Reverse transcriptase from avian myeloblastosis virus: A zinc metalloenzyme

Vol.

57, No.

4, 1974

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

REVERSE TRANSCRIPTASE FROM AVIAN MYELOBLASTOSIS

COMMUNICATIONS

VIRUS:

A ZINC METALLOENZYME D. S. Auld,*

H. Kawaguchi,*

D. M. Livingston?

and B. L. Vallee*tt

*The

Biophysics Research Laboratory, Department of Biological Chemistry, Harvard Medical School, Division of Medical Biology, Peter Bent Brigham Hospital, Boston, Massachusetts and the Viral Lymphoma and Leukemia Branch, National Cancer Institute, National Institute of Health, Bethesda, Maryland, 20014 Received

March

4,1974

Previous postulates of a relationship between a zinc enzyme and the leukemic process (1,2) have led to the identification of the RNA dependent DNA polymerase -- reverse transcriptase -- of avian myeloblastosis virus as a zinc metalloenzyme. Microwave induced emission spectrometry provides a microanalytical system capable of measuring precisely lo-l1 to lo-l4 g atoms of metal in microgram amounts of enzyme, orders of magnitude more sensitive than other, conventional methods. The chromatographic fraction with highest enzymatic activity contains 1.5 x lo-11 g atoms of zinc per 1.4 pg of protein, corresponding to 1.7 to 1.9 g atoms of zinc per mole of enzyme for a molecular weight previously determined either as 1.6 or 1.8 x 105. The Zn/activity ratio is constant in the active fractions. detectio~fo;O~f~ rnrq?&"~~e,a;;,;~sent, i.e., at or below their limits of Cower, Agents known to chelate zinc inhibit the enzyme while their nonchelating isomers do not. The data underline the participation of zinc in nucleic acid metabolism and bear importantly upon the lesions which accompany leukemia and zinc deficiency. Introduction Differences

in zinc

led

to the postulate

cal

to the pathophysiology

Many enzymes

(3)

metabolism

that

disturbance

including

The existence

of an RNA dependent

given

new perspectives

other

malignant

zinc

in

tPresent ttTo

address:

zinc

essential

virus

for

(AMV) and other

regarding

of normal

Children's

whom correspondence

dependent

enzyme is

and lymphatic

a biochemical

leukemia

catalytic --

basis

and leukemic

leukocytes

Research

be addressed.

Foundation,

(4,5)

transcriptase

viruses

of leukemic

indications

criti-

activities.

reverse

RNA tumor

first

(1,2).

of E. coli

The earlier

Cancer should

their

DNA polymerase

transformations.

the metabolism

leukocytes

the DNA and RNA polymerases

to contain

myeloblastosis

and leukemic

of a zinc

of myelogenous

are now known

in avian

of normal

(6,7)

Boston,

has

and

of a role (1,2)

--

led

for to

Massachusetts.

BIOCHEMICAL

Vol. 57, No. 4, 1974 the present merase

kinetic

of AMV.

Materials

and spectrographic

The data

purification

that

Concentrations

MnC12,

2.4 nM; DTT,

2 mM; KCl,

at 60 min with Poly

it

of RNA dependent is

a zinc

were

rA and oligo Mass.,

poration.

All

1.11 aliquots obtained

DNA poly-

metalloenzyme.

(lo),

of zinc

to initiate

the

were

were

reagent method

in a series

grade. (9).

0.11

Approximately

M. which

was terminated

Microgram were

Nuclear

Cor-

was measured quantities

analyzed

on 15

of enzyme,

for

zinc,

copper,

discussed

designed Laboratories,

was established

metalloenzymes

Research,

to principles

Dayton

of the method

Collaborative

New England

facilities

Corporation,

of zinc

100 ~1

1 uM; 'HTTP,

Protein

according

instrumental

and accuracy

in any of the

dT12-1g,

from from

chromatography,

Research

used standard

reaction

obtained

70 C per mole,

using

by a modifica-

acid.

on 5 ~1 aliquots but

Monsanto

The validity

0.1 I-IM; oligo

added

exclusion

and manganese

in the

pH 7.8,

Lowry

virus

EDTA was not

M; Tris-Cl,

chemicals

by gel

previously

rA,

dT12-1g

the

(8).

