Activity and biospecificity of proteolyzed forms and dimeric combinations of recombinant human and murine nerve growth factor.
Purified recombinant human nerve growth factor (rhNGF) and submaxillary gland-derived murine NGF (muNGF) were characterized by amino acid composition, polyacrylamide gel electrophoresis (PAGE), reversed-phase HPLC (RP-HPLC), and high-performance ion-exchange chromatography (HPIEC). Limited tryptic digest of the N and C termini of the 120-residue form of rhNGF produced a species of 109 residues (10-118). The previously observed natural murine analogue of this variant, muNGF lacking the first eight N-terminal amino acids, was also isolated as a homodimer. Both species were purified using HPIEC and characterized by amino acid analysis, N-terminal sequence, PAGE, and RP-HPLC analysis. Each of the four homodimeric species was evaluated in some or all of the following biological assays for NGF: chick dorsal root and sympathetic ganglion assays and rat pheochromocytoma-12 cell line neurite extension assay. The 118-residue homodimeric versions of both rhNGF and muNGF displayed equivalent bioactivity, whereas the N terminal-modified molecules presented activity reduced by 50- to 100-fold. Utilizing HPIEC, we have examined the ability of the monomeric forms of any two of the homogeneous dimeric species of rhNGF to recombine. We have shown that not only can all of the previously described species form dimers by recombination, but an interspecies dimer can be created between muNGF and rhNGF.
Burton
L E
LE
Genentech, Inc., South San Francisco, California.
Schmelzer
C H
CH
Szonyi
E
E
Yedinak
C
C
Gorrell
A
A
D000595:Amino Acid Sequence; D000818:Animals; D002642:Chick Embryo; D010450:Endopeptidases / Q000494:pharmacology; D005727:Ganglia, Spinal / Q000737:chemistry; D005728:Ganglia, Sympathetic / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D051379:Mice; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000737:chemistry* / Q000378:metabolism; D016716:PC12 Cells; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013045:Species Specificity; D013329:Structure-Activity Relationship; D013363:Submandibular Gland / Q000737:chemistry
D009414:Nerve Growth Factors; D011994:Recombinant Proteins; D010450:Endopeptidases