Purification of rat pro-atrial natriuretic factor: a simplified scheme using reversed-phase high-performance liquid chromatography.
A simple scheme for the rapid and efficient isolation of rat pro-atrial natriuretic factor (pro-ANF) has been developed. An isolated rat adrenal cell bioassay for ANF was established to optimize heart tissue extraction and chromatography conditions. This assay is based on the ability of ANF to inhibit angiotensin II-stimulated aldosterone secretion. IC50 values for ANF were approximately 320 pM. The protocol that was established consisted of extraction of rat atria in 5 N acetic acid containing protease inhibitors. The extract was lyophilized, resolubilized in 0.1% trifluoroacetic acid containing 1% (w/v) sodium chloride, and subjected to RP-HPLC. Extraction of small batches of atria (i.e., from 10 or 20 rats) resulted generally in a yield of 2 nmol per rat (i.e., approximately 30 micrograms). The identity and purity of the pro-ANF were confirmed by the determination of both the amino acid composition and the amino-terminal sequence. Purified pro-ANF was radioiodinated and the efficiency of the extraction and purification procedure was assessed by adding labeled peptide to the initial tissue extract. The structural integrity and overall recovery of radioactivity were determined by RP-HPLC. The purification scheme provides undamaged pro-ANF of high purity. Purified pro-ANF was compared with synthetic rat ANF in the rat adrenal glomerulosa cell and isolated rat aortic strip bioassays. The peptides were apparently equally active in the adrenal cell system and approximately threefold less potent in relaxing aortic strips. The apparent equipotency in the adrenal cell bioassay may be due to the conversion of pro-ANF to ANF-like peptides during the bioassay incubation.
Belcourt
D
D
Endocrine Laboratory, Royal Victoria Hospital, Montreal, Quebec, Canada.
Varma
D R
DR
Toney
K
K
Bennett
H P
HP
D000450:Aldosterone / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001013:Aorta, Thoracic / Q000187:drug effects; D009320:Atrial Natriuretic Factor / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D001681:Biological Assay / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006325:Heart Atria / Q000737:chemistry; D008297:Male; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D051381:Rats; D011919:Rats, Inbred Strains; D015384:Zona Glomerulosa / Q000187:drug effects / Q000378:metabolism
D011498:Protein Precursors; D000450:Aldosterone; D009320:Atrial Natriuretic Factor