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Sequential Limited Proteolysis of Myelin Basic Protein by Neutral Protease Activities of Bovine Brain
Author(s) -
Berlet Hans H.,
Ilzenhöfer Heike
Publication year - 1985
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1985.tb05482.x
Subject(s) - proteolysis , myelin basic protein , pepstatin , protease , biochemistry , chemistry , phenylmethylsulfonyl fluoride , peptide , myelin , gel electrophoresis , sodium dodecyl sulfate , proteolytic enzymes , microbiology and biotechnology , enzyme , biology , neuroscience , central nervous system
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 α 2 macroglobulin. 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.