Polypeptide structure of human terminal transferase

Vol. 108, No. 3, 1982 October

BIOCHEMICAL

Istilqto

STRUCTURE

OF

HUMAN TERMINAL

Y Capucci , Gianfranco Giorpio Cattoretti' Sacchi*,

Plevani*,

Received

RESEARCH COMMUNICATIONS Pages 1196-1203

15, 1982

POLYPEPTIDE Pa010

AND BIOPHYSICAL

Lorenzo

TRANSFERASE

9 , Diego

Badaracco

and Enrico

Breviario*, 1c .

Ginelli

Nicoletta

di Biologia, Universits di Milano and E.U.L.O., Brescia, 'Istituio di Fisiolopia Generale, Universith di Geneva, Italy. Clinica Pediatrica, Universitb di Milano, Italy.

August

24,

Italy.

1982

SUMMARY. The polypeptide structure of terminal transferase purified from human lymphoblasts was examined with an immunoblot procedure using rabbit anti-calf thymus terminal transferase antibodies. Two doublets of bands of Mr 58-56,000 and M, 44-42,000 are the major immunoreactive polypeptides. Only the M, 44-42,000 polypeptides can be efficiently renatured polyacrylamide gel electrophoresis in the presence of --in situ after Controlled degradation with trypsin produces fully ,ctive ensodium dodecyl sulfate. zyme containing the c1 and B polypeptides typical of the low molecular weight terminal transferase, suggesting that the different forms of purified terminal transferase may arise by proteolysis of the M, 58,000 polypeptide.

INTRODUCTION. normally

localized

of bone levels

Terminal

deoxynucleotidyl

in a major lymphocytes

marrow

Terminal native

transferase molecular

weight

dium dodecyl

sulfate

has been purified a variability

(3).

were

city

(6).

tide

present

cell

lines

from

and M, 45,000

isolated

A M, 5%56,000 in the

thymus

thymus

and in a minor

by gel

Recently,

lymphoblasts

for

a number

polypeptide

has been

of a variety

(7.8).

0006-291X/82/191196-08$01.00/0 Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.

1196

observed

of leukemic

patients has been

lymphoblasts M, 26,000 raised

species,

(1,2)

peptide)

in the presence

as well

of soin a puri-

of M, 62,000 (5).

However,

observed (6).

when the

Forms exhi-

and M, 10,000

to explain

shown to be the major

of animal

of high

leukemia

( B

polypeptide

have been

population

shown to have a

a single

and containing

hypotheses

certain

was initially

of human leukemic

polypeptides

in

enzyme

finding,

of M, 26,500

transferase

terminal

the

was originally

electrophoresis

(4).

from

and marrow

glands

structure

from

and several

thymocytes in TdT stems

subunit

cells

forms

a DNA polymerizing

and two polypeptides

A similar

of polypeptide

were

calf

detected

to homogeneity

M, 62,000

peptides

from

human leukemic

enzyme has been purified biting

interest

is

lymphocytes

of M, 32,000

( ~1 peptide)

enzyme from

Clinical

purified

(TdT)

of cortical

in circulating

and M, 6,000

fied

fraction

(1).

of enzyme activity

Transferase

this

multipli-

immunoreactive as in

poly-

TdT positive

pep-

Vol. 108, No. 3, 1982 'To investigate of

whether

leukemic

the

enzyme

by that

Mr

and

--in

degradation

MATERIALS chased

then

AND from

a

related activity,

to

METHODS.

Radiochemical

the

we

have

both

species active

with

to

terminal common

can

low

Mr

be

58,000 the

M,

blasts

structure In

converted

molecular

of

5&56,000

and first

weight

form

detected polypeptide

technique. from

the

RESEARCH COMMUNICATIONS transferase

examined

purified

contains

the

of

immunochemical

transferase

55-56,000

AND BIOPHYSICAL forms

sensitive

leukemia

peptides vitro

all

terminal

predominant

different

TdT

using

lymphoblastic

The

are

expressing

monstrate te

the

patients

tissues

human

BIOCHEMICAL

to of

the

in

all

purified

presentation

a single M,

blasts

found of

this

in

patient

44-42,000 the

we

M,

with

acu-

polypeptides. 44-42,000

terminal

de-

poly-

transferase

by

trypsin.

