Binding properties of rat prorenin and renin to the recombinant rat renin/prorenin receptor prepared by a baculovirus expression system

Abstract

The rat recombinant renin/prorenin receptor (AB188298 in DDJB), which conjugated with FLAG epitope in its N-terminus, was expressed in a baculovirus expression system. The recombinant receptor, prepared from the cytoplasmic fraction of the insect cells, was identified by Western blotting using anti-FLAG antibody. Prorenin as well as renin bound to the receptor with different binding affinities. Their Kd values were estimated at 8.0 and 20 nM, respectively. The amounts of prorenin and renin bound to the immobilized receptors were 1.0 and 0.2 pmole, respectively. The prorenin bound to the receptor had renin activity and the renin kept the activity at similar level to that before the binding. The Km of their complexes was the same at 3.3 μM when sheep angiotensinogen was used as the substrate. Their V max values were 5.5 and 10 nM·h-1, respectively. The molecular activities of prorenin and renin bound to the receptor were 1.1 and 10 h-1, respectively. From these findings, rat prorenin as well as renin was indicated to bind to the recombinant receptor and express the enzymatic activity in vitro.