Methods for the Detection of D-Amino-Acid Oxidase

Four methods (an enzyme activity assay, western blotting, RT-PCR, and northern hybridization) to detect the enzyme D-amino-acid oxidase are described.

1 ). This enzyme is present in a wide variety of organisms (2 ). However, the physiological role of this enzyme has been unclear because its substrate D-amino acids have been considered to be rare in eukaryotic organisms (2 , 3 ). In previous work, my colleagues and I concluded that D-amino-acid oxidase is not present in the mouse liver (13 ). This paper examines in greater detail the methods used in reaching that conclusion.

4 ). Organs, tissues, or tissue culture cells were homogenized in 7 mM pyrophosphate buffer (pH 8.3). The homogenates were centrifuged at 550 x g for 5 minutes. The supernatant solutions were used for the assay. The reaction mixture consisted of 0.3 ml of 0.133 M pyrophosphate buffer (pH 8.3) containing catalase at 700 IU/ml, 0.3 ml of 0.1 M D-alanine, 0.2 ml of 0.1 mM FAD, and 0.1 ml of 70% (V/V) methanol. The reaction was started by the addition of 0.1 ml of the supernatant solution. The reaction was carried out at 37°C for 15-60 minutes depending on the activity of the samples. It was terminated by the addition of 1 ml of 10% trichloroacetic acid. In a blank, trichloroacetic acid was added to the reaction mixture before the enzyme reaction was started. The precipitate was removed by centrifugation (700 x g, 20 min). To 0.5 ml of the supernatant solution were added 0.5 ml of 5 N KOH and 0.5 ml of 0.5% 4-amino-3-hydrazino-5-mercapto-1, 2, 4-triazole in 0.5 N HCl. The mixture was kept standing at room temperature for 15 min. After 0.5 ml of 0.75% KIO₄ in 0.2 N KOH were added to the mixture with vigorous shaking, absorbance at 550 nm was D-Amino-acid oxidase activity is calculated with this formula: Activity (μmol min^-1 = 2.584A /t , where A is a differential absorbance at 550 nm between the sample and the blank, and t is the reaction time in minutes. This activity value was further divided by the quantity of the protein present in the first reaction. The protein concentration in the supernatant solution was determined according to the method of Lowry et al. (5 ) using bovine serum albumin as a standard. It was also determined using a Protein Assay Kit (Bio-Rad, Hercules, CA). D-Amino-acid oxidase activity is finally expressed as the amount of D-alanine oxidized per min per milligram of protein. Hog kidney D-amino-acid oxidase (Sigma, St. Louis, MO or Boehringer Mannheim, Germany) was used as a control.

Organs, tissues, or tissue culture cells were homogenized in distilled water and centrifuged at 550 x g for 5 min. The supernatant solutions were mixed with an equal volume of the sample buffer (63 mM Tris-HCl, pH 6.8, 2% SDS, 5% 2-mercaptoethanol, 10% glycerol and 0.002% bromophenol blue) and were heated in a boiling water for 3 min. The samples (5~50 μg protein per lane) were electrophoresed according to the method of Laemmli (6 ) on a polyacrylamide-gradient (8-16%) slab gel (Tefco, Tokyo) with the electrophoresis buffer (25 mM Tris, 192 mM glycine, and 0.1% SDS) at 20 mA for about 1.5 hr. Hog kidney D-amino-acid oxidase (10 ng, Sigma) and prestained protein standards (SeeBlue, Novex, San Diego, CA) were run together. The proteins were electrophoretically transferred from the gel to a nitrocellulose membrane (BA85, Schleicher and Schuell, Dasel) with the blotting buffer (25 mM Tris-HCl, 192 mM glycine, and 20% methanol) at 180 mA for 1 hr.

The membrane was incubated in 5% non-fat dried milk (Bio-Rad) in TBS-T (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, and 0.1% Tween-20) for 1 hr and then quickly rinsed twice with TBS-T and washed in TBS-T once for 15 min then twice for 5 min. It was incubated for 1 hr in TBS-T containing rabbit anti-hog D-amino-acid oxidase IgG (1/3,000 dilution) and quickly rinsed twice with TBS-T and further washed in TBS-T once for 15 min then twice for 5 min. The membrane was incubated for 1 hr in TBS-T containing horseradish peroxidase-labeled antibody against rabbit IgG raised in a donkey (ECL western blotting detection set, Amersham