Chemicals:

radioactixre

Center

deoxynticleoside

(Amersham).

5'-triphosphates

Unlabeled

deoxynucleoside The polymer from Sigma. according to published assay mixture in a final

were

pur-

5'-triphosphates of deoxyadenilyc

and phenylmethylsulfonylfluoride (PMSF) were acid poly(dA)-$ was prepared in our laboratory procedures (10). Assay for terminal transferase: the standard volume of 0.10 ml contained 0.2 M potassium cacod late, pH 7.5, 0.5 mM MnCl , 1 mM 2-mercaptoethanol, 0.01 mM poly(dA)--$ and 1 mM 3H -dGTP (100 cpm/pmol). The2reactions were incubated at [.5 35OC for varying times and terminated by application of aliquots to GF/C glass fiber disks (Whatman) as described by Bollum (11). Gel electrophoresis was conducted on cribed by Laemmli cellulose filters anti-calf thymus

and detection of the immunoreactive peptides: a 12% polyacrylamide gel containing 0.1% sodium (12). After electrophoresis the proteins were as described by Towbin et al. (13) and labeled terminal transferase antibodies kindly provided

gel electrophoresis dodecyl sulfate as transferred to nitrowith specific by Prof.F.J.Bollum,

U.S.U.H.S., Bethesda, U.S.A. The immunoreactive polypeptides were visualized radish peroxidase:goat anti-rabbit IgG conjugate and benzidine:H 0 staining 22 bed by Bollum and Chang (8). + Renaturation of human terminal transferase: renaturation of terminal transferase polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate

des-

rabbit with horseas descri-

and

after assay

of the enzymatic activity was carried out as previously described (9). --in situ Purification of terminal transferase from blasts of a patient acute lymphohlastic --- with --leukemia (ALL): human leukemic lymphoblasts were collected from a patient undergoing therapeutic leukapheresis. A suspension of packed lymphoblasts (600 g of cells) was diluted with three volumes of extraction buffer (0.25 M potassium phosphate, pH 7.2, 1 mM 2-mercaptoethanol, 1 mM PMSF), mixed for 1 hour at 4°C and then further homogeneized with a Polytron. The suspension was centrifuged at 12,000 g for 1 hour to give the crude extract supernatant (Fraction I). Fraction I was diluted with three volumes of 1 mM 2-mercaptoethanol, 1 mM PMSF and added to 1 1 of phosphocellulose previously equilibrated with 50 mM potassium phosphate, pH 7.2, 1 mM PMSF. The mixture was stirred for two hours at 4°C and then ter paper in a Buchner funnel without was suspended in 3 1 of 75 mM potassium collected by filtration as above. The column and the bound enzyme was eluted 2-mercaptoethanol, 0.5 M NaCl to yield two changes of 20 1 of 30 mM potassium the precipitate formed was removed by a hydroxylapatite washing with

75

column (14) mM potassium

(5 x 10 phosphate,

P-11 slurry (Whatman) 1 mM 2-mercaptoethanol, filtered through fil-

allowing the phosphocellulose to dry. phosphate, pH 7.2, 1 mM Z-mercaptoethanol phosphocellulose was then poured into using 75 mM potassium phosphate, pH Fraction phosphate, centrifugation.

II.

cake

and a p,lass 7.2, 1 mM

II was dialyzed against 1 mM 2-mercaptoethanol and The supernatant was loaded onto cm) equilibrated with the dialysis buffer. After pH 7.2, I miil 2-mercap-toethanol, the bound en-

1197

Fraction pH 7.2,

The

Vol. 108, No. 3, 1982 zyme was eluted with The active fractions added to 75% saturation. the centrifuge tubes filters in a Buchner of the enzymatic activity

BIOCHEMICAL

AND BIOPHYSICAL

a 2 1 gradient from were pooled to give The majority of after centrifugation funnel. This procedure (see Table I).

