Pyridoxal phosphate

SPL expression was determined in lung tissue lysates by western blotting: anti-S1PL (H-300); rabbit; 1:1,000; sc-67368 (Santa Cruz Biotechnology). SPL activity was determined as previously described (REF 61) with some modifications in sample preparation using a fluorogenic SPL substrate. Briefly, lung tissue samples were homogenized in 0.25% solution of Triton-X100 in potassium phosphate buffer 0.5 M (pH 7.4) and then adjusted to 10 mg protein/ml in 0.5 M potassium phosphate buffer (pH 7.4). The fluorogenic substrate (Cayman, Ann Arbor, USA) was prepared in advance by removing the solvent from 2.5 μl of a 5 mM stock solution per reaction and solubilization in 15 μl of potassium phosphate buffer (0.5 M, pH 7.4). Incubation mixtures per sample contained 75 μl of tissue lysate, 15 μl of substrate solution (125 μM final concentration), 5 μl of 0.5 mM sodium orthovanadate (25 μM final concentration, Sigma-Aldrich, Steinheim, Germany), and 5 μl of 5 mM pyridoxal phosphate (250 μM final concentration, Sigma-Aldrich, Steinheim, Germany). The reactions were incubated in black 96-well plates with clear bottoms (Greiner, Frickenhausen, Germany) for 6 h at 37 °C in the dark, stopped with 50 μl methanol, and incubated for an additional 2 h in the dark. Fluorescence intensities were measured at ex. 365 nm/em. 460 nm.

l-Cystine, l-homocystine, l-serine, cystathionine, iodoacetamide, pyridoxal phosphate, potassium cyanide and potassium thiocyanate were purchased from Sigma-Aldrich. All other chemicals were purchased from Fisher Scientific. Cystinotic fetal lung fibroblasts (GM00090, donor age: 24 weeks) and normal fetal lung fibroblasts (GM01379, donor age: 12 weeks) were purchased from the Coriell Institute Biorepository (Camden, New Jersey).