Biosynthesis of renin in mouse kidney tumor As4.1 cells.
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 oligosaccharides 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 +/- 1.4% (mean percentage of total precipitable radioactivity +/- SD) 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, beta-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.
Jones
C A
CA
Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
Petrovic
N
N
Novak
E K
EK
Swank
R T
RT
Sigmund
C D
CD
Gross
K W
KW
D000818:Animals; D002451:Cell Compartmentation; D003594:Cytoplasmic Granules / Q000378:metabolism; D004792:Enzyme Precursors / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006023:Glycoproteins / Q000096:biosynthesis; D007668:Kidney / Q000201:enzymology*; D051379:Mice; D008970:Molecular Weight; D012083:Renin / Q000096:biosynthesis* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D004792:Enzyme Precursors; D006023:Glycoproteins; D012083:Renin