Purification of calcium-activated neutral proteinase (CANP) from purified myelin of bovine brain white matter.
A calcium-activated neutral proteinase was purified from myelin of bovine brain white matter. Myelin purified in the presence of EDTA (2 mM) was homogenized in 50 mM Trisacetate buffer at pH 7.5, containing 4 mM EDTA, 1 mM NaN3, 5 mM beta-mercaptoethanol and 0.1% Triton X-100 for two hours. After centrifugation at 87,000 g for 1 hour, the supernatant was subjected to purification through successive column chromatography as follows: i) DEAE-cellulose, ii) Ultrogel (AC-34) filtration, iii) Phenyl-Sepharose, iv) a second DEAE-cellulose. The enzyme activity was assayed using azocasein as substrate. The myelin enzyme was purified 2072-fold and SDS-PAGE analysis of the purified enzyme revealed a major subunit of 72-76 K. The enzyme was inhibited by iodoacetate (1 mM), leupeptin (1 mM), E-64C (1.6 mM), EGTA (1 mM), antipain (2 mM) and endogenous inhibitor calpastatin (2 micrograms). It required 0.8 mM Ca2+ for half-maximal activation and 5 mM Ca2+ for optimal activation. Mg2+ (5 mM) was ineffective while Zn2+ and Hg2+ were inhibitory. The pH optimum was ranged from 7.5-8.5. Treatment of myelin with Triton X-100 increased the enzyme activity by 10-fold suggesting it is membrane bound whereas the purified enzyme was not activated by Triton X-100 treatment. The presence of CANP in myelin may mediate the turnover of myelin proteins and myelin breakdown in degenerative brain diseases.
Chakrabarti
A K
AK
Department of Neurology, Medical University of South Carolina, Charleston 29425.
Banik
N L
NL
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D002154:Calpain / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D007477:Ions; D008670:Metals / Q000494:pharmacology; D009186:Myelin Sheath / Q000032:analysis*; D017830:Octoxynol; D009994:Osmolar Concentration; D011092:Polyethylene Glycols / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature
D007477:Ions; D008670:Metals; D011480:Protease Inhibitors; D011092:Polyethylene Glycols; D017830:Octoxynol; D002154:Calpain; D002118:Calcium