Type
Text
Type
Dissertation
Advisor
Dale G. Deutsch. | Berhane Ghebrehiwet | Stella E. Tsirka | Carol A. Carter | Marian J. Evinger.
Date
2011-08-01
Keywords
Molecular biology -- Biochemistry | 2-arachidonylglycerol, monoacylglycerol lipase, spectrophotometric assay, transcript variants
Department
Department of Genetics
Language
en_US
Source
This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree.
Identifier
http://hdl.handle.net/11401/71719
Publisher
The Graduate School, Stony Brook University: Stony Brook, NY.
Format
application/pdf
Abstract
Monoacylglycerol lipase (MAGL) is the principal enzyme responsible for hydrolysis of the endocannabinoid, 2-arachidonylglycerol (2-AG). MAGL is universally expressed as a ~33 kDa protein. However, a ~35 kDa form of MAGL is also expressed in brain and testes while being absent in other tissues. Interestingly, the MAGL mRNA sequence contains two putative translation initiation sites upstream of the start codon originally used for MAGL cloning, which may explain the ~35 kDa form. Examination of whole mouse brain and brain region cDNA revealed the presence of three MAGL transcript sequences. These sequences were isolated and characterized in neuronal and non-neuronal cell lines. It was determined that all three constructs are enzymatically active, are expressed by all cell lines, and have similar sub-cellular localizations. These data indicate that despite their differing N-termini peptide sequences, these MAGL variants possess redundancy in their activity and cellular trafficking. Since the characteristics of the MAGL variants examined did not differ between non-neuronal and neuronal cell lines, the expression of ~35kDa protein may be a result of other modes of protein regulation such as protein interactions only stimulated by incubation with 2-AG or post-translational modifications. MAGL activity is usually measured by costly radiometric or time-consuming mass spectrometric assays. An assessment of a spectrophotometric assay was carried out to determine its usefulness as an alternative method. MAGL obtained from transiently transfected COS-7 cells was incubated with arachidonoyl-1-thio-glycerol, a thioester-containing analog of 2-AG, which results in the release of a thiolate ion that has measurable absorbance at 412 nm. This spectrophotometric assay was used to measure MAGL activity and measure inhibition by the sulfhydryl inhibitor, N-arachidonyl maleimide (NAM). Most results obtained were comparable to those reported in previous studies. The spectrophotometric assay, therefore, is a useful tool for general purposes such as initial high-throughput screening of inhibitors.
Recommended Citation
Ulloa, Nadine, "Variants of Monoacylglycerol Lipase (MAGL) and an Assessment of a Spectrophotometric Assay for MAGL Activity" (2011). Stony Brook Theses and Dissertations Collection, 2006-2020 (closed to submissions). 924.
https://commons.library.stonybrook.edu/stony-brook-theses-and-dissertations-collection/924