NAD biosynthesis in human placenta: purification and characterization of homogeneous NMN adenylyltransferase.
Nicotinamide mononucleotide (NMN) adenylyltransferase has been purified to homogeneity from human placenta. The purification procedure consists of several chromatographic steps, including dye-ligand, adsorption, and hydrophobic interaction chromatography. The final enzyme preparation is homogeneous as judged by a single silver stainable band on both nondenaturating and denaturating polyacrylamide gels. The native enzyme shows a molecular weight of about 132,000, as determined by gel filtration on a Superose 12 HR 10/30 fast protein liquid chromatography column. The protein possesses a quaternary structure and is composed of four apparently identical M(r) 33,000 subunits. Isoelectrofocusing experiments give multiple pI values ranging from pH 4.7 to 6.6. Optimum pH study shows a plateau extending from pH 6.0 to pH 9.0. Km values for NMN, ATP, NAD+, and PPi are 38, 23, 67, and 125 microM, respectively. Kinetic analysis reveals a behavior consistent with an ordered sequential Bi-Bi mechanism. Among several metabolites tested only ADP-ribose and beta-NMNH were found to significantly inhibit the enzyme activity.
Emanuelli
M
M
Istituto di Biochimica, Facoltà di Medicinae Chirurgia, Università di Ancona, Italy.
Natalini
P
P
Raffaelli
N
N
Ruggieri
S
S
Vita
A
A
Magni
G
G
D000596:Amino Acids / Q000032:analysis; D002412:Cations / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000096:biosynthesis*; D009612:Nicotinamide-Nucleotide Adenylyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010920:Placenta / Q000201:enzymology*
D000596:Amino Acids; D002412:Cations; D009243:NAD; D009612:Nicotinamide-Nucleotide Adenylyltransferase