Purification of basic fibroblast growth factor from rat brain: Identification of a Mr 22,000 immunoreactive form

Vol. 155, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1161-1172

September 30, 1988

PURIFICATION OF BASIC FIBROBLAST GROWTH FACTOR FROM RAT BRAIN: IDENTIFICATION OF A Mr 22,000 IMMUNOREACTIVE FORM

M. Presta,

M. Rusnati,

J.A.M.

Maier,

and G. Ragnotti

Sciences and Chair of General Pathology, Dept. Biomedical Biotecnology, School of Medicine, University of B r e s c i a , via Valsabbina 19, 25124 Brescia, Italy Received August 5, 1988

A 18,000-dalton protein which stimulates plasminogen activator (PA) activity in endothelial GM 7373 cells has been purified from rat brain by using heparin affinity chromatography and ion-exchange chromatography. The purified molecule stimulates PA activity in a dose-dependent manner between 1 and 30 ng/ml. It also stimulates proliferation of GM 7373 cells and DNA synthesis in NIH 3T3 cells in a similar concentration range. The molecule has been identified as a bFGF-like molecule on the basis of its biological activity, its affinity for heparin-Sepharose, and its cross-reactivity with anti-human bFGF antibodies. In the final preparation of the rat brain bFGF, trace amounts (less than 5%) of a contaminant were detectable. This contaminant has a molecular weight of 22,000 and cross reacts with several antihuman placental bFGF antibodies. On the basis of its affinity for heparin-Sepharose and its immunological characteristics, this protein appears to be an high molecular weight form of bFGF. © 1988 Academic Press, Inc.

Basic

fibroblast

growth

factor

(bFGF)

is an

mitogen

with a molecular weight of 16-18,000.

purified

from bovine pituitary

a basic mitogen

isolated

It was originally

and later shown to be identical

from bovine brain

(i).

of the same molecular weight has been purified tissues has

been

(2)

and its presence

demonstrated

in relatively

in the central

different

animal

species,

stimulates

neurite outgrowth

heparin-binding

from a variety

high

nervous

including

man

of hippocampal

Since then,

bFGF of

concentrations

system (2-4).

(CNS) Basic

and cerebral

ABBREVIATIONS: basic fibroblast growth factor: activator: PA; central nervous system: CNS.

to

of FGF

cortical

bFGF; plasminogen

0006-291X/88 $1.50 1161

Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 155, No. 3, 1988

neurons

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and it exerts a t r o p h i c

non-cholinergic expression

of

neurons bFGF

embryonic development

in vitro

a

Structural

single

chain

and in vivo

in brain appears (3).

to

(5-9).

be

differentiation,

studies have shown that bovine p i t u i t a r y bFGF is polypeptide of 146 amino acids w h i c h

amino acid bFGF has been p u r i f i e d

from human placenta

of

purified weight

bFGF

(i0,ii).

with a m o l e c u l a r weight

from guinea pig brain

(14).

rat brain

(15). Therefore,

molecular

form of bFGF may exist.

of

can

also

However,

25,000

Moreover,

immunoreactive bFGF-like proteins

high

a 157

(12,13) and has

been

molecular

have been identified in

these data indicate that more than one On these basis,

c h a r a c t e r i z a t i o n of bFGF forms in the CNS, the p u r i f i c a t i o n of bFGF from rat brain. purification

during

and trophism of the

forms

form

Moreover,

modulated

exist in amino terminal t r u n c a t e d

a

and

These data suggest that bFGF might be

involved in the development, CNS.

function on both cholinergic

for a

better

we decided to attempt Here,

we describe the

of a Mr 18,000 form of bFGF from rat brain and

the

presence in the same p r e p a r a t i o n of an i m m u n o l o g i c a l l y related Mr 22,000 form.

