Secretory vesicle aminopeptidase B related to neuropeptide processing: molecular identification and subcellular localization to enkephalin- and NPY-containing chromaffin granules.
Biosynthesis of peptide hormones and neurotransmittters involves proteolysis of proprotein precursors by secretory vesicle cathepsin L. Cathepsin L generates peptide intermediates with basic residues at their NH(2)-termini, indicating that Arg/Lys aminopeptidase is needed to generate the smaller biologically active peptide. Therefore, this study identified the Arg/Lys aminopeptidase that is present in secretory vesicles of adrenal medulla and neuroendocrine tissues, achieved by molecular cloning and localization in 'model' neuropeptide-containing secretory vesicles (bovine). Molecular cloning of the bovine aminopeptidase B (AP-B) cDNA defined its primary sequence that allowed selection of antisera for immunolocalization studies. AP-B was present in secretory vesicles that contain cathepsin L with the neuropeptides enkephalin and neuropeptide Y. The AP-B in several neuroendocrine tissues was detected by western blots. Recombinant bovine AP-B showed preference for Arg-methylcoumarinamide substrate. AP-B was inhibited by arphamenine, an inhibitor of aminopeptidases. Bovine AP-B showed similar activities for Arg-(Met)enkephalin (ME) and Lys-ME neuropeptide substrates to generate ME, while rat AP-B preferred Arg-ME. Furthermore, AP-B possesses an acidic pH optimum of 5.5-6.5 that is similar to the internal pH of secretory vesicles. The significant finding of the secretory vesicle localization of AP-B with neuropeptides and cathepsin L suggests a role for this exopeptidase in the biosynthesis of neuropeptides.
Hwang
Shin-Rong
SR
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California 92093-0744, USA.
O'Neill
Audrey
A
Bark
Steven
S
Foulon
Thierry
T
Hook
Vivian
V
D000313:Adrenal Medulla / Q000648:ultrastructure; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000626:Aminopeptidases / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D000818:Animals; D001483:Base Sequence; D056668:Cathepsin L; D002403:Cathepsins / Q000378:metabolism; D002417:Cattle; D002837:Chromaffin Granules / Q000201:enzymology / Q000378:metabolism*; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004745:Enkephalins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D009478:Neuropeptide Y / Q000378:metabolism*; D010902:Pituitary Gland / Q000648:ultrastructure; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D022142:Secretory Vesicles / Q000201:enzymology / Q000378:metabolism*; D013379:Substrate Specificity
D000596:Amino Acids; D004745:Enkephalins; D009478:Neuropeptide Y; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D002403:Cathepsins; D000626:Aminopeptidases; C019721:aminopeptidase B; D003546:Cysteine Endopeptidases; D056668:Cathepsin L; C534280:Ctsl protein, rat