A specific antibody was prepared against purified pig spleen heme oxygenase. This antibody cross-reacted well with the microsomal heme oxygenase of pig alveolar macrophages. By combined use of the immunochemical method and the radiochemical method, it was shown that the hemin-induced increase of the heme oxygenase activity in alveolar macrophages was actually due to increased synthesis of the heme oxygenase protein. In either the hemin-induced or uninduced macrophages the specific activity of heme oxygenase was much higher in smooth microsomes than in rough microsomes, suggesting that the smooth part of the endoplasmic reticulum may be the major site for the physiological functioning of heme oxygenase. Free and bound polysomes were isolated from macrophages and nascent peptides on these polysomes were analyzed by means of labeling with [3H]-puromycin. The results obtained indicated that free polysomes are the major site of heme oxygenase synthesis. Cell-free synthesis of heme oxygenase was also performed in the reticulocyte lysate system with the free and bound polysomes isolated from hemin-treated macrophages, and the results obtained again suggested that heme oxygenase is synthesized predominantly on free polysomes. The polysome-directed cell-free synthesis of heme oxygenase revealed that functional mRNA for heme oxygenase was actually increased in the hemin-induced cells. It was concluded that hemin acts at the transcription level to stimulate synthesis of the heme oxygenase-specific mRNA in alveolar macrophages.
ASJC Scopus subject areas
- Molecular Biology