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Calcium-Mediated Neurofilament Protein Degradation in Rat Optic Nerve In Vitro: Activity and Autolysis of Calpain Proenzyme

Authors
Journal
Experimental Eye Research
0014-4835
Publisher
Elsevier
Publication Date
Volume
65
Issue
1
Identifiers
DOI: 10.1006/exer.1997.0286
Keywords
  • Calpain
  • Calpastatin
  • Neurofilament Proteins
  • Optic Nerve Degeneration
  • Optic Neuritis
Disciplines
  • Biology

Abstract

Abstract 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.

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