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.

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 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.

journal_name

Endocrinology

journal_title

Endocrinology

authors

Wildey GM,Fischman AJ,Fallon JT,Matsueda GR,Zisfein JB,Preibisch G,Seipke G,Homcy CJ,Graham RM

doi

10.1210/endo-123-4-2054

subject

Has Abstract

pub_date

1988-10-01 00:00:00

pages

2054-61

issue

4

eissn

0013-7227

issn

1945-7170

journal_volume

123

pub_type

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