Gastrin-amidating enzyme in the porcine pituitary and antrum. Characterization of molecular forms and substrate specificity.
As is the case with many other peptide hormones of the brain and intestine, the formation of biologically active gastrin from a glycine-extended processing intermediate occurs via the action of a peptidylglycyl alpha-amidating monooxygenase (PAM). The observation that gastrin exists primarily as unamidated precursors in the pituitary but as amidated gastrin in the antrum prompted this study to examine whether the amidating enzymes in the two organs were different in their characteristics. Amidating activity was quantified by measuring the conversion of glycine-extended tridecagastrin (G13-Gly) to amidated tridecagastrin and glycine-extended hexapancreatic polypeptide (PP6-Gly) to amidated hexapancreatic polypeptide by radio-immunoassay. Two molecular forms of amidating activity were identified in both the porcine antrum and pituitary. The first, PAM-A, had an apparent Mr of 51,000 and a net negative charge at pH 7.0, whereas PAM-B was smaller (Mr approximately 30,000) and had a net positive charge at pH 7.0. Both molecular forms were similar in their cofactor requirements (copper, ascorbic acid, and catalase) and pH optima in the antrum and pituitary. The Km was significantly lower and the Vmax higher for PP6-Gly than for G13-Gly in the pituitary and antrum. These data suggest that although there is no difference between antral and pituitary PAM, the selective affinity of PAM for certain substrates may provide a mechanism for the differential amidation of different hormones within a given tissue or cell.
Dickinson
C J
CJ
Department of Pediatrics, University of Michigan Medical Center, Ann Arbor 48109-0800.
Yamada
T
T
D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D005753:Gastric Mucosa / Q000201:enzymology*; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008970:Molecular Weight; D009097:Multienzyme Complexes; D010902:Pituitary Gland / Q000201:enzymology*; D011706:Pyloric Antrum / Q000201:enzymology; D013379:Substrate Specificity; D013552:Swine
D007527:Isoenzymes; D009097:Multienzyme Complexes; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase