A group of growth factors, designated heparin-binding brain mitogens (HBBMs), is disclosed. The HBBMs are isolated from brain tissue by a sequence of purification steps.

SUMMARY OF THE INVENTION 
This invention relates to a group of novel protein growth factors which are 
believed to promote angiogenesis and, therefore, should be useful in wound 
healing, bone healing and the treatment of burns. The proteins induce 
mitogenesis in endothelial cells and, as such, may be considered to be 
growth factors for those cells. The proteins are also believed to promote 
the formation, maintenance and repair of tissue, in particular, neural 
tissue. 
The proteins are isolated from brain cells and may each be termed a 
heparin-binding brain mitogen (HBBM). The proteins are single chain and 
highly basic. Three such proteins have been isolated from bovine brain, 
and have been designated HBBM-1, HBBM-2 and HBBM-3, with molecular weights 
of 18, 16 and 15kd, respectively. The proteins possess a common 19 amino 
acid N-terminal sequence which differs from that of other known proteins. 
The same three HBBMs have also been isolated from human brain tissue and 
have the same N-terminal sequence and the same type of mitogenic activity 
as bovine HBBMs. Rat and chicken brains have also been found to contain 
HBBMs. 
The proteins are isolated and purified from brain tissue by a combination 
of steps, which include extraction from the tissue, heparin affinity 
chromatography, and cation-exchange chromatography. Hydrophobic 
interaction chromatography may be used as an auxiliary method of 
purification. 
BACKGROUND OF THE INVENTION 
Numerous protein growth factors have been isolated and characterized in 
recent years. These growth factors include epidermal growth factor, 
fibroblast growth factors, insulin-like growth factors, transforming 
growth factors, platelet-derived growth factor and interleukins. For 
example, fibroblast growth factor ("FGF") was first purified by 
Gospodarowicz in 1975 from bovine pituitary and had an estimated molecular 
weight of 13,300 daltons (Reference 1). 
FGF was later purified from bovine brain (2). FGF can be isolated in either 
an acidic ("aFGF") or basic ("bFGF") form, depending on the isolation 
procedures used (3,4). A complete amino acid sequence for bovine pituitary 
bFGF (5) and bovine and human brain aFGF (6,7) has been published, 
together with the N-terminal sequences for bovine and human brain bFGF 
(5). The N-terminal sequences for bovine pituitary and bovine brain bFGF 
are identical (5,8). 
It has now been found that, when the FGFs are purified from brain tissue 
using heparin-Sepharose affinity chromatography (9,10), a significant 
quantity of unknown proteins may also be present. Because proteins that 
bind with particularly high affinity to heparin are rare, further study of 
these unknown proteins was undertaken. This investigation revealed these 
proteins to be the HBBMs, which differ from the FGFs in their N-terminal 
sequence and amino acid compositions. 
Accordingly, it is an object of this invention to isolate, purify and 
characterize the HBBMs from brain tissue. It is a further object of this 
invention to establish the physiological activity of the HBBMs.

DETAILED DESCRIPTION OF THE INVENTION 
The novel growth factors of this invention are single chain, basic, 
heparin-binding brain mitogens. HBBMs have been identified in the brain 
tissues of all species tested, which include human, bovine, rat and 
chicken. Based on this distribution, it is expected that other species 
will also contain HBBMs. 
The N-terminal sequences of the HBBMs differ markedly from those published 
for bovine or human brain aFGF and bFGF (5,6,7). The N-terminal sequences 
of the first 19 amino acids have been found to be identical for human and 
bovine HBBMs. The N-terminal sequences are as follows: 
Gly-Lys-Lys-Glu-Lys-Pro-Glu-Lys-Lys-Val-Lys-Lys-Ser-Asp-Cys-Gly-Glu-Trp-Gl 
n. The rat sequence is identical with the exception of the cysteine residue 
in position 15, because a determination of the presence or absence of 
cysteine was not conducted. However, the fact that no other amino acid was 
identified at position 15 is consistent with the presumed presence of 
cysteine. To date, the first ten N-terminal residues of chicken HBBMs have 
been sequenced; they are identical to those of human, bovine and rat 
HBBMs. 
The HBBMs have been found in three forms in tissue from bovine brain. 
