Characterization and purification of a protease in serum that cleaves proatrial natriuretic factor (ProANF) to its circulating forms

Journal Article · Tue Dec 29 04:00:00 EST 1987 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00400a030· OSTI ID:5272146

Zisfein, J B; Graham, R M; Dreskin, S V; Wildey, G M; Fischman, A J; Homcy, C J

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. This reports describes the purification and characterization of this proANF-cleaving protease from rat serum. The cleavages both of /sup 35/S-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. 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. Incubation of the purified enzyme with (/sup 3/H)DFP followed by SDS-PAGE and autoradiography revealed a specifically labeled 38-kDa peptide, the substrate binding subunit. Analysis by high-performance liquid chromatography of the 3-kDa products resulting from the cleavage of /sup 35/S-labeled proANF by the purified enzyme revealed, as previously described with whole serum, two radiolabeled peptides which coeluted with the 28 and 24 amino acid C-terminal peptides. These observations imply a precursor-product relationship, with the initial cleavage of proANF to the 28 amino acid peptide, which is then cleaved to the 24 amino acid peptide. These studies indicate that the majority of proANF cleavage activity found in rat serum is represented by that of a distinct serine protease whose properties are different from a variety of well-characterized proteases. The role of this protease in the in vivo processing of proANF remains to be defined.

• Research Organization: Massachusetts General Hospital, Boston (USA)
• OSTI ID: 5272146
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:26; ISSN BICHA
• Country of Publication: United States
• Language: English

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59 BASIC BIOLOGICAL SCIENCES
62 RADIOLOGY AND NUCLEAR MEDICINE
ANIMALS
AUTORADIOGRAPHY
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMISTRY
BLOOD SERUM
CHEMISTRY
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
ELECTROPHORESIS
ENZYME ACTIVITY
ENZYMES
EVEN-ODD NUCLEI
HYDROLASES
ISOTOPES
LABELLED COMPOUNDS
LIGHT NUCLEI
LIQUID COLUMN CHROMATOGRAPHY
MAMMALS
NUCLEI
ORGANIC COMPOUNDS
PEPTIDE HYDROLASES
PEPTIDES
POLYPEPTIDES
PROTEINS
PURIFICATION
RADIOISOTOPES
RATS
RODENTS
SEPARATION PROCESSES
SULFUR 35
SULFUR ISOTOPES
TRITIUM COMPOUNDS
VERTEBRATES