Multiple forms of peptidyl alpha-amidating enzyme: purification from rat medullary thyroid carcinoma CA-77 cell-conditioned medium.
Conditioned medium from cultures of rat medullary thyroid carcinoma CA-77 cells was used as a source for purification of the secreted forms of peptidyl alpha-amidating enzyme. The alpha-amidating enzyme activity was partially purified using a combination of weak anion exchange and gel filtration chromatography. Subsequent strong anion exchange chromatography at pH 6.0 resolved this partially pure enzyme into four distinct peaks of activity, termed Ia, Ib, II, and III. Peaks Ia and Ib exhibited broad pH optima between pH 6.0-8.5, whereas peaks II and III both exhibited pH optima at approximately pH 5.0. The peak III activity was further purified to electrophoretic homogeneity using hydrophobic interactive chromatography followed by strong anion exchange chromatography at pH 8.0. The enzyme exhibited an apparent molecular mass of 75K, a pH optimum of approximately pH 5.0, and a maximal turnover number of 580 min-1 in the presence of L-ascorbate. Kinetic studies demonstrated that the enzyme probably functions through a ping-pong mechanism with respect to the binding of the glycine-extended peptide substrate and the L-ascorbate cofactor. The peak III enzyme exhibits several distinctive characteristics compared to amidating enzymes isolated and characterized by other laboratories.
Gilligan
J P
JP
Unigene Laboratories, Inc., Fairfield, New Jersey 07006.
Lovato
S J
SJ
Mehta
N M
NM
Bertelsen
A H
AH
Jeng
A Y
AY
Tamburini
P P
PP
D000818:Animals; D002460:Cell Line; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003470:Culture Media; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D006899:Mixed Function Oxygenases; D009097:Multienzyme Complexes; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D013964:Thyroid Neoplasms / Q000201:enzymology*
D003470:Culture Media; D007527:Isoenzymes; D009097:Multienzyme Complexes; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D000587:Oxidoreductases Acting on CH-NH Group Donors