Abstract
Kallikrein-binding protein was purified to apparent homogeneity from rat serum by Affi-Gel Blue, DEAE-Sepharose CL-6B, Sephacryl S-200 chromatography, and preparative gel electrophoresis or high performance liquid chromatography. The purified protein migrates as a single band of 60 kDa in a sodium dodecyl sulfate-polyacrylamide gel under reducing conditions. It is an acidic protein with isoelectric points ranging from 4.2 to 4.6. The amino terminus of the binding protein is an Asp residue as determined by sequence analysis. It forms a 92-kDa sodium dodecyl sulfatestable complex with kallikrein with a t1/2 of 18 min. Western blot and radioimmunoassay showed a distribution of the kallikrein-binding protein in serum, urine, and various tissues with a 5-10-fold lower amount in spontaneously hypertensive rats (SHR) than in Wistar-Kyoto rats (WKY). A full length cDNA clone encoding the kallikrein-binding protein was isolated from a rat liver cDNA library by immunoscreening and the translated amino acid sequence matches the amino-terminal 29-amino acid sequence of the binding protein. The cDNA sequence shares 68.8% identity with human alpha 1-antichymotrypsin and is identical to that of a rat hepatic protein. Dot blot analysis shows that kallikrein-binding protein is expressed at high levels in the liver and at low levels in the lung, salivary gland, and kidney. Its mRNA level in the liver decreases by 2-fold after acute phase inflammation and is higher in male than in female rats. Genomic Southern blot analyses reveal restriction fragment length polymorphisms between SHR and WKY rats in the binding protein locus. The results indicate that rat kallikrein-binding protein belongs to the serpin superfamily and its level is significantly reduced in the spontaneously hypertensive rats.
Highlights
The purified protein migrates as a single band of 60 kDa in a sodium dodecyl sulfate-polyacrylamide gel under reducing conditions
The results indicate that rat kallikrein-binding protein belongs to the serpin superfamily and its level is significantly reduced in the spontaneously hypertensive rats
The calculated molecular weight of mature Rat kallikrein binding protein levels (RKBP) based on the translated amino acid sequence is 44,587, which is less than the molecular weight of 60,000 as determined by SDS-polyacrylamide gel electrophoresis (PAGE)
Summary
Materials-The following reagents were obtained from commercial sources: DEAE-Sepharose. The gels were stained with 0.2% Coomassie Brilliant Blue and destained in a solution containing 10% acetic acid and. The gel eluates were collected in 0.2-ml volumes and were examined for both protein staining and binding activity to kallikrein. The remainder of the slab gel was fixed in 25% trichloroacetic acid overnight, stained in 0.2% Coomassie Brilliant Blue solution for 15 min and destained in methanol/acetic acid/water (300/120/1100). The nitrocellulose membranes were washed three times with BLOTTO and once with phosphate-buffered saline (0.01 M sodium phosphate, pH 7.4, 0.14 M NaCl), air-dried, and exposed to Kodak X-Omat film. Protein-A specific radioimmunoassay for RKBP was developed according to the previously described procedures (Chao et al, 1989a). Kallikrein-binding Protein in Hypertensive Rat washing, the blots were exposed at -70 “C with intensifying screens to Kodak X-Omat film
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
