Calcium-mediated neurofilament protein degradation in rat optic nerve in vitro: activity and autolysis of calpain proenzyme.
In this study, we examined calcium-mediated degradation of a neurofilament protein (NFP), and autolytic activation of calpain in Lewis rat optic nerve in vitro. After incubation with calcium, homogenized optic nerve samples were analysed by SDS-PAGE in association with ECL immunoblot techniques. 68 kD NFP, calpain, and calpastatin antibodies were used for identification of the respective proteins. The extent of calcium-mediated 68 kD NFP degradation compared to EGTA controls, served to quantify calpain activity, while the extent of calpain autolysis measured the activation of the enzyme. A progressive loss of 68 kD NFP was observed at 15 min (42.1%), 1 hr (52.7%) and 6 hr (73.4%) incubation periods compared to EGTA controls. The immunoreactive calpain bands showed progressive autolysis after 15 min (26.6%), 1 hr (31.4%) and 6 hr (43.4%) incubations. We also found degradation of low molecular weight isoforms of calpastatin (43 kD and 27 kD) in the presence of calcium compared to controls. These results indicate that calpain is present in optic nerve in its inactive form but when calcium is added, it undergoes autolysis and becomes active. Thus, active calpain is capable of degrading endogenous substrates (e.g. cytoskeletal and myelin proteins) and may promote the degeneration of optic nerve in optic neuritis.
Shields
D C
DC
Department of Neurology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, S.C., 29425, USA.
Leblanc
C
C
Banik
N L
NL
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000502:physiology; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology; D016900:Neurofilament Proteins / Q000378:metabolism*; D009900:Optic Nerve / Q000201:enzymology*; D051381:Rats; D011917:Rats, Inbred Lew
D002135:Calcium-Binding Proteins; D004792:Enzyme Precursors; D016900:Neurofilament Proteins; C033668:calpastatin; D002154:Calpain; D002118:Calcium