RESEARCH COMMUNICATIONS

75 mM to 350 mM potassium phosphate, pH 7.2. Fraction III and solid ammonium sulfate was the precipitate formed floated on the top of and was collected by filtration through GF/C caused a considerable loss in the recovery Alternative ways, such as dialysis against a

solution of concentrated ammonium sulfate or dialysis against the next column also caused a loss of enzyme activity. The precipitate was resuspended in 10 sium phosphate, pH 7.2, 1 mM 2-mercaptoethanol, 10% glycerol and loaded onto phadex A-50 column (4 x 11 cm) equilibrated with 30 mM potassium phosphate, The column was washed with the same buffmcr 2-mercaptoethanol, 10% glycerol.

buffer mM potasa CM-SepH 7.2, 1 mM containing

50 mM potassium phosphate,, pH 7.2 and eluted with a 1.5 1 gradient from 531 mM to 350 mM potassium phosphate, pH 7.2. The active fractions (Fraction IV) were pooled and precipitated by addition of ammonium sulfate to 75% saturation. The precipitate was collected by centrifugafion, resuspended in 4 ml of 10 mM potassium phosphate, pH 7.7, 0.25 M NaCl, 1 mM 2-mercaptoethanol, 10% glycerol and fractionated on a Sephadex G-150 column (2 x 80 cm) equilibrated in the resuspension buffer. The active fractions were pooled (Fraction V and directely loaded onto a hydroxylapatite column (1.4 x 5 cm) equilibrated in the previous buffer. After washing the enzyme was eluted with a 160 ml gradient from 10 mM to 130 mM potassium phosphate, pH 7.2. Fractions containing the TdT activity were pooled to give Fraction VI and concentrated by precipitation with 75% ammonium sulfate. Fraction VI was resuspended in 3 ml of 50 mM potassium phosphate, pH 7.2, 1 mM 2-mercaptoethanol, 10% glycerol and dialyzed against the same buffer. To the dialyzed Fraction VI, M&l was added to a final concentration of 10 M and the enzyme was loaded onto an oligo f dT)cellulose column (Type 7, PL Biochemicals lnc.)(l.l x 4 cm) equilibrated in the dialysis buffer. After in the above 50 mM potassium -2OOC.

Terminal

washing, the enzyme was buffer. Fractions containing phosphate, pH 7.4, 10

transferase

blastic

has

leukemia. and

mica1

immunological

positive

("spot",

(0.5%,

TdT

with a 70 TdT activity mM 2-mercaptoethanol,

RESULTS

AND

DISCUSSION

purified

from

the

been

Diagnosis

eluted

of

T cell

ALL

determinations: 90%),

positive

Sudan 51%.

By

was

blasts made

of

negative,

to

with the

(granular,

E rosettes

immunofluorescence

a patient

according

PAS positive

Blak

ml gradient from 0 to were pooled, dialyzed 50% glycerol and

SmIg

determination

(1)

acute

lympho-

following

51%),

87.5%,

1.2 M KC1 against stored at

cytoche-

acid

phosphatase

< 0.5%, TdT

had

shown

in

anti-CALL a nuclear

lo-

calization. A summary final

of

the

purification

scheme

oligo(dT)-cellulose

mg when signed ges

assayed for

of

activity

fraction as

terminal

enzyme

se

fraction

bed

for

in

purification

(15).

of

has homogeneous

using

a specific human

TdT and

polymerization

polymerization By

human

determination dATP

of

3 H -dATP L3 activity TdT

dATP as

the of

transferase

terminal

Materials

transferase

because

lization

described

for

has

linear labeled

95,310

(5,6).

1198

a specific

Methods.

