The isolation and purification of two anionic endothelial cell growth factors from human brain

Vol. 124, No. 1, 1984 October

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 262-268

15, 1984

Greg Corm and Victor

B. Hatcher

Department of Biochemistry Albert Einstein College of Medicine Bronx, NW York 10461 Received August 23, 1984 Tw anionic polypeptides which stimulate the proliferation unbilical vein endothelial cells have been isolated and purified to ity fran hunan brain using heparin affinity chranatography. The weights of the polypeptides are 18,500 and 19,300; the isoelectric both polypeptides is at @I 5.2. The purified polypeptides differed The half ability to stimulate hunan endothelial cell growth. activities observed for the 18.5 and 19.3 kilodalti polypeptides concentrations of 10.8 rig/ml and 8.9 &ml reSpeCtiVely. @1984 Academic

We have previously factor

fran

onbilical

bovine

brain

vein endothelial

isoelectric

point

purified

endothelial

increases

the

rate

matrix

of

which bind tightly hanogeneity division

al.

endothelial

cell

population purified

growth

factor

endothelial

mitogen

cell

(IN, Bianed.

to heparin.

We report

of two acidic

polypeptides

of hunan endothelial

cells.

0 1984 bx Academic Press, Inc. of reproduction in any form reserved.

by fran

endothelial

cell

that

Recently,

chondrosarm

the isolation

and

262

but

Shing et affinity

extracellular

Rlagsbrun and Shing

growth supplement Inc.)

heparin

growth factor,

heparin-Sepharcse

Tech.,

(2) and

proliferation

levels.

growth.

hunan

as the partially

(EGS, Collab.

contain

carrponents

and purification

fran hunan brain which stimulate

0006-291X/84 $1.50 Copyright All rights

doubling

and

of

possesses a similar

(4) have demonstrated

amount of anionic

Inc.

growth

by Maciag et al.

cell

capillary

an anionic

heat and protease described

Press.

propagation

endothelial

a cationic

and endothelial

et

serial

vein

isolated

that stimulates

the

purified

The growth factor

factor

unbilical

(6) have also observed that Res.)

(1).

growth

the cumulative

have

chraaatography

permits

to acid,

Thornton

decreases the required

(5)

which calls

cell

(3).

does not affect

and partially

and sensitivity

Lemmn et al.

al.

identified

of hunan hamqenen&ecular point for in their maximal were at

to cell

Vol. 124, No. 1, 1984

BIOCHEMICAL

Materials

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and Methods

Materials: Porcine heparin and type I gelatin were obtained from Sigma Chemical. Falcon tissue culture plates were used. The glutamine, fetal bovine serLPn and trypsin sed were from Grand Island Biological Company. 'The heparin affinity resin was produced using Sepharose 4B frcxn Pharmacia Fine Chemicals (7). Reagents for electrophoresis and protein assays were obtained from Bio-Rad Laboratories. Isolation and Purification of Gru&h Factors: Four human brains and one human hypothalanus (approx. 4000 grams) which were obtained 24-48 h postmarten were homogenized, extracted and the extract fractionated by ammonion sulfate precipitation as described by Gordon et al. (1). The protein which was precipitated fran the extract with saturated ammonium sulfate 30-95% was dissolved in 0.15 M sodium phosphate buffer, pB 6.0, and applied to a Sephadex CM-50 column (5 x 20 cm). Proteins were eluted with a step gradient of 0, 0.15 M and 1.0 M YaCl in 0.15 ti sodiun phosphate buffer, pH 6.0. Active fractions fran the column were dialyzed, lyophilized and 100 mg of the active protein was dissolved in 4 ml of 0.15 M Hepes buffer, pE 7.5. The solubilized protein was applied to a heparin-Sepharose colunn (1 x 8 cm). The co1un.n was eluted using a step gradient of increasing NaCL concentrations up to 3.0 H and 0.5 ml fractions were collected. The active fractions were stored at 4% and retained significant activity for at least four weeks. Endothelial Cell GrosJth Assay: Human unbilical endothelial cells were isolated and maintained in culture by the method of Gordon et al. (1). Cell gro+ith assays were performed by plating 5000 cells per Cm2 on gelatin coated 8 cm2 tissue culture dishes containing growth media supplemented with 90 ug/ml porcine heparin (4). Samples to be assayed for mitogenic activity ere added to the dishes and the dishes were incubated for 72 h. The cells were detached with 0.1% trypsin and quantitated with a hemocytaneter. Control plates received an amount of elution buffer equal to the volune of the saqle assayed. The cell number per plate recorded for each concentration of gro.&h factor was the average of four replicate plates. One unit of mitogenic activity was defined as the amount of protein which stimulates half maximal cell growth. Gel Electrophoresis: SDS-PAGEwas performed in 16% gels by the method of Laenmli (8). Gels were silver stained according to the methcd of tierrill (9). Isoelectric Focusing: Electrofocusing was performed in L mm slab gels mounted on Gelbond supports (FW Corporation, Rockland, Qine). Gels were 5% acrylamide, 4% crosslinker and contained 2% Servalyt ampholytes (pH 3-10 or 4-6) and 5% urea as solubilizer. Samples were prepared by dialyzing protein eluted fron the heparin affinity colcann against 0.1 M amniun acetate pH 6.5, lyophilizing the sample and dissolving the protein in 5% urea, 2% ampholytes. In this manner approximately 1 ug of protein could be applied to the gel in 510 ul., Conpletion of isoelectric focusing was determined by monitoring the rate of decrease in the current flm through the gel. Protein standards were used to determine tine pH gradient and isoelectric points of the unknowns. Results Our protocol growth

factors

fractionation chromatography;

for

fran of

isolation

and purification

human brain a

and Discussion

human

and heparin

consists

brain affinity

of

of three

extract;

steps:

