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A pH SENSITIVE LINKER REGION INFLUENCES FUNCTION AND TRAFFICKING OF PAM
Author(s) -
Vishwanatha Kurutihalli S,
Maciejewski Mark,
Mains Richard E,
Betty Eipper A
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.690.4
PAM (Peptidylglycine α‐Amidating Monoxygenase, EC 1.14.17.3) has 2 catalytic domains that function sequentially to produce amidated peptides from Gly‐extended precursors. In vertebrates, a single gene encodes a type I integral membrane protein that includes both enzymes. The linker region separating the enzymes includes a conserved His‐rich region; in PAM isoforms with exon16, a cleavage site separates the enzymes. PAM is one of the few membrane‐spanning proteins concentrated in secretory granules and affects regulated secretion. Antibody binding studies revealed pH dependent epitope masking of the linker region. Since PAM traverses sub‐ cellular compartments ranging from pH7.4–4.5, the effects of pH on the conformation of this linker region were explored. Recombinant exon14/15/16, exon15/16 and exon16 were prepared by cleavage of purified GST‐fusion proteins. MALLS analysis of exon16 subjected to gel filtration at pH 7.0 revealed a monomer. Circular dichroism revealed that exon16 is unstructured at pH 7.4 and 4.5. Fluorescence spectroscopy revealed pH‐dependent shifts in λmax and RFI. NMR analysis of 15N‐labeled exon16 indicated that the majority of backbone protons and carbons exhibit chemical shifts characteristic of random‐coil configurations at pH7.0. At the concentration needed for NMR analysis, reversible aggregation occurred between pH7.0 and 5.0. This work is funded by NIH grant DK32949.