MATERIALS

AND

METHODS

Cell culture. Fetal bovine aortic endothelial GM 7373 cells (16) were obtained from the N.I.G.M.S. Human Genetic Mutant Cell Repository (Institute for Medical Research, Camden, NJ, U.S.A.). They were grown in Eagle' Minimal Essential medium (MEM) supplemented with 10% fetal calf serum (FCS), vitamins, essential and non essential amino acids. Plasminogen activator assay. GM 7373 cells were seeded at the density of 70,000 cells/sq, cm. After 24 h, confluent m o n o l a y e r cultures were washed twice with MEM and incubated in fresh m e d i u m containing 0.4% FCS and the material to be tested. After incubation at 37°C for 16 h, the cell layers were washed twice with cold p h o s p h a t e - b u f f e r e d saline , pH 7.5 (PBS). Then, PA activity was m e a s u r e d in the cell extracts by using the plasmin chromogenic substrate H-D-norleucyl-hexahydrotyrosil-lisine-pnitroanilide-acetate (American Diagnostica, Greenwich, CT) according to the m a n u f a c t u r e r ' s instructions. Purification of the P A - i n d u c i n g factor. Frozen brains (approximately 100 gr) from male Sprague Dawley rats (Charles River, Calco, Italy) were h o m o g e n i z e d in a food processor. After homogenization, all subsequent steps were p e r f o r m e d at 4°C. The 1162

Vol. 155, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

homogenized brains were added (1:3, vol:vol) with cold 20 mM Tris-HCl (pH 7.5) / 3 m M EDTA (Tris/EDTA buffer) and sonicated for 15 min at 50 W. Then, NaCI was added to a final c o n c e n t r a t i o n of 0.5 M and the sonicate was c e n t r i f u g e d at 15,000 g for 30 min. The supernatant was loaded onto a 30x300 mm column of heparinSepharose (Pharmacia Fine Chemicals, Uppsala, Sweden) e q u i l i b r a t e d with 0.5 M NaCI in T r i s / E D T A buffer. The column was w a s h e d first w i t h the same buffer, then s e q u e n t i a l l y eluted with 0.8 M and 2.0 M N a C l , b o t h in T r i s / E D T A buffer. Fractions were assayed for P A - i n d u c i n g activity, and the active fractions were pooled together and diluted w i t h T r i s / E D T A buffer until the c o n c e n t r a t i o n of NaCl was 0.5 M. Then, the sample was loaded onto a second smaller h e p a r i n - S e p h a r o s e column (15x150 mm). The column was w a s h e d first w i t h 0.95 M NaCI in T r i s / E D T A buffer and then eluted with 2.0 M NaCI in T r i s / E D T A buffer. The active fractions from the second h e p a r i n - S e p h a r o s e column were dyalized overnight in 0.2 M NaCI/15% glycerol/0.1 M sodium phosphate (pH 6.0) and c l a r i f i e d by c e n t r i f u g a t i o n at 15,000 g for 30 min. The sample was then loaded onto a 7x20 mm column of CM-Sephadex C-50 (Pharmacia) e q u i l i b r a t e d with the dyalisis buffer. The column was w a s h e d with 0.2 M NaCI in the same buffer and then sequentially eluted with 0.5 and 2.0 M NaCI, each in 0.i M sodium phosphate (pH 6.0)/15% glycerol. Fractions were assayed for PA-inducing activity and active fractions were pooled. Purity was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE. SDS-polyacrylamide gels (3% stacking gel and 12% resolving gel) were p r e p a r e d and run as described (17). Proteins were detected with the silver stain procedure (18). Protein determination. Protein concentrations were determined with the Bio-Rad protein assay (Bio-Rad Laboratories, Richmond, CA) or with the Pierce protein assay (Pierce Europe B.V., OudBeijerland, The Netherlands) using bovine serum albumine as standard. W e s t e r n blot analysis. Samples were run on SDS-12% p o l y a c r y l a m i d e gels. Then, the proteins were e l e c t r o p h o r e t i c a l l y t r a n s f e r r e d to a n i t r o c e l l u l o s e membrane. Membranes were incubated with a 1:100 dilution of rabbit polyclonal a n t i s e r u m raised against human placental bFGF (obtained from D.B. Rifkin, New York U n i v e r s i t y Medical Center, New York, NY) or with a 1:12 dilution of affinity purified rabbit antibodies raised against peptides representing synthetic fragments of human placental bFGF (obtained from A. Sommer, Synergen Inc., Boulder, CO). Immunocomplexes were visualized by successive incubations with alkaline phosphatase conjugated anti-rabbit IgG and BCIP/NBT substrates (Promega Biotec, Madoson, WI), according to m a n u f a c t u r e r ' s instructions. Immuno dot-blot analysis. Aliquots of the fractions from the different columns were assayed for the presence of immunoreactive bFGF-like material by immuno dot-blot analysis. Aliquots of the fractions were filtered through a 96-well vacuum filtration apparatus onto a n i t r o c e l l u l o s e membrane. Then, membranes were incubated with anti-human placental bFGF antiserum and stained as described for W e s t e r n blot. Cell proliferation assay. GM 7373 cells were seeded at the density of 70,000 cells/sq, cm. After 24 h, confluent m o n o l a y e r cultures were w a s h e d twice with serum-free m e d i u m and incubated in fresh m e d i u m c o n t a i n i n g 0.4% FCS and different concentrations of p u r i f i e d rat brain bFGF. 24 h later, cells were trypsinized and counted by using a Burker chamber. DNA synthesis assay. The p u r i f i e d rat brain bFGF was tested for its ability to stimulate 3H-thymidine i n c o r p o r a t i o n into the DNA of quiescent NIH 3T3 cells as d e s c r i b e d p r e v i o u s l y (19).

