Identification and characterization of an aromatic amino acid decarboxylase from the filarial nematode, Dirofilaria immitis.
A novel secreted aromatic amino acid decarboxylase-like molecule was identified in the excretory/secretory products of L3/L4 larvae as well as in an extract of adult Dirofilaria immitis. The secretion of the enzyme was developmentally regulated. Peak enzyme activities were detected in the culture medium before and after the molting of L3 larvae in vitro. The enzyme was purified from D. immitis adult extracts and the excretory/secretory products of L3/L4 larvae using different chromatographic methods followed by isoelectric focusing and SDS-PAGE. The enzyme has a molecular mass of 48 kDa and a pI of 5.6, and shows a specific enzymatic activity towards the aromatic amino acid substrates phenylalanine, tyrosine and tryptophan. The enzyme's activity did not show an absolute requirement for exogenous pyridoxal-5-phosphate. However, addition of pyridoxal-5-phosphate at 5 microM in the reaction increased the enzyme activity greatly. The enzyme had the ability to catalyze the formation of dopamine from L-dopa. Studies on the effects of inhibitors on the enzyme activity showed that the enzyme was sensitive to Pefabloc and p-chloromercuribenzoic acid, but not to diisopropyl flurophosphate. The Km values of the enzyme for H-Phe-AMC, H-Tyr-AMC and H-Trp-AMC were calculated to be 32.1 microM, 35.1 microM and 29.1 microM, respectively.
Heska Corporation, Fort Collins, Colorado 80525, USA.
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D000818:Animals; D065105:Aromatic Amino Acid Decarboxylase Inhibitors; D001142:Aromatic-L-Amino-Acid Decarboxylases / Q000737:chemistry* / Q000302:isolation & purification; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004183:Dirofilaria immitis / Q000201:enzymology* / Q000254:growth & development; D004285:Dogs; D004298:Dopamine / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D007814:Larva; D008969:Molecular Sequence Data; D011732:Pyridoxal Phosphate / Q000378:metabolism; D013379:Substrate Specificity
D065105:Aromatic Amino Acid Decarboxylase Inhibitors; D004791:Enzyme Inhibitors; D011732:Pyridoxal Phosphate; D001142:Aromatic-L-Amino-Acid Decarboxylases; D004298:Dopamine