NMN adenylyltransferase: its association with chromatin and with poly(ADP-ribose) polymerase.
The nuclear location of NMN adenylytransferase, which catalyses the formation of NAD and pyrophosphate from ATP and NMN, has been examined to ascertain if the enzyme is bound to the domains of chromatin which undergo poly(ADP-ribos)ylation. This latter reaction utilizes much of the cellular NAD. A radioisotope assay using [alpha-32P]ATP was developed to enable precise measurement of picomole amounts of NAD. With this assay, it appeared that the reaction catalysed by NMN adenylyltransferase proceeded with a rapid, early 'burst' of NAD before steady-state velocities were established. From this it was calculated that there could be 10- active sites of NMN adenylyltransferase per HeLa nucleus in asynchronously growing cells: that is, approximately one per 10-20 nucleosomes. Very little enzyme activity was liberated by digesting HeLa nuclei with micrococcal nuclease in 80 mM NaCl, and the enzyme which was solubilized was not bound to oligonucleosomes separated by electrophoresis on polyacrylamide gels. In contrast, poly(ADP-ribose) polymerase activity was clearly demonstrated on these particles. The enzyme was readily liberated by DNase I digestion, especially when the digestion was carried out in low-ionic-strength buffer. The results demonstrated that the enzyme was neither bound to oligonucleosomes nor part of the nuclear envelope or matrix. Preliminary results suggested that there could be some direct channelling of NAD between the two enzymes in intact nuclei. It appears that NMN adenylyltransferase is bound within rather than to chromatin.
D002467:Cell Nucleus / Q000201:enzymology; D002843:Chromatin / Q000201:enzymology*; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D009244:NAD+ Nucleosidase / Q000378:metabolism*; D009612:Nicotinamide-Nucleotide Adenylyltransferase / Q000378:metabolism*; D009707:Nucleosomes / Q000201:enzymology; D009713:Nucleotidyltransferases / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*
D002843:Chromatin; D009707:Nucleosomes; D011065:Poly(ADP-ribose) Polymerases; D009713:Nucleotidyltransferases; D009612:Nicotinamide-Nucleotide Adenylyltransferase; D009244:NAD+ Nucleosidase