0.10

and 'HTTP,

using

the purified

of agents

10% trichloroacetic

Waltham,

Wooten,

mM; poly

0.2

0.2 pg of protein

from

of Ross et al

steps.

are:

iron

demonstrate

was prepared

of the procedure

assay

studies

and Methods

AMV polymerase tion

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

by Dr.

George

Dayton,

Ohio.

by determination

of known metal

content

and

stoichiometry. Results

and Discussion

With rate is

poly

rA as the template

of the AMV polymerase linear

over

a 20-fold

assay pmoles

(see methods). TTP incorporated Metal

tion

both

complexing reversibly

catalyzed range

of DTT, KC1 and Mn2+ were

and oligo

The Km for per minute

and also

incorporation

as the

inhibit

to result TTP is

in optimal

conditions

the 12

at 25'.

the AMV polymerase

968

for

1 x 10 -5 M and the V is max

dependent,

the

Concentrations

per pig of protein

in a time

initiator,

of TMP at 25' pH 7.8

of enzyme concentration.

chosen

agents

dT12-18

catalyzed

irreversible

reacmanner.

Vol. 57, No. 4, 1974

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Effect

of Metal

Binding

Dependent

I Agents

DNA Polymerase

Metal Binding

on the Activity*

Concentration Agent

xlO,M

l,lO-phenanthroline

%

40 4

2

101

8

95

4,7-phenanthroline

3

98

8 hydroxyquinoline-5-sulfonate

1

33

5

15

3

30

5

2

*All

rates

measured

at 25".

**Enzyme activities inhibitor, v.

In the absence either

7 x 10

-5

at 25" or 37".

M.

intervals

decreases

At 37',

See methods

in the absence,

of such agents

1 mM, the enzyme is

the At 25',

inhibited

up to 60 minutes

other

conditions.

v c, and in the presence

enzyme is

completely

in the presence

instantaneously

preincubation

for

both

for

60 min-

of l,lO-phenanthroline,

and reversibly

of enzyme with causes

stable

of

with

a KI of

l,lO-phenanthroline

instantaneous

at

and irreversible

in activity.

The metal the

4

2

EDTA

time

v/v ** -c

1

1,7-phenanthroline

utes

(AMV) RNA

inhibition:

complexing its

isomers

properties 1,7

of l,lO-phenanthroline

and 4,7-phenanthroline

969

account neither

bind

for metals

BIOCHEMICAL

Vol. 57, No. 4, 1974 nor

do they

where

inhibit

the nucleotide

l,lO-phenanthroline

number

AND BIOPHYSICAL RESEARCH COMMUNKATIONS

of other,

polymerization

inhibits

structurally

it

activity that turally

agents

bind

indispensible

the presence essential

quantitative Microwave

induced

detection

limits

tion

emission

metal

to us for of either

zinc,

after

removal

tein

contaminants

were

analyzed

in a sharp, enzyme.

for narrow

copper

transition

band with

10 -13

of the most

were

from in

(Table

active

enzyme placed pur ,if ied

II).

on the

preparations

for

This of the

The pre-

the observed

with

were

high

g atoms.

enzyme

inhibi-

deterweight

pro-

emerged

precision

Zinc

(Table

absent,

than

is

II),

i.e.,

to less

activity,

present but

times

higher

activity

is

closely

enzyme employed

for

the

the amounts 0.06

g atoms

7.5 pmole/min/ug

three

970

pre-

v, of 2.1 pmoles/min/ul

virtually

is

means.

Enzyme activity

to 10 -14

correspond

thus,

The 45 ~1 fractions

quantitatively

the purified

the usual

and low molecular

activity,

10 -11

to allow

of enzyme

by this

and protein.

No. 19, column.

of

of enzyme.

amounts

chromatography.

The specific

fraction,

sensitive

and manganese

agents

Cu, Fe and Mn are g atoms,

struc-

of course,

g atoms and,

account

elements

measured

vary

to lo-l4

to 10 -14

could

maximal

quantities

metals,

of enzyme

of highly

activity

amounts

in stoichiometric

detected,

metals,

is,

can extend

seemed feasible

quenching

exclusion

and/or

The demonstration

amounts

on the microgram

and these

by gel

metal.

however,

or iron

of metal

The elements

when absolute

the

purpose

EDTA

interpretation

functional

small

of magnitude

of the AMV polymerase,

mined

mole

this

the

been sufficiently

on very

analyses

e.g.,

a

the polymerase

of such a metal

spectrometry,

by 6 orders

Moreover,

agents,

support

IIB

conditions

hypothesis,

have not

determinations

quantitative

sence

of this

I).