HBBM-1, HBBM-2 and HBBM-3 have molecular weights of 18, 16 and 15 kD, 
respectively. Their amino acid compositions (determined as hereinafter 
described) are set forth in Table I below and also differ from the amino 
acid compositions of the bovine brain FGFs (5,6,7). 
Based on the molecular weights and the identical N-terminal sequences, it 
is concluded that the three HBBMs probably differ at their C-termini. The 
available data strongly suggest that HBBM-2 and HBBM-3 are C-terminally 
truncated forms of HBBM-1, lacking approximately 13 and 29 amino acids, 
respectively. 
It was found that the ratios of the three forms varied between different 
isolation batches. However, on average, based on quantitative amino acid 
analysis, the overall isolation yield of HBBMs was estimated at 
approximately 30, 10 and 40 .mu.g/kg brain tissue for HBBM-1, HBBM-2, and 
HBBM-3, respectively. The ratios may depend on variables during tissue 
storage and extraction, in particular proteolysis. Proteolysis during 
tissue extraction may cause carboxy-terminal truncation of HBBM-1, the 
largest of the HBBM forms, which would yield HBBM-2 and HBBM-3 in varying 
amounts. 
The process for isolating the novel HBBMs of this invention in 
substantially pure form from a source of brain tissue comprises the 
sequence of steps of: 
(a) extraction from the source tissue; 
(b) cation-exchange chromatography; 
(c) heparin affinity chromatography; 
(d) cation-exchange chromatography; and, optionally, 
(e) hydrophobic-interaction chromatography. 
The extraction is accomplished by treating the tissue sequentially with 
0.15M ammonium sulfate, adjusting to pH 4.5 with hydrochloric acid, 
stirring and centrifugation, treating with ammonium sulfate after the pH 
is adjusted to 6-6.5 with sodium hydroxide, stirring and centrifugation, 
followed by dialysis and centrifugation. 
The first cation-exchange chromatography step comprises batch adsorption on 
a carboxymethyl-Sephadex column, followed by washing with 100 mM sodium 
phosphate buffer at pH 6 and elution with 100 mM sodium phosphate, pH 
6.0/0.6M NaCl. 
The heparin affinity chromatography is performed on a heparin-Sepharose 
(Pharmacia) column by washing with a buffer containing 10 mM Tris-HCl, pH 
7.0/0.6M NaCl, followed by elution with a linear gradient of from 0.6 to 
2.0M NaCl in 10 mM Tris-HCl, pH 7.0. 
The second cation-exchange chromatography step is performed on a Mono-S 
(Pharmacia) column equilibrated and, after loading of the sample, washed 
with 50 mM sodium phosphate, pH 6.8, followed by elution of HBBMs with a 
linear gradient of from 0 to 0.6M NaCl in 50 mM sodium phosphate, pH 6.8. 
The hydrophobic-interaction chromatography step comprises preequilibrating 
a HIC column (LKB Ultropac-TSK-Phenyl-5PW) with a buffer of 100 mM sodium 
phosphate, pH 7.0, and 1.5M sodium sulfate, followed by elution with a 
linear gradient of from 1.5 to 0.6M sodium sulfate. 
Although the HBBMs may be separated easily and quantitatively from the FGFs 
by reverse-phase HPLC, this procedure reduces the biological activity of 
the FGFs due to the conditions used in reverse-phase HPLC. Therefore, in 
order to separate and isolate the HBBMs and the FGFs in biologically 
active form for comparative testing, the process set forth above was 
developed. The use of reverse-phase HPLC was limited to the testing of 
small aliquots of fractions from heparin-Sepharose affinity chromatography 
and cation-exchange chromatography for the presence of HBBMs and FGFs. The 
reverse-phase HPLC steps were performed on a C4 column (The Separations 
Group) eluted with a shallow gradient of acetonitrile in 0.1% 
trifluoroacetic acid. 
The biological activity of the HBBMs has been established by tests 
evidencing the induction of mitogenesis in endothelial cells. Although 
HBBM-2 was not tested, the activity demonstrated for HBBM-1 and HBBM-3 
suggests that HBBM-2 will also possess this activity. The testing was 
performed on bovine aortic arch endothelial cells. 
