The Role of Calcium in Semi-alkaline Proteinase from Aspergillus melleus

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The role of calcium in the thermal stability of semi-alkaline proteinase (SAP) from Aspergillus melleus was investigated. As removal of calcium from active SAP induced an autolytic degradation, phenylmethylsulfonyl fluoride-modified SAP (PMS-SAP), having no proteolytic activity, was used. The calcium binding isotherm obtained by equilibrium gel chromatography indicated that PMS-SAP bound a maximal amount of 2 mol of calcium per mol of enzyme. The results of high-performance liquid chromatography and differential scanning calorimetry of the EDTA-treated PMS-SAP suggested that removal of calcium lowered the transition temperature of the thermal denaturation, accompanied with a decrease in the enthalpy change (ΔH) and in the entropy change (ΔS) for the denaturation. No significant change was observed in the circular dichroism spectrum or immunological properties when one of the two calciums was removed from PMS-SAP. The other one was not removed by dialysis against various concentrations of EDTA or EGTA at pH 8.0,but was removed when PMS-SAP was irreversibly denatured under acidic conditions (pH 2.0). It seems likely that the calcium atom which is removed by dialysis is present on the surface of the enzyme molecule and stabilizes the enzyme against thermal denaturation, whereas the remaining calcium atom seems to be firmly bound to the enzyme or buried in the molecular interior.
社団法人日本薬学会の論文
1985-02-25