Kinetics assay development of SAM-dependent methyltransferase and characterization of dimer formation of aged protein isoaspartyl methyltransferase (PIMT)
Abstract
An adenosine deaminase-related growth factor homolog was isolated as a minor component in a commercial preparation of AMP deaminase from Aspergillus melleus. Anticipating its application to coupled assays of SAM-dependent methyltransferases, the enzyme was purified 40-fold to homogeneity in a two-step chromatographic procedure by following putative deaminase activity on S-adenosyl homocysteine and comparing with activity on S-adenosyl methionine. The product of reaction with S-adenosyl homocysteine was identified as S-inosyl homocysteine by UV spectroscopy, thin layer chromatograph separation and mass spectrometry. The purified protein has a molecular weight of 69,137 Da. Edman and MALDI-TOF/TOF sequencing identified the purified enzyme as one of the two adenosine deaminase-related growth factor homologs found in several genomic sequences of Aspergillus species. With identification as a member of adenyl deaminase superfamily, kinetic constants were determined on several adenine group containing substrates and compared with literature values. Possessing activity suitable for use as a coupled enzyme where S-adenosyl homocysteine is produced, the Aspergillus adenosine deaminase-related growth factor was used to develop both continuous and discontinuous spectrophotometric assays for S-adenosyl methionine-dependent methyltransferases, using human protein isoaspartyl methyltransferase as the spectrophotometric assays for S-adenosyl methionine-dependent methyltransferases, using human protein isoaspartyl methyltransferase as the methyltransferase activity of interest. The continuous format allowed the first analysis of the steady state kinetics of human protein isoaspartyl methyltransferase in a continuous assay. The 96-well plate discontinuous format enabled screening of potential inhibitors. Confirming earlier reports of inhibition of methylation in Xenopus oocyte, the coupled assay specifically identified sinefungin as a micromolar inhibitor of the human protein isoaspartyl methyltransferase, further validating the coupled assays. In the third part of our research, we pursued the existence and characteristics of dimer formation in in vitro aged rat protein isoaspartyl methyltransferase under physiological conditions upon the detection of a 50 kDa protein in rat lung cytosol by purified protein isoaspartyl methyltransferase antibody. We characterized dimer accumulation rates and investigated the crosslinks responsible for dimer formation. Other than dimerization, protein precipitation, peptide cleavage and oligomerization with more than two monomers were observed during rat protein isoaspartyl methyltransferase in vitro aging. These phenomena may play a role in the inactivation and degradation of protein isoaspartyl methyltransferase in vivo.
Degree
M.S.
Advisors
Friedman, Purdue University.
Subject Area
Molecular biology|Biochemistry
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.