Three separate tests were performed, both to confirm the existence of 
mitogenic activity and to demonstrate that the activity was due to the 
HBBMs and not to the FGFs. 
First, the eluent fractions from Mono-S chromatography which contained 
HBBM-3 were pooled, rechromatographed on a Mono-S column and then tested 
for their ability to stimulate the growth of bovine vascular endothelial 
cells. For comparison, samples of aFGF and bFGF were also tested. FIG. 5 
presents the results of this test. 
Next, the available HBBM-3 fractions were rechromatographed for sample 
concentration on a Mono-S column using a steeper gradient and lower flow 
rate. The HBBM-3 was then compared with aFGF and bFGF in dose-response 
analyses. The upper panel of FIG. 6 presents the results of this test. 
This procedure was then repeated for HBBM-1. Those results are presented 
in the lower panel of FIG. 6. 
Finally, HBBM-3 fractions concentrated by Mono-S chromatography were placed 
on an hydrophobic interaction column, which has different selectivities 
than a Mono-S column. FIG. 7 presents the results of this test. 
These tests established that the HBBMs stimulate the proliferation of 
cultured bovine vascular endothelial cells in a dose-dependent manner. The 
ED.sub.50 was approximately 20 ng/ml for HBBM-3 and 180 ng/ml for HBBM-1 
The ED.sub.50 was calculated as follows: ED.sub.50 =conc [cell 
no.(dose=0)+cell no.(dose=max)/2]. These mitogens were comparable to the 
FGFs in terms of biological activity and high affinity to heparin. HBBM-1 
and HBBM-3 are each equipotent to aFGF. The ED.sub.50 are in the range of 
50-150 ng/ml. 
The test results also make plain that the biological activity associated 
with HBBM-3 was genuine and was not the result of contamination with an 
FGF. HBBM-3 and the FGFs are clearly separated by highly resolutive Mono-S 
chromatography, as shown in FIG. 5, and are also clearly separated by a 
second high resolution technique with different selectivities, hydrophobic 
interaction chromatography, as shown in FIG. 7. 
Moreover, the HBBMs did not contain measurable quantities of aFGF as 
determined by quantitative reverse-phase HPLC. As further evidence that 
the activity was due to HBBMs, the HBBMs were not cross-reactive in an 
immuno-dot assay using polyclonal antibodies raised against synthetic 
peptides corresponding to the N-terminal 15 residues of aFGF (1-15) and 
bFGF (30-50), the latter yielding an antibody which cross-reacts with aFGF 
(antibodies provided by A. Baird, Salk Institute; growth factor 
nomenclature according to refs. 5,7). Finally, the concentrations of HBBM 
and aFGF preparations were carefully determined by quantitative amino acid 
analysis as a basis for potency comparisons (FIG. 6). Based on those 
measurements, an inherently inactive HBBM-3 would have to be contaminated 
with an equal amount of aFGF in order to produce the biological response 
seen. The results rule out such a contamination. In summary, the results 
of those studies clearly show that the activity of HBBM-3 is not a 
consequence of aFGF contamination. 
Several lines of evidence indicate, furthermore, that HBBM-3 activity is 
not due to bFGF contamination. In theory, a few percent contamination of 
HBBM-3 with bFGF would suffice to produce the activities observed. 
However, such a contamination is not indicated, because HBBMs and bFGF are 
widely separated on heparinSepharose chromatography. Moreover, HBBM-3 and 
bFGF are also well-separated in Mono-S chromatography (FIG. 5). Finally, 
when HBBM-3 was subjected to chromatography on a third highly resolutive 
system, hydrophobic interaction chromatography, it was evident that 
biological activity was still associated with HBBM-3 and not with the 
well-separated bFGFs (FIG. 7). There is no evidence that the two entities 
would copurify, even in trace amounts, in three widely disparate and 
resolutive chromatographic systems. 
Based on those results, it is concluded that the activity of HBBM-3 is 
genuine and not the result of contamination with the known heparin-binding 
FGFs. 
The HBBMs are further distinguished from the FGFs by a lack of amino acid 
sequence homology between the two groups of proteins. In addition to the 
difference in N-terminal sequences described earlier, the presently 
available sequence information (approximately 75 of 130 residues of 
HBBM-3) shows no sequence homology to the published 146 residue sequence 
of the FGFs (5). 
