICAL

less

was restored isomers,

GTP-binding

983

was present than

effective

3 x 10-4

The effect

in M.

in inhibiting

to 90% of the

1,7-phenanthroline activity.

complex

control

and 4,7-phenanof l,lO-phenan-

VoL67,No.

Figure

3, 1975

2.

BIOCHEMICAL

Recorder

Chart

Tracing

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

from Atomic

Absorption

Analysis

of EFlL.

The ordinate calibration comes from Zn2' standard solutions run on the same day. The sample consisted of a slice from a polyacrylamide electrophoresis gel containing the EFlL band (see Figure 1); the blank corresponds to an equivalent slice of a protein-free gel. Both slices were digested as described in the text and dissolved in 1% HN03 for analysis.

throline

cannot

be attributed

present

in large

excess

l,lO-phenanthroline EDTA inhibited tively

high

50%.

Since

by chelating

EFlL

reaction

much more strongly

much less

effectively

than

buffer

during

isolation

must represent

removal

since

mixture

and it

with

Zn2+ than

GTP-binding

contained of tightly

add ition

of Mg2+,

is

Mg2+ is

known that

with

Mg2+ (23).

l,lO-phenanthroline.

of EDTA (1 mM) reduced

by l,lO-phenanthroline,

restored

removal

complexes

the final

the inhibition

possible

(10 mM) in the

concentration

agents

to its

10-4 bound

A comparaactivity

by only

M EDTA, inhibition Zn2+ ions.

As with

of 10m4 M Zn2+ to EDTA-inh ibited

enzyme activity. DISCUSSION

Because EFlL

of the relatively

preparations

and the

contamination,

analytical

with

as a zinc

certainty

small rather data

absolute

considerable alone

metalloenzyme.

would

not Thus,

984

amounts

of Zn2+ ( 0.1

chances

of adventitious

suffice although

to identify our

atomic

ppm) in Zn2+ this

protein

absorption

Vol.67,No.3,

1975

TABLE III.

Effect

BIOCHEMICAL

of Chelating

AND BIOPHYSICAL

and Non-chelating Binding

RESEARCH COMMUNICATIONS

Isomers

on EFl-Nucleotide

Activity

Each assay (0.25 ml total volume) consisted of 25 ~1 enzyme solution which contained 80 Pg EFl, 25 ~1 reagent (incubated with EFl for 45 minutes prior to reaction), and GTP-binding mixture of 150 ~1, containing 0.10 M Tris-HCl (pH 7.4), 0.10 M NH4C1, 0.02 M MiC12, 3.75 x 10-3 M phosphoenolpyruvate, 10 ug pyruvate kinase and 2.5 x loM [3H] - GTP. System

pmol

16.7

0

+ 1.0 mM l,lO-phenanthrolinea

0.0

100

+ 0.3 mM l,lO-phenanthroline

0.0

100

+ 0.1 mM l,lO-phenanthroline

1.7

90

13.4

20

5.0

70

8.3

50

15.0

10

+ 1.D mM EDTA + 0.1 mM Zn2+

15.9

5

+ 1.0 mM 1,7-phenanthroline

17.0

0

+ 1.0 mM 4,7-phenanthroline

16.7

0

Control

+ 0.01

mM l,lO-phenanthroline

+ 10.0

mM EDTA

+ 1.0 mM EDTA +O.l

mM l,lO-phenanthroline + 0.1 mM ZnPtb

aThe phenanthroline isomers were 10% methanol. A methanol blank the reported values. b

(Table

Zn2'/mole

EFlL,

II)

consistently

showed

the demonstration

or EDTA (Table

of inhibition control

added from stock solutions containing (0.5 pmol GTP) was subtracted from

25 ~1 of 1 mM ZnC12 was added to the reaction mixture for 3 min. incubation at 37" before terminating the reaction.

analyses

line

% Inhibition

GTP Bound

III)

the presence

of inhibition

represents

by the non-chelating

essential

of approximately

of GTP-binding supporting

phenanthroline

an additional

by l,lO-phenanthro

evidence.

isomers

1.0 g-atom

provides

The absence an important

(4). Further

and to establish

experiments the

are

required

stoichiometry

to specify with

certitude.

985

the precise The Zn2'

role

of Zn2+ in EFlL

may merely

aid

in

Vol. 67, No. 3, 1975

maintaining

the

(11, so that

might

native

its

GTP-binding

activity.

provide

to almost

full

Zn2+ also

influences

in a subunit

of this

recovery the

growth

Co2+ for

Zn2+ in EFl poses

the

system

in which

peptidyl-tRNA,

exploration

occurs,

aminoacyl-tRNA,

EFlL,

of this

practical under

to chelating

agents

Drews et al.

(6,29))

stages

from other etc.)

as opposed

v&,

because

and GTP.

of Zn2+

been reported

for

in attempts

structural

--

role

complexity

of

of 80s ribosome,

A presumptive

resonance

role

GTP to H20 (or

for

mRNA, the metal

perhaps

techniques

might

first allow

liver

EFIL as a zinc

that

future

experiments

of well-defined

is

addition probably

with

metal

such as EDTA may be harmful and deliberate

metalloenzyme

ion

(as,

this

brain

be examined

for

protein

for

example,

Exposure in the work

of 0.1 mM Zn2+ to buffers advisable

in order

(14),

rabbit

the

presence

reticulocyte and possible

Charles

Terner

for

986

a generous

supply

of

in the

to to maintain (30). (lo),

EFl preparawheat

functional

ACKNOWLEDGEMENTS Professor

im-

should

concentration.

to the case of T7 RNA polymerase (calf

has the

of zinc.

We thank

the

(28).

conditions

should

that

of Zn2+ favoring

of the

an aggregate

Magnetic

of rat

sources

leads

involvement

to merely

(25-27)).

analogous

Zn2+

experiments.

from

of purification

maximum activity,

of the

been unsuccessful

transfer

consequence

be performed

of

dichroism

indicate

has recently far

loss

replacement

removal

A similar

phosphoryl

possibility

The identification

(31,32),

versa.

difficulties

catalysis

protein

of EFl,

We have thus

considerable

since

experiments

process

--

removal

in structure,

and vice

consequent

such as circular

In any event,

Zn2+ in reactivation

be to assist

to a ribosomal

probe

of aggregation

(24).

aspartate transcarbamylase

with

Preliminary

of a functional

for

tions

state

factor

Demonstration

latter

denaturation

an alteration of activity.

RESEARCH COMMUNICATIONS

as in E. coli

hypothesis.

of EFlH to EFlL,

to substitute

mediate

partial

aggregation/disaggregation

mouse nerve

would

conformation

causes

too drastic

conversion

AND BIOPHYSICAL

A conformation-sensitive

a test

cause

ion

protein

removal

must not

the

BIOCHEMICAL

of rat

livers,

germ role

Vol.67,No.3,1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Professor Standish C. Hartman for the use of his laboratory facilities, fessor Arno Heyn for assistance with the atomic absorption instrument, Marcus Horn for densitometer scans. National Cancer Institute grant 02 supported this research.

Proand Dr. CA 14397-

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