Fluorimetric assay for D-amino acid oxidase activity in rat brain homogenate by using D-kynurenine as a substrate

Kozaki, Anna

doi:10.5582/bst.2012.v6.5.241

Cited by 10 publications

(6 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Determination of ᴅ‐Kyn in tissues can be also performed with HPLC–FD. This method employs enzymatic oxidation of ᴅ‐Kyn by ᴅ‐amino acid oxidase and subsequent fluorescent detection of the generated Kyna .…”

Section: Chromatographic Analysis Of Tryptophan Metabolitesmentioning

confidence: 99%

Chromatographic analysis of tryptophan metabolites

Sadok

Gamian

Staniszewska

2017

J of Separation Science

View full text Add to dashboard Cite

The kynurenine pathway generates multiple tryptophan metabolites called collectively kynurenines and leads to formation of the enzyme cofactor nicotinamide adenine dinucleotide. The first step in this pathway is tryptophan degradation, initiated by the rate‐limiting enzymes indoleamine 2,3‐dioxygenase, or tryptophan 2,3‐dioxygenase, depending on the tissue. The balanced kynurenine metabolism, which has been a subject of multiple studies in last decades, plays an important role in several physiological and pathological conditions such as infections, autoimmunity, neurological disorders, cancer, cataracts, as well as pregnancy. Understanding the regulation of tryptophan depletion provide novel diagnostic and treatment opportunities, however it requires reliable methods for quantification of kynurenines in biological samples with complex composition (body fluids, tissues, or cells). Trace concentrations, interference of sample components, and instability of some tryptophan metabolites need to be addressed using analytical methods. The novel separation approaches and optimized extraction protocols help to overcome difficulties in analyzing kynurenines within the complex tissue material. Recent developments in chromatography coupled with mass spectrometry provide new opportunity for quantification of tryptophan and its degradation products in various biological samples. In this review, we present current accomplishments in the chromatographic methodologies proposed for detection of tryptophan metabolites and provide a guide for choosing the optimal approach.

show abstract

Section: Chromatographic Analysis Of Tryptophan Metabolitesmentioning

confidence: 99%

Chromatographic analysis of tryptophan metabolites

Sadok

Gamian

Staniszewska

2017

J of Separation Science

View full text Add to dashboard Cite

show abstract

“…The prepared compounds were tested as DAAO inhibitors with the KYNA enzyme inhibitory assay [18]. Although d -serine is a natural substrate of DAAO, its metabolite is not suitable for fluorometric evaluation.…”

Section: Resultsmentioning

confidence: 99%

“…d -2-Amino-4-(2-aminophenyl)-4-oxobutanoic acid (D-KYN) was used to measure d -amino acid oxidase activity based on the protocol described in ref. [18,20]. Human DAAO (purchased from TargetEx Ltd. (Dunakeszi, Hungary)) was used for the measurements.…”

Section: Methodsmentioning

confidence: 99%

Synthesis and Biochemical Evaluation of Lid-Open d-Amino Acid Oxidase Inhibitors

et al. 2019

View full text Add to dashboard Cite

Most of the known inhibitors of d-amino acid oxidase (DAAO) are small polar molecules recognized by the active site of the enzyme. More recently a new class of DAAO inhibitors has been disclosed that interacts with loop 218−224 at the top of the binding pocket. These compounds have a significantly larger size and more beneficial physicochemical properties than most reported DAAO inhibitors, however, their structure-activity relationship is poorly explored. Here we report the synthesis and evaluation of this type of DAAO inhibitors that open the lid over the active site of DAAO. In order to collect relevant SAR data we varied two distinct parts of the inhibitors. A systematic variation of the pendant aromatic substituents according to the Topliss scheme resulted in DAAO inhibitors with low nanomolar activity. The activity showed low sensitivity to the substituents investigated. The variation of the linker connecting the pendant aromatic moiety and the acidic headgroup revealed that the interactions of the linker with the enzyme were crucial for achieving significant inhibitory activity. Structures and activities were analyzed based on available X-ray structures of the complexes. Our findings might support the design of drug-like DAAO inhibitors with advantageous physicochemical properties and ADME profile.

show abstract

“…Previously, we have reported that DAO produced a fluorescent substance, kynurenic acid (KYNA), from d -kynurenine ( d -KYN) as a substrate. − According to the enzymatic reaction of DAO using d -KYN, DAO activity can be measured directly without using additives such as POD and some colorants. However, since KYNA emits weak fluorescence with a wavelength below 400 nm, as seen in previous research, it is necessary to add excess zinc ions (>100 mM) to form a more intensely fluorescent Zn 2+ –KYNA complex − (Scheme a). After all, even if such an assay using d -KYN is used, it seems impossible to assess intracellular DAO activity because the fluorescent complex, Zn 2+ -KYNA, cannot be formed under intracellular zinc ion levels.…”

mentioning

confidence: 85%

Direct Fluorescence Evaluation of d-Amino Acid Oxidase Activity Using a Synthetic d-Kynurenine Derivative

et al. 2022

View full text Add to dashboard Cite

D-Amino acid oxidase (DAO) has been suggested to be associated with the central nervous system diseases, such as schizophrenia. We newly synthesized a nonfluorescent 5-methylthio-D-kynurenine (MeS-D-KYN), which was converted to blue-fluorescent 6-MeS-kynurenic acid (MeS-KYNA, λ ex = 364 nm, λ em = 450 nm) through a one-step reaction by incubation with DAO. It was revealed that fluorescence intensity increased accompanied by commercial porcine kidney DAO activity (unit) with a good correlation (R 2 = 0.9972), suggesting that the fluorometric evaluation of DAO activity using MeS-D-KYN is feasible. MeS-D-KYN was applied to fluorescent DAO imaging in cultured LLC-PK1 cells, and the blue fluorescence of MeS-KYNA overlapped considerably with the location of peroxisomes, which was suggested to be the location of DAO in the cells. Because fluorescence was diminished in the presence of 6-chloro-1,2benzisoxazol-3(2H)-one (CBIO), a DAO inhibitor, it was considered that DAO activity in cells could be directly evaluated using MeS-D-KYN as the substrate.

show abstract

Fluorimetric assay for D-amino acid oxidase activity in rat brain homogenate by using D-kynurenine as a substrate

Cited by 10 publications

References 29 publications

Chromatographic analysis of tryptophan metabolites

Chromatographic analysis of tryptophan metabolites

Synthesis and Biochemical Evaluation of Lid-Open d-Amino Acid Oxidase Inhibitors

Direct Fluorescence Evaluation of d-Amino Acid Oxidase Activity Using a Synthetic d-Kynurenine Derivative

Contact Info

Product

Resources

About