Characterization and purification of a protease in serum that cleaves proatrial natriuretic factor (ProANF) to its circulating forms.
Atrial natriuretic factor (ANF) is synthesized and stored in atrial cardiocytes as a 17-kilodalton (kDa), 126 amino acid polypeptide, proANF, but circulates as smaller, 24 and 28 amino acid peptide fragments of the carboxy terminus of proANF. It has previously been shown that proANF is secreted intact from cultured atrial cardiocytes and can be cleaved by a serum protease to smaller, 3-kDa peptides believed to be the circulating forms. This report describes the purification and characterization of this proANF-cleaving protease from rat serum. The cleavages both of 35S-labeled proANF derived from rat atrial cell cultures, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/autoradiography, and of a synthetic p-nitroanilide-containing substrate were used as assays for the detection of enzyme activity. ProANF-cleaving activity was found in rat serum, with no such activity detectable in rat plasma. Cleavage in serum was not dependent on the presence of platelets or other cellular elements. Complete inhibition of proANF cleavage was obtained with the protease inhibitors benzamidine, leupeptin, phenylmethanesulfonyl fluoride, and diisopropyl fluorophosphate (DFP) but not with aprotinin, soybean trypsin inhibitor, pepstatin, or hirudin. Unlike the vitamin K dependent plasma proteins, the proANF-cleaving protease did not adsorb to barium sulfate. With the sequential application of ion-exchange, hydroxylapatite, lectin affinity, and gel filtration chromatography, a 5000-6000-fold purification of the enzyme from rat serum was achieved. Fractionation of either whole serum or the purified enzyme by gel filtration chromatography revealed a single peak of activity corresponding to a protein with a Stokes radius of 45 A.(ABSTRACT TRUNCATED AT 250 WORDS)
Zisfein
J B
JB
Cellular and Molecular Research Laboratory, Massachusetts General Hospital, Boston 02114.
Graham
R M
RM
Dreskin
S V
SV
Wildey
G M
GM
Fischman
A J
AJ
Homcy
C J
CJ
D000818:Animals; D009320:Atrial Natriuretic Factor / Q000097:blood* / Q000378:metabolism*; D007531:Isoflurophate / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000097:blood* / Q000302:isolation & purification; D011480:Protease Inhibitors / Q000494:pharmacology; D011485:Protein Binding; D011498:Protein Precursors / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D011480:Protease Inhibitors; D011498:Protein Precursors; D007531:Isoflurophate; D009320:Atrial Natriuretic Factor; D010447:Peptide Hydrolases