Sequential limited proteolysis of myelin basic protein by neutral protease activities of bovine brain.
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-polyacrylamide gel electrophoresis and densitometry. At pH 7 greater than 50% of total protein and about 75% of MBP were degraded after 48 h, whereas at pH 3 it was less than 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-ethylmaleimide, phenylmethylsulfonyl fluoride, o-phenanthroline, and EDTA, as well as pepstatin A and alpha 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 endopeptidases of which a cysteine protease appears to initiate the breakdown of MBP.
Berlet
H H
HH
Ilzenhöfer
H
H
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D004676:Myelin Basic Protein / Q000378:metabolism*; D015260:Neprilysin; D010436:Pepstatins / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism; D010618:Phenanthrolines / Q000494:pharmacology; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D000511:alpha-Macroglobulins / Q000494:pharmacology
D004676:Myelin Basic Protein; D010436:Pepstatins; D010446:Peptide Fragments; D010618:Phenanthrolines; D011480:Protease Inhibitors; D000511:alpha-Macroglobulins; C014195:Streptomyces pepsin inhibitor; D010664:Phenylmethylsulfonyl Fluoride; D004492:Edetic Acid; D010450:Endopeptidases; D015260:Neprilysin; D005033:Ethylmaleimide; C031375:pepstatin; C025205:1,10-phenanthroline