Fluorimetric determination of D-amino acid oxidase.
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ABSTRACT:
SUBMITTER: Verity MA
PROVIDER: S-EPMC1270418 | biostudies-other | 1967 May
REPOSITORIES: biostudies-other
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Project description:L-amino acid oxidase (LAAO) is attracting increasing attention due to its important functions. Diverse detection methods with their own properties have been developed for characterization of LAAO. In the present study, a simple, rapid, sensitive, cost-effective and reproducible method for quantitative in-gel determination of LAAO activity based on the visualization of Prussian blue-forming reaction is described. Coupled with SDS-PAGE, this Prussian blue agar assay can be directly used to determine the numbers and approximate molecular weights of LAAO in one step, allowing straightforward application for purification and sequence identification of LAAO from diverse samples.
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Project description:D-Amino acid oxidase (DAAO) is an FAD-containing flavoenzyme that catalyzes with absolute stereoselectivity the oxidative deamination of all natural D-amino acids, the only exception being the acidic ones. This flavoenzyme plays different roles during evolution and in different tissues in humans. Its three-dimensional structure is well conserved during evolution: minute changes are responsible for the functional differences between enzymes from microorganism sources and those from humans. In recent years several investigations focused on human DAAO, mainly because of its role in degrading the neuromodulator D-serine in the central nervous system. D-Serine is the main coagonist of N-methyl D-aspartate receptors, i.e., excitatory amino acid receptors critically involved in main brain functions and pathologic conditions. Human DAAO possesses a weak interaction with the FAD cofactor; thus, in vivo it should be largely present in the inactive, apoprotein form. Binding of active-site ligands and the substrate stabilizes flavin binding, thus pushing the acquisition of catalytic competence. Interestingly, the kinetic efficiency of the enzyme on D-serine is very low. Human DAAO interacts with various proteins, in this way modulating its activity, targeting, and cell stability. The known properties of human DAAO suggest that its activity must be finely tuned to fulfill a main physiological function such as the control of D-serine levels in the brain. At present, studies are focusing on the epigenetic modulation of human DAAO expression and the role of post-translational modifications on its main biochemical properties at the cellular level.
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Project description:L-amino acid oxidase (LAAO) has important biological roles in many organisms, thus attracting great attention from researchers to establish its detection methods. In this study, a new quantitative in-gel determination of LAAO activity based on ferric-xylenol orange (Fe(III)XO) formation was established. This method showed that due to the conversion of Fe(II) to Fe(III) by H2O2 and subsequent formation of Fe(III)XO complex halo in agar medium, the logarithm of H2O2 concentration from 5 to 160 µM was linearly correlated to the diameter of purplish red Fe(III)XO halo. By extracting the LAAO-generated H2O2 concentration, the LAAO activity can be quantitatively determined. This Fe(III)XO agar assay is highly sensitive to detect H2O2 down to micromolar range. More importantly, it is easy to handle, cheap, reproducible, convenient and accurate. Coupled with SDS-PAGE, it can directly be used to determine the number and approximate molecular weight of LAAO in one assay. All these features make this in-gel Fe(III)XO assay useful and convenient as a general procedure for following enzyme purification, assaying fractions from a column, or observing changes in activity resulting from enzyme modifications, hence endowing this method with broad applications.
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Project description:pLG72 is a small, primate-specific protein of 153 amino acids. It is the product of the G72 gene, expressed in testis, spinal cord, and brain. The presence of G72 transcript and pLG72 has recurrently been called into question, however G72 mRNA and pLG72 protein levels were higher in blood and brain of patients with schizophrenia than in healthy controls. On the one hand, the SNP rs2391191 corresponding to the R30K substitution in pLG72 was genetically linked to schizophrenia, reduced thickness of the brain cortex in schizophrenia-affected individuals, and altered memory function. Various lines of evidence indicated that pLG72 is a mitochondrial protein, specifically an extrinsic protein bound on the outer membrane. Over the years, pLG72 was proposed to be involved in different functions: (a) overexpression induces mitochondria fragmentation, increasing the numbers of shorter and more mobile ones which could be delivered faster to regions of intense growth and facilitating the dendritic complexity; (b) it might induce oxidative stress by interacting with methionine-R-sulfoxide reductase B2; and (c) it binds and modulates the activity of FMN-containing oxidoreductase of the respiratory complex I. The main role of this protein, however, is related to its binding to the human flavoenzyme D-amino acid oxidase (hDAAO), i.e., the main catabolic enzyme for D-enantiomer of serine. This D-amino acid is a main endogenous coagonist of the N-methyl-D-aspartate type glutamate receptor (NMDAR) involved in main functions such as synaptic plasticity, learning, memory, and excitotoxicity. For this work, we reviewed the recent literature concerning the hDAAO-pLG72 interaction, focusing on the molecular details of the interaction, the effect of hDAAO function and stability, and the cellular effects, especially on D-serine concentration. The main effects related to the pathological R30K substitution are also reported. We have highlighted the gaps in our knowledge of this human protein as well as the relevance of clarifying the molecular details of hDAAO-pLG72 interaction in order to design molecules to modulate hDAAO activity/stability and thus NMDAR function acting at the D-serine cellular level.
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Project description:L-amino acid oxidases (LAAO) are flavoproteins that catalyze the oxidative deamination of L-amino acids to a keto-acid along with the production of H₂O₂ and ammonia. Interleukin 4 induced gene 1 (IL4I1) is a secreted LAAO expressed by macrophages and dendritic cells stimulated by microbial derived products or interferons, which is endowed with immunoregulatory properties. It is the first LAAO described in mammalian innate immune cells. In this work, we show that this enzyme blocks the in vitro and in vivo growth of Gram negative and Gram positive bacteria. This antibiotic effect is primarily mediated by H₂O₂ production but is amplified by basification of the medium due to the accumulation of ammonia. The depletion of phenylalanine (the primary amino acid catabolized by IL4I1) may also participate in the in vivo inhibition of staphylococci growth. Thus, IL4I1 plays a distinct role compared to other antibacterial enzymes produced by mononuclear phagocytes.
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