Tumor growth-inhibitory glycoprotein secreted from the mouse brain at terminal stage of the ontogeny: molecular homogeneity and requirement of the retained protein conformation for exhibition of the cytotoxic action.
The mouse brain at the neonatal stage but not at the adult or fetal stage, secretes a protein which inhibits growth and DNA synthesis of malignant cells preferentially over those of normal cells, and so is termed neonatal brain-derived carcinostatic factor (NBCF). In the present study, NBCF was prepared from conditioned medium cultured from neonatal brain, and was obtained by HPLC as a homogeneous protein (62 kDa, pI 9.1); physico-chemical and biological properties of NBCF differ from those of cerebral proteins known. A 1,2-cis-diol affinity column retained NBCF, which was thereafter eluted with D-sorbitol; NBCF treated with neuraminidase lost part of activity without emergence of new N-terminal amino acids, suggesting glycan moieties required for the activity exhibition. This is supported by repression of NBCF secretion by tunicamycin of doses as low as is not cytotoxic. NBCF did not hydrolyze target proteins; cytotoxic action of NBCF was not counteracted by a diversity of proteinase inhibitors. An ether extract of NBCF was not cytotoxic, whereas the ether-insoluble residuum retained part of the initial activity. NBCF was inactivated with immobilized trypsin or with dithiothreitol combined with guanidine more markedly than with either agent. Thus cytotoxicity exhibition of NBCF, mediated through actions other than proteolysis, is attributed to the proteinic principle but not to protein-bound lipophilic ligands, and requires retention of the protein conformation and intramolecularly buried SS bonds.
Miwa
N
N
Department of Antibiotics, National Institute of Health, Tokyo, Japan.
Mizuno
S
S
Suitani
Y
Y
D000818:Animals; D000831:Animals, Newborn / Q000378:metabolism*; D001921:Brain / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D006023:Glycoproteins / Q000302:isolation & purification / Q000494:pharmacology*; D006801:Humans; D007526:Isoelectric Point; D051379:Mice; D008970:Molecular Weight; D009369:Neoplasms / Q000473:pathology*; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D009439:Neuraminidase / Q000494:pharmacology; D009447:Neuroblastoma; D011134:Polysaccharides; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds; D014407:Tumor Cells, Cultured; D014415:Tunicamycin / Q000494:pharmacology
D006023:Glycoproteins; D009419:Nerve Tissue Proteins; D011134:Polysaccharides; D013438:Sulfhydryl Compounds; D014415:Tunicamycin; D009439:Neuraminidase