1163

Vol. 155, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Inhibition of rat b r a i n bFGF a c t i v i t y b y anti-human placental bFGF antibodies. Rat brain bFGF was incubated with a 1:20 dilution of a n t i - h u m a n placental bFGF a n t i s e r u m for 2 h at 4°C. Then, the incubation mixture was added to c o n f l u e n t GM 7373 cells. After a 24 h incubation, cells were assayed for PA activity as d e s c r i b e d above.

RESULTS

Purification The

of rat b r a i n bFGF

ability

of bFGF to stimulate

endothelial of

this

factor

hepatoma cell

cells

cells

had been used

as an assay

from human p l a c e n t a (14,20).

associated

PA a c t i v i t y

an high affinity

chromatography bFGF.

When

Sepharose column peak

a first

most

reactivity

shown by immuno dot-blot of the P A - i n d u c i n g step

afforded

pooled which salt.

and

concentration

overlapped

with

of the

smaller

a

factor

further

(Fig.

SDS-PAGE,

approximately the

very

100 ng/ml)

only

a

with

18,000 was d e t e c t a b l e

low specific suggested

activity that m a j o r

bFGF

brain

heparin-

of

The

immuno

IA).

This

first

fractions

purification

the active

a

M and

fractions

weight

not shown).

a

of

However,

(ED50 higher with

2

and analyzed

molecular

sample

were

column

being eluted with

3-fold

as

the identity

heparin-Sepharose

contaminants,

1164

affinity

antibodies,

The active

(results

of this

bFGF

bound to the

a peak

column were p o o l e d

triplet

an

a 2 M NaCI wash.

(Fig.

IB). When

of

as

of rat

supporting

rat bFGF

from the second h e p a r i n - S e p h a r o s e by

thus

0.95 M NaCI before in

human

Because

activity

with

placental

a 50-fold purification.

resulted

GM 7373 cells

was p a s s e d over a

peak with

analysis,

molecule

was w a s h e d w i t h

cultured

we used heparin

of the P A - i n d u c i n g

bound to a second

This

(2),

with anti-human

in

we used the stimulation

homogenate

activity

(PA)

purification

of rat b r a i n bFGF.

as a single

PA-inducing

in the

step in the p u r i f i c a t i o n

the rat b r a i n column,

activator

from

in endothelial

for heparin

and was eluted of

cross

as

and

Therefore,

assay to follow the p u r i f i c a t i o n has

plasminogen

than

molecular

Vol. 155, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Dot blot analysis with anti-bFGF Ab l

•

12

•

:

2M~Cl ...........

I I

A

to.~

t

1,.0

.=._.

]o.3~.

