[Frontiers in Bioscience E2, 1234-1240 June 1, 2010]

Species specificity of prorenin binding to the (pro)renin receptor in vitro

Kazal Boron Biswas1, AHM Nurun Nabi2, Yoshie Arai2, Tsutomu Nakagawa1,2, Akio Ebihara1,2, Fumiaki Suzuki1,2

1United Graduate School of Agricultural Science, Gifu University, Gifu 501-1193, Japan, 2Faculty of Applied Biological Sciences, Gifu University, Gifu 501-1193, Japan

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and Methods
3.1. Preparation of human and rat prorenin
3.2. Expression of human and rat (pro)renin receptor on the membrane of COS-7 cells
3.3. Bindings of prorenin molecules to the (pro)renin receptor expressed on the membrane of COS-7 cells
3.4. In vitro expression of recombinant human and rat (pro)renin receptor
3.5. Binding assay of prorenin molecules to the (pro)renin receptor using BIAcore assay system
3.6. Determination of the molecular activities of receptor-bound prorenin molecules
4. Results
4.1. Binding of prorenin to (pro)renin receptor expressed on the membrane of COS-7 cells
4.2. Real-time bindings of prorenin molecules to the immobilized (pro)renin receptors in BIAcore
4.3. Molecular activities of the receptor-bound prorenin
5. Discussion
6. Acknowledgements
7. References

1. ABSTRACT

In this study, binding properties of human prorenin to rat (pro)renin receptor (r(P)RR) and rat prorenin to human (pro)renin receptor (h(P)RR) were elucidated in vitro to investigate species specificity of prorenin bindings to (P)RR. Both (P)RRs were expressed in vitro based on wheat germ lysate and purified using His trap column. The association and dissociation rate constants of human and rat prorenin for the immobilized r(P)RR and h(P)RR were 6.64x106 and 1.01x106 M-1.s-1 and, 0.024 and 8.45x10-3 s-1, respectively. Their KD values were 3.7nM (3-fold higher than that of human prorenin vs h(P)RR (1.2nM)) and 8.3nM (1.2-fold lower than that of rat prorenin vs r(P)RR (10.2nM)), respectively. Additionally, human and rat prorenin bound to the pre-adsorbed rat and human (P)RR, respectively, performed enzymatic activities. Their molecular activities were 4.1h-1 (almost similar (3.8h-1) to human prorenin vs h(P)RR) and 0.98h-1 (~2-fold lower than rat prorenin vs r(P)RR), respectively. Thus, these results suggest the species specificity for bindings of prorenin to (P)RR, which could be useful in elucidating the biochemical properties of prorenin binding to the receptor.