The purification procedure of NMN adenylyltransferase from bull testis
presented here consists of a heat step and an acidic precipitation fo
llowed by four chromatographic steps, including dye ligand, adsorption
and hydrophobic chromatography. The final enzyme preparation subjecte
d to non-denaturating and denaturating PAGE with silver nitrate staini
ng exhibited a single band. At this step the enzyme appeared to be hom
ogeneous. The M(r) value of the native enzyme calculated by gel filtra
tion was about 133000. The protein appeared to possess a quaternary st
ructure with four subunits of apparent M(r) 33000 without disulphide i
nterchain bonds. Isoelectric experiments gave a pI of 6.2, and pH stud
ies showed the possible presence of an acidic group in the active site
having a pK(a) of 4.9. Analysis of the amino acid composition showed
the presence of more acidic residues than basic ones, according to the
pI value calculated by Mono P FPLC. The E(a) calculated by Arrhenius
plot gave an apparent value of 55.7 kJ/mol. The K-m values for NMN, AT
P, NAD(+) and PPi were 0.11, 0.023, 0.37 and 0.16 mM respectively. The
polyclonal antiserum produced against the NMN adenylyltransferase rea
cted with the purified enzyme at different dilutions and recognized th
e enzyme in the homogenate as well.