Calf intestine alkaline phosphatase

The assay of CYP735A activity was performed according to the method of Sasaki et al. (2013) (link) with minor changes. The enzyme extract (0.2 mL) was incubated with 0.2 mL of the reaction mixture (100 mM sodium phosphate, 10% sucrose, 3 mM triphosphopyridine nucleotide, 1 mg/mL bovine serum albumin, pH 7.5) and 0.08 mL iPMP (10 mM) at 20°C for 2 h. The reaction was terminated by the addition of 0.2 mL of termination buffer (50 mM CHES-NaOH, 0.5 mM MgCl₂, pH 10.0). The mixture was incubated with 0.01 mL of calf-intestine alkaline phosphatase (1 u/μL, Sigma) at 37°C for 40 min and centrifuged at 18,000 × g for 20 min. The supernatant was subjected to HPLC. The content of tZR was quantified by measuring absorbance at 269 nm and comparison with the standard curve for tZR. CYP735A activity (nmol mg^-1 protein h^-1) was defined as the amount of tZR produced per hour per mg protein under the selected reaction conditions.