The mitogenic activity of human HBBMs was generally found to be 
indistinguishable from that of bovine HBBM, although the human mitogens 
were less extensively characterized owing to limited quantities of 
material available. This is consistent with other findings: identical 
amino-terminal sequence, presence of three forms, and retention behavior 
on ion-exchange, heparin-Sepharose, and reverse-phase chromatography. 
The identity of N-terminal sequences among the various sources of the HBBMs 
suggests that evolutionary pressure may have prevented mutations, at least 
during the evolution from birds to mammals. Sequence conservation as a 
result of evolutionary pressure is strongly suspected with many 
well-known, biologically active proteins that are highly homologous 
between species. 
The HBBMs were not present in bovine kidney tissue that was extracted using 
the procedure for brain tissue. Therefore, the HBBMs are novel proteins 
which may also play a role in the formation, maintenance and/or repair of 
tissue, in particular, neural tissue. 
The bases for this statement about the role of HBBMs are the following: (1) 
the HBBMs have the same biological and heparin-binding activities as aFGF; 
(2) the HBBMs are brain-specific; (3) higher amounts of the HBBMs than 
aFGF are found in the brain; and (4) aFGF and bFGF are known to have very 
prominent neural activities, such as neurotrophic (neuron survival) 
activity in vivo and in vitro, they are mitogenic for neuroblasts and 
glial cells, they promote neurite outgrowth and induce brain-specific 
protein synthesis. 
Although the protein structures of the HBBMs and the FGFs are dissimilar, 
their similarity in terms of heparin binding and biological activity 
suggests that both groups of mitogens act through a similar mechanism. For 
example, the mitogens could bind to cell surface or extracellular, 
matrix-associated heparinlike structures. 
Therapeutic compositions in accordance with this invention include HBBMs, 
either singly or in mixtures, dispersed in a conventional pharmaceutically 
acceptable liquid or solid carrier. The therapeutic compositions may be 
administered topically in the form of creams, lotions and so forth, or 
orally in such forms as tablets, capsules, dispersible powders, granules 
or suspensions, or parenterally in the form of sterile injectable 
solutions or suspensions. These therapeutic compositions may be 
administered to human or veterinary patients to promote angiogenesis and 
the repair and maintenance of neural tissue. 
EXAMPLE 
In this example, the isolation and characterization of bovine HBBM are set 
forth in detail. Results using human material were very similar and will 
not be described in detail unless differences from bovine material were 
found. 
1) Tissue Extraction 
Human brains were obtained less than 24 hours post-mortem from the 
Department of Pathology, University of Zurich. Bovine brains were obtained 
at a slaughterhouse. Tissues were frozen immediately after receipt, stored 
at -80.degree. C., and processed within two weeks after receipt. Batches 
of 3-5 kg of brain tissue were extracted at a time. 
Brain tissue was extracted following the procedure developed by 
Gospodarowicz and co-workers (2) as described (6,10): Frozen brains were 
crushed with a hammer. Six-hundred gram portions of tissue were 
homogenized in 1.2 1 of 0.15M ammonium sulfate for 3 minutes in a Waring 
Blender. The pH of the homogenate was adjusted immediately to pH 4.5 with 
concentrated HCl and the mixture was further homogenized using a Polyton 
homogenizer until the tissue was finely dispersed. The homogenate was then 
extracted by stirring of the suspension for 2 hours at 4.degree. C. 
followed by centrifugation at 4.degree. C. for 60 minutes at either 11,500 
rpm (GSA rotor) or 9000 rpm (GS-3 rotor) to remove cells and debris. The 
supernatant was adjusted to pH 6-6.5 with NaOH, ammonium sulfate (230 g/l) 
was added slowly, and the resulting suspension was stirred for at least 30 
minutes at 4.degree. C. and centrifuged again as described above. The 
pellet was discarded. More ammonium sulfate (300 g/l) was slowly added to 
the supernatant, the suspension was stirred for at least 30 minutes at 
4.degree. C. and centrifuged as above. The resulting pellet was dissolved 
in cold water (100 ml per kg of starting material) and dialysed at 
4.degree. C. for 20 hours against 20 l of water using a Spectrapor 
membrane (molecular weight cut-off 6-8 kD, diameter 31.8 mm). The 
dialysate was centrifuged again to remove precipitated material and the 
supernatant subjected to chromatographic purification (see below) after 
determining its conductivity. 
