Cloning, expression and characterization of mouse (Mus musculus) nicotinamide 5'-mononucleotide adenylyltransferase-2.
Nicotinamide adenine dinucleotide (NAD+) is synthesized by the action of nicotinamide mononucleotide adenylyltransferase (NMNAT) from NMN and ATP. The mouse homolog of NMNAT-2 (mmNMNAT-2) was cloned, expressed, and subsequently identified using MALDI-TOF in conjunction with the ProFound database. Circular dichroism analyses of recombinant mmNMNAT-2 showed α helical and β sheet secondary structures, consistent with the known structure of the human isoform. Competition experiments using mouse pancreatic tissue lysates with recombinant mmNMNAT-2 demonstrated that the activity of the expressed protein was similar to the human isoform. Immunohistochemistry of mouse embryonic tissues with hNMNAT-2 also showed a tissue- and cellular-specific expression of this isoform. Therefore, our studies demonstrate for the first time the clear biological evidence for the existence of a mouse isoform of hNMNAT-2. These studies may help in future investigations aimed at understanding the regulation of this gene and its pathway, and in turn, will spur the development of novel therapies for diseases such as cancer and diabetes since mice are the most frequently used experimental system for in vivo studies.
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, USA.
David W
DW
Raches
Suhong
S
Xiao
Praveen
P
Kusumanchi
Joel
J
Yalowitz
Paresh
P
Sanghani
Eric C
EC
Long
Aśok C
AC
Antony
Hiremagalur N
HN
Jayaram
D000818:Animals; D002942:Circular Dichroism; D003001:Cloning, Molecular; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D009243:NAD / Q000096:biosynthesis / Q000737:chemistry; D009612:Nicotinamide-Nucleotide Adenylyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D011994:Recombinant Proteins; D009243:NAD; C484008:NMNAT2 protein, human; D009612:Nicotinamide-Nucleotide Adenylyltransferase