That

in

fractions

crude is

is

is

activity assay

was (1).

of

monitoring

to

than

80%

Units/me,

the a value

final similar

56,952

For

for

substrate,

I.

of

The Units/

specifically

preferable greater

Table

de-

the

final the

substrate

sta-

enzyme uti-

oliRo(dT)-celluloto

that

descri-

Vol. 108, No. 3, 1982

BIOCHEMICAL

1. Purification

Table

AND BIOPHYSICAL

ot‘ Terminal

Transferase

Total Enzyme

Crude extract Phosphocellulose Hydroxylapatite I Cb!-Sephadcx A-50 Sephadex G-150 Hydroxylapatite II Oli~o(tlT)-cellulose

II III IV VI VII

The

biochemical

detail

by

tion as

of

properties

M.S.Coleman

purine are

by

molecular

weight between

aminoacid

decyl

ALL

a doublet

reful

examination calf

pIs

of

the

was

in

human

raised

as

the

bands

The

discrepancy

the

immunoblot of

gel

to

the

not

significantly

A)

M,

gel.

enzyme

by

rabbit

against

reagent

between is

due protein

nitrocellulose

a contaminant

weight

of

to

with in

human M,

the

terminal enzyme

and

Comassie of

partial

the

blue

stained

peroxidase

differential 72,000

transferase preparation.

1199

protein

By

sodium

do-

twomajor

sets

72,000.

detectable

of

the

cahuman

polypeptides

using

monospecific weight

TdT

calf were

(Figure bands

and

at

thymus two

1,

high

of

proteins

detected

in

the

it

is

as

terminal

and

bands:

after

between

transfer

considering

of

A doublet

also

stain

antibodies

their

of

polypeptides 44-42,000

in

presence

molecular

M,

discre-

of

immunoreactive (13)

low

5%56,000

M,

the

immunoreactive

of

The

detect

The

high

blasts

M,

were

that

the

differences

cross-reactivity

procedure

non-linearity

filter. react

to

the

homogeneous

intensities

and/or

bands

tried

the The

the

minor

shown).

the

the

of

for

from

showed

band

immunoblot

(8).

the to

the

stain

not with

(9).

purified in

data

found

some

great

poly(dA)$

identical

= 8.6)

reflect

gels

of

and

that

(p1

a single

Because we

than

may

blue

other

(16

in

polymeriza-

using

a value

transferase

and and

(81,

lower

enzyme

of

preparation

transferase

Comassie

5%56,000

analyses

shown),

5 10 5 10 5 IO lo5 105 LO5 IO5

x x x x x x x

analyzed

been

findings

not but

(6),

have

enzyme

polyacrylamide

the

stained

(data

calf

7.7 7.3 2.7 2.1 1.7 1.3 0.3

kinetic

previous

terminal

44-42,000

a molecular

antigen

as

in

primary with

the

and

on

transferase

the

antibodies

human

lane

human

terminal

analyzed

Mr

8.2

thymus

The

those

enzyme

human

to

of

is

the

1,

to

TdT human

When

terminal

present

dered

calf

corresponding corresponding

tions

of

(Figure

bands

of

the

our

with

Blasts. Total Enzyme Units

transferase

(5,6,16). with

Leukemic

10 76 353 1,612 6,106 25,417 56,952

terminal

coworkers

human

for

patient

sulfate

and

of

composition.

a single

human

agreement

form the

the

her

full

others

7:i,o50 9,600 767 1 3: ? 28 2 1.4

obtained

point

determined

TdT

in

isoelectric

pancy

and

nucleotides

initiator

The

of

from

Specific ,1ctivity inmol per hour)

Prl>tein (mp 1

Fraction

RESEARCH COMMUNlCATlONS

doublets lane

B).

those

on

concentrafrom

stained therefore

the gel

does

consitransfera-

Vol. 108, No. 3, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

-92

K

-68 -55 -45

K K K

-14

K

Figure 1. Polyacrylamide gel and immunoblot analysis of human terminal transferase. Two 6 JL~ samples of purified terminal transferase were separated on a 12% polyacrylamithe gel de slab gel in the presence of sodium dodecyl sulfate. After electrophoresis was cut and one sample was stained used for immunoblotting as described were phosphorylase B, bovine serum

se polypeptides estimate note

only

that

that

our

the

Because the

Mr 5b-56,000 and the

molecular a variability

weight

te the

60 minutes

retained

by --in vitro

proteolysis

the

immunoblot

sensitive typical After (Figure

B polypeptide times

2, lanes

of different

purified

it

is very

both

we

interesting

to

present

in the

by proteolysis

of incubation, and a

resistant

shown).

