Purification and properties of a human nicotinamide ribonucleoside kinase

Nicotinamide ribonucleoside kinase (NRK) phosphorylates at least two nucleoside analogs of potential clinical interest, tiazofurin and 3-deazaguanosine. In this study NRK has been purified to near homogeneity from human placenta. The purification procedure consists of several chromatographic steps including salt precipitation, DE-52 chromatography, sucrose density gradient fractionation, hydroxylapatite chromatography, and anion exchange FPLC. The final enzyme preparation is homogeneous as judged by a single silver-stainable band on both nondenaturing and denaturing polyacrylamide gels. The molecular weight of the enzyme, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration on Superdex 75 HR 10/30, is approximately 29 and 32 kDa, respectively. The isoelectric pH for NRK is 5.6. The reaction requires ATP. The pH optimum is in the region 6.5-9.0. NRK in the purified preparations, with added bovine serum albumin, was stable for days at 4 degrees C and for months at -70 degrees C. The enzyme is very unstable at low protein concentration. NRK phosphorylated several substrates including nicotinamide ribonucleoside, guanosine, tiazofurin, and 3-deazaguanosine with apparent Km values of 9.6, 115, 90, and 16.5 microM, respectively.