Calcium‐activated neutral proteinase (calpain) activity in C6 cell line: Compartmentation of μ and m calpain

Abstract

Calcium-activated neutral proteinase (calpain) activity was determined, including in cytosol and membrane fractions, in rat glioma C6 cell line. The mu and m forms of calpain were separated by DEAE and phenylsepharose column chromatography and with removal of the endogenous inhibitor calpastatin. C6 cells contained more mcalpain than the mu isoform. More than 70% of mcalpain activity was membrane-associated and 20% was cytosolic. Isolated plasma membrane also contained 69% of the mcalpain activity. In contrast, approximately 80% of mucalpain activity was cytosolic and 16% was membranous. Half-maximal activity for mu and mcalpain was obtained at 1 microM and 0.2 mM CaCl2, respectively. Trypsin dissociation of cells reduced activity. Triton X-100 stimulated mcalpain activity of the whole homogenate and the membrane pellet but not of the cytosol. Activity of the myelin marker enzyme adenosine 2'3'-cyclic nucleotide 3'-phosphohydrolase (CNPase), was also found in C6 cells. The identification of calpain and CNPase in C6 cells is in keeping with an interpretation that C6 differentiation resembles, at least in part, that of the myelin-forming oligodendroglial cells.

Myelin Recovery in Multiple Sclerosis: The Challenge of Remyelination

“…Both ubiquitous and tissue-specific calpains have been identified. Ubiquitously expressed ones exist in CNS in two forms: as μ-calpain and m-calpain requiring μM and mM Ca 2+ concentrations for activation, respectively [180], and are regulated by calpastatin (its endogenous inhibitor), lipids and activator proteins [181,182]. Calpains play roles in many molecular processes including cell proliferation and differentiation, T cell activation [183], immune cell migration [184], signal transduction [185], necrosis [186] and apoptosis [187].…”

Immunolocalization of cytoplasmic and myelin mCalpain in transfected Schwann cells: I. effect of treatment with growth factors

“…Our results indicated mcalpain to be associated mainly with membrane of both C6 cells and Schwann cells (Banik et al, , 1992b. Immunoelectron microscopic (morphologic) localization studies of calpain in the peripheral nervous system (PNS) using cytosolic calpain antibodies did not show a significant amount of calpain in myelin while immunocytochemical studies with myelin mcalpain antibody revealed the presence of calpain in myelin (Li and Banik, 1995).…”

“…Since mcalpain is associated with myelin, we have previously investigated the distribution of calpain in the PNS myelin-forming Schwann cells in culture and in the C6 glioma cell line cloned from rat glioma (Banik et al, 1992b) which have properties similar to CNS oligodendrocytes and astrocytes. Our results indicated mcalpain to be associated mainly with membrane of both C6 cells and Schwann cells (Banik et al, , 1992b.…”

Heat shock induction of a 65 kDa ATP-binding proteinase in rat C6 glioma cells

“…Concluded from their location in the cells and optimum-pH of activity, they were very different from the reHeat shock induction of a 65 kDa ATP-binding proteinase ported lysosomal proteinases [7] and calpain [6]. However, a comparison of properties of the 65 kDa ATP-BPase, with the 45, 55 and 100 kDa ATP-BPases in C6 cells showed that, apart from their difference of molecular masses and time of expression in heat shock response of the C6 cells (unpublished data), they are similar in the following aspects: they all can be inhibited by serine proteinase inhibitor a1-antitrypsin, dissociated by ATP-affinity chromatography and activated by Mg 2+ and ATP; their content in the C6 cells is rather low and their maxinal activity around pH 8.0.…”

“…There are many reports on their culture, growth and metabolism [3][4][5]. However, up to now, we know nothing about their ATP-BPases except calpain [6] and acidic lysosomal proteinases [7].…”