~ u

o

25o

~o

7.

ioo

EFFLUENTVOLUME(rot) Dot blot analysis with anti-bFGF Ab

[

1

" ° _"

--

0

Dot blot analysis with anti-bFGF Ab

30

60 90 EFFLUENT VOLUME(ml)

",,

B

~

120

0

1

i

8

~

° 21M

10 12 EFFLUENT VOLUME(ml)

14 '

Fig. i. A) H e p a r i n - S e p h a r o s e i. Rat brain homogenate (ca. 2 g of protein) was loaded on a h e p a r i n - S e p h a r o s e column equilibrated with 0.5 M NaCI in T r i s / E D T A buffer. After w a s h i n g with the same buffer, the column was eluted with 0.8 M and 2.0 M NaCI, each in Tris/EDTA buffer. Fractions were tested for their ability to stimulate PA activity in GM 7373 cells and to cross react with anti-human bFGF antibodies by immuno dot-blot analysis. The material pooled for subsequent p u r i f i c a t i o n is indicated by the horizontal striped bar. B) Heparin-Sepharose 2. The active fractions from the previous column were loaded on a second, smaller heparin-Sepharose column. The column was eluted sequentially with 0.95 M and 2.0 M NaCI, both in T r i s / E D T A buffer. Fractions were assayed as for panel A. C) CM-Sephadex. The active fractions from the second heparin-Sepharose column were dyalized in 0.2 M NaCI/15% glycerol/0.1 M sodium phosphate (pH 6.0), clarified by centrifugation, and loaded on a CMSephadex column e q u i l i b r a t e d in the same buffer. Then, the column was sequentially eluted with 0.5 M and 2.0 M NaCI, both in dyalisis buffer, and the fractions were assayed as for panel A. The material pooled for SDS-PAGE analysis and biological studies is indicated by the striped horizontal .bar.

1165

,,, i C

16

Vol. 155, No. 3, 1988

weight

very

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

similar

to

bFGF,

were

still

present.

These

contaminants were t e n t a t i v e l y identified as histones on the basis of their m o l e c u l a r weight and their strong affinity for Therefore,

to

attempt a further p u r i f i c a t i o n of rat brain bFGF,

we took advantage of the c a p a c i t y of bFGF to bind to (12). When the active fractions column

(12).

the

a

from the second h e p a r i n - S e p h a r o s e

The

applied

precipitate was obtained,

precipitate which

was removed by

contained

the

to the CM-Sephadex column. to

as already

p u r i f i c a t i o n p r o c e d u r e of bFGF from

supernatant,

bound

CM-Sephadex

were dyalized against a low salt buffer before binding to

CM-Sephadex, during

described

human

centrifugation

PA-inducing

1

column

ug by

the

activity,

was

All the P A - i n d u c i n g activity

the resin and was eluted with 0.5 M

of the pooled active fractions SDS-PAGE

weight

of

procedure

recombinant human bFGF gave

from

showed the presence of a

apparent m o l e c u l a r weight of 18,000,

placenta and

NaCI

(Fig.

These steps resulted in a further 100-fold purification. of

heparin.

the

2).

doublet

The

Analysis

CM-Sephadex

identical to the (Fig.

IC).

with

molecular

purification

a p p o x i m a t e l y a 15,000-fold p u r i f i c a t i o n

with

,-66 45 • 36 29 24

20.1

....

14.2

Fig. 2. SDS-PAGE. Ca. 1 ug of purified rat brain bFGF was run on a 12% polyacrylamide gel in reducing conditions. Proteins were visualized with silver staining. Molecular weight standards are indicated in thousands. 1166

an

a

Vol. 155, No. 3, 1988

yield

of

were

20 u g of b F G F

obtained

rat brain

proteinase

inhibitors

Biological

activities

purified

7373

cells

for

also when

acidified

The

100 gr of r a t brain.

the purification

homogenate

in a d o s e

stimulated dependent

at a c o n c e n t r a t i o n

activity

was

Synergen

Inc.

anti-human of

rat bFGF,

like

PA activity

in

bFGF

The PA-inducing by preincubation

antiserum, with

171

thus

rat bFGF

the recombinant

A

3A).

(obtained

from

activity

of r a t b r a i n

of

sample

the

confirming

the

with

identity

not

shown).

h u m a n bFGF,

was

also

The

able

to

~c

j/

0.~

,l~

16j

9

_> a4

,3

A similar

i

o =." o~

<

GM

stimulation

(results

B

I

of v a r i o u s

endothelial

half maximal

human

molecule

an

r a t b r a i n bFGF.

by recombinant

bFGF

on

shown)

(Fig.

quenched

results

performed

ng/ml

CO).

