NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase: immunochemical characterization of the lung enzyme from pregnant rabbits.
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.
Okita
R T
RT
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
Sinning
A R
AR
Okita
J R
JR
Tai
H H
HH
Markwald
R R
RR
Bergholte
J M
JM
D000527:Alprostadil / Q000031:analogs & derivatives / Q000096:biosynthesis; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000936:Antigen-Antibody Complex; D015153:Blotting, Western; D003600:Cytosol / Q000201:enzymology; D004848:Epithelium / Q000201:enzymology; D005260:Female; D006911:Hydroxyprostaglandin Dehydrogenases / Q000276:immunology* / Q000378:metabolism; D008168:Lung / Q000201:enzymology*; D008970:Molecular Weight; D010053:Ovary / Q000201:enzymology; D011247:Pregnancy; D011270:Pregnancy, Animal / Q000378:metabolism*; D011817:Rabbits
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; C011008:15-ketoprostaglandin E; D006911:Hydroxyprostaglandin Dehydrogenases; C035859:15-hydroxyprostaglandin dehydrogenase; D000527:Alprostadil