2) Cation Exchange Chromatography 
The tissue extract resulting from the above procedure (approximately 150 ml 
for each kg of brain tissue) was subjected to batch adsorption/elution by 
cation exchange chromatography as follows: The sample was diluted with 
water as required to bring the conductivity below that of a 0.1M sodium 
phosphate buffer (pH 6)/0.15M NaCl solution. The sample was then loaded 
onto a column of carboxymethyl-Sephadex (5.5.times.3 cm) which was 
preequilibrated with 100 mM sodium phosphate (pH 6)/0.15M Cl. The column 
was washed with the equilibration buffer and a protein fraction was eluted 
with 100 mM sodium phosphate, pH 6.0/0.6M NaCl and collected. All 
operations were carried out at room temperature and a flow rate of 500 
ml/hour. 
3) Heparin-Sepharose Affinity Chromatography 
The 0.6M NaCl eluate from cation exchange chromatography was loaded on a 
heparin-Sepharose column (Pharmacia, 5.times.1.5 cm) at a flow rate of 125 
ml/hour. The column was washed with 200 ml of a buffer containing 10 mM 
Tris-HCl, pH 7.0/0.6M NaCl until the absorbance of the column eluate at 
280 nm became negligible. Protein bound to the column was eluted with a 
120-minute linear gradient from 0.6M to 2M NaCl in 10 mM Tris-HCl, pH 7.0 
at a flow rate of 35 ml/h. Chromatography was performed at room 
temperature using a LKB peristaltic pump and a low pressure LKB 
programmable gradient former. Fractions of 1.4 ml were collected and 
aliquots subjected to bioassay. 
The results of this Heparin-Sepharose chromatography are shown in FIG. 1. 
The fractions subjected to further purification, using steps described 
below, are indicated by the horizontal bar A. The fractions containing 
aFGF eluted between 1.1-1.3M NaCl (as determined by HPLC analysis of 
individual heparin-Sepharose column fractions; data not shown) and 
correspond to the shoulder of the larger peak eluting at 1-1.2M NaCl, and 
to the horizontal bar B. The reverse-phase HPLC analysis was conducted on 
a C4 column (25.times.0.46 cm, 5 um particle size, 300 angstroms pore 
size, The Separations Group, Hesperia, Calif.). The proteins were eluted 
in a shallow gradient of acetonitrile (10%/hour) in 0.1% trifluoroacetic 
acid at a flow rate of 0.7 ml/minute. The heparin-Sepharose chromatography 
was performed at room temperature. 
4) Mono-S Cation Exchange Chromatography 
In order to separate the contaminating aFGF from the other protein 
material, fractions eluting at 1-1.2M NaCl were pooled, diluted 
sufficiently with a buffer containing 50 mM sodium phosphate, pH 6.8 (to 
reduce their ionic strength to approximately that of the diluent) and 
subjected to cation-exchange chromatography on a Mono-S column (Pharmacia) 
equilibrated with 50 mM sodium phosphate, pH 6.8. This material was pumped 
onto a Mono-S column (Pharmacia) equilibrated with 50 mM sodium phosphate, 
pH 6.8. After washing the column with the same buffer until the absorbance 
at 210 nm reached a minimum value, protein was eluted with a gradient from 
0 to 0.6M NaCl in 50 mM sodium phosphate, pH 6.8. 
Several well-discernible peaks were eluted from the Mono-S column under 
these conditions, as shown in FIG. 2. Each peak was analyzed by 
reverse-phase HPLC using LKB HPLC equipment at room temperature with a 
flow rate of 0.7 ml/minute. 
The first and quantitatively minor peak which eluted from the Mono-S column 
at 0.3M NaCl corresponded to aFGF as evidenced by co-elution with a 
reference standard of authentic aFGF in the same system under identical 
conditions (see arrow in FIG. 2), as well as by co-elution in 
reverse-phase HPLC on a previously-described C4 column, eluted under 
highly resolutive, shallow gradient conditions (data not shown). The 
presence of aFGF in this Mono-S column fraction was expected, because the 
originating fraction from heparin-Sepharose chromatography is known to 
contain aFGF. The major part of the material eluted from the Mono-S column 
at approximately 0.45-0.65M NaCl as a relatively well-resolved triplet of 
peaks. Since those peaks were determined to contain HBBM, as hereinafter 
described, they were designated as HBBM-3, HBBM-2, and HBBM-1, in the 
order of their elution (FIG. 2). The elution behavior of human HBBM on a 
Mono-S column is similar in general, but has been less well studied than 
bovine HBBM. 
