Cellular processing of pro-atrial natriuretic factor (pro-ANF): studies using an antiserum that selectively binds ANF-(99-126) after its cleavage from pro-ANF.
Atrial natriuretic factor (ANF) is stored in atrial myocytes as a 15-17K prohormone, but circulates in plasma as a 3K, carboxy (C)-terminal fragment of the prohormone. The tissue location at which the cleavage of pro-ANF to its hormonal form occurs is unknown. In the present study, an immunological approach was taken to address this question. A polyclonal antiserum was generated which recognizes the hormonal form of ANF [ANF-(99-126)] only after its cleavage from the prohormone. This was accomplished by immunizing rabbits with a synthetic peptide corresponding to the seven amino (N)-terminal residues of ANF-(99-126) coupled to carrier protein via a C-terminal cysteine. This antiserum, anti-ANF-(99-105), demonstrated high affinity for ANF-(99-126) (IC50 = 170 pM), but displayed 100-fold less affinity for recombinant pro-ANF [ANF-(2-126)]. The N-terminal specificity of anti-ANF-(99-105) was evident by its failure to bind ANF-(103-126) at concentrations up to 100 nM. The specificity of anti-ANF-(99-105) for the hormonal form of ANF was examined by using thrombin to cleave pro-ANF and testing for the generation of anti-ANF-(99-105) immunoreactivity. Cleavage of atrial pro-ANF or 35S biosynthetically-labeled pro-ANF resulted in the production of immunoreactive material from the prohormone, whereas pro-ANF itself demonstrated no cross-reactivity with anti-ANF-(99-105). Anti-ANF-(99-105) could also recognize ANF released from the isolated perfused rat heart. When anti-ANF-(99-105) was used in immunohistochemical studies of rat atrial myocardium, no staining was observed in unfixed frozen sections. This suggests that proteolytic processing of pro-ANF is not an intracardiocytic event.
Wildey
G M
GM
Cellular and Molecular Research Laboratory, Massachusetts General Hospital, Boston 02114.
Fischman
A J
AJ
Fallon
J T
JT
Matsueda
G R
GR
Zisfein
J B
JB
Preibisch
G
G
Seipke
G
G
Homcy
C J
CJ
Graham
R M
RM
D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D000936:Antigen-Antibody Complex; D009320:Atrial Natriuretic Factor / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D002478:Cells, Cultured; D005260:Female; D006325:Heart Atria / Q000378:metabolism; D007106:Immune Sera; D007124:Immunoenzyme Techniques; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D011499:Protein Processing, Post-Translational; D011863:Radioimmunoassay; D051381:Rats; D011919:Rats, Inbred Strains
D000936:Antigen-Antibody Complex; D007106:Immune Sera; D011498:Protein Precursors; D009320:Atrial Natriuretic Factor