B12 METABOLISM IN HUMANS By NICOLE AURORA LEAL A ...

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5’- triphosphate (ATP), and dithiothreitol (DTT) were purchased from ICN Biomedicals,
Inc. (Aurora, Ohio). Pefabloc SC PLUS was from Roche Diagnostics (Penzberg,
Germany). All other chemicals and reagents were from Fisher Scientific (Norcross, GA).
Bacterial Strains and Growth Media
The bacterial strains used were E. coli DH5α and BL21 (DE3) RIL (Stratagene, La
Jolla, CA). LB was the rich medium used (Difco, Detroit, MI) (Miller 1972). LB was
supplemented with 25 µg/ml kanamycin and 20 µg/ml chloramphenicol or as indicated.
General Molecular and Protein Methods
Agarose gel electrophoresis and restriction enzyme digests were performed using
standard protocols (Maniatis et al. 1982, Johnson et al. 2001). PCR products and plasmid
DNA were gel purified using QIAquick ® gel extraction kit (QIAGEN, Inc., Valencia,
CA). DNA ligation was accomplished using T4 DNA ligase according to manufacturer’s
instructions. Bacterial transformation and SDS-PAGE were performed as previously
described (Johnson et al. 2001).
ATP:cob(I)alamin Adenosyltransferase Assays
ATR assays were performed as previously reported with some modifications
(Johnson et al. 2001). Reaction mixtures contained: 200 mM Tris-HCl (pH 8.0), 2.8 mM
MgCl2, 10 mM KCl, 0.05 mM HOCbl, 0.4 mM ATP, and 1 mM titanium(III) citrate in a
total volume of 2 ml. Reaction components (except for the ATR) were dispensed into
cuvettes inside an anaerobic chamber (Coy Laboratories, Ann Arbor, MI). The cuvettes
were sealed, removed from the chamber, and incubated at 37°C for 2 minutes. Reactions
were initiated by addition of purified recombinant ATR, and AdoCbl formation was
measured by following the decrease in absorbance at 388 nm (∆ε388 = 24.9 cm -1 mM -1 ).