Affiliations: MRF Biochemistry Laboratory, Department of Surgery,
University of the Witwatersrand, Johannesburg, ZA 2193, South Africa
Note: [] Address for correspondence: M.M.J. Oosthuizen, Clinical Biochem
Institute, P.O. Box 73231, Fairland, Johannesburg, ZA 2030, South Africa. Tel.:
+27 11 679 2447; Fax: +27 11 475 0020; E-mail: mmjo@finit.co.za
Abstract: Hepatoproliferin (HPF), a liver regeneration factor, was isolated
initially as an aggregated molecule (big-HPF) and was purified into two
homogeneous, bioactive species of 14 kDa and 18.5 kDa. These two big-HPFs were
disaggregated to completion into two monomeric forms (small-HPFs) when
incubated for 10 days in 0.15 M ammonium bicarbonate at 25°C. Both
monomeric forms were purified to homogeneity as active entities, one with a
molecular mass of 944 Da and one with a molecular mass of 1066 Da. Each of the
two ^{35}S-labelled small-HPFs was found, by enzymic
analysis, to contain a charged sulfonated saccharide, which was neutralized by
a specific amine. Monomeric HPF is therefore a stable ionic complex formed
between these two ionic species. So strong was the electrostatic association
that small-HPF remained intact in solution and no amine was displaced by the
ammonium ions of the buffer. Small-HPF remained unimpaired during purification,
since all activity was retained despite alternating acidic and basic
conditions. However, when small-HPF was brought into contact with either a
cationic or an anionic resin, it was dissociated to completion when mixed
continuously with the resin for 4 days. The ionic entity that was released had
no bio-activity and was either a pure radioactively labeled saccharide or a
non-labeled amine, depending on the kind of resin used. When incubated
together, the separated counterions combine to regain full activity after 2
days of reassociation. However, with incubation for longer, this reassociated
small-HPF formed different oligomeric HPFs by aggregation. Small-HPF is therefore a new kind of growth enhancer, consisting of
an acidic sulfonated saccharide and a basic amine assembled into a stable
active ionic complex that has a tendency to aggregate.
