PEPTIDE AMIDATION IN AN INVERTEBRATE - PURIFICATION, CHARACTERIZATIONAND INHIBITION OF PEPTIDYLGLYCINE ALPHA-HYDROXYLATING MONOOXYGENASE FROM THE HEADS OF HONEYBEES (APIS-MELLIFERA)
Tm. Zabriskie et al., PEPTIDE AMIDATION IN AN INVERTEBRATE - PURIFICATION, CHARACTERIZATIONAND INHIBITION OF PEPTIDYLGLYCINE ALPHA-HYDROXYLATING MONOOXYGENASE FROM THE HEADS OF HONEYBEES (APIS-MELLIFERA), Archives of insect biochemistry and physiology, 26(1), 1994, pp. 27-48
Peptidylglycine alpha-hydroxylating monooxygenase (PHM), an enzyme inv
olved in formation of neuropeptides with a C-terminal amide functional
ity in mammals and amphibians, was isolated from the head of an invert
ebrate, the honeybee, Apis mellifera, and purified 220-fold in 1% over
all yield. The bee PHM has a molecular weight of 71,000, is membrane a
ssociated but can be solubilized with a detergent (n-octyl-beta-D-gluc
opyranoside), and cross-reacts with rabbit antibodies generated toward
bacterially expressed rat PHM. In the presence of copper, oxygen, and
ascorbic acid, the enzyme hydroxylates model tripeptides such as dans
yl-L-Phe-L-Phe-Gly on the methylene carbon of the glycine residue with
retention of configuration. Using this tripeptide as substrate, the K
-m is 1.7 mu M and the V-max is 2.3 nmol.mu g(-1) h(-1). Treatment of
the insect PHM with D-Phe-L-Phe-D-vinylglycine, a substrate analogue a
nd mechanism-based inactivator of PHM from pig pituitary, results in i
rreversible loss of activity. The diastereomeric analogue, D-Phe-L-Phe
-L-vinylglycine, is only a competitive inhibitor (IC50 = 320 mu M). (C
) 1994 Wiley-Liss, Inc.