title: Acetoxy drug: protein transacetylase of buffalo liver-characterization and mass spectrometry of the acetylated protein product.
abstract: The purification and characterization of the buffalo liver microsomal transacetylase (TAase) catalyzing the transfer of acetyl groups from a model acetoxy drug: 7,8-diacetoxy-4-methylcoumarin (DAMC) to GST3-3 has been described here. The enzyme was routinely assayed using DAMC and cytosolic GST as the substrates and was partially purified from microsomes of the buffalo liver. The enzyme was found to have approximate molecular of weight 65 kDa. The action of TAase and DAMC on liver cytosolic GST resulted in the formation of monoacetoxymonohydroxy-4-methylcoumarin (MAMHC) and 7,8-dihydroxy-4-methylcoumarin (DHMC), although the former was the major metabolite. The buffalo liver microsomal TAase exhibited hyperbolic kinetics and yielded K(m) (1667 microM) and V(max) (192 units) when the concentration of DAMC was varied keeping the concentration of GST constant. After having characterized the nature of the substrates and a product of the TAase-catalyzed reaction, we set out to identify the acetylated protein which is another product of the reaction. GST3-3 was used as a model protein substrate for the action of TAase using DAMC as the acetyl donor. The subunit of control and modified GST3-3 were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and digested with trypsin. The tryptic peptides were extracted from the gel pieces and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOFMS). The data search for calibrated and labeled mass peaks of peptides was performed on the Matrix Science Server using the search engine Mascot. The peptide maps so obtained covered 97% of the GST3-3 sequence. On comparison of MALDI peptide maps of modified and control GST, seven new peaks were recognized corresponding to the potentially acetylated peptides in peptide map. The mass value of each of them was 42 Da higher than the theoretical mass of a non-modified GST3-3 tryptic peptide, strongly suggesting acetylation. By examining the fragmentation patterns and by comparing experimental and predicted values for MS/MS daughter ions, the identity of the seven acetylated GST tryptic peptides could be confirmed by the application of LC/MS/MS. In the modified GST, N-terminal proline and six lysines (Lys(51), Lys(82), Lys(123), Lsy(181), Lys(191) and Lys(210)) were found to be acetylated. The structure of acetylated GST revealed that the lysines that underwent acetylation were peripheral in positions.
authors.affiliation: Department of Biochemistry, V.P. Chest Institute, University of Delhi, Delhi 110 007, India.
authors.forename: Ekta
authors.initials: E
authors.lastname: Kohli
authors.forename: Marco
authors.initials: M
authors.lastname: Gaspari
authors.forename: Hanumantharao G
authors.initials: HG
authors.lastname: Raj
authors.forename: Virinder S
authors.initials: VS
authors.lastname: Parmar
authors.forename: Sunil K
authors.initials: SK
authors.lastname: Sharma
authors.forename: Jan
authors.initials: J
authors.lastname: van der Greef
authors.forename: Ranju
authors.initials: R
authors.lastname: Kumari
authors.forename: Garima
authors.initials: G
authors.lastname: Gupta
authors.lastname: Seema
authors.forename: Pulkit
authors.initials: P
authors.lastname: Khurana
authors.forename: Yogesh K
authors.initials: YK
authors.lastname: Tyagi
authors.forename: Arthur C
authors.initials: AC
authors.lastname: Watterson
authors.forename: Carl E
authors.initials: CE
authors.lastname: Olsen
mesh_terms: D000107:Acetylation; D000123:Acetyltransferases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002020:Buffaloes / Q000378:metabolism*; D003374:Coumarins / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: C114369:7,8-diacetoxy-4-methylcoumarin; D003374:Coumarins; D010455:Peptides; D011506:Proteins; D000123:Acetyltransferases; D005982:Glutathione Transferase