inhibit

unidentified

quantities

procedures

metal

markedly

or group

of stoichiometric

binding

Such data

to a hitherto

to the verification

available

I).

transition

Conventional

cise

(Table

under

(Table

metal

also

instantaneously these

completely

different

and 8-hydroxyquinoline-5-sulfonate

reaction

than

per

protein that

similar molecular

of to that

Vol. 57, No. 4, 1974

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Analysis

of Gel Exclusion (45 ~1)

Fraction

II Chromatography

Exhibiting

Maximal

Activity*

Protein

Zn/Protein

1-lg

g at/mole

0.7

0.7

1.8

c.001

<.Ol

.06

19

1.5

1.4

1.9

<.OOl

<.Ol

.05

20

1.2

1.1

1.9

<.OOl

<.Ol

.05

Zn

No.

g at x 10

18

11

Cu

determinations

zinc

in 1.4 ug of protein

of zinc

per mole

1.8 x lo5 control

is

cates ing

Zinc

structure

in

function

on the reverse

in the formation a leukemic

on the induction

process, of zinc

of leukemia

to 1.9

found

of DNA from

under

g atoms 1.6 or

(3).

or

For the

to be determined. from AMV show that

RNA templates a hypothesis

acid

of

in catalysis

or both remain

confirming

1.7

to be either

directly

transcriptase

in nucleic are

to from

indirectly

alternatives

of this polymerase are expressed as Analyses were

g atoms

weights

participate

Mn

of 1.5 x lo-l1

corresponds

molecular

these

studies

The roles

II)

may either

and affect

essential

zinc (1,2).

(Table

polymerases,

The present

The detection

of enzyme for

(11-13).

nucleotidyl

zinc

(11-13).

Fe

g at/mole

*Calculations are based on a molecular weight Zinc and protein content of 1.8 x lo5 (13). g atoms or vg per 5 ~1 aliquot, respectively. performed in triplicate.

weight

Fractions

metabolism

and impliof long

stand-

and its

bearing

Todaro,

Edward

study.

Acknowledgements We appreciate Scolnick

and Jeffrey

the advice

and counsel

Ross.

971

of Drs.

George

Vol.

BIOCHEMICAL

57, No. 4, 1974

This Institutes

work

was supported

of Health,

AND

BIOPHYSICAL

by Grant-in-Aid

of the Department

RESEARCH

GM-15003 of Health,

from

Education

COMMUNICATIONS

the National and Welfare.

References 1. Vallee, B.L., Fluharty, R.G. and Gibson, J.G., 2nd, --~ Acta Union Intern. Contre Cancer 6, 869 (1949). 2. Gibson, J.G., 2nd, Vallee, B.L., Fluharty, R.G. and Nelson, J.E., Acta Union Intern. 6 1102 (1950). -Contre ~Cancer -9 3. Vallee, B.L. and W.E.C. Wacker, -The Proteins, Vol. 5, 2nd Ed., H. Neurath (Ed.), Academic Press, New York (1970). 4. Slater, J.P., Mildvan, A.S. and Loeb, L.A., Biochem. Biophys. -*Res Commun. 64, 37 (1968). 5. Scrutton, M.C., Wu, C.W. and Goldthwait, D.A., --Proc. Natl. Acad. --Sci. U.S.A. 68, 2497 (1971). 6. Temin, H.M. and Mizutani, S., Nature 226, 1211 (1970). 7. Baltimore, D., Nature 22, 1209m). 8. Ross, J., ScolnG.F, Todaro, G.J. and Aaronson, S.A., Nature New Biology 231, 163 (1971). A.L. and Randall, R.S., --J. Biol. 9. Lowry, O.M., Rosenbrough, N.T., Farr, Chem. 193, 265 (1951). 10. Kawaguchi, H., Hasegawa, M. and Mizuike, A., Spectrochimica Acta Vol. z, 205 (1972). 11. Gian, D.L., Watson, K.F., Burner, A. and Speigelman, S., Biophys. Biochem. Acta 246, 365 (1971). -__ 12. Hurwitz, J. and Lies, J.P., 2. __ Vir. 8, 116 (1972). 13. Grandgenett, D.P., Gerard, G.F. and Green, M., --Proc. Natl. Acad. m. U.S.A. 3, 230,(1973).

972