title: Metalloendoprotease cleavage of 18.2- and 14.1-kilodalton basic proteins dissociating from rodent myelin membranes generates 10.0- and 5.9-kilodalton C-terminal fragments.
abstract: Rat and guinea pig myelin membranes were incubated at physiological ionic strength with millimolar concentrations of Ca2+/Mg2+ ions (37 degrees C; pH 7.4). After 1-3 h, electrophoresis of the membranes revealed loss of 50% of 18.2- and 14.1-kilodalton (kDa) forms of myelin basic protein (MBP). Concomitantly, peptides representing 25% of the original membrane-associated MBP were detected in incubation media. Roughly equal amounts of MBP fragments with molecular masses of 10.0 and 8.4 kDa were found in media from guinea pig myelin incubations. Media from rat myelin experiments contained a major 8.4-kDa and minor 10.0- and 5.9-kDa MBP peptides. Kinetic studies implied that proteolysis occurred subsequent to MBP dissociation from the membranes. Immunoblotting studies indicated that both the 18.2- and 14.1-kDa forms of MBP were cleaved near residue 73 to produce a 10.0- and 5.9-kDa C-terminal fragment, respectively. Degradation of MBP in myelin membranes was partially inhibited by only 5-20% using leupeptin (20 microM) but up to 50% by dithiothreitol mM), phenylmethylsulphonyl fluoride (1 mM), and phosphoramidon (50 microM) but up to 50% by dithiothreitol (DDT, 10 mM). Only DDT and 1,10-phenanthroline substantially blocked the formation of the characteristic 10.0-and 5.9-kDa C-terminal fragments. This suggests that MBP, dissociating from myelin membrane preparations, is cleaved near residue 73 by a metalloendoprotease distinct from N-ethylmaleimide/leupeptin-sensitive calpains and phosphoramidon-sensitive endopeptidase 24.11.
mesh_terms: D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D003470:Culture Media; D006168:Guinea Pigs; D008566:Membranes / Q000378:metabolism; D008666:Metalloendopeptidases / Q000378:metabolism*; D008970:Molecular Weight; D004676:Myelin Basic Protein / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009186:Myelin Sheath / Q000378:metabolism*; D010446:Peptide Fragments / Q000096:biosynthesis*; D010447:Peptide Hydrolases / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains
chemical_list: D003470:Culture Media; D004676:Myelin Basic Protein; D010446:Peptide Fragments; D010447:Peptide Hydrolases; D008666:Metalloendopeptidases