Quantitative Electroimmunoblotting Study of the Calcium‐Activated Neutral Protease in Human Myelin

Abstract

Degradation of myelin basic protein (MBP) in human myelin was monitored by electroimmunoblotting. Problems of variation between, as well as within, electroimmunoblots were overcome by the introduction of an internal standard in each sample, thus allowing reproducible quantification of MBP. The Ca²⁺-dependent protease acting on MBP was active at endogenous levels of Ca²⁺ (∼300 μg/g myelin) and was inhibited in the presence of Ca²⁺ chelators. Extensive degradation of MBP occurred rapidly in the presence of added Ca²⁺, reaching a plateau after a 1 h incubation (80–85% degradation). The proteolytic activity was not enhanced in the presence of 2-mercaptoethanol. It was most active at neutral pH and at temperatures approaching physiological conditions. No difference was observed between proteolytic activities of control and multiple sclerotic myelin. It is suggested that fluctuations in the accessibility of free Ca²⁺ to the protease may lead to the regulation of Ca²⁺-activated myelinolysis.