Amidation of joining peptide, a major pro-ACTH/endorphin-derived product peptide.

An antibody specific for an endoproteolytic cleavage site provides evidence that pro-opiomelanocortin is packaged into secretory granules in AtT20 cells before its cleavage.

“…In ART20 cells, a line of murine anterior pituitary tumor cells (10), cleavage of POMC liberates ACrH, ~lipotropic hormone (I]LPH), and an amino-terminal peptide (5,17,25). Some of the I3LPH is cleaved further to yield 13-endorphin while the amino-terminal peptide is cleaved to yield from its carboxy end the joining peptide whose carboxy terminus is ~t-amidated (8). The ACTH does not, however, undergo further cleavage.…”

Peptidylglycine α‐amidating monooxygenase as a therapeutic target or biomarker for human diseases

“…The inhibitory effects of divalent metal ion chelators on peptide amidation became apparent as soon as different buffers were tested for use in enzyme assays of tissue lysates (Eipper, Mains, & Glembotski, 1983). Inhibition by diethyldithiocarbamate was reversed only by the addition of Cu (II), providing the first evidence that PAM/PHM catalysis was copper‐dependent; no other divalent metal, including Zn (II), restored activity (Eipper, Mains, et al, 1983; Eipper et al, 1986). Other metal chelators like EDTA (Merkler et al, 1993) and disulfiram inhibit PAM/PHM reversibly (Mains et al, 1986) (Figure 3a).…”

Multiple roles for peptidylglycine α‐amidating monooxygenase in the response to hypoxia

“…Signals in the linear range were captured and quantified using Gene Tools software (Syngene). In-house antibodies used included: affinity-purified antibodies to specific regions of PAM (JH471 to PAL; JH629 to Exon 16; CT267 to PAM-CD); Russo-Savage et al, 2020); antibodies to aminoterminal POMC processing products (antiserum Georgie, to 18/16 kDa fragment and affinity-purified antibody Jamie, specific for amidated joining peptide (JP-NH 2 ), the COOH-terminal region that distinguishes 18 kDa from 16 kDa fragment (Eipper et al, 1986); affinity-purified antibody to prohormone convertase 1 (PC1; Pcsk1) (JH888) (Russo-Savage et al, 2020). Commercial antibodies used included antibodies to: Hif1a (10009269; Cayman Chemical; Simpson et al, 2015); chromogranin A-Gly (Chga-Gly) (ab52983/ EP1031Y; Abcam) and chromogranin A-Tot (ab15160; Abcam; Simpson et al, 2015); pErk1/2 (pMapk3/pMapk1) and total-Erk1/2 (total-Mapk3/total-Mapk1) (9102 and 9102, respectively); Akt (4691); pAkt (4060); p70 S6 kinase (9204), all from Cell Signaling Technologies.…”

“…ProPC1 is rapidly cleaved while in the ER, producing PC1; a cleavage that occurs in secretory granules generates PC1Δ, a more active, COOH-terminally truncated protein (Ramos-Molina et al, 2016). N-glycosylated and nonglycosylated POMC can be seen, along with Nterminal fragments (18/16 kDa) produced by the endoproteolytic cleavages shown in the diagram (Eipper et al, 1986). Equal samples (6% of cell extracts and of medium) were analyzed.…”