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.
Sasiak
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Department of Biogenic Amines, Polish Academy of Sciences, Lodz, Poland.
Saunders
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PP
D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005260:Female; D006801:Humans; D007525:Isoelectric Focusing; D007700:Kinetics; D008970:Molecular Weight; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011489:Protein Denaturation; D012265:Ribonucleotides / Q000378:metabolism; D012710:Serum Albumin, Bovine; D013379:Substrate Specificity
D012265:Ribonucleotides; D012710:Serum Albumin, Bovine; D017853:Phosphotransferases (Alcohol Group Acceptor); C061783:nicotinamide riboside kinase