Biosynthesis of Renin in Mouse Kidney Tumor As4.1 Cells

Open Access
Publisher Website
Google Scholar
Abstract
As4.1, a renin-expressing cell line isolated from a mouse renal tumor, was characterized for synthesis, processing, storage and secretion of renin polypeptides. Metabolic labeling, immunoprecipitation and SDS/PAGE analysis revealed that renin was secreted into the culture supernatant predominantly in the form of prorenin which migrated as products of 42–47 kDa. The predominant intracellular renin was processed into two chains, of 33–34 and 5 kDa. N-glycanase treatment removed N-linked oligosaccha-rides and yielded products of 41 kDa for prorenin and 31–32 kDa for the heavier chain of two-chain renin. The N-terminus of the constitutively secreted prorenin was determined by automated Edman degradation to be Leu22 while the N-terminus of the heavy chain was Ser72. Renin polypeptides constituted 3.1 Z1.4% (mean percentage of total precipitable radioactivity2SD) of de-novo-synthesized protein secreted into the medium and 0.2±0.17% retained intracellularly. Extrapolation of renin activity assays suggest that a single cell stores approximately 680 fg of active renin. A slow incremental release into the medium of processed renin heavy chain was detected by immunoprecipitation and SDS/PAGE. Renin activity assays confirmed the release of approximately 4 fg prorenin and 0.32 fg active renin cell−1 h−1. Indirect immunofluorescence demonstrated intracellular renin to be distributed in a punctate pattern. Renin was found to be colocalized with the lysosomal marker, β-glucuronidase, by double-fluorescent labeling. These cells have enabled characterization of glycosylated mouse renin-1 and may prove a valuable tool for studying intracellular trafficing of renin and associated processing enzymes.