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Purification and molecular cloning of mouse renal dipeptidase
1Product Development Laboratories, Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan.
Abstract:
Mouse renal dipeptidase (mouseRDP, EC 3.4.13.11) was purified from the membrane fraction of kidney. The molecular mass of the enzyme was 115 kDa by size-exclusion HPLC and SDS-PAGE under non-reduced conditions and 58 kDa by SDS-PAGE under reduced conditions. The mouseRDP cDNA fragment was amplified from mouse kidney total RNA by reverse transcription-polymerase ŏffin reaction (RT-PCR). The mouseRDP cDNA was isolated from a kidney cDNA library using the probe. The primary structure of mouseRDP deduced from the cDNA showed a high homology with renal dipeptidase from various mammals, except for the amino-terminal and carboxy-terminal domains. Recombinant mouseRDP obtained from transfected mouse L929 cells containing the expression plasmids has the same Km value and molecular mass as native mouse renal dipeptidase. From Northern blotting analysis, expression of the mouseRDP gene was recognized in both kidney and liver.
Insights
Researchers purified mouse renal dipeptidase (mouseRDP), a kidney enzyme. They characterized its structure and confirmed recombinant mouseRDP matches the native enzyme, finding expression in kidney and liver.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Renal dipeptidases play crucial roles in kidney function.
- Understanding the structure and expression of mouse renal dipeptidase (mouseRDP) is essential for comparative studies.
Purpose of the Study:
- To purify and characterize mouse renal dipeptidase (mouseRDP).
- To determine the molecular mass and primary structure of mouseRDP.
- To analyze the expression pattern of the mouseRDP gene.
Main Methods:
- Purification of mouseRDP from kidney membrane fractions.
- Molecular mass determination using size-exclusion HPLC and SDS-PAGE (reduced and non-reduced conditions).
- cDNA amplification via reverse transcription-polymerase chain reaction (RT-PCR) and isolation from a kidney cDNA library.
- Analysis of recombinant mouseRDP expressed in transfected L929 cells.
- Gene expression analysis using Northern blotting.
Main Results:
- Purified mouseRDP exhibited molecular masses of 115 kDa (non-reduced) and 58 kDa (reduced).
- The deduced primary structure showed homology to mammalian renal dipeptidases, with variations in terminal domains.
- Recombinant mouseRDP displayed identical Km values and molecular mass to the native enzyme.
- Northern blotting confirmed mouseRDP gene expression in both kidney and liver tissues.
Conclusions:
- Mouse renal dipeptidase (mouseRDP) has been successfully purified and characterized.
- The recombinant enzyme serves as a valid model for studying native mouseRDP.
- The mouseRDP gene is expressed in both kidney and liver, suggesting potential roles beyond the kidney.