Reeves M L, Leigh B K, England P J
Biochem J. 1987 Jan 15;241(2):535-41. doi: 10.1042/bj2410535.
Four cyclic nucleotide phosphodiesterase (PDE) activities were separated from low-speed supernatants of homogenates of human cardiac ventricle by DEAE-Sepharose chromatography, and designated PDE I-PDE IV in order of elution with an increasing salt gradient. PDE I was a Ca2+/calmodulin-stimulated activity, and PDE II was an activity with a high Km for cyclic AMP which was stimulated by low concentrations of cyclic GMP. Human ventricle PDE III had Km values of 0.14 microM (cyclic AMP) and 4 microM (cyclic GMP), and showed simple Michaelis-Menten kinetics with both substrates. PDE IV is a previously unrecognized activity in cardiac muscle, the human enzyme having Km values of 2 microM (cyclic AMP) and 50 microM (cyclic GMP). PDE III and PDE IV were not activated by cyclic nucleotides or calmodulin. Four PDE activities were also isolated from guinea-pig ventricle, and had very similar kinetic properties. By gel filtration, the Mr of PDE III was 60,000, and that of PDE IV 45,000. The drug SK&F 94120 selectively and competitively inhibited PDE III with a Ki value of 0.8 microM (human), showing simple hyperbolic inhibition kinetics. Rolipram (Schering ZK 62711) and Ro 20-1724 (Roche), which have previously been reported to inhibit PDE III-like activities strongly, were shown to be weak inhibitors of human and guinea-pig PDE III enzymes (Ki values greater than 25 microM), but potent inhibitors of PDE IV [Ki values 2.4 microM (Rolipram) and 3.1 microM (Ro 20-1724) with human PDE IV]. The inhibition in all cases demonstrated simple hyperbolic competition. These observations suggest that the previously reported complex inhibition of PDE III-type activities from cardiac muscle was caused by incomplete separation of the PDE III from other enzymes, particularly PDE IV.
通过DEAE - 琼脂糖凝胶色谱法从人心脏心室匀浆的低速上清液中分离出四种环核苷酸磷酸二酯酶（PDE）活性，并按照盐梯度增加的洗脱顺序将其命名为PDE I - PDE IV。PDE I是一种受Ca2+/钙调蛋白刺激的活性，PDE II是一种对环磷酸腺苷（cAMP）具有高Km值且受低浓度环磷酸鸟苷（cGMP）刺激的活性。人心室PDE III对cAMP的Km值为0.14微摩尔/升，对cGMP的Km值为4微摩尔/升，并且对两种底物均表现出简单的米氏动力学。PDE IV是心肌中一种先前未被识别的活性，人源该酶对cAMP的Km值为2微摩尔/升，对cGMP的Km值为50微摩尔/升。PDE III和PDE IV不受环核苷酸或钙调蛋白的激活。还从豚鼠心室中分离出四种PDE活性，并且它们具有非常相似的动力学特性。通过凝胶过滤法测得，PDE III的相对分子质量为60,000，PDE IV的相对分子质量为45,000。药物SK&F 94120以0.8微摩尔/升（人源）的Ki值选择性地竞争性抑制PDE III，表现出简单的双曲线抑制动力学。罗匹尼罗（先灵葆雅公司的ZK 62711）和Ro 20 - 1724（罗氏公司），先前报道它们能强烈抑制PDE III样活性，现显示它们是人源和豚鼠源PDE III酶的弱抑制剂（Ki值大于25微摩尔/升），但却是PDE IV的强效抑制剂[人源PDE IV的Ki值分别为2.4微摩尔/升（罗匹尼罗）和3.1微摩尔/升（Ro 20 - 1724）]。在所有情况下，抑制作用均表现出简单的双曲线竞争。这些观察结果表明，先前报道的对心肌中PDE III型活性的复杂抑制是由于PDE III与其他酶，特别是PDE IV未完全分离所致。
