Reversible cryoactivation of recombinant human prorenin.
Cleavage of prorenin's prosegment causes irreversible formation of renin. In contrast, renin activity is reversibly exposed when prorenin is acidified to pH 3.3. Nonetheless, acidification of plasma results in irreversible activation of prorenin, because endogenous proteases cleave the prosegment of acid-activated prorenin. Chilling of plasma results in irreversible cryoactivation of prorenin. In this study we investigated whether cryoactivation of purified prorenin is reversible. The intrinsic renin activity of recombinant human prorenin was measured by an enzyme kinetic assay using partially purified human angiotensinogen as substrate. Results are expressed as a percent (mean +/- S.E.) of the maximal activity exposed after limited proteolysis by trypsin. The intrinsic renin activity of two pools (0.3 and 0.06 Goldblatt units/ml) was 1.5% +/- 0.3 and 1.2% +/- 0.6 at 37 degrees C. Activity increased to 19% +/- 0.3 and 26% +/- 0.5 after incubation at 0 degrees C and to 5.4% +/- 0.5 and 2.1% +/- 1.2 at room temperature. Cryoactivation did not occur in buffers containing more than 1 M NaCl. It took 8 min at 37 degrees C or 180 min at room temperature for cryoactivated prorenin to lose half of its intrinsic renin activity. It took 48 and 26 h, respectively, at 0 degree C for the two pools of prorenin at 37 degrees C to regain half of their maximum intrinsic activity at 0 degrees C. A direct immunoradiometric assay that detects active renin but not prorenin was able to detect cryoactivated prorenin. These results show that human prorenin can be reversibly cryoactivated in buffers of low ionic strength and has greater intrinsic activity at room temperature than at 37 degrees C.
Pitarresi
T M
TM
Cardiovascular Center, Cornell University Medical College, New York, New York 10021.
Rubattu
S
S
Heinrikson
R
R
Sealey
J E
JE
D002384:Catalysis; D003080:Cold Temperature; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009994:Osmolar Concentration; D011994:Recombinant Proteins / Q000378:metabolism; D012083:Renin / Q000378:metabolism*; D012965:Sodium Chloride / Q000378:metabolism; D000092502:Renin Inhibitors
D004792:Enzyme Precursors; D011994:Recombinant Proteins; D012083:Renin; D012965:Sodium Chloride; D000092502:Renin Inhibitors