General Salting-out and Distinctive Mg-ion Effects on Processing and Maturation of Halophilic Thermolysin-like Proteinase, Salilysin

抄録

Pro-form of halophilic thermolysin-like proteinase, salilysin, was processed three times to form intermediate 1 (I1), intermediate 2 (I2) and final mature form (M). Halophilic enzymes are generally characterized by enhanced stability and catalytic activity in the presence of high concentration of salts. Autolytic processing of I1-form salilysin to I2-form and M-form in vitro was enhanced with the addition of 1.0 M and 0.5 M salts in parallel to valency and general salting-out effects of salts: NaCl ⁄ KCl < NH₄Cl < (NH₄)₂SO₄ ⁄ Na₂SO₄ ⁄ MgSO₄. Mg salts at lower concentration, 0.1 M, regardless of salting-out (MgSO₄: exerting stabilizing effects on protein structures) or salting-in (MgCl₂: exerting destabilizing effects) salts, showed the highest efficiency in enhancing autolysis of I1 to mature form, suggesting importance of Mg ion in addition to the general salting-out effects of high concentration of salts. Even 5 ～ 10 mM Mg salts clearly enhanced autolysis of I1 and processing of inactive pro-E167A salilysin by wild-type mature salilysin, thus again suggesting distinct effects of Mg ion. Processing and maturation of halophilic salilysin was both enhanced by general salting-out effects of various salts and by specific effects, most likely possible binding to salilysin, of Mg ion.