Amidating monooxygenase

28-Jun-2019 12:29 by 9 Comments

Amidating monooxygenase

Experiments were conducted according to the the mutated fusion proteins are estimated to be coexpressed in a ratio of at least 1:2 with endogenous pro ANP.

could play a role in its segregation to secretory granules.Accordingly, there are thousands of studies on ANP release and action, in both animal and man.In contrast to this wealth of knowledge, the cell biology of ANP-containing secretory vesicles is far less understood.Secretory vesicles were visualized by fluorescence microscope imaging in live rat atrial myocytes expressing pro ANP–enhanced green fluorescent protein (EGFP), or N-terminal–mutated fusion proteins thought to suppress the calcium-dependent aggregation of pro ANP.Results showed the following: (1) aggregates of pro ANP and coexpressed pro ANP-EGFP recruited peptidylglycine α-amidating monooxygenase (PAM)-1, an abundant atrial integral vesicle membrane protein; (2) coexpressed N-terminal–mutated (Glu23,24→Gln23,24) and N-terminal–deleted pro ANP-EGFP inhibited recruitment of PAM-1 by up to 60%; (3) 4-phenyl-3-butenoic acid (PBA) (10 μmol/L), a pharmacological inhibitor of the lumenal peptidylglycine α-hydroxylating monooxygenase domain of PAM proteins, inhibited recruitment of endogenous PAM-1 and of coexpressed pro-EGFP–PAM-1; (4) PBA had no effect on exocytosis of the potassium inward rectifier KIR2.1; (5) PBA induced a deformation of the secretory vesicles but did not inhibit docking.If PAM interacted with lumenal proteins of the TGN, such as pro ANP, PAM could be involved in forming the atrial secretory vesicles.

The purpose of this study was to determine whether PAM proteins were implicated in shaping and docking the atrial pro ANP vesicles. Are PAM proteins recruited to pro ANP-EGFP–expressing secretory vesicles?We found that peptidylglycine α-amidating monooxygenase (PAM), an abundant protein family in the cardiac atrium, is localized on atrial pro ANP–enhanced green fluorescent protein (EGFP)–expressing vesicles and could thus be a viable candidate.PAM posttranslationally activates approximately half of all mammalian neuropeptides by converting their COOH-terminal glycine into an essential α-amide moiety (see Prigge et al for review).These findings suggest that recruitment of PAM-1 to secretory vesicles depends on intact N-terminal pro ANP and on the lumenal domain of PAM-1.Conversely, PAM-1 participates in shaping the pro ANP-secretory vesicles.Do mutations of the N-terminal calcium-binding sequence of pro ANP suppress recruitment of PAM?