The purification procedure of NMN adenylyltransferase from bull testis presented here consists of a heat step and an acidic precipitation followed by four chromatographic steps, including dye ligand, adsorption and hydrophobic chromatography. The final enzyme preparation subjected to non-denaturating and denaturating PAGE with silver nitrate staining exhibited a single band. At this step the enzyme appeared to be homogeneous. The M(r) value of the native enzyme calculated by gel filtration was about 133 000. The protein appeared to possess a quaternary structure with four subunits of apparent M(r) 33 000 without disulphide interchain bonds. Isoelectric experiments gave a pI of 6.2, and pH studies 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, ATP, NAD+ and PP(i) were 0.11, 0.023, 0.37 and 0.16 mM respectively. The polyclonal antiserum produced against the NMN adenylyltransferase reacted with the purified enzyme at different dilutions and recognized the enzyme in the homogenate as well.
NMN ADENYLYLTRANSFERASE FROM BULL TESTIS: PURIFICATION AND PROPERTIES
BALDUCCI, Enrico;POLZONETTI, Valeria;VITA, Alberto;RAFFAELLI, Nadia;RUGGIERI, Silverio;NATALINI, Paolo
1995-01-01
Abstract
The purification procedure of NMN adenylyltransferase from bull testis presented here consists of a heat step and an acidic precipitation followed by four chromatographic steps, including dye ligand, adsorption and hydrophobic chromatography. The final enzyme preparation subjected to non-denaturating and denaturating PAGE with silver nitrate staining exhibited a single band. At this step the enzyme appeared to be homogeneous. The M(r) value of the native enzyme calculated by gel filtration was about 133 000. The protein appeared to possess a quaternary structure with four subunits of apparent M(r) 33 000 without disulphide interchain bonds. Isoelectric experiments gave a pI of 6.2, and pH studies 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, ATP, NAD+ and PP(i) were 0.11, 0.023, 0.37 and 0.16 mM respectively. The polyclonal antiserum produced against the NMN adenylyltransferase reacted with the purified enzyme at different dilutions and recognized the enzyme in the homogenate as well.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