Similar

in t h e p r e s e n c e

manner,

of

was

3-10

Boulder,

the p u r i f i e d

pure

0.?

shown

placental

not

of t h e p u r i f i e d

molecule

was

and/or

(results

occurring

preparation

E

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

+t

O~

-I,

4-

0.:

0.1

3j+

13

/

0.1

0.3

1

3

10 30

60

i

i

i

i

i

0.1

0.3

1

3

10

1

r

i

I

I

t

0.1

0.3

1

3

10

b-FGF (ng/ml) Fig. 3. Biological activity of purified rat brain bFGF. A) Stimulation of PA activity in endothelial GM 7373 cells. Confluent cultures of GM 7373 cells were given the indicated concentrations of the purified rat brain bFGF or recombinant human bFGF. After 16 h, cells were extracted and extracts were assayed for PA activity. B) Stimulation of proliferation of GM 7373 cells. Cells were treated as for panel A. After 24 h, cells were trypsinized and counted in a Burker chamber. C) Stimulation of DNA synthesis in NIH 3T3 cells. Quiescent cultures Of NIH 3T3 cells were incubated in fresh medium with 0.4% calf serum containing 3H-thymidine and the indicated concentrations of rat brain bFGF or recombinant human bFGF. After 24 h, the amount of radioactivity incorporated in the trichloroacetic acidprecipitable material was measured. The white or black arrows indicate the value of the measured paramether in control cultures given only 0.4% or 10% serum, respectively. Purified rat brain bFGF: • ; recombinant human bFGF: o . 1167

Vol. 155, No. 3, 1988

stimulate

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

proliferation

in GM 7373 cells and DNA

quiescent NIH 3T3 cells with a similar potency

synthesis

(Fig.

in

3B and C).

Immunological c h a r a c t e r i z a t i o n of p u r i f i e d rat b r a i n bFGF Amino acid sequence has shown a very high homology between bovine and human bFGFs preparation

(13).

Here we have shown that the rat brain bFGF

cross-reacts

polyclonal antiserum. human

and

analysis

rat

with

anti-human

bFGFs,

rat bFGF was

synthetic

control.

placental All

confirming bFGF

bFGF

its

Western

raised against

40-63,

63-75,

blot

peptides bFGF

(13)

and 143-157.

p o l y c l o n a l antiserum was used

immunological

preparation

also

a

m o l e c u l a r weight of 22,000. silver

by

as

a

these antibodies recognize the Mr 18,000 rat bFGF,

(Fig. 4). Surprisingly,

same

assayed

fragments of human placental

c o r r e s p o n d i n g to amino acid residues Anti-human

bFGF

To evaluate the degree of homology between

with immunopurified antibodies

representing

placental

staining

(approximately

A

2.5

on

similarity with

human

placental

all the antibodies recognized in the second

doublet,

with

an

apparent

This doublet was barely detectable by

SDS-PAGE

only

when

an

ug) of the rat bFGF p r e p a r a t i o n

high was

amount loaded

8

ilaii ¸¸¸!bill¸

Fig. 4. Western blot analysis of rat brain bFGF. A) Ca. 0.5 ug of purified rat brain bFGF were probed with affinity purified antibodies raised against synthetic bFGF fragments corresponding to amino acid residues 40-63 (a), 63-75 (b), and 143-157 (c). B) Ca. 0.5 ug of rat brain bFGF (a) and 0.i ug of recombinant human bFGF (b) were probed with an anti-human placental bFGF antiserum. Molecular weight standards are indicated in thousands. 1168

Vol. 155, No. 3, 1988

onto

the

silver

gel

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(results

stained

approximately

gel

not shown).

Soft-laser

scanning

showed that the Mr 22,000

form

of

the

represents

5% of the total protein of the sample.

DISCUSSION

Here, from

we have shown the purification

rat

brain.