and the

same of

that

form

1200

initial

proteins is

are

of human terAfter

enzymatic fragments

2. By using are

together (Figure completely

observed,

activi-

generated

proteins

generated

(M, 8,000)

of M, 24,000

form

shown in Figure

is

to investiga-

degradation.

the M, 5B-56,000

c1 polypeptide

B polypeptide

weight

when

and a low mole-

we wanted

of the

is

was found

(6)

to trypsin

analysis

trypsin

M, 44-42,000

(4),

90% of the

A kinetic

the M, 44-42,000

First

patients

molecular

of trypsin,

can be visualized

(Mr 26,000)

E-G)

is

antibodies

transferase

leukemic

on the high

35 p&ml not

terminal

from human cells

proteolysis

with

for

of the human TdT with

to degradation.

longer

structure

from blasts

(data

technique

are

It

was used

form.

Human TdT activity

ty was still

75% pure.

may be generated

species

the other sample B). The markers and lysozyme.

anti-TdT

Mr 44-42,000

of controlled

of incubation

with

proteins

TdT was originally

effect

immunocomplexes

is approximately

of polypeptide

transferase.

minal

to form

and the Mr 44-42,000

weight

enzyme was purified

cular

able

enzyme preparation

enzyme preparation the higher

those

with Comassie blue (lane A) and in Materials and Methods (lane albumin, rabbit IgG, ovoalbumin

with

very the

2, lanes

A-D).

degraded

and this

pattern

Vol. 108, No. 3, 1982

BIOCHEMICAL

G

F

E

AND BIOPHYSICAL

D

C

RESEARCH COMMUNICATIONS

I3

A

-

68 K K K

- 55 - 45

25 K

Fipure 2. Degradation of human terminal transferase by trypsin. Terminal transferase at 150 p&n1 was incubated with 35 p&ml of pancreatic trypsin at 35OC in 50 mM Hepes, pH 7.5. At various times of incubation 50 ~'1 aliquots were removed and added to 5 ~1 of 1 mg/ml of soybean trypsin inhibitor. After electrophoresis on a 12% polyacrylamide gel in the presence of sodium dodecyl sulfate, the proteins were transferred to a nitrocellulose filter and the immunoreactive polypeptides detected as described in Materials Lanes A,B,C,D,E,F,G represent the immunoblot of samples taken at 0,0.5,1, and Methods. 3,6,30,60 minutes of incubation respectively. A band of M, 23,500 is stained in all the samples and represents trypsin. Trypsin is stained by the immunoblot procedure probably because anti-carbohydrate antibodies are present in the anti-calf thymus TdT antiserum used (20). When column-purified ant-TdT IgG was reagent, trypsin used as the primary did not stain in the peroxidase reaction (9).

is

characteristic

By

careful

of

extensively

examination

of

nerated

during

proteolysis,

version

of

calf

When --in

the

human situ

lane

on B),

enzyme

M,

TdT

preparation

previous in

human

from

the 'TdT,

3, higher and

1,

calf

on lane

the

A,

the

detection

for

the

are

renaturation

of

to

a control

ones.

M, This

feature

enzymatically

active

1201

(9),

also

pe-

proteolytic

con-

as

further

56-56,000

renaturation

detected in

both

the

Mr

3,

human 58-56,000 purified

3, well

(Figure the

a partially

renature

be

(9). is

after

(Figure

proteins

M,

the

were

in

TdT

can

32,000

polypeptides

represented

44-42,000

thymus

TdT.

experiment,

thymus

Mr for

proteins

renatured

calf

calf

activity

stained

positively

the

human

enzymatic

the

equally

of that

weight

In

A).

were

thymus,

of

of

proposed

44-42,000

M,

5-10%

which

indicate

molecular

analyzed

from

fragment

scheme

applied

only

lane

the

also

only

proteins,

results

Figure

than

44-42,000

be

was

represent (Figure

that

can

gel,

they

preparation the

(9)

preparations

a transient

immunoblot,

transferase

if

enzyme

suggesting

a polyacrylamide

even

and

the

enzyme

terminal

degraded

lane

as

the

more emphasized polypeptides

A).

Our

data

shown

efficiently for may

the be

Vol. 108, No. 3, 1982

BIOCHEMICAL

AND BIOPHYSICAL

A

RESEARCH COMMUNICATIONS

B

c

Figure 3. Renaturation of terminal transferase on polyacrylamide gels. 800 Units of partially purified calf thymus terminal transferase (lane A) and 700 Units of purified human terminal transferase (lane B) were separated on a 12% polyacrylamide gel contaiof sodium dodecyl sulfate. ning 150 pg/ml of activated calf thymus DNA in the presence Renaturation of the active polypeptides and their detection by autoradiography --in situ have been previously described (9).

hindered

by

the

limited

sensitivity

of

A general

conclusion

purified

human

tained

in

of

calf

as

is

By

using

Both

human

tion

and

vitro

by

and the

during

two

differ

may

be

their

loaded

onto

the

at

gel

and

by

the

leukemic

the

patients to

the

the the

However,

also

partially, very

(61,

processing

of

1202

enzyme

aminoacid

shown

to may

be

polypeptide

represent the

enzyme

human

composition point.

the

enzymes

different proteolytic the

result

pattern structures

a biologically during

native

isoelectric that

degradation of

may

been

ob-

the the

and

sensitive

specific

variability

or

the

results

represent

immunochemically

purified very

the

of

similarities,

efficiency

has

are

for

as

structural

may

structure

it

structures with

protein

renaturation

least

polypeptide agreement

58,000

tridimensional

transferases

well

the in

their

in

described

related

M,

Besides

are,

purification.

or

be

comparable,

antibodies,

terminal

as

can

results:

a common

differences

structures

our

are

and

in

species

calf

that

from

enzyme

anti-TdT

tissues and

drawn

(8)

their

degradation,

different nomenon

may

the

protein

transferase.

monospecific from

be (9)

terminal

enzymes

rified

--in

can calf

of

itself.

fractions

suggested

lysis

method

and

crude

structure and

the

amount

(6). degradaof

proteo-

observed detected relevant

differentiation

pu-

on in pheand/

Vol. 108, No. 3, 1982 or

to

its

rify

the

tiate

from

intracellular structure TdT

localization of

positive

BIOCHEMICAL

terminal

(6,17). transferase

prelymphocytes

ACKNOWLEDGMENTS. We are indebted of anti-calf thymus TdT antiserum njugate. We wish to thank Mr.M.Chiari partially della

supported by the grant Crescita Neoplastica",C.N.R.,Rome

AND BIOPHYSICAL It in tc

will cells

a TdT

to Prof.F.J.Bollum and horseradish for expert No 81.01379.96

RESEARCH COMMJNlCATlONS be

that

negative

of

particular

have state

interest

been

to

induced

to

ve-

differen-

(18,19).

and Prof.L.M.S.Chang for peroxidase:goat anti-rabbit technical assistance. This of the Progetto Finalizzato

the pift IpG cowork was "Controllo

(Italy).

REFERENCES 1. 2. 3. 4. 5. 6. 7. ?'. 9. 10. 11.

Bollum,F.J.(1979),Blood,~,l2O3-I2l5 Kung,P.C.,Long,J.C.,McCaffrey,R.P.,R.P.,Ratliff,R.L.,Harrison,T.A. (1976),Am.J.Med.,g,788-794 and Bollum,F.J.(1971),J.Biol.Chem.,246,909-916 Chang,L.M.S.

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