Expression, Purification, and Characterization of Rat Aromaticl-Amino Acid Decarboxylase inEscherichia coli

Structure modelling and site-directed mutagenesis of the rat aromatic L-amino acid pyridoxal 5´-phosphate-dependent decarboxylase: A functional study

“…The AADC His‐Tag fusion was previously shown to be expressed efficiently in E. coli in an active form 25. This system was used to produce different AADC mutants which were then tested for enzymatic activity.…”

“…The pET‐20b+ expression vector and E. coli strain BL21 (DE3) (F‐ dcm − OmpT − r B − m B − lon − λ(DE3)) (Novagen, Madison, WI) were used for expression of wild type and mutant AADC with a C‐terminal of six histidine residues (AADC‐His 6 ) 25 . E. coli strain JM109 was used in the first step of the mutagenesis procedure for all genetic manipulations.…”

“…Anti‐rabbit IgGs coupled with alkaline phosphatase were supplied by Promega and the Immobilon‐P (PVDF) membrane was from Millipore Corp. (Bedford, MA). AADC was measured by competitive ELISA assay25 using two different anti‐AADC sera, polyclonal anti‐βgal/AADC serum30 diluted 1/4000 or polyclonal anti‐AADC serum prepared against a pig kidney protein diluted 1/2000. AADC concentrations in the pseudo‐linear portion of the curve (1–30 ng for anti‐βgal/AADC, 30–100 ng for anti‐AADC) were taken into account and analyzed by linear regression (r = 0.98, n = 5).…”

Current Advances on Structure-Function Relationships of Pyridoxal 5′-Phosphate-Dependent Enzymes

“…The K m values of the Pseudomonas putida (K m = 0.092 mM) and mammalian aromatic amino acid decarboxylases (K m = 0.19 mM for hog kidney enzyme, K m = 0.086 mM for rat enzyme) are in the similar range, but the insect 3,4-dihydroxyphenylalanine decarboxylase K m value is bigger (Christenson et al, 1970; Hayashi et al, 1993; Han et al, 2010b; Koyanagi et al, 2012) (Table 1). Different from mammalian and insect enzymes, which catalyze decarboxylation of both 5-hydroxytryptophan and 3,4-dihydroxyphenylalanine at similar catalytic efficiency level, this Pseudomonas putida DDC has high catalytic efficiency (k cat /K m = 21 mM −1 s −1 ) on 3,4-dihydroxyphenylalanine and the catalytic efficiency on 5-hydroxytryptophan is quite low (k cat /K m = 0.1 mM −1 s −1 ) (Gene locus tag: PP_2552) (Christenson et al, 1970; Hayashi et al, 1993; Jebai et al, 1997; Han et al, 2010b; Koyanagi et al, 2012). The activity on tyrosine is also negligible (Koyanagi et al, 2012), which is also in contrast to the similar activity level toward both tyrosine and 3,4-dihydroxyphenylalanine in plant 3,4-dihydroxyphenylalanine/tyrosine decarboxylases (Facchini and De Luca, 1995; Facchini et al, 2000).…”

Nonexocytotic serotonin release tonically suppresses serotonergic neuron activity

“…Firing suppression is mediated by 5-HT release from surrounding neurites Finally, because fi ndings of whole-cell experiments indicated that 5-HT o underlying autoinhibition of a given serotonergic neuron originates principally in surrounding of 5-HT normally present in the cytosol is unknown, we used 5-HT at concentrations of 0.3 and 3 mM, assumed to be one to two orders of magnitude higher than normal, based on the rationale explained in Materials and methods. Similarly, 5HTP was applied at 0.1 and 1 mM, concentrations expected to partially and nearly completely saturate AADC in the recorded neuron, given that AADC K m for 5HTP is ‫ف‬ 18-66 μM ( Rahman et al, 1981 ;Jebai et al, 1997 ). In these experiments, after establishment of gigaseal, the cell-attached confi guration was typically maintained for 5-7 min before rupturing the patch to allow for washout of 5-HT or 5HTP, which leaked out from the pipette tip.…”