Sequential Limited Proteolysis of Myelin Basic Protein by Neutral Protease Activities of Bovine Brain

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Abstract
Acid extracts of delipidated white matter of bovine brain were prepared, and their proteolytic activities toward myelin basic protein (MBP) were evaluated at pH 3 and pH 7. This was done by measuring changes in total protein using a selective dye-binding assay, and by evaluating peptide patterns by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis and densitometry. At pH 7 >50% of total protein and about 75% of MBP were degraded after 48 h, whereas at pH 3 it was <20% altogether. Neutral proteolysis of MBP entailed up to 12 different proteolytic peptide fragments in the molecular weight range of 17.5 to 6 kd. Its enzymatic nature was verified using protease inhibitors, including N-ethylmale-imide, phenylmethylsulfonyl fluoride, o-phenanthroline, and EDTA, as well as pepstatin A and α2macroglobulin. Both transient changes in percentages of some intermediate peptides and differential effects of individual inhibitors on electrophoretic peptide patterns strongly suggest a sequential type of limited proteolysis. The results also indicate that acid extracts contained several endopepti-dases of which a cysteine protease appears to initiate the breakdown of MBP.