The HBBMs are clearly distinguishable by chromatographic retention not only 
from aFGF but also bFGF (FIG. 2). 
Moreover, when analyzed by reverse-phase HPLC (FIG. 3), the HBBMs were also 
found to differ clearly from the FGFs with respect to retention times 
(data not shown). Finally, reverse-phase HPLC separated all three HBBMs 
from each other (FIG. 3) and thus provided a means for their preparation 
in high purity (as needed for structural characterization) and for 
relatively unambiguous identification. 
Human brain also yielded three forms of HBBMs with reverse-phase HPLC 
elution patterns identical to those of their bovine counterparts. 
5) Hydrophobic Interaction Chromatography 
Fractions from Mono-S chromatography containing samples of interest were 
made 1.5M in sodium sulfate and applied to an HIC column (LKB Ultropac 
TSK-Phenyl-5PW, 7.5.times.75 mm) which was preequilibrated with a buffer 
of 100 mM sodium phosphate, pH 7.0/1.5M sodium sulfate. Protein was 
chromatographed at room temperature using a 30-minute linear gradient from 
1.5 to 0.6M sodium sulfate at a flow rate of 1.0 ml/min. Hydrophobic 
interaction chromatography is a separation system with selectivities quite 
different from Mono-S chromatography. Consequently, the separation of the 
HBBMs from the FGFs by HIC (as shown in FIG. 7), reinforces the result of 
Mono-S chromatography (as shown in FIG. 5) that the HBBMs are different 
chemical entities from the FGFs. 
6) Amino Acid N-Terminal Sequence Analysis 
Usually, sequence analyses were conducted without chemical modification of 
the proteins. However, as a preliminary step in conducting some analyses 
of the amino acid N-terminal sequence of the HBBMs, the cysteine residues 
of the HPLC-purified proteins were treated according to the procedure of 
Gautschi-Sova et al. (6). Briefly, the cysteine residues were reduced with 
a five-fold molar excess of dithiothreitol and alkylated by 
carboxymethylation using a three-fold molar excess of iodo-[2-.sup.14 
C]-acetic acid. 
The amino acid N-terminal sequence analyses of proteins (100-500 pmol) were 
performed on an Applied Biosystems (Foster City, Calif.) Model 470A 
gas/liquid phase protein microsequenator as described by Esch et al. (5). 
In addition, phenylthiohydantoin (PTH) derivatives of amino acids were 
identified by reverse-phase HPLC on an Applied Biosystems Model 120A 
On-line PTH amino acid analyzer. Both procedures were carried out 
according to protocols from the instrument manufacturer using chemicals 
supplied by Applied Biosystems. 
The N-terminal sequences of all three HBBMs were found to be identical to 
each other for the first 19 amino acids. The sequences were determined as 
Gly-Lys-Lys-Glu-Lys-Pro-Glu-Lys-Lys-Val-Lys-Lys-Ser-Asp-Cys-Gly-Glu-Trp-Gl 
n. Human HBBMs (probably analyzed as a mixture) were found to possess the 
same N-terminal sequence as bovine HBBMs. 
While sequencing data clearly indicate that the three bovine HBBM proteins 
are structurally related to each other, amino acid compositions and 
molecular weights further suggest that those proteins may differ in the 
carboxy-terminal region. All data are compatible with the alternative 
interpretations that either HBBM-2 and HBBM-3 are carboxy-terminally 
truncated forms of HBBM-1, lacking approximately 13 and 29 amino acids, 
respectively, at their carboxy-terminals. 
7) Molecular Weights of HBBMs 
Protein samples were analyzed using sodium dodecyl sulfate polyacrylamide 
gel electrophoresis ("SDS-PAGE") as described by Gospodarowicz et al. (9). 
