Okita R T, Sinning A R, Okita J R, Tai H H, Markwald R R, Bergholte J M
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
Arch Biochem Biophys. 1990 Jun;279(2):242-8. doi: 10.1016/0003-9861(90)90488-k.
A polyclonal antibody was produced in guinea pig against the lung NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) purified from pregnant rabbits. Western blot analysis demonstrated that the protein identified by this antibody in the 105,000g supernatant fraction of lung tissue from pregnant rabbits had a molecular mass of 30 kDa and comigrated with the purified PGDH. The specific activity of the lung PGDH in pregnant rabbits (25- to 28-day gestations) was 36.7 nmol NADH formed/min/mg protein compared to 0.3 nmol NADH formed/min/mg protein in nonpregnant rabbits. Although the PGDH activity in the lung cytosol of nonpregnant rabbits was inhibited by the anti-lung PGDH antibody, the 30-kDa protein was not detected by Western blot analysis. An examination of this 30-kDa protein during the gestational period indicated that the protein was present after 10 days and the amount of the protein increased from Day 10 to Day 28. This increase in the immunochemically reactive protein correlated with the marked increase in PGDH specific activity between 10 and 28 days. An immunochemically reactive protein also was observed in the ovary of 25- to 28-day pregnant rabbits and the specific activity of the ovary PGDH was 19.3 nmol NADH formed/min/mg protein. Only trace levels of the PGDH activity were detected in the ovaries of nonpregnant rabbits. A 30-kDa protein was not detected by the anti-rabbit lung PGDH in brain, kidney, bladder, uterus, liver, and heart tissue of pregnant or nonpregnant rabbits. When rabbit or human placental cytosol was examined with the anti-rabbit lung PGDH only faint 30-kDa bands were observed by Western blot analysis. A monoclonal antibody prepared against human placental PGDH did not recognize the 30-kDa band in the pregnant rabbit lung. Localization studies indicated a marked increase in immunochemical staining in pulmonary epithelial cells of pregnant rabbits as compared to nonpregnant rabbits. Lung epithelial cells but not endothelial cells were identified as containing the PGDH.
用从妊娠兔肺中纯化的NAD(+)依赖性15-羟基前列腺素脱氢酶(PGDH)在豚鼠体内制备了一种多克隆抗体。蛋白质印迹分析表明，该抗体在妊娠兔肺组织105,000g上清液组分中识别出的蛋白质分子量为30 kDa，与纯化的PGDH迁移率相同。妊娠兔(妊娠25至28天)肺PGDH比活性为36.7 nmol NADH生成/分钟/毫克蛋白质，而未妊娠兔为0.3 nmol NADH生成/分钟/毫克蛋白质。虽然未妊娠兔肺胞质溶胶中的PGDH活性受到抗肺PGDH抗体的抑制，但蛋白质印迹分析未检测到30 kDa的蛋白质。对妊娠期这种30 kDa蛋白质的检测表明，该蛋白质在10天后出现，其含量从第10天到第28天增加。这种免疫反应性蛋白质的增加与10至28天期间PGDH比活性的显著增加相关。在妊娠25至28天的兔卵巢中也观察到一种免疫反应性蛋白质，卵巢PGDH比活性为19.3 nmol NADH生成/分钟/毫克蛋白质。在未妊娠兔卵巢中仅检测到微量的PGDH活性。在妊娠或未妊娠兔的脑、肾、膀胱、子宫、肝和心脏组织中，抗兔肺PGDH未检测到30 kDa的蛋白质。当用抗兔肺PGDH检测兔或人胎盘胞质溶胶时，蛋白质印迹分析仅观察到微弱的30 kDa条带。针对人胎盘PGDH制备的单克隆抗体不能识别妊娠兔肺中的30 kDa条带。定位研究表明，与未妊娠兔相比，妊娠兔肺上皮细胞中的免疫化学染色显著增加。肺上皮细胞而非内皮细胞被确定含有PGDH。
