Related Experiment Videos
Characterization of hog kidney renin-binding protein: interconversion between monomeric and dimeric forms.
S Takahashi1, R Miura, Y Miyake
1Department of Biochemistry, National Cardiovascular Center Research Institute, Osaka, Japan.
Biochemistry International
|June 1, 1988
Summary
Hog kidney renin-binding protein (RnBP) exists as a dimer. Sulfhydryl-alkylating reagents cause RnBP to dissociate into a monomer, revealing its dimeric structure and cysteine residue involvement.
Area of Science:
- Biochemistry
- Renal Physiology
Background:
- Renin-binding protein (RnBP) plays a role in regulating renin activity.
- Understanding RnBP's structure is crucial for elucidating its function in the kidney.
Purpose of the Study:
- To develop a radioimmunoassay for hog kidney RnBP.
- To characterize the molecular properties and structure of hog kidney RnBP.
Main Methods:
- Radioimmunoassay development and application.
- Gel filtration chromatography (Ultrogel AcA 44) for molecular weight estimation.
- Chemical modification with N-ethylmaleimide (NEM) and DTNB to assess sulfhydryl group reactivity.
- Cross-linking studies using dimethyl suberimidate.
Main Results:
- A sensitive radioimmunoassay for hog kidney RnBP was established with a lower detection limit of 24 fmol.
- Native hog kidney RnBP has an estimated molecular weight of 65,000 Da, suggesting a dimeric form.
- Treatment with sulfhydryl-reactive agents (NEM, DTNB) reduced the molecular weight to 38,000 Da, indicating dissociation into monomers.
- Reconversion to the dimeric form was achieved with dithiothreitol.
- Native RnBP contains twelve cysteine residues, with three being alkylated by NEM.
Conclusions:
- Hog kidney RnBP exists primarily as a dimer.
- The dimeric structure is stabilized by disulfide bonds or other interactions involving cysteine residues.
- Sulfhydryl-modifying reagents disrupt these interactions, leading to monomer formation.