Abstract
The production of alpha-amidated peptides from their glycine-extended precursors is a two-step process involving the sequential action of two catalytic domains encoded by the bifunctional peptidylglycine alpha-amidating monooxygenase (PAM) precursor. The NH2-terminal third of the PAM precursor contains the first enzyme, peptidylglycine alpha-hydroxylating monooxygenase (PHM), a copper, molecular oxygen, and ascorbate-dependent enzyme. The middle third of the PAM precursor contains the second enzyme, peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL). The COOH-terminal third of the PAM precursor encodes a transmembrane domain and a hydrophilic domain that may form a cytoplasmic tail. Antisera to a peptide within the PAL domain were used to identify a 50-kDa protein as the major form of PAL in bovine neurointermediate pituitary granules. This 50-kDa PAL protein was purified and found to begin at Asp434 of bPAM, indicating that it could arise through endoproteolytic cleavage of the bPAM precursor at Lys432-Lys433. With alpha-N-acetyl-Tyr-Val-alpha-hydroxyglycine as the substrate, PAL exhibits a pH optimum of 5.0; enzymatic activity is inhibited by high concentrations of salt but is relatively resistant to thiol reagents and urea. PAL activity is inhibited by EDTA and restored by a number of divalent metals, including Cd2+, Cu2+, Zn2+, and Ca2+. Kinetic studies using alpha-N-acetyl-Tyr-Val-alpha-hydroxyglycine indicate that PAL has a Km of 38 microM and a turnover number of 220/s. Expression vectors encoding only the soluble PHM domain or the PAM precursor from which the PHM domain had been deleted were constructed. hEK293 cells transfected with the PHM vector exhibited a 10-fold increase in secretion of PHM activity with no PHM activity detectable in control or transfected cells. hEK293 cells transfected with the PAL vector exhibited a 2-fold increase in secretion of PAL activity and a 15-fold increase in cellular PAL activity. Most of the PAL activity produced by the transfected cells remained membrane-associated.
Highlights
Enzymatic a-amidation is a key step in the biosynthesis of glycine-extended precursors is a two-step process in- many neuroendocrine peptides
Volving the sequentialaction of two catalyticdomains kDa peptidylglycine a-amidating enzyme from bovine neuencoded by the bifunctional peptidylglycine a-amidat- rointermediate pituitary led to the identification of a cDNA
Ing monooxygenase (PAM) precursor.TheNH2-ter- encoding a 108-kDa precursor protein (Fig. 1); based on the minal third of the PAM precursor contains the first ability of the purified protein to catalyze the conversion of D
Summary
Were determined using the BCA protein assay reagent (Pierce Chemical Co.), and PHM and PAL assays were carried out as described below. Enzyme Assays-PHM activity was measured using 0.5 p~ a-N-acetyl-Tyr-Val-Gly, mono-'251-a-N-acetyl-Tyr-Val-G0l.y5,p~ cuso4, 0.18 mg/ml catalase, 0.5 mM ascorbate, 120-150 mM sodium. A-N-Acetyl-Tyr-Vala-hydroxyglycine was prepared enzymatically; the reaction mixture (1.8 ml) contained 100 p~ a-N-acetyl-Tyr-Val-Gly, 0.18 mg/ml catalase, 0.5 pM &SO4, 1.0 mM ascorbate, 140 mM sodium MES, pH 4.5, and 25 p1 containinga mixture of purified bPHM-A and B (approximately 12 ng). The forms of PAL found in bovine neurointer-enzymatically from mono-'251-a-N-acetyl-Tyr-Val-Galnyd purified mediate and anterior pituitary granules and the endoproteolyticcleavage site used to generate the NH2 terminus of by adsorption to a C,, Sep-Pak cartridge. Direct iodination of the a hydroxyglycine intermediate with Iodobeads (Pierce Chemical Co.) using the conditions described for labeling D-Tyr-Val-Gly[3] resulted soluble PAL were identified. Analysis by RP-HPLC indicated that the '2sI-labeled-a-N-acetylTyr-Val-a-hydroxyglycinwe as 97% monoiodinated
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