3 -Phosphoadenosine 5 -phosphate assay

The assay for monoamine oxidase contained in a final volume of 100 pL to 30 pL of homogenate and 50 pAf [7-14C] dopamine (0.93 mCi/mmol) or [7-,4C] tyramine (3.11 mCi/mmol) in 0.5 M phosphate buffer (pH 7.4). The assay for phenol sulfotransferase was also initiated by adding 30 pL of homogenate. The mixture contained 1.7 pAf [35S] 3 -phosphoadenosine-5 -phosphosulfate (1.51 Ci/mmol) and 50 pAf dopamine, 3,4-dihydroxyphenylacetic acid, or phenol in 10 mAf phosphate buffer (pH 6.4). After various incubation periods, the activity of either enzyme was stopped by addition of 30 pL of 2 N HC1. The resulting mixtures were centrifuged or filtered before analysis by HPLC. 

The reaction mixture in a total volume of 250 yL contained 50 /xL of 10 mM sodium phosphate buffer (pH 7.2), 50 /xL of enzyme preparation, and 100 /xL of 50 fiM 3 -phosphoadenosine 5 -phosphosulfate. The reaction was started by adding 50 /xL of 12.5 mM p-nitrophenol (when assaying the thermo-labile form) or 50 /xL of 50 yM p-NP (when determining the thermostable form). After incubation for 30 minutes at 37°C, the reaction was stopped by the addition of 25 /xL of 4 M perchloric acid. After centrifugation, a 20 /xL aliquot was injected into the HPLC system. Formation of product was linear with time for both forms of enzyme for up to 45 minutes, and with protein amount up to 0.5 mg. 

The enzyme was assayed in a final volume of 200 /xL containing 73 fiM 3 -phosphoadenosine 5 -phosphosulfate, 50 fiM A-acetyldopamine, and 50 mM phosphate buffer (pH 6.0). The reaction was started by addition of 10 to 60 /xL of the enzyme preparation. The reaction was stopped after 15 minutes of incubation at 37°C by boiling for 1 minute, followed by the addition of 50 fiL of 2 M Tris-HCl buffer (pH 8.6). Unreacted A-acetyldopamine was extracted twice by adsorption onto 10 mg of activated alumina. The supemate obtained after centrifugation was filtered, and 5 to 100 /xL aliquots were analyzed by HPLC. 

Assay mixtures contained 200 fiM 3 -phosphoadenosine 5 -phosphosulfate, 0.25 M phosphate buffer (pH 7.0), 8.3 mM 2-mercaptoethanol, acceptor substrate (e.g., 1-naphthalenemethanol) in acetone (< 5% (v/v) final concentration in assay), and 0.5 to 3.0 fig of aryl sulfotransferase IV in a final volume of 30 fiL. The reaction was initiated by addition of enzyme. After 30 minutes of incubation, the reaction was terminated by adding 30 fiL of methanol and... 

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