Isolation and properties of lung 15-hydroxyprostaglandin dehydrogenase from pregnant rabbits.
Isolation and properties of lung 15-hydroxyprostaglandin dehydrogenase from pregnant rabbits.
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怀孕兔肺15-羟基前列腺素脱氢酶的分离和性质。
DOI：
10.1016/0003-9861(86)90221-3
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发表时间：
1986
影响因子：
3.9
通讯作者：
Okita,RT
中科院分区：
文献类型：
--
作者：
Bergholte,JM;Okita,RT
从28日龄孕兔肺中纯化出一种NADH依赖性的15-羟基前列腺素脱氢酶（PGDH），以50 μmprostaglandin E1as为底物，形成的NADH比活性超过25000 nmol /min/mg。这意味着酶纯化了2600倍，恢复了6%的起始酶活性。使用怀孕兔的肺，因为妊娠20天后观察到PGDH活性诱导42至55倍。该酶通过cm -纤维素、deae -纤维素、Sephadex G-75、辛胺琼脂糖和羟基磷灰石层析纯化。该酶不能用NAD-或蓝葡聚糖结合树脂亲和层析纯化。纯化后的酶对NAD具有特异性，亚基分子量为29,000。以3-（环己胺）丙磺酸为缓冲液，前列腺素e1氧化的最佳pH范围为10.0 ~ 10.4。前列腺素e1的kmandvmaxvalue分别为33 μ和40,260 nmol/min/mg蛋白，前列腺素e2的kmandvmaxvalue分别为59 μ和43,319 nmol/min/mg蛋白。前列腺素f2 α的k值是前列腺素E1的4倍。PGDH活性被氯基苯磺酸抑制，但添加二硫苏糖醇后酶活性恢复。n-乙基马来酰亚胺也使酶活性迅速下降，但当NAD加入孵育系统时，没有观察到抑制作用。
A NAD-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) was purified to a specific activity of over 25,000 nmol NADH formed/min/mg protein with 50 μmprostaglandin E1as substrate from the lungs of 28-day-old pregnant rabbits. This represented a 2600-fold purification of the enzyme with a recovery of 6% of the starting enzyme activity. The lungs of pregnant rabbits were used because a 42- to 55-fold induction of the PGDH activity was observed after 20 days of gestation. The enzyme was purified by CM-cellulose, DEAE-cellulose, Sephadex G-75, octylamino-agarose, and hydroxylapatite chromatography. The enzyme could not be purified by affinity chromatography using NAD- or blue dextran-bound resins. The purified enzyme was specific for NAD and had a subunit molecular weight of 29,000. The optimal pH range for the oxidation of prostaglandin E1was between 10.0 and 10.4 using 3-(cyclohexylamino)propanesulfonic acid as the buffer. TheKmandVmaxvalues for prostaglandin E1were 33 μmand 40,260 nmol/min/mg protein, respectively, while theKmandVmaxvalues for prostaglandin E2were 59 μmand 43,319 nmol/min/mg protein, respectively. TheKmfor prostaglandin F2αwas four times the value for prostaglandin E1. The PGDH activity was inhibited byp-chloromercuriphenylsulfonic acid but the enzymatic activity was restored by the addition of dithiothreitol.n-Ethylmaleimide also produced a rapid decline in enzymatic activity but when NAD was included in the incubation system, no inhibition was observed.