A calcium-dependent protease associated with the neural cytoskeleton. Purification and partial characterisation.
Calcium-dependent protease activity was found associated with a neurofilament-enriched cytoskeleton isolated from the bovine spinal cord. The protease was extracted from the cytoskeleton by 0.6 M KCl, and purified to apparent homogeneity (3300-fold) by chromatography on organomercurial-Sepharose 4B, casein-Sepharose 4B, and Sepharose CL-6B. A cytosolic calcium-dependent protease was similarly purified from the bovine spinal cord, after the cytosol was fractionated on DEAE-cellulose. Both cytoskeleton-bound and cytosolic enzymes had an apparent molecular mass of 100 kDa as judged by gel filtration, and consisted of two subunits (79 kDa and 20 kDa) upon sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Both enzymes exhibited caseinolytic activity with 0.5 mM Ca2+ and above, and the activity was strongly inhibited by various thiol protease inhibitors. In the presence of 0.1-0.2 mM Ca2+, the 68-kDa and 160-kDa components, and to a lesser extent the 200-kDa component, of the neurofilament triplet polypeptides were degraded by the cytosolic protease, whereas the cytoskeleton-bound protease needed two-fold higher concentration of Ca2+ to degrade the neurofilaments. Nevertheless, the cytoskeleton-bound protease in situ, i.e. before its extraction form the cytoskeleton by 0.6 M KCl, preferentially degraded the 160-kDa component in the presence of 0.1-0.2 mM Ca2+, suggesting that a proper locational relation of this enzyme to the neurofilament structure is a prerequisite to its preference for the 160-kDa component. It appears that a factor or factors involved in such an interaction between the protease and the neurofilament were eliminated during the course of enzyme purification. The glial fibrillary acidic protein was almost insensitive to the proteases purified in the present study.
Ishizaki
Y
Y
Kurokawa
M
M
Takahashi
K
K
D000818:Animals; D002021:Buffers; D002154:Calpain; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity; D003599:Cytoskeleton / Q000201:enzymology*; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D010455:Peptides / Q000302:isolation & purification; D013116:Spinal Cord / Q000201:enzymology*
D002021:Buffers; D010455:Peptides; D010450:Endopeptidases; D002154:Calpain