Improved purification and characterization of Equine DNase I

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Deoxyribonuclease I (DNase I) from equine parotid gland was purified by reformation with a yield of 48 % to electrophoretic homogeneity using three-step conventional column chromatography. The purified enzyme showed a molecular mass of about 37kDa and maximum activity at pH 7.0. It required divalent cations Mg2+ and Ca2+ for its activity and was inhibited by EDTA and EGTA. The purified enzyme preparation was found to contain no other detectable nucleases. An antibody against the purified enzyme was found to be monospecific against the equine parotid gland and the pure antigen, and completely blocked the activity of the purified enzyme. Inhibition by globular actin (G-actin) was investigated in seven mammalian enzymes. Activity inhibition by G-actin was very strong in an equine enzyme. In G-actin-inhibited human DNases I, four amino acid residues, Tyr-65, Val-66, Val-67 and Ala-114, were involved in actin binding. In equine, Tyr-65 is substituted with Phe-65. Considering that the enzymatic activity of a rabbit enzyme with Phe-65 and Ala-67 was less efficiently inhibited by G-actin, the structural involvement of Phe-65 and Val-67may result in a strong DNase I and G-actin complex.

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