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Kinetic characterization of arylamine N‐acetyltransferase 2
Author(s) -
Baker Inesha,
Parker Chasta L
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1008.5
Subject(s) - nat , acetyltransferase , arylamine n acetyltransferase , enzyme , enzyme kinetics , gene , microbiology and biotechnology , acetylation , biochemistry , chemistry , biology , active site , computer network , computer science
N‐acetyltransferase 2 (NAT 2) is an enzyme that catalyzes the acetylation of harmful arylamines and has been implicated in various types of cancer. NAT 2 is primarily expressed in the hepatic system and intestinal epithelium and is encoded at two polymorphic loci that give the phenotypic characteristics of slow and fast acetylation. The purpose of this experiment was to study the stability of the NAT 2 over time in order to determine if rate data was affected by a time lapse between purification and rate determination. The NAT 2 gene was amplified from both pENTR 221 and genomic DNA using PCR. NAT 2 was ligated into a pET28b+ vector containing an N‐terminal hexahistidine tag and transformed into E. coli Rosetta 2 cells. Protein expression was carried out under different conditions to determine the correct culture and IPTG concentrations necessary for protein production. The protein was isolated from the cells and purified using FPLC. The enzyme was then kinetically characterized using sulfamethazine and aspartame immediately after purification as well as one week post – purification. Preliminary results indicate that a one week time lapse results in a 2 to 4 fold decrease in the activity. This project was supported by the Winthrop University Research Council and NIH Grant Number P20 RR‐16461 from the NCRR for support of the program entitled “South Carolina IDeA Networks of Biomedical Research Excellence (SC‐INBRE)”.