NAD pyrophosphorylase from yeast chromatin. Purification and properties.
NAD pyrophosporylase has been purified to homogeneity from baker's yeast. The purification procedure is relatively simple and consists of high salt extraction of enzyme activity, precipitation with polyethylenimine followed by ion exchange and by ligand chromatography separations. The final enzyme preparation is homogeneous as judged by a single Coomassie blue-stainable band when run on non-denaturing and denaturating polyacrylamide gels. The native enzyme shows a molecular weight of about 200,000 calculated by gel filtration and sucrose gradient centrifugation. The protein possess quaternary structure and is composed by four apparently identical Mr 50,000 subunits. The absorption spectrum shows a maximum at 280 nm and a minimum at 253 nm. Isoelectric point is 6.2. Amino acid composition shows the presence of 28 half-cystine residues, in agreement with the results obtained by titrating the enzyme in denaturing conditions with Ellman's reagent upon previous incubation with sodium borohydride. NAD pyrophosphorylase is a glycoprotein containing 2% sugar, 2 moles of alkali-labile phosphate per enzyme mol, and 1 mol of adenine moiety per enzyme mol. Therefore the possibility that the enzyme is ADP-ribosylated exists. Km for ATP, NMN and NaMN are 0.11 mM, 0.19 mM and 5 mM respectively. Kinetic analysis reveals a behaviour which is consistent with an ordered sequential Bi-Bi mechanism. pH optimum is in the range 7.2-8.4.
Magni
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G
Dipartimento di Biologia Cellulare, Università di Camerino, Italy.
Natalini
P
P
Santarelli
I
I
Vita
A
A
Raffaelli
N
N
Ruggieri
S
S
D000225:Adenine / Q000032:analysis; D000596:Amino Acids / Q000032:analysis; D002241:Carbohydrates / Q000032:analysis; D011232:Chemical Precipitation; D002843:Chromatin / Q000201:enzymology*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D009612:Nicotinamide-Nucleotide Adenylyltransferase / Q000302:isolation & purification* / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000302:isolation & purification*; D010710:Phosphates / Q000032:analysis; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000032:analysis; D013696:Temperature
D000596:Amino Acids; D002241:Carbohydrates; D002843:Chromatin; D010710:Phosphates; D013438:Sulfhydryl Compounds; D009713:Nucleotidyltransferases; D009612:Nicotinamide-Nucleotide Adenylyltransferase; D000225:Adenine