d-Amino-acid oxidase is not present in the mouse liver

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Abstract

Since there are conflict reports on the presence of d-amino-acid oxidase in the mouse liver, this problem was examined. d-Amino-acid oxidase activity was not detected in the homogenates of the mouse liver, lung, or heart, whereas it was detected in the homogenates of the mouse kidney and brain. Western blotting showed that a protein which reacted with the antiserum against pig d-amino-acid oxidase was present in the homogenates of the mouse kidney and brain but not in those of the liver or heart. Northern hybridization using a d-amino-acid oxidase cDNA probe detected a hybridizing signal in poly(A)+ RNAs extracted from the mouse kidney and brain but not in those from the liver, heart, or lung. Reverse transcription–polymerase chain reaction using three primer pairs always amplified d-amino-acid oxidase cDNA fragments of expected sizes in the mouse kidney and brain but very rarely did so in the liver, heart, or lung. The results indicate that d-amino-acid oxidase is not present in the mouse liver in a measurable amount.

Introduction

d-Amino-acid oxidase catalyzes oxidative deamination of d-amino acids (stereoisomers of naturally occurring l-amino acids) to the corresponding 2-oxo acids, producing ammonia and hydrogen peroxide in the course of the reaction [1]. This enzyme is present in various organisms: mammals, birds, reptiles, amphibians, fish, insects, molluscs, fungi, and yeasts [2]. In higher animals, it is present mainly in their kidneys, livers, and brains. Although the physico-chemical properties and the kinetic mechanism of d-amino-acid oxidase has been determined in detail 3, 4, the physiological role of this enzyme has been unclear because its substrate d-amino acids have been considered to be rare in eukaryotic organisms for a long time 2, 5. However, recent improvements in analytical methods to separate and quantitate stereoisomers of amino acids have revealed the presence of a considerable amount of d-amino acids in a variety of organisms including humans 6, 7, 8, 9, 10, 11, 12. Investigations using the mutant mice lacking d-amino-acid oxidase activity have shown that this enzyme is involved in the metabolism of d-amino acids of a microbial origin [13]. However, this enzyme should have some other functions because it is present in the kidneys and livers more than enough to metabolize these d-amino acids. It is unlikely that d-amino-acid oxidase present in the brain works for the metabolism of microbial origin.

d-Amino-acid oxidase is a single polypeptide which uses flavin adenine dinucleotide as a prosthetic group. A mouse enzyme consists of 345 amino acid residues, having a molecular mass of 38 699 Da [14]. mRNA for this enzyme consists of a coding region of 1035 nucleotides, 5′- and 3′-untranslated region and poly(A) tail, comprising about 2 kilobase pair (kb). d-Amino-acid oxidases of the pig, rabbit, and human have two more amino acid residues and are slightly larger than the mouse enzyme 15, 16, 17, 18. The homology of these mammalian enzymes is high and about 80% of the amino acid residues in the sequence are the same among these enzymes [18].

Clark et al. [19]and Shack [20]reported that d-amino-acid oxidase activity was not detected in the mouse liver. Many investigators have confirmed their results 21, 22, 23, and it has long been accepted that the mouse is an exceptional animal in that it does not have d-amino-acid oxidase in its liver [2]. However, Dabholkar [24]showed the presence of this enzyme in the liver of the fetal mouse using a histochemical method and an electron microscope. Furthermore, Nagata and Akino [25]and Nagata et al. [26]indicated that this enzyme is present in the liver of adult mice as well. They used an enzyme-linked immunosorbent assay and two spectrophotometric methods to demonstrate the presence of the enzyme protein and its activity, respectively. However, using Northern hybridization, Tada et al. [14]showed that mRNA for d-amino-acid oxidase is not present in the mouse liver though it is present in the mouse kidney and brain. Due to these conflicting evidence, it should be important to determine whether or not this enzyme is present in the mouse liver. In this study, we examined d-amino-acid oxidase activity, the presence of the d-amino-acid oxidase molecule by Western blotting, and the presence of the message for this enzyme using Northern hybridization and reverse transcription–polymerase chain reaction (RT-PCR). None of these methods showed the presence of detectable level of d-amino-acid oxidase in the mouse liver.

Section snippets

Mice

The ddY/DAO+ mice were maintained in the animal facility of Dokkyo University School of Medicine. BALB/cAnN, C3H/HeN Jcl and C57BL/6N Jcl mice were purchased from Doken (Shimodate, Ibaraki).

Assay of d-amino-acid oxidase activity

The kidneys, brains, livers, hearts, and lungs were removed from the ddY/DAO+ and BALB/c mice under the chloroform anesthesia. They were kept frozen at −40°C until use. These organs were thawed by the addition of 7 mM pyrophosphate buffer (pH 8.3) and homogenized in a Potter-Elvehjem-type glass-Teflon

d-Amino-acid oxidase activity in mouse organs

d-Amino-acid oxidase activity was measured in homogenates of the kidney, brain, liver, heart, and lung of the adult ddY/DAO+ mice (Table 1). The kidney and brain had enzyme activity. The activity in the brain was about 1/27 of that in the kidney. These results are consistent with the previous report [38]. In contrast to these organs, the liver, heart, and lung did not have any detectable d-amino-acid oxidase activity. This was not just for the ddY/DAO+ mice. The livers of BALB/c mice did not

Discussion

The presence of d-amino-acid oxidase in the mouse liver was examined using four different methods: enzyme activity assay, Western blotting, Northern hybridization, and RT-PCR. Every test resulted positive in the mouse kidney and brain. However, none of these tests produced any clear evidence of the presence of this enzyme in the liver.

In d-amino-acid oxidase assay using two spectrophotometric methods, Nagata et al. [26]showed that the liver and heart of adult mice had higher specific activity

Acknowledgements

We wish to thank Ms. Y. Shinozaki for taking care of the animals and Ms. Rosanne Villeneuve for her assistance in preparing the manuscript. This work was supported in part by Grant-in-Aid for Co-operative Research (A) (No. 07308048) and Grant-in-Aid for General Scientific Research (C) (No. 05670959) from the Ministry of Education, Science and Culture of Japan.

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