The role(s) of arginine-specific mono(ADP-ribosyl)transferase in skeletal muscle cells

dc.contributor.advisor Donald J. Graves Huang, Hui-Yu
dc.contributor.department Biochemistry, Biophysics and Molecular Biology 2018-08-23T15:50:29.000 2020-06-30T07:08:39Z 2020-06-30T07:08:39Z Sun Jan 01 00:00:00 UTC 1995 1995
dc.description.abstract <p>The overall goal of this research project was to examine the biological function of arginine-specific mono(ADP-ribosyl) transferase in skeletal muscle cells. To better understand the function of this enzyme in skeletal muscle, the experimental approach to accomplish this goal consisted of two specific objectives. The first objective was to identify the protein substrates for this enzyme in myogenic cell cultures and cellular fractions. Embryonic chick primary muscle cell cultures were used as a working system for this purpose. In membrane fractions of 96-h myotube cultures incubated with [[superscript]32 P]NAD under conditions selected for mono(ADP-ribosyl)ation, proteins of 127,000, 56,000 and 36,000 Da were predominately labeled. Based on the inhibitory effects of two inhibitors, novobiocin and meta-iodobenzylguanidine (MIBG), as well as the reducing agent, DTT, on ADP-ribosylation of muscle membrane proteins, two potential intracellular target substrates with molecular masses of 56 kDa and 36 kDa were identified. The result of an immunoblot assay where rabbit antiserum specific for desmin reacted with 56 kDa protein strongly suggests that 56 kDa protein might be desmin;The second objective was to examine the enzymology of muscle ADP-ribosyltransferase using the defined protein substrate(s) and to characterize the enzyme by expressing it in bacterial systems. Muscle specific intermediate filament protein type III, desmin, was found to be an excellent substrate for the purified muscle transferase in vitro. Results from sedimentation and electron microscopy experiments showed that ADP-ribosylation of desmin resulted in a remarkable inhibition of desmin polymerization into 10 nm filaments. Additionally, ADP-ribosylation caused a significant inhibition of phosphorylation of desmin by the catalytic subunit of phosphokinase A. Fragmentation of desmin by lysyl endopeptidase revealed that the ADP-