NMN ADENYLYLTRANSFERASE FROM BULL TESTIS - PURIFICATION AND PROPERTIES

Citation
E. Balducci et al., NMN ADENYLYLTRANSFERASE FROM BULL TESTIS - PURIFICATION AND PROPERTIES, Biochemical journal, 310, 1995, pp. 395-400
Citations number
34
Categorie Soggetti
Biology
Journal title
ISSN journal
02646021
Volume
310
Year of publication
1995
Part
2
Pages
395 - 400
Database
ISI
SICI code
0264-6021(1995)310:<395:NAFBT->2.0.ZU;2-C
Abstract
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.