A photoaffinity probe covalently modifies the catalytic site of the cGMP-binding cGMP-specific phosphodiesterase (PDE-5).
A photoaffinity probe covalently modifies the catalytic site of the cGMP-binding cGMP-specific phosphodiesterase (PDE-5).
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光亲和探针共价修饰 cGMP 结合 cGMP 特异性磷酸二酯酶 (PDE-5) 的催化位点。
DOI：
10.1007/bf02737833
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发表时间：
1998
影响因子：
2.6
通讯作者：
Sekhar,KR
中科院分区：
文献类型：
--
作者：
Corbin,JD;Beasley,A;Turko,IV;Haik,TL;Mangum,KA;Wells,JN;Francis,SH;Sekhar,KR
cGMP 结合 cGMP 特异性磷酸二酯酶 (PDE-5) 包含不同的催化和变构结合位点，并且每个位点都是 cGMP 特异性的。环核苷酸磷酸二酯酶抑制剂，如 3-异丁基-1-甲基黄嘌呤 (IBMX)，被认为在这些酶的催化位点与环核苷酸竞争，但尚未确定导致这些抑制剂或底物本身与酶催化结构域相互作用的 PDE-5 部分。 IBMX 被衍生化产生光亲和探针 8([3-125I,-4-叠氮基]-苄基)-IBMX，称为 8(125IAB)-IBMX。该探针与部分纯化的重组牛 PDE-5 一起孵育。 UV 照射和 SDS-PAGE 后，在凝胶上可见与 PDE-5 位置一致的单个放射性标记条带，并且 PDE-5 的光亲和标记随着 8(125IAB)-IBMX 浓度的增加呈线性关系。除 PDE-5 之外的显着考马斯蓝染色条带没有被显着标记。当浓度高于 10 μM 时，光亲和标记逐渐被 cGMP 阻断，而 cAMP 或 5'-GMP 仅表现出微弱的抑制作用。其他被认为与 PDE-5 催化位点相互作用的化合物，包括 IBMX、clMP 和 β-苯基-1,N2-etheno-cGMP (PET-cGMP)，也以浓度依赖性方式抑制光亲和标记。 PET-cGMP 抑制光亲和标记的 IC50 为 10 μM，优于该化合物抑制 PDE-5 催化活性的 IC50 5 μM。结论是，这种光亲和探针与 PDE-5 的相互作用对于 PDE-5 变构结合位点上的催化位点具有高度特异性，并且可能在绘制 PDE-5 催化位点的研究中有用。
The cGMP-binding cGMP-specific phosphodiesterase (PDE-5) contains distinct catalytic and allosteric binding sites, and each is cGMP-specific. Cyclic nucleotide phosphodiesterase inhibitors, such as 3-isobutyl-1-methylxanthine (IBMX), are believed to compete with cyclic nucleotides at the catalytic sites of these enzymes, but the portion of PDE-5 that accounts for interaction of either of these inhibitors or the substrates themselves with the catalytic domain of the enzymes has not been identified. IBMX was derivatized to yield the photoaffinity probe 8([3-125I,-4-azido]-benzyl)-IBMX, which is referred to as 8(125IAB)-IBMX. This probe was incubated with partially purified recombinant bovine PDE-5. After UV irradiation and SDS-PAGE, a single radiolabeled band that coincided with the position of PDE-5 was visualized on the gel, and the photoaffinity labeling of PDE-5 was linear with increasing concentration of the 8(125IAB)-IBMX. Prominent Coomassie blue-stained bands other than PDE-5 were not labeled significantly. The photo-affinity labeling was progressively blocked by cGMP at concentrations higher than 10 μM, whereas cAMP or 5′-GMP exhibited only weak inhibitory effects. Other compounds that are believed to interact with the PDE-5 catalytic site, including IBMX, clMP, and β-phenyl-1,N2-etheno-cGMP (PET-cGMP), also inhibited the photoaffinity labeling in a concentration-dependent manner. The IC50of PET-cGMP for inhibition of photoaffinity labeling was 10 μM, which compared favorably with an IC50of 5 μMfor inhibition of PDE-5 catalytic activity by this compound. It is concluded that the interaction of this photoaffinity probe with PDE-5 is highly specific for the catalytic site over the allosteric binding sites of PDE-5 and could prove useful in studies to map the catalytic site of PDE-5.