Calcium-stimulated proteolysis in myelin: evidence for a Ca2+-activated neutral proteinase associated with purified myelin of rat CNS.
Incubation of myelin purified from rat spinal cord with CaCl2 (1-5 mM) in 10-50 mM Tris-HCl buffer at pH 7.6 containing 2 mM dithiothreitol resulted in the loss of both the large and small myelin basic proteins (MBPs), whereas incubation of myelin with Triton X-100 (0.25-0.5%) and 5 mM EGTA in the absence of calcium produced preferential extensive loss of proteolipid protein (PLP) relative to MBP. Inclusion of CaCl2 but not EGTA in the medium containing Triton X-100 enhanced degradation of both PLP and MBPs. The Ca2+-activated neutral proteinase (CANP) activity is inhibited by EGTA (5 mM) and partially inhibited by leupeptin and/or E-64c. CANP is active at pH 5.5-9.0, with the optimum at 7-8. The threshold of Ca2+ activation is approximately 100 microM. The 150K neurofilament protein (NFP) was progressively degraded when incubated with purified myelin in the presence of Ca2+. These results indicate that purified myelin is associated with and/or contains a CANP whose substrates include MBP, PLP, and 150K NFP. The degradation of PLP (trypsin-resistant) in the presence of detergent suggests either release of enzyme from membrane and/or structural alteration in the protein molecule rendering it accessible to proteolysis. The myelin-associated CANP may be important not only in the turnover of myelin proteins but also in myelin breakdown in brain diseases.
N L
NL
Banik
W W
WW
McAlhaney
E L
EL
Hogan
D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D002154:Calpain; D004533:Egtazic Acid / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism / Q000502:physiology*; D005615:Freezing; D006863:Hydrogen-Ion Concentration; D004676:Myelin Basic Protein / Q000378:metabolism; D009185:Myelin Proteins / Q000378:metabolism; D009186:Myelin Sheath / Q000201:enzymology* / Q000378:metabolism; D017830:Octoxynol; D010447:Peptide Hydrolases / Q000378:metabolism / Q000502:physiology*; D011092:Polyethylene Glycols / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013116:Spinal Cord / Q000201:enzymology
D004676:Myelin Basic Protein; D009185:Myelin Proteins; D011092:Polyethylene Glycols; D004533:Egtazic Acid; D017830:Octoxynol; D010450:Endopeptidases; D010447:Peptide Hydrolases; D002154:Calpain; D002118:Calcium