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

G. M. Wildey, A. J. Fischman, J. T. Fallon, G. R. Matsueda, J. B. Zisfein, G. Preibisch, G. Seipke, C. J. Homcy, R. M. Graham

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10 Scopus citations

Abstract

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 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.

Original languageEnglish
Pages (from-to)2054-2061
Number of pages8
JournalEndocrinology
Volume123
Issue number4
DOIs
StatePublished - 1 Oct 1988
Externally publishedYes

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