Couchie D, Erneux C, Dumont J E
Biochem J. 1981 Nov 1;199(2):441-6. doi: 10.1042/bj1990441.
Chromatography on hexyl-agarose resolved a partially purified cyclic GMP-activated phosphodiesterase from rat liver into two peaks of activity: the first was eluted with 0.5 M-KCl and was cyclic AMP-specific. The second was tightly bound to hexyl-agarose and was not eluted with KCl (0--2.0 M), which enhanced the hydrophobic interactions of this form with the matrix. It was eluted with 0.5 M-Tris, hydrolysed cyclic AMP and cyclic GMP and was specifically activated by cyclic GMP. The cyclic GMP-activated phosphodiesterase was immobilized on hexyl-agarose. Enzyme activity, quantitatively bound to hexyl-agarose, was not released from the hydrophobic matrix in the presence of cyclic AMP or cyclic GMP, under our assay conditions. The immobilized form of the enzyme retained catalytic activity, was inhibited by 0.1 mM-cyclic AMP and was activated by micromolar concentrations of cyclic GMP to a lesser extent (7-fold) than the control, i.e. the enzyme mixed with unsubstituted agarose (15-fold). When the enzyme was immobilized, inhibition of cyclic AMP phosphodiesterase activity was only observed in the presence of cyclic GMP (at 3 microM); in its absence, activity remained unchanged. The kinetic behaviour of the immobilized enzyme is consistent with the hypothesis of a binding site distinct from the hydrolytic and activating sites.
用己基琼脂糖进行色谱分离，可将大鼠肝脏中部分纯化的环鸟苷酸激活的磷酸二酯酶分离为两个活性峰：第一个峰用0.5M - KCl洗脱，对环腺苷酸具有特异性。第二个峰与己基琼脂糖紧密结合，不能用KCl（0 - 2.0M）洗脱，这增强了这种形式与基质的疏水相互作用。它用0.5M - Tris洗脱，可水解环腺苷酸和环鸟苷酸，并被环鸟苷酸特异性激活。环鸟苷酸激活的磷酸二酯酶被固定在己基琼脂糖上。在我们的测定条件下，定量结合到己基琼脂糖上的酶活性在存在环腺苷酸或环鸟苷酸时不会从疏水基质中释放出来。固定化形式的酶保留了催化活性，受到0.1mM - 环腺苷酸的抑制，并被微摩尔浓度的环鸟苷酸激活，但激活程度（7倍）低于对照，即与未取代琼脂糖混合的酶（15倍）。当酶被固定化时，仅在存在环鸟苷酸（3μM）时才观察到环腺苷酸磷酸二酯酶活性的抑制；在其不存在时，活性保持不变。固定化酶的动力学行为与存在一个与水解和激活位点不同的结合位点的假设一致。
