High impact information on PAM

• The subcellular distribution of glutaminyl cyclase corresponded to the one of peptidylglycine alpha-amidating monooxygenase believed to catalyze C-terminal amidations during posttranslational precursor processing [1].
• Carboxyl-terminal amidation, a required post-translational modification for the bioactivation of many neuropeptides, entails sequential enzymatic action by peptidylglycine monooxygenase (PAM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PGL, EC 4.3.2.5) [2].
• The monooxygenase, PAM, first catalyzes conversion of a glycine-extended pro-peptide to the corresponding alpha-hydroxyglycine derivative, and the lyase, PGL, then catalyzes breakdown of this alpha-hydroxyglycine derivative to the amidated peptide plus glyoxylate [2].
• Kinetic experiments with the ascorbate-dependent copper monooxygenases, PAM and dopamine-beta-monooxygenase, established that these compounds also bind competitively with respect to ascorbate; however, pyruvate-extended N-acyl-amino acid derivatives possessing hydrophobic side chains are much more potent inhibitors of PGL than of PAM [2].
• 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 [3].

Biological context of PAM

• The enzyme responsible for this essential posttranslational modification is known as peptidyl-glycine alpha-amidating monooxygenase or PAM [4].
• Finally, PGL has no effect on the substrate or inhibition kinetics of PAM, and purified pituitary PAM has an acidic pH optimum consistent with its known localization in secretory granules [5].
• 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 [3].
• A novel enzyme from bovine neurointermediate pituitary catalyzes dealkylation of alpha-hydroxyglycine derivatives, thereby functioning sequentially with peptidylglycine alpha-amidating monooxygenase in peptide amidation [6].

Anatomical context of PAM

• High levels of PAM mRNA were found in bovine pituitary and cerebral cortex [4].
• In marked contrast, an AtT-20 cell line transfected with a cDNA encoding a truncated, soluble form of bPAM had elevated levels of PAM activity, but levels of SPAM activity were not increased compared to wild-type cells [7].
• A new facile trinitrophenylated substrate for peptide alpha-amidation and its use to characterize PAM activity in chromaffin granules [8].
• We now report that the two enzymes essential for amidation, peptidylglycine alpha-monooxygenase (PAM) and peptidylamidoglycolate lyase (PGL), are present in both the cytosol and membrane fractions of cultured bovine aortic endothelial cells [9].
• Qualitative and quantitative expression of m.RNA coding for Peptidyl-Glycine alpha-Amidating Monooxygenase (PAM) in the developing rat pancreas was investigated by Northern and dot blot hybridization, with a bovine PAM c.DNA probe (0.7 kb fragment) [10].

Associations of PAM with chemical compounds

• An NH2-terminal signal sequence and short propeptide precede the NH2 terminus of purified PAM [4].
• The monooxygenase first catalyzes formation of the alpha-hydroxyglycine derivative of the glycine-extended precursor, and the lyase subsequently catalyzes breakdown of the PAM product to the amidated peptide and glyoxylate [5].
• In addition, we report that the olefinic substrate analogues trans-benzoylacrylic acid and 4-phenyl-3-butenoic acid are potent time-dependent inactivators of PAM, with inactivation exhibiting the characteristics expected for mechanism-based inhibition [11].
• Finally, we introduce several small non-peptide substrates for PAM by demonstrating that PAM catalyzes the transformation of hippuric acid and several ring-substituted derivatives to the corresponding benzamides and glyoxylic acid, with the most facile substrate of this class being 4-nitrohippuric acid [11].
• We report here that in the presence of molecular O2, copper and PAM substrate, NN-dimethyl-1,4-phenylenediamine (DMPD) serves as the requisite electron donor for the mono-oxygenase, being oxidized in the process to a stable and highly chromophoric cation radical [12].

Other interactions of PAM

• One of the questions addressed was the suitability of the AF islet neuropeptide Y-like peptide, aPY-Gly, as a substrate for the islet PAM [13].

Analytical, diagnostic and therapeutic context of PAM

• Following differential centrifugation of rat atrial homogenates, most of the PAM activity was associated with crude granule fractions, with lesser amounts of activity associated with crude microsomal fractions [14].
• In rat anterior pituitary this factor (denoted SPAM for stimulator of PAM activity) was a soluble protein with a mol wt of 44 K by gel filtration; its stimulatory activity could be reduced or eliminated by trypsin digestion or boiling [7].
• Partially purified PAM from AF islet secretory granules was incubated with [125I] aPY-Gly and the resulting products were analyzed by HPLC [13].
• A colorimetric assay for measuring peptidylglycine alpha-amidating monooxygenase using high-performance liquid chromatography [15].

References