This

molecule

molecular weight of 18,000, human recombinant antiserum, forms

bFGF.

appears

identical

(10,11).

naturally

Microheterogeneity

may

be

generated

procedure been

These N-terminus

occurring

presence

both

neutral

second doublet,

the

weight of 22,000.

affinity

for heparin-Sepharose antibodies

representing

human

both

raised

placental of

amino

forms may represent extent or the

was performed

they

extraction

on

conditions

inhibitors

rat and

(results

has

brain in

the

not shown),

to be contaminated

The Mr 22,000

form shows

and cross-reactivity against

synthetic

with a

(13).

This

a

strong

with affinity

bFGF

(amino acid residues

(amino acid residues

bFGF molecule

relatedness

18,000 bFGF. activity

of its

has

related to bFGF, with an apparent

the middle part

and the C-terminus

degree

of the bFGF molecule

Mr 18,000 bFGF-doublet

appeared

immunologically

molecular

63-75)

with an anti-bFGF

that the two forms may naturally occur in vivo.

The Mr 18,000 bFGF-doublet

purified

of

to different

or acidic

or absence of proteinase

suggesting

to the molecular weight

released during

In our hands,

in

a

truncated

observed when the purification

extracted

with

on variability

forms processed by proteases

(10,11).

doublet

that they represent microheterogeneous

already been reported and it depends terminus

as a

Both bands cross-react

thus indicating

of rat bFGF.

of a bFGF-like molecule

fragments 40-63 and

143-157)

indicates

of the

between the Mr 22,000 doublet and the

In all the biological

assays performed,

high Mr

the specific

of the rat brain bFGF preparation was identical

1169

the

or even

Vol. 155, No, 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

higher than that of purified recombinant human bFGF. 22,000

form represents

seems

very

likely

biological

less than 5% of the Mr

Since the Mr

18,000

form,

that the latter form is responsible

activity

of

the

preparation.

However,

it

of

we

the

cannot

exclude that also the Mr 22,000 doublet is endowed of a bFGF-like activity.

The

purification

of this form

to

homogeneity

will

clarify this point. A

form

purified

of bFGF with a m o l e c u l a r weight of 25,000

from

biochemical, 18,000 bFGF proteins (15).

guinea pig brain.

This

protein

shares

(14).

Here,

immunoreactive

been identified in different areas of

rat

we

Mr

have

shown

the

presence

of

in the CNS. At present,

support or reject this hypothesis. that

the

immunoreactive

presence

bFGF-like

characteristic

of the CNS.

immunoreactive

high

molecules

relationship

a

is

However, that

from amino

this

amino

acids

(13).

The

that

pointed

weight

not

unique

an

In fact, both Mr 18,000 and Mr 22,000 are detectable by Western blot

between

the

various

(results not shown). forms

of

clones

has

N-terminal to the

proposed

to the

proposed

initiation

1170

initiation

been

shown

including

codon

2

methionine

clones of bFGF contained an open reading 5'

is

(21,22).

acid sequence of human placental bFGF has

protein is composed of 157 amino acids,

extended

bFGF

Both bovine and human bFGFs have been cloned,

the nucleotide sequence of the

cDNA

out,

molecular

p r i m a r y translation product of 155 amino acids

predicted

It

forms may exert

It must be

of

bFGF-like proteins

at present unclear. and

22,000

no data are available

analysis in the extract of RAT-I fibroblasts The

a

brain

bFGF-like protein in a rat brain homogenate.

functions

however,

several

High m o l e c u l a r weight immunoreactive bFGF-like

is tempting to speculate that different bFGF-like

to

been

biological and immunological properties with the Mr

have

different

has

frame

(21,22),

Vol. 155, No. 3, 1988

suggesting Indeed,

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

that

N-terminus

extended

forms of bFGF

amino acid sequence data has confirmed

form of bFGF purified

from guinea pig brain

extended

(ii).

bFGF

form of bFGF

gene,

products

of

product

of

alternative

the

different

the

pathway

(14) is an N-terminal

of

transcript. different

protein.

They mRNAs

splicing or they might derive

synthetic

that the Mr 25,000

forms of bFGF should

high molecular weight precursor the

exist.

Since there is a single copy of

same initial

translation

may

all

represent

might

be

the

generated

by

from processing

Further

of bFGF is required

to

the

of

an

investigation elucidate

of

this

point.

