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Biosynthesis of porcine kidney D-amino acid oxidase
Abstract:
The biosynthesis of a porcine kidney peroxisomal enzyme, D-amino acid oxidase (EC 1.4.3.3., DAO), was investigated. Pig kidney mRNA as well as free and membrane-bound polysomes were used to investigate in vitro protein synthesis using a rabbit reticulocyte lysate. mRNA and free polysomes, but not membrane-bound polysomes, directed the synthesis of DAO. To examine the in vivo synthesis of the enzyme, a pig kidney cell line (LLC-PK1) was biosynthetically labelled. Both the in vitro and in vivo synthesized DAO had the same molecular weight, 38,000, as that of the purified enzyme. These results indicate strongly that DAO is synthesized on free ribosomes and transferred to the interior of peroxisomes without any proteolytic modification.
Insights
D-amino acid oxidase (DAO) in pig kidneys is synthesized on free ribosomes. This peroxisomal enzyme is then transferred into peroxisomes without modification, as confirmed by in vitro and in vivo studies.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Peroxisomal enzymes play crucial roles in cellular metabolism.
- Understanding the biosynthesis of enzymes like D-amino acid oxidase (DAO) is essential for comprehending peroxisome function.
Purpose of the Study:
- To investigate the biosynthesis pathway of porcine kidney D-amino acid oxidase (DAO).
- To determine the site of DAO synthesis (free vs. membrane-bound ribosomes) and its post-translational processing.
Main Methods:
- In vitro protein synthesis using pig kidney mRNA and polysomes with a rabbit reticulocyte lysate system.
- In vivo biosynthetic labeling of DAO in a pig kidney cell line (LLC-PK1).
- Analysis of synthesized DAO molecular weight and comparison with purified enzyme.
Main Results:
- Pig kidney mRNA and free polysomes directed the in vitro synthesis of DAO.
- Membrane-bound polysomes did not synthesize DAO.
- Both in vitro and in vivo synthesized DAO exhibited a molecular weight of 38,000, matching the purified enzyme.
- DAO is synthesized on free ribosomes.
Conclusions:
- D-amino acid oxidase (DAO) is synthesized on free ribosomes in porcine kidney cells.
- The synthesized DAO is translocated into peroxisomes without undergoing proteolytic modification.