On the measurement of inactive renin in rat plasma: activation by trypsin generates interfering tetradecapeptide-like material and destroys angiotensinogen.
Trypsin cleaved plasma angiotensinogen with apparent first-order kinetics and generated an angiotensin I (Ang I) immunoreactive material. Size exclusion high-performance liquid chromatography (HPLC) of rat plasma proteins demonstrated that the Ang I immunoreactive material was formed in those fractions which contained angiotensinogen. The Ang I immunoreactive material was higher in nephrectomized rat plasma than normal plasma, in accordance with the higher angiotensinogen concentration. These findings indicated that angiotensinogen could be the source of the Ang I immunoreactive material. Purification of the Ang I immunoreactive material by cation-exchange chromatography followed by reverse-phase HPLC demonstrated an elution pattern close to that of human tetradecapeptide. The purified Ang I immunoreactive material was cleaved by pure mouse submandibular renin to Ang I, exclusively. Incubation at 37 degrees C of the Ang I immunoreactive material with plasma partially destroyed the angiotensin immunoreactive material. These findings demonstrated that the angiotensin immunoreactive material was an Ang I containing tetradecapeptide (TDP)-like peptide, unstable during a renin incubation step, leading to erroneous values for plasma inactive renin if not removed. The Ang I immunoreactive material was removed by cation-exchange chromatography of trypsin-activated plasma allowing for a determination of inactive renin. The presence of inactive renin in plasma from normal and nephrectomized rats was confirmed, and identified by neutralization and immunoprecipitation with antirenins. These findings should enable us to develop a routine assay for plasma inactive renin in rat plasma.
Johannessen
A
A
Institute of Biochemistry, Immunology and Microbiology, Royal Dental College, Copenhagen N, Denmark.
Nielsen
A H
AH
Jacobsen
J
J
Poulsen
K
K
D000803:Angiotensin I / Q000032:analysis / Q000378:metabolism*; D000808:Angiotensinogen / Q000032:analysis / Q000378:metabolism*; D000818:Animals; D004792:Enzyme Precursors / Q000097:blood*; D008297:Male; D009392:Nephrectomy; D051381:Rats; D011919:Rats, Inbred Strains; D012083:Renin / Q000097:blood*; D014357:Trypsin / Q000378:metabolism
D004792:Enzyme Precursors; D000808:Angiotensinogen; D000803:Angiotensin I; D014357:Trypsin; D012083:Renin