Characterization of hog kidney renin-binding protein: interconversion between monomeric and dimeric forms.
A radioimmunoassay for hog kidney renin-binding protein (RnBP) was developed. Using this assay method, we investigated the properties of hog kidney RnBP. The lower limit of detection was 24 fmol RnBP. The molecular weight of RnBP in hog kidney extract, as well as the purified RnBP, was estimated to be 65,000 by gel filtration on Ultrogel AcA 44. When the purified RnBP was treated with N-ethylmaleimide (NEM) or 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), the molecular weight was reduced to 38,000. DTNB-treated RnBP was reconverted to the 65,000-dalton species with dithiothreitol. Cross-linked high molecular weight species of RnBP were produced by the reaction of native RnBP with dimethyl suberimidate, but formation of such species was much less with NEM-treated RnBP. These results suggest that the native RnBP exists as a dimeric form and dissociates to a monomeric form by sulfhydryl-alkylating or -oxidizing reagent. It was shown from analysis of amino acid composition of S-carboxymethylated RnBP and titration of sulfhydryl groups of native and NEM-treated RnBP with DTNB that native RnBP contained twelve cysteine residues and that three cysteine residues were alkylated by NEM under the conditions employed.
D000478:Alkylation; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002238:Carbohydrate Epimerases; D002352:Carrier Proteins / Q000032:analysis*; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000032:analysis; D007668:Kidney / Q000032:analysis*; D008970:Molecular Weight; D010084:Oxidation-Reduction; D011863:Radioimmunoassay; D012083:Renin / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D013552:Swine
D000596:Amino Acids; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D013438:Sulfhydryl Compounds; D012083:Renin; D002238:Carbohydrate Epimerases; C024519:N-acyl-D-glucosamine 2-epimerase; D003545:Cysteine