Stress response of lysosomal cysteine proteinases in rat C6 glioma cells.
Acid proteinases of C6 rat glioma cells were analyzed by means of gelatine polyacrylamide electrophoresis with respect to their responses to stress (heat shock and butanol). Proteinase activities on gelatine gels were characterized by their molecular masses. pH-optima, isoelectric points and reactions to inhibitors. Four bands of 25, 35 and 65/85 kDa most probably represent active and proforms as well as precursor complexes of lysosomal cysteine proteinases with pH optima between 4.0 and 5.0. The 25-kDa band seems to contain cathepsin L and B, the 35-kDa band proforms of cathepsin L and B and the 65/85-kDa bands possibly precursor complexes of cathepsin L and B. After 30-min heat shocks of different temperatures (40-50 degrees C), the 35-kDa activity increased, whereas the 65/85-kDa activity decreased after exposure to 42 and 44 degrees C, which also caused a strong increase in the level of the inducible heat shock protein of 68 kDa (HSP 68). The alterations of the proteinase activities and the increases of the HSP 68 levels occur at heat shock treatments that cause cell death in about 25-40% of the population as determined by Trypan blue staining. HSP 68 induction and proteinase activity changes were also observed 12 hr after a 1-hr treatment with different butanol concentrations (0.14-0.16 M). Kinetics of the response to a 30-min heat shock (44 degrees C) revealed a maximal decrease of the 35-kDa and a maximal increase of the 65/85-kDa activities after 12 hr recovery. When cells were exposed to repeated heat shocks (44 degrees C) at 12-hr intervals, the HSP 68 level further increased, whereas the 35-kDa and 65/85-kDa proteinase activities did not change. This result indicates a role of HSP 68 (or other HSPs) in the processing or stability of the putative cathepsin precursors (65/85-kDa complexes).
Xu
C
C
Institute of Cell Biology, Biochemistry and Biotechnology, University of Bremen, Germany.
Fracella
F
F
Richter-Landsberg
C
C
Rensing
L
L
D000818:Animals; D015153:Blotting, Western; D000440:Butanols / Q000494:pharmacology*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005910:Glioma / Q000201:enzymology*; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology*; D008970:Molecular Weight; D051381:Rats; D013312:Stress, Physiological; D014407:Tumor Cells, Cultured
D000440:Butanols; D018840:HSP70 Heat-Shock Proteins; C097210:Hspa1a protein, rat; D003546:Cysteine Endopeptidases