Rossi P, Giorgi M, Geremia R, Kincaid R L
Dipartimento di Sanitá Pubblica e Biologia Cellulare, II Universitá di Roma, Italy.
J Biol Chem. 1988 Oct 25;263(30):15521-7.
A cell-specific isozyme of calmodulin (CaM)-dependent phosphodiesterase that exhibits micromolar affinity for cAMP has been purified 900-fold from mouse testis by DEAE chromatography, gel filtration, affinity chromatography with CaM-Sepharose 4B, and isoelectric focusing. The highly purified enzyme is stimulated 5-6-fold by CaM in the presence of Ca2+ and hydrolyzes both cAMP and cGMP with anomalous substrate dependence, i.e. high and low affinity components (Km 2 and 20 microM) are observed either in the presence or absence of CaM. Each of the substrates acts as a noncompetitive inhibitor of the other, suggesting the presence of two distinct catalytic sites on the enzyme. Hydrodynamic studies suggest that the testis phosphodiesterase is an asymmetric monomer of 68-70 kDa that forms a dimer after interaction with Ca2+ and CaM; the tetrameric complex exhibits an apparent molecular size of 180 kDa. These enzymatic and biophysical properties differ in many respects from those of the brain isozyme, suggesting that they are different proteins. Nevertheless, common epitopes do exist, since the testis enzyme interacted with rabbit antibodies raised against bovine brain CaM-dependent phosphodiesterase. The major peptide of 68 kDa was strongly reactive on immunoblots, and was distinguished unambiguously from the 60-kDa species from mouse brain. A comparison of the immunoreactive fragments produced by limited proteolysis with staphylococcal V-8 protease indicated several similarities in the domains of these polypeptides. Thus, although differing in several important physical and biochemical parameters, the testis enzyme appears immunologically related to CaM-dependent phosphodiesterase from brain. On the basis of these data, we conclude that common elements of the structural genes for these isozymes have been conserved, whereas certain biological properties, including substrate specificity, have diverged substantially.
一种对环磷酸腺苷（cAMP）具有微摩尔亲和力的钙调蛋白（CaM）依赖性磷酸二酯酶的细胞特异性同工酶，已通过DEAE色谱、凝胶过滤、用CaM - Sepharose 4B进行的亲和色谱以及等电聚焦从小鼠睾丸中纯化了900倍。在Ca2 +存在的情况下，该高度纯化的酶被CaM刺激5 - 6倍，并且以异常的底物依赖性水解cAMP和cGMP，即在有或没有CaM的情况下都观察到高亲和力和低亲和力成分（Km分别为2 μM和20 μM）。每种底物都作为另一种底物的非竞争性抑制剂，这表明该酶上存在两个不同的催化位点。流体动力学研究表明，睾丸磷酸二酯酶是一种68 - 70 kDa的不对称单体，在与Ca2 +和CaM相互作用后形成二聚体；四聚体复合物的表观分子大小为180 kDa。这些酶学和生物物理性质在许多方面与脑同工酶不同，表明它们是不同的蛋白质。然而，由于睾丸酶与针对牛脑CaM依赖性磷酸二酯酶产生的兔抗体相互作用，所以确实存在共同的表位。68 kDa的主要肽段在免疫印迹上具有强烈反应性，并且与小鼠脑的60 kDa条带明显区分开来。用葡萄球菌V - 8蛋白酶进行有限蛋白水解产生的免疫反应性片段的比较表明，这些多肽的结构域有几个相似之处。因此，尽管在几个重要的物理和生化参数上有所不同，但睾丸酶在免疫学上似乎与脑CaM依赖性磷酸二酯酶相关。基于这些数据，我们得出结论，这些同工酶结构基因的共同元件得以保留，而某些生物学特性，包括底物特异性，已经有了很大的差异。
