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 Cterminal 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 ...