Calcium‐Stimulated Proteolysis in Myelin: Evidence for a Ca2+‐Activated Neutral Proteinase Associated with Purified Myelin of Rat CNS

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Abstract
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 μM. 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.