Sephadex

chrcmatography.

the endothelial

cell

anrnoniun sulfate CM-50

Virtually

ion-exchange all

of the

Vol. 124, No. 1, 1984

BIOCHEMICAL

Table 1 Purification

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of Anionic Endothelial Cell Growth Factors Fold Purification

Protein Recovery w

l/2 Maximal. mtal Activity Activity units

Specific Activity units/mg

(=I4) 2=4 Fractionation

3.20 x lo4

63.0 ug/ml

5.1 x 105

15.9

Sephadex al-50

1.95 x 103

4.0 us/ml

4.9 x 105

2.5 x lo2 15.7

0.212

10.0 rig/ml

2.1 x 104

1.0 x 105

6.3 x lo3

0.125

0.9 rig/ml

1.4 x 105

1.2 x 106

7.6 x lo4

Hepar in Affinity

(I)

Hepar in Affinity

(II)

endothelial precipitated

cell

grcwth

pranoting

activity

with annrxiiun sulfate

from the hm

fro

4 kg of hunan brain

was 32 g, approximately

fran

4 kg of bovine

brain

(1) .

was similar

to

brain

extract

preparation

that

The elution

(Table 1) .

obtainable profile

a-lied

in the active

40

.lSM N&I

recovered fran

1.

extract

was

of protein

the amount obtained fran

the hunan

an equivalent

of honan brain

is shown in Figure

was recovered

The yield

twice

activity

Sephadex CM-50 chranatography protein

brain

at 30-95% saturation.

The total

1.0

extract

Approximately

fractions.

bovine following 6% of the

The interaction

which

IM NoCl

1

1

30 B 20% x . .% s 10 f 2

0

zoo

400

Eoo ml. effluenl

Sephadex (M-50 chranatcgraphy of hunan brain extract precipitated ZZ?&.un sulfate at 30-95% saturation The soluble portion of the precipitate (5 grams) was applied to the bolum. Practicna eluted were measured for absorbance at 280 nn () and for activity in the endothelial cell growth assay (m). 264

Vol. 124, No. 1, 1984

I,

I

BIOCHEMICAL

I

5 IO 25

I

50

I

1

loo

prorein

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

150

I 5

II IO

II 15 20

cont. (Jig/ml)

protein

I 30

25

cont.

the

anionic

polypeptides

to

bind

to

Sephadex

I 45

(nghl)

Figure 2. Mitogenic resparse of hunan uubilical vein endothelial grwth factors isolated frcan the humn brain. A. Plot of protein amcentraticn vs. cell nmher for the mm&m precipitate of huum brain extract ( rJ ) and the active fraction fran CM-50 chranatography ( 0 ). B. Plot of protein vs. oell nuker for the Wo active fractions I ( II ( 0 ) isolated by hfqarin affinity chranatogra@y.

permits

II 35 40

(X-50

cells

to

sulfate Sephdex 0 ) and

at

@-I 6.0

is

unknown. The

erdothelial

samples are

after

shown

Figure

2h.

while

reached mined

sulfate

reached

cells/plate

growth

of

addition assays

active

response

The

in the

of

protein

the

fran

miun

fractions

would

the

Sephadex

amnoniun

sulfate

(X-50

2.7

lower

x 10’

chromatography

We have

sulfate

active

CM-50 chrcnnatography

at approximately

x 105 Cells/plate.

the

with

after

curve

following 4.0

CM-50

fractions

growth

fractions

generated

and Sephadex

active

approximately of

curves

fractionation

a plateau

the

a maximun if

growth

ammoniun in

precipitation

cell

not

deter-

precipitate the

to

maximal

the

growth

attainable. The

pattern

of

heparin-Sepharose

elution

affinity

of

protein

and

CO~I.XIEI is

presented

binding

growth

factors

were

recovered

following

raphy.

Both

growth

factors

were

hcamogeneous

analysis

(Fig.

3).

kilodalton

polypeptide

1.2

M &Cl

colunn

at

An

18.5

and exhibited

half 265

maximal

mitogenic in

fran

3.

‘Vitro heparin

Figure

heparin as

activity

affinity

determined eluted

activity

fran at

10.0

the

chromatogby

SIX-PAGE

the

affinity

rig/ml.

(Fig.

