Isolation from bovine brain and structural characterization of HBNF, a heparin-binding neurotrophic factor.
A heparin-binding protein with neurotrophic activity for perinatal rat neurons, termed HBNF, was purified to homogeneity from bovine brain utilizing pH 4.5 extraction, ammonium sulfate precipitation, cation exchange and heparin-Sepharose affinity chromatographies, and reverse phase HPLC. In the presence of protease inhibitors during extraction, a protein with an apparent molecular weight of 18 kDa was obtained in a yield of approximately 0.5 mg/kg brain tissue. The amino acid sequence of the first 114 residues of HBNF was determined and found to highly homologous to the cDNA-derived amino acid sequence of human HBNF, a 136-residue protein. Bovine and human HBNFs have identical molecular weights as judged by SDS gel electrophoresis and very similar amino acid compositions. This and overall sequence conservation suggest that bovine HBNF is also a 136 amino acid protein with a calculated molecular weight of approximately 15.5 kDa. The apparent discrepancy between calculated and observed molecular weights of bovine HBNF (and of human HBNF of which the complete sequence is known) is most likely a result of the highly basic nature of HBNF. If protease inhibitors were omitted during tissue extraction, two additional proteins with lower apparent molecular weights and identical N-terminal sequences were isolated, with the smallest forms being the major product. Amino acid analysis showed that the smaller forms correspond to C-terminally truncated HBNFs with calculated molecular weights of 13.6 and 12.4 kDa, lacking approximately 14 and 22 residues. Comparison of the HBNF protein sequence with sequences stored in the Protein Identification Resource/Genbank databases reveals high homology to the translation product of the MK-1 gene, which is retinoic acid-inducible in embryonic carcinoma cells and developmentally expressed during gestation in mice.
Böhlen
P
P
Medical Research Division, American Cyanamid Company, Pearl River, NY 10965.
Müller
T
T
Gautschi-Sova
P
P
Albrecht
U
U
Rasool
C G
CG
Decker
M
M
Seddon
A
A
Fafeur
V
V
Kovesdi
I
I
Kretschmer
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001369:Axons / Q000187:drug effects / Q000378:metabolism; D001923:Brain Chemistry; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002417:Cattle; D016207:Cytokines / Q000737:chemistry / Q000302:isolation & purification*; D051379:Mice; D000078792:Midkine; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000235:genetics; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000302:isolation & purification*; D011506:Proteins / Q000235:genetics
D002352:Carrier Proteins; D016207:Cytokines; C066254:MDK protein, human; D009363:Neoplasm Proteins; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D011506:Proteins; C066845:pleiotrophin; D000078792:Midkine