Briefly, molecular weight determinations were performed by SDS-PAGE as 
follows: Aliquots containing 40-80 ng of protein were added to the sample 
buffer composed of 30% (v/v) glycerol, 0.2M dithiothreitol, 4% (w/v) 
sodium dodecyl sulfate, 4 mM EDTA, and 75 mM Tris-HCl, pH 6.8. Samples 
were boiled for 3 minutes and then applied to a 20% polyacrylamide gel 
slab (1.5 mm) with a 3% stacking gel. Electrophoresis under non-reducing 
conditions was performed in identical fashion except that dithiothreitol 
was omitted from the sample buffer. A protein standard mixture containing 
lysozyme (14.4 kD), trypsin inhibitor (21.5 kD), carbonic anhydrase (31 
kD), ovalbumin (45 kD) and serum albumin (66.2 kD) was also applied to 
each gel. SDS-PAGE revealed the molecular weights of HBBM 1, -2, and -3 to 
be 18, 16 and 15 kD, respectively (FIG. 4). The molecular weights did not 
differ significantly regardless of whether the samples were 
electrophoresed in the presence or absence of reducing agent, indicating 
that the proteins are single chain polymers. The molecular weights of the 
human HBBMs have not yet been determined by SDS-PAGE. 
8) Amino Acid Composition Analysis 
A 10-20 pmol protein sample was hydrolyzed according to the high 
sensitivity methodology of Bohlen and Schroeder (11). Briefly, the protein 
sample was added to a hydrolysis tube and dried in vacuo. Fifty .mu.l 
constant boiling HCl containing 2% (v/v) thioglycollic acid were added. 
The tube was then evacuated with high vacuum (less than 50 mm Torr) after 
freezing the sample in liquid nitrogen. The sample was then allowed to 
melt while under vacuum and the tube was flame-sealed. 
The tube was hydrolyzed by heating at 110.degree. C. for 20 hours. After 
hydrolysis, the tube was opened, dried in vacuo, and the residue dissolved 
in 130 .mu.l citrate buffer (0.067 sodium citrate, pH 2.20) prior to 
loading on the cation exchange chromatography column. 
The protein hydrolysates were chromatographed on a Chromakon 500 amino acid 
analyzer (Kontron, Zurich, Switzerland) equipped with a polystyrene-based 
cation exchange column and an o-phthalaldehyde fluorescence detection 
system for high-sensitivity detection (11). 
Quantitation of amino acids was by the external standard method using an 
amino acid standard mixture. Based on quantitative amino acid analysis, 
the overall isolation yield of HBBMs was estimated at approximately 30, 
10, and 40 .mu.g/kg brain tissue for HBBM-1, HBBM-2, and HBBM-3, 
respectively. 
Amino acid compositions of the three HPLC-purified proteins are shown in 
Table I. 
TABLE I 
______________________________________ 
Amino acid compositions of heparin-binding brain 
mitogens 
HBBM-1 HBBM-2 HBBM-3 
______________________________________ 
Molecular weight.sup.1 
18,000 16,000 15,000 
Order of elution 
reverse-phase HPLC 
1 2 3 
Mono-S 3 2 1 
Amino acid (number of residues) 
Asparagine & aspartic acid 
10 10 9 
Threonine 14 14 13 
Serine 9 9 6 
Glutamine & glutamic acid 
23 20 17 
Proline nd.sup.2 nd nd 
Glycine 16 15 13 
Alanine 10 8 8 
Cysteine nd nd nd 
Valine 4 4 4 
Methionine 1 1 1 
Isoleucine 2 2 2 
Leucine 8 7.sup.3 8 
Tyrosine 2 2 1 
Phenylalanine 2 2 2 
Histidine 1 1 1 
Lysine 35 28 23 
Tryptophan 3 nd.sup.3 3 
Arginine 8 7.sup.3 8 
______________________________________ 
.sup.1 as determined by SDSPAGE 
.sup.2 nd: not determined 
.sup.3 in accordance with the hypothesis of Cterminal truncation, it is 
believed that the following are the proper values for HBBM2: Leucine 8, 
Tryptophan 3, Arginine 8. Amino acid compositions are calculated from 3-5 
determinations. 
