CHARACTERIZATION OF A MEMBRANE-BOUND METALLOENDOPROTEASE OF RAT C6 GLIOBLASTOMA CELLS

C6 rat glioblastoma cells are able to attach to and to spread on cultu re dishes which are coated with purified central nervous system myelin , in contrast to normal astrocytes, fibroblasts or neurons which adher e poorly and are unable to spread on this substrate. The metalloprotea se blockers o-phenanthroline and a newly developed oligopeptide could specifically inhibit C6 cell spreading on central nervous system myeli n, suggesting a crucial role for a metalloprotease. Here we characteri ze this metalloproteolytic activity of C6 cells using a peptide degrad ation assay with the iodinated tetrapeptide carbobenzoxy-Phe-Ala-Phe- I-125-Tyr-amide as a substrate. Purified, salt-washed C6 plasma membra nes cleaved the peptide between alanine and phenylalanine, an effect w hich is strongly inhibited by o-phenanthroline, but not by thiol-block ing agents or aspartic and serine protease inhibitors. The metalloendo protease is highly sensitive to phosphoramidon but insensitive to thio rphan. The enzyme is tightly bound to the plasma membrane but not G pr otein-phosphatidylinositol linked. It can be solubilized in part by th e detergents 3-cholamidopropyldimethylamino)-1-propanesulfonate or Tri ton X-114. Gel filtration chromatography using the Triton X-114-solubi lized proteins or the proteins removed by a short trypsin treatment re vealed a molecular weight range for the C6 enzyme of 60,000-100,000. P olymerase chain reaction with primers corresponding to endopeptidase 2 4.11 or to the highly conserved motif of the ''astacin family'' showed that both enzymes were not detectable in the C6 glioblastoma cells.