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Intracellular site of synthesis of microsomal heme oxygenase in pig spleen
Abstract:
In the pig spleen the specific activity of heme oxygenase was two to three times higher in smooth microsomes than in rough microsomes, whereas the total heme oxygenase activities recovered in the two microsomal fractions were similar. Free and bound polysomes were isolated from pig spleen and nascent peptides on these polysomes were analyzed by employing [3H]puromycin and a heme oxygenase-specific rabbit antibody (IgG). It was shown that free polysomes are the major site of heme oxygenase synthesis. In addition, cell-free synthesis of heme oxygenase was performed in a reticulocyte lysate system with free and bound polysomes isolated from pig spleen, and the results obtained again indicated that heme oxygenase is synthesized predominantly on free polysomes. The heme oxygenase newly synthesized on free polysomes may be incorporated first into the rough portion of endoplasmic reticulum either before or after its release from polysomes, although the specific activity of this enzyme at the steady state is considerably higher in the smooth region.
Insights
Heme oxygenase, an enzyme crucial for heme breakdown, is primarily synthesized on free polysomes in pig spleen cells. This newly synthesized enzyme may then be processed through the endoplasmic reticulum.
Area of Science:
- Biochemistry
- Cell Biology
- Enzymology
Background:
- Heme oxygenase (HO) is a key enzyme in heme catabolism.
- Microsomal fractions of pig spleen exhibit differential HO activity.
- Understanding HO synthesis location is vital for cellular heme homeostasis.
Purpose of the Study:
- To determine the primary site of heme oxygenase synthesis in pig spleen.
- To investigate the role of free and bound polysomes in HO production.
- To elucidate the initial cellular localization of newly synthesized HO.
Main Methods:
- Isolation of free and bound polysomes from pig spleen.
- Analysis of nascent peptides using [3H]puromycin incorporation.
- Immunological detection of heme oxygenase using specific rabbit antibodies (IgG).
- Cell-free synthesis assays in reticulocyte lysate systems.
Main Results:
- Specific activity of heme oxygenase was significantly higher in smooth microsomes compared to rough microsomes.
- Free polysomes were identified as the major site of heme oxygenase synthesis.
- Cell-free synthesis experiments confirmed predominant HO production on free polysomes.
- Newly synthesized HO may be initially targeted to the rough endoplasmic reticulum.
Conclusions:
- Heme oxygenase synthesis predominantly occurs on free polysomes in pig spleen.
- The enzyme's distribution between smooth and rough microsomes suggests post-translational processing or compartmentalization.
- These findings contribute to understanding the regulation of heme metabolism and enzyme localization within the cell.