Vr. Amberger et al., CHARACTERIZATION OF A MEMBRANE-BOUND METALLOENDOPROTEASE OF RAT C6 GLIOBLASTOMA CELLS, Cancer research, 54(15), 1994, pp. 4017-4025
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.