Abstract:
Chicl( brain growth factor (CBGF) is a mitogen isolated from
embryonic chick brains thought to have a potential role as a trophic factor
involved in nerve dependent amphibian limb regeneration. In addition,
CBGF stimulates 3H-thymidine incorporation in chick embryo brain
astrocytes in vitro.
In this study, cultured chick embryo brain non-neuronal cells were
employed in a bioassay to monitor CBGF activity throughout various stages
of its pllrification. Cell culture and assay conditions were optimized. Nonneuronal
cells grew best on collagen-coated culture dishes in complete
medium, were most responsive to a growth stimulus [10% fetal bovine
serum (FBS)] at the second and third subcultures, and were healthiest
when rendered "quiescent" in medium supplemented with 1% FBS. The
most effective bioassay conditions consisted of a minimum 14.5 hour
"quiescence" time (24 hours was used), a 6 hour "prestimulation" time, and
a 24 hour 3H-thymidine labeling time. Four-day subconfluent primary
non-neuronal cells consisted of 6.63% GFAP positive cells; as a result
cultures were thought to be mainly composed of astroblasts.
CBGF was purified from 18-day chick embryo brains by
ultrafiltration through Amicon PM-30 and YM-2 membranes, size exclusion
chromatography through a Biogel P6 column, and analytical reverse-phase
high-performance liquid chromatography (rp-HPLC). The greatest activity
resided in rp-HPLC fraction #7 (10 ng/ml) which was as effective as 10%
FBS at stimulating 3H-thymidine incorporation in chick embryo brain nonneuronal
cells.
Although other researchers report the isolation of a mitogenic
fraction consisting of 5'-GMP from the embryonic chick brain, UV
absorbance spectra, rp-HPLC elution profiles, and fast atom bombardment
(FAB) mass spectra indicated that CBGF is neither 5'-GMP nor 51-AMP.
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Moreover, commercially available 5t-GMP was inhibitory to 3H-thymidine
incorporation in the chick non-neuronal cells, while Sf-AMP had no effect.
Upon treatment with pronase, the biological activity of fraction P6-3
increased; this increase was nearly 30% greater than what would be
expected from a simple additive effect of any mitogenic activity of pronase
alone together with P6-3 alone. This may suggest the presence of an
inhibitor protein. The bioactive component may be a protein protected by
a nucleoside/nucleotide or simply a nucleoside/nucleotide acting alone.
While the FAB mass spectrum of rp-HPLC fraction #7 did not reveal
molecular weight or sequence information, the ion of highest molecular
weight was observed at m/z 1610; this is consistent with previous
estimations of CBGF's size.
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