An investigation of the assay of dopamine using trinitrobenzensulphonic acid

Charteris, Alan; John, Robert A.

doi:10.1016/0003-2697(75)90604-1

Cited by 66 publications

(35 citation statements)

References 6 publications

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“…The enzyme concentration was determined by using a molar absorption coefficient of 1.3i10& M V ":cm V " [7]. DDC activity was determined by measuring amine production with the spectrophotometric assay outlined by Sherald et al [8] and modified by Charteris and John [9]. The concentrations of -dopa and -5-HTP was determined at 280 nm by using molar absorption coefficients of 2630 and 5500 M V ":cm V " respectively.…”

Section: Enzyme Purification and Assaysmentioning

confidence: 99%

Reaction of dopa decarboxylase with L-aromatic amino acids under aerobic and anaerobic conditions

Bertoldi

Voltattorni

2000

Biochemical Journal

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Analysis of the reaction of dopa decarboxylase (DDC) with L-dopa reveals that loss of decarboxylase activity with time is observed at enzyme concentrations approximately equal to the binding constant, Kd, of the enzyme for pyridoxal 5ƀ-phosphate (PLP). Instead, at enzyme concentrations higher than Kd the course of product formation proceeds linearly until complete consumption of the substrate. Evidence is provided that under both experimental conditions no pyridoxamine 5ƀ-phosphate (PMP) is formed during the reaction and that dissociation of coenzyme occurs at low enzyme concentration, leading to the formation of a PLP-L-dopa Pictet–Spengler cyclic adduct. Taken together, these results indicate that decarboxylation-dependent transamination does not accompany the decarboxylation of L-dopa proposed previously [O'Leary and Baughn (1977) J. Biol. Chem. 252, 7168–7173]. Nevertheless, when the reaction of DDC with L-dopa is studied under anaerobic conditions at an enzyme concentration higher than Kd, we observe that (1) the enzyme is gradually inactivated and inactivation is associated with PMP formation and (2) the initial velocity of decarboxylation is approximately half of that in the presence of O2. Similar behaviour is observed by comparing the reaction with L-5-hydroxytryptophan occurring in aerobiosis or in anaerobiosis. Therefore the reaction of DDC with L-aromatic amino acids seems to be under O2 control. In contrast, the reactivity of the enzyme with D-aromatic amino acids does not change in the presence or absence of O2. These and other results, together with previous results on the effect exerted by O2 on reaction specificity of DDC towards aromatic amines [Bertoldi, Frigeri, Paci and Borri Voltattorni (1999) J. Biol. Chem. 274, 5514–5521], suggest a productive effect of O2 on an intermediate complex of the reaction of the enzyme with L-aromatic amino acids or aromatic amines.

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Section: Enzyme Purification and Assaysmentioning

confidence: 99%

Reaction of dopa decarboxylase with L-aromatic amino acids under aerobic and anaerobic conditions

Bertoldi

Voltattorni

2000

Biochemical Journal

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“…Enzyme U S S~J -Dopa decarboxylase activity was measured by the assay method of Sherald et al [ll], according to the modification introduced by Charteris and John [12].…”

Section: N Terialsmentioning

confidence: 99%

Affinity labeling of pig kidney 3,4‐dihydroxyphenylalanine (Dopa) decarboxylase withN‐(bromoacetyl)pyridoxamine 5′‐phosphate

Dominici¹,

Maras²,

Mei³

et al. 1991

European Journal of Biochemistry

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Pig kidney 3,4-dihydxroxyphenylalanine (Dopa) decarboxylase is inactivated by N-( bromoacety1)pyridoxamine 5'-phosphate (HAPMP) in a reaction which follows first-order kinetics at pH 7.5 and 25 'C. The concentration dependence of inactivation reveals saturation kinetics with an apparent Ki of 0.16 mM and kinact of 0.086 min-' at saturating inhibitor concentration. Enzyme can be protected from inactivation by pyridoxal 5'-phosphate. Inactivation of enzyme by [ 14C]BAPMP proceeds with the incorporation of a stoichiometric amount of labeled inhibitor. Proteolytic digestions of the radioactively labeled enzyme followed by high-performance liquid chromatography allow the isolation of the modified peptide corresponding to the sequence Ala-Ala-Ser-Pro-AlaCys-Thr-Glu-Leu in which cysteine (Cyslll) is the modified residue. The conservation of this residue and also of an extended region around it in all Dopa decarboxylases so far sequenced is underlined. The overall conclusion of these findings is that Cysl11 may be at, or near, the pyridoxal-5'-phosphate binding site of pig kidney Dopa decarboxylase and plays a critical role in the catalytic function of the enzyme. Furthermore, fluorescence studies of BAPMP-modified apoenzyme provide useful information on the microenvironment of the affinity label at its binding site.

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“…Enzymatic activity was measured by the assay method of Sherald et al [S], according to the modification introduced by Charteris and John [6]. The standard reaction mixture contained, in a final volume of 250 pl, 10 pmol of potassium phosphate buffer (pH 6 .…”

Section: Assay Of Enzymatic Activitymentioning

confidence: 99%

“…2), which shows a single symmetrical peak, with a sedimentation constant S~O , , , , equal to 6.1. From the same ultracentri- Percentage of decarboxylation of 0.5 mM ~-3,4-dihydroxyphenyIalanine by different enzyme concentrations measured in the absence O'Leary and Baughn [4] of added pyridoxal-P (A) as Charteris and John [6] [2]. The coenzyme content of our preparation, determined as described elsewhere [9], ranges from 0.86 to 1.02 rnol of pyridoxal-P per 103 000 g of protein.…”

Section: Purity Of the Enzymementioning

confidence: 99%

Purification and Characterization of 3,4‐Dihydroxyphenylalanine Decarboxylase from Pig Kidney

Voltattorni

Minelli

Vecchini

et al. 1979

European Journal of Biochemistry

107

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A procedure for 3,4-dihydroxyphenylalanine decarboxylase from pig kidney purification is described in detail.The preparation has no detectable impurity on electrophoresis and on ultracentrifugation and has a coenzyme content and a specific activity comparable with the same enzyme purified by other authors.However two significant differences are observed: a different stimulation of activity by added pyridoxal 5'-phosphate and a nearly complete decarboxylation of ~-3,4-dihydroxyphenylalanine in absence of added coenzyme.Absorption, fluorescence and circular dichroism properties of the coenzyme-apoenzyme interaction are also described.The results are consistent with the existence of at least four coenzyme-apoenzyme complexes, three of them active.

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An investigation of the assay of dopamine using trinitrobenzensulphonic acid

Cited by 66 publications

References 6 publications

Reaction of dopa decarboxylase with L-aromatic amino acids under aerobic and anaerobic conditions

Reaction of dopa decarboxylase with L-aromatic amino acids under aerobic and anaerobic conditions

Affinity labeling of pig kidney 3,4‐dihydroxyphenylalanine (Dopa) decarboxylase withN‐(bromoacetyl)pyridoxamine 5′‐phosphate

Purification and Characterization of 3,4‐Dihydroxyphenylalanine Decarboxylase from Pig Kidney

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