Kuciel R, Mazurkiewicz A, Ostrowski W S
Institute of Medical Biochemistry, Collegium Medicum, Jagiellonian University, Poland.
Int J Biol Macromol. 1996 Apr;18(3):167-75. doi: 10.1016/0141-8130(95)01065-3.
Human prostatic acid phosphatase (hPAP) [EC 3.1.3.2], a homodimer of ca. 50 kDa subunit molecular weight, shows reversible denaturation in 6 M urea at pH 2.5. Rapid dilution of the denatured enzyme allowed partial renaturation of hPAP, as measured by enzyme activity, to a level which depended on the composition of the dilution solution employed and time of the reaction. The renaturation reaction of hPAP was examined using spectral analysis (circular dichroism and fluorometry), fast size-exclusion chromatography and proteolysis by trypsin. The observed results are in agreement with the concentration-dependent kinetics of hPAP reactivation, assuming that the reconstitution of the active enzyme requires the association of subunits in dimeric form. Moreover, it suggests formation of an inactive intermediate during refolding of the denatured PAP. A mechanism of renaturation of the active enzyme from denatured PAP is proposed.
人前列腺酸性磷酸酶（hPAP）[EC 3.1.3.2]，一种亚基分子量约为50 kDa的同型二聚体，在pH 2.5的6 M尿素中表现出可逆变性。通过酶活性测定，对变性酶进行快速稀释可使hPAP部分复性，复性水平取决于所用稀释溶液的组成和反应时间。利用光谱分析（圆二色性和荧光法）、快速尺寸排阻色谱法和胰蛋白酶蛋白水解法对hPAP的复性反应进行了研究。观察结果与hPAP重新激活的浓度依赖性动力学一致，假设活性酶的重构需要亚基以二聚体形式缔合。此外，这表明在变性PAP重折叠过程中形成了无活性中间体。本文提出了一种从变性PAP复性为活性酶的机制。
