Protein disulfide isomerase (PDI), a member of the thioredoxin superfamily,
contains two domains with significant sequence homology to the active sites
in thioredoxin. PDI facilitates the folding of nascent proteins in the
endoplasmic reticulum (ER), binds hormones and Ca�����, catalyzes the
glutathione dependent reduction of dehydroascorbate, serves as a major
chaperone molecule in the ER and serves as a subunit for prolyl-4-hydroxylase and microsomal triglyceride transferase. Because of its abundance in the ER and association with disease and chemically induced
toxicity, the goal of this research was to investigate the relative susceptibility
of PDI thiols to alkylation. The sensitivity of PDI to 1-chloro-2,4-dinitrobenzene (CDNB), iodoacetamide (IAM) and biotinoylated iodoacetamide (BIAM) was explored. The relative susceptibility of the thiolate
anions present in the two active sites of PDI each containing the -CGHC-
sequence was investigated with mass spectrometric techniques. PDI was
inactivated by CDNB but was not found as sensitive as thioredoxin reductase
as shown by Amer and coworkers (1995). IAM and BIAM were used as
model alkylating agents to explore the two active sites of PDI and determine
the residues most susceptible to alkylation. Alkylation by IAM and BIAM was
first detected at the N-terminal cysteine in each active site (-C*GHC-)
followed by alkylation at the second cysteine residue (-C*GHC*-) as shown
by tandem mass spectrometry. Mass spectroscopy showed that the
episulfonium ion derived from the glutathione conjugate of 1,2-dichloroethane, S-(2-chloroethyl)glutathione (CEG), decreased activity and protein thiols of PDI. CEG produced two protein adducts at very low
excesses of CEG over PDI; however, higher concentrations resulted in
several protein adducts. Only one modification in each active site at the N-terminal
cysteine residue can be identified, indicating that while these thiolate
anions of PDI are susceptible, it would appear that the episulfonium ion may
present itself to other sites as well. This may have important toxicologic
significance regarding the mechanism of 1,2-dichloroethane toxicity and the
role of PDI in the redox status of the cell. / Graduation date: 2003
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/31479 |
| Date | 04 March 2003 |
| Creators | Kaetzel, Rhonda Sue |
| Contributors | Reed, Donald J. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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