Amino acid compositional data agree with the elution order of the three 
proteins on Mono-S: the least basic protein (HBBM-3) elutes first, while 
the most basic protein (HBBM-1) elutes last. Expected molecular weights 
calculated from amino acid analyses are somewhat lower than those 
determined by SDS-PAGE. The discrepancies may be accounted for by proline 
and cysteine residues which were not quantitated by amino acid analysis. 
However, based on the N-terminal sequence analysis, it is known that 
proline and cysteine are in fact present. Amino acid analyses of human 
HBBMs are not yet available. 
9) Biological Activity 
The ability of certain of the HBBMs to induce mitogenesis in endothelial 
cells was tested by measuring the effect of those proteins on the 
proliferation in vitro of bovine aortic arch endothelial cells which had 
been cultured as described by Bohlen et al. (2,10). Briefly, the bovine 
endothelial cells were seeded with column fractions obtained as described 
below at low density (10,000-20,000 cells/35 mm dish) in Dulbecco's 
modified Eagle's medium containing 10% calf serum (Hyclone, Sterile 
Systems, Logan, Utah). Cultures were grown for 5 days in the presence of 
various concentrations of column fraction aliquots (added on days 0 and 2) 
and then counted in a Coulter particle counter. The biological activities 
of the proteins tested are indicated in the number of vascular endothelial 
cells grown in each test well for each fraction in FIGS. 5-7. 
In particular, activity of the HBBMs was compared with that of the FGFs. 
Using published procedures (9,12), aFGF and bFGF were isolated and their 
authenticity was verified by N-terminal sequence analysis and molecular 
weight determination (SDS-PAGE). 
The eluant fractions corresponding to the peak for HBBM-3 from Mono-S 
chromatography, as shown in FIG. 2, were pooled, diluted three times with 
starting buffer and rechromatographed on the same system. Aliquots of 
these fractions were tested as described above for their ability to 
stimulate the growth of bovine vascular endothelial cells. Comparative 
tests were run with aFGF and bFGF. The results, as shown in FIG. 5, 
indicated that HBBM-3 stimulated mitogenic activity for bovine endothelial 
cells. Furthermore, the HBBM-3 and its activity were separable from the 
FGFs. 
In further test of activity, the available HBBM-3 fractions were 
concentrated on a Mono-S column using the same buffer system as described 
for FIG. 2, but with a steeper gradient (0-1.0M NaCl in 20 minutes) and 
lower flow rate (0.4 ml/minute). Comparative dose-response analyses were 
run with aFGF and bFGF. 
The upper panel of FIG. 6 indicates that HBBM-3 stimulated bovine aortic 
endothelial cells in a dose-dependent manner. The ED.sub.50 was in the 
order of 20-50 ng/ml, with the minimally stimulating dose being 
approximately 3 ng/ml. The dose-response curves of HBBM-3 and aFGF were 
indistinguishable, both qualitatively and quantitatively, under the assay 
conditions used. The response of HBBM-3 appeared to be distinguishable 
from that of bFGF. In the assay system used, bFGF possessed much higher 
potency and apparently higher intrinsic activity than HBBM-3. It should be 
cautioned, however, that the question of intrinsic activity of HBBM-3 
could not be addressed adequately in this test because doses sufficiently 
high to allow assessment of the intrinsic activity of HBBM-3 could not be 
used in the assay due to limitations with respect to the amount of salt 
that could be added to the cells without adverse effects on cell growth. 
Preliminary results indicate, however, that the intrinsic activity of 
HBBM-3 is identical to that of aFGF under the assay conditions used. 
The dose-response analysis was repeated for HBBM-1. The results, as shown 
in the lower panel of FIG. 6, indicate that HBBM-1 was also biologically 
active in the same test system. Human HBBM, comprising a mixture of 
HBBM-1, -2 and -3, was tested in the same manner and was similarly active 
(data not shown). However, results have not yet been obtained for 
individual fractions of the human HBBMs. Furthermore, bovine HBBMs were 
also tested on human umbilical cord endothelial cells and found to be 
active (data not shown). 
Finally, aliquots of HBBM-3 concentrated by Mono-S chromatography were made 
1.5M in sodium sulfate and placed on an hydrophobic interaction column. 
The results, as shown in FIG. 7, indicated that HBBM-3 was active and was 
well-separated from both aFGF and bFGF. 
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