Degradation of exogenous MBP by myelin Ca(2+)-activated neutral protease and effect of extraction of myelin on enzyme activity.

Time-course of degradation of exogenous myelin basic protein (MBP) by myelin calcium-activated neutral protease (CANP) and the effect of different treatments with organic solvents and salt solutions on CANP activity were studied. Activity was estimated from the supernatants of incubation mixtures as primary amino groups released during proteolysis by a spectrophotometric method or from the protein patterns on SDS gels. Analysis of the time-course of bovine 18.3 K MBP proteolysis revealed a defined sequence of degradation, at least twelve peptides being observed. Bovine and mouse myelin CANPs degraded both bovine 18.3 K MBP and mouse 14.0 K MBP in a similar manner. On SDS gels the banding patterns of degradation of these proteins revealed differences as well as similarities. Delipidation of myelin with acetone did not affect on myelin CANP activity. Removal of lipid with ether:ethanol (3:2) slightly reduced activity, and after chloroform:methanol (2:1) extraction the residue had no CANP activity. The activity could not be established in the chloroform-methanol supernatant either. These findings together with extraction experiments with buffered salt solutions suggest that the enzyme molecule is tightly bound in the membrane interior and may require certain myelin lipids for activity. Analysis of the time-course experiment suggests that MBP in the membrane-bound form acts as a substrate for CANP, and soluble degradation products are released from the membrane.