ACKNOWLEDGMENTS The authors wish to thank Dr. A. Sommer and Dr. D.B. Rifkin for their helpful discussion and criticism and Miss M. Belleri for her skilled technical assistence. This work was supported by a grant from the Associazione Italiana per la Ricerca sul Cancro and grant n.87.01349.04 from C.N.R. to M.P. and by grants from Ministero della Pubblica Istruzione (M.P.I. 40%, National Project of Experimental Oncology) and from C.N.R. (Progetto Finalizzato Oncologia) to G.R.

REFERENCES

i) Gospodarowicz D., Cheng J., Lui G.M., Baird A., and Bohlen P. (1984) Proc. Natl. Acad. Sci USA 81: 6963-6967. 2) Gospodarowicz D., Ferrara N., Schweigerer L., and Neofeld G. (1987) Endocrine Reviews 8: 95-114. 3) Mascarelli F., Raulais D., Counis M.F., and Courtois Y. (1987) Biochem. Biophys. Res. Commun. 146: 478-486. 4) Bohlen P., Esch F., Baird A., Jones K.L., and Gospodarowicz D. (1985) FEBS Lett. 185: 177-181. 5) Unsicker K., Reichert-Preibsch H., Schmidt R.,Pettmann B., Labourdette G., and Sensenbrenner M. (1987) Proc. Natl. Acad. Sci. USA 84: 5459-5463. 6) Walicke P., Cowan W.M., Ueno N., Baird A., and Guillemin R. (1986) Proc. Natl. Acad. Sci. USA 83: 3012-3016. 7) Morrison R., Sharma A., De Vellis J., and Bradshaw R. (1986) Proc. Natl. Acad. Sci. USA 83: 7537-7541. 8) Otto D., Unsicker K., and Grothe C. (1987) Neurosci. Lett. 83: 156-160. 9) Anderson K.J., Dam D., Lee S., and Cotman C.W. (1988) Nature (London) 332: 360-361. 10) Esch F., Baird A., Ling N., Ueno N., Hill F., Deneroy L., Klepper R., Gospodarowicz D., Bohlen P., Guillemin R. (1985) Proc. Natl. Acad. Sci. USA 82: 6507u6511. Ii) Moscatelli D., Joseph-Silverstein J., Presta M., and Rifkin D.B. (1988) Biochimie 70: 83-87. 117l

Vol. 155, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

12) Moscatelli D., Presta M., and Rifkin D.B. (1986) Proc. Natl. Acad. Sci. USA 83: 2091-2095. 13) Sommer A., Brewer M.T., Thompson R.C., Moscatelli D., Presta M., and Rifkin D.B. (1987) Biochem. Biophys. Res. Commun. 144: 543-550. 14) Moscatelli D., Joseph-Silverstein., Manejias M., and Rifkin D.B. (1987) Proc. Natl. Acad. Sci. USA 84: 5778-5782. 15) Presta M., Foiani M., Rusnati M., Joseph-Silverstein J., Maier J.A.M., and Ragnotti G. (1988) Neurosci. Lett., in press. 16) Grinspan J.B°, Stephen N.M., Levine E.M. (1983) J. Cell. Physiol. 114: 328-338. 17) Laemmli U.K. (1970) Nature (London) 277: 680-685. 18) Wray W., Boulikas T., Wray W.P., and Hancock R. (1981) Anal. Biochem. 118: 197-203. 19) D'Amore P.A., Glaser B.M., Brunson S.K., and Fenselau A.H. (1981) Proc. Natl. Acad. Sci. USA 78: 3068-3072. 20) Presta M., Moscatelli D., Joseph-Silverstein J., and Rifkin D.B. (1986) Mol. Cell. Biol. 6: 4060-4066. 21) Abraham J.A., Mergia A., Whang J.L., Tumolo A., Friedman J., Hjerrild K.A., Gospodarowicz D., and Fiddes J.C. (1986) Science 233: 545-548. 22) Abraham J.A., Whang J.L., Tumolo A., Mergia A., Friedman J., Gospodarowicz D., and Fiddes J. (1986) EMBO J. 5: 2523-2528.

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