BIOCHEMICAL

Vol. 124, No. 1, 1984

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

t

Zl.OkDa

t t

19.3kDa 18.5kDa

t

16.5 kDo

0.8 4

lo-

98I

7-

s 69 .5 5e g 4-

,) 4,

32-,, \ l-

5

0

15 20 25 ml. effluent

IO

30

35

Figure 3. Heparin affinity chranatcgraphy of tbs active fraction (hunan brain) fran Sephadex U4-50 chraaatography. ‘Iwc peaks of mitogenic activity eluted fran tha colum at 1.2 M and 1.4 M NaCl (labeled I and II respactively) . The uppar part of the figure shows an SCS-PAQIanalysis of the twc active fractions isolated fran huaan brain (HI and HII) along with corres@ing fractions isolated ty heparin affinity draaatography fran bovine A preparatiur of rabbit skeletal muscle myosin light brain (BI and BII). chains was used for molecular weight standards.

a *

The 19.3 kilodalton

activity

at concentrations

The fold

polypeptide

eluted

purification

for

the 18.5 and 19.3 kilodaltons

respectively

both polypeptides

focused as sharp single

Sulfate

the 19.3 kilodalton fractionation

maximal

of 0.9 rig/ml.

6,300 and 76,000

purified

at 1.4 M NaCl showed half

(Table

1).

polypeptide

and heparin

affinity 266

In analytic

isoelectric

bands at p+I 5.2. fran

hunan brain

chromatography.

cunponents

was

focusing

We have recently using only

atnaxicm

We have not been

Vol.

124,

No.

able to purify procedure.

fron

bovine

molecular

weights

cell

COMMUNICATIONS

using a two step factors

isolated

methodology

is also

shown in

fran the bovine brain

exhibited

similar

using similar

purified

RESEARCH

in high yield

of endothelial

brain

The polypeptides

BIOPHYSICAL

polypeptide

The SDS-PAGEanalysis

3.

gro&h

and focused at pH 5.2.

In ccmparing the acidic are

polypeptides

the isoelectric

points

factor

purified

by Thomas et al.

blast

growth

purified

AND

the 18.5 kilodalton

and purified Figure

BIOCHEMICAL

1, 1984

factor,

to that

similar

which

of the acidic

(10) from bovine brain.

is

as two unseparated

with other purified

a mitogen

forms with

for

PXi3/C

reported

gru&

factors,

fibroblast

growth

The acidic

fibro-

3T3 fibroblasts,

nkolecular weights

was

of 16,000

and 16,800. The molecular

basis for the differences

between the two anionic

tides

remains to be determined.

The relationship

other

and the acidic

fibroblast

grcMzh factor

target

cells

as well as further

characterization.

The isolation yet exhibiting

and purification differences

in heparin difference

cell

an analysis

both heparin

will

permit

binding

between the he-rin to

endothelial

stimulate

Klagsbrun and Shing's interaction

proposal

require

affinity, ability

activity.

was observed

process of endothelial

in

features

which affect

A direct

correlation

and their which

ability

substantiates

(6) of a growth factor:heparin:endothelial

in the stimulatory

weight endothelial

stimulate

of these polypeptides growth

functions

molecular to

to each of their

analysis

with similar

of the structural

promoting

affinity cell

binding

in their

and growth binding

will

of two polypeptides

SDS-PAGE, and a ten-fold growth,

of the polypeptides

polypep-

cell

cells.

AckncMedgements The authors assistance

thank Dr. Portia

in the preparation

American Heart Associations.

and Joan 3yrne for

of this manuscript.

This research was supported (AG 01732 and HL 16387),

Gordon for constitation

by grants

the Cystic

Fibrosis

fran the TJ.S. Public Health Service Foundation,

and the New York and

Vol. 124, No. 1, 1984

Victor

B. Hatcher

tion and a recipient

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is ah Established

Fellow of the New York Heart Associa-

of the AM Weinberg Cystic

Fibrosis

Research Scholarship

Award.

References 1.

Gordon, P.G., Sussman, I., ahd Hatcher, V.B. (1983) In Vitro 19, 661671. 2. Maciag, T., Hoover, G.A., Steinermah, M.B., and Weinstein, R. (1981) J. Cell Biol. 91, 420-426. 3. Lemon, SK., Riley, M.C., Thomas, K.A., Hoover, G.A., Maciag, T., and Bradshaw, R.A. (1982) J. Cell Biol. 9S, 162-169. 4. Thornton, S.C., Mueller, S.N., and Levine, E.M. (1983) Science 623-625. 5. Shing, Y., Folkman, J., Sullivan, R., Butterfield, C., Murray, J., and Klagsbrun, M. (1984) Science 223, 1296-1298. 6. Klagsbruh, M., and Shing, Y. (1984) Fed. Proc. 43, 3, Abst. 1376. (1975) Biochim. Biophys. Acta 410, 7. Elgart, E.S., and Rosenberg, M.D. 178-192. 8. Laemnli, U.K. (1970) Nature 227, 680. 9. Merril, C.R., Goldman, D., Sedman, S.A., and Ebert, M.H. (1981) Science 211, 1437-1439. 10. Thomas, K.A., Rios-Candelore, M., and Fitzpatrick, S. (1984) Proc. Natl. Acad. Sci. USA, 81, 357-361.

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