D Kloor1, K Yao, U Delabar, H Osswald. 1. Department of Pharmacology, Faculty of Medicine, University of Tübingen, Wilhelmstrasse 56, D-72074 Tübingen, Germany. doris.kloor@uni-tuebingen.de
Abstract
BACKGROUND: Adenosine has been suggested to play an important role in the regulation of renal function. We developed a simple and sensitive binding assay for the detection of adenosine based on the displacement of [(3)H]adenosine from S-adenosylhomocysteine (SAH) hydrolase in its reduced form. METHODS: SAH hydrolase was purified to apparent homogeneity from bovine kidney by standard chromatographic methods. SAH hydrolase was converted in its reduced form, which had the advantage that the SAH hydrolase is enzymatically inactive. This reduced enzyme retains its ability to bind adenosine with high affinity. To determine adenosine in urine or tissues, samples must be deproteinized (e.g., with 10 g/L sulfosalicylic acid or 0.6 mol/L perchloric acid). RESULTS: The reduced SAH hydrolase bound adenosine with a dissociation constant of 33.0 +/- 2 nmol/L. Displacement of adenosine binding by the adenine 5'-nucleotides, adenine and hypoxanthine, required >1000-fold higher concentrations than adenosine itself. The intra- and interassay imprecision (CV) was <3.9% and 7.8%, respectively, and the values obtained showed acceptable correlation with those by HPLC. CONCLUSIONS: The highly sensitive adenosine-binding protein assay is a simple test that allows detection of adenosine in samples with small volumes without purification, and is in this respect superior to HPLC.
BACKGROUND:Adenosine has been suggested to play an important role in the regulation of renal function. We developed a simple and sensitive binding assay for the detection of adenosine based on the displacement of [(3)H]adenosine from S-adenosylhomocysteine (SAH) hydrolase in its reduced form. METHODS:SAH hydrolase was purified to apparent homogeneity from bovine kidney by standard chromatographic methods. SAH hydrolase was converted in its reduced form, which had the advantage that the SAH hydrolase is enzymatically inactive. This reduced enzyme retains its ability to bind adenosine with high affinity. To determine adenosine in urine or tissues, samples must be deproteinized (e.g., with 10 g/L sulfosalicylic acid or 0.6 mol/L perchloric acid). RESULTS: The reduced SAH hydrolase bound adenosine with a dissociation constant of 33.0 +/- 2 nmol/L. Displacement of adenosine binding by the adenine 5'-nucleotides, adenine and hypoxanthine, required >1000-fold higher concentrations than adenosine itself. The intra- and interassay imprecision (CV) was <3.9% and 7.8%, respectively, and the values obtained showed acceptable correlation with those by HPLC. CONCLUSIONS: The highly sensitive adenosine-binding protein assay is a simple test that allows detection of adenosine in samples with small volumes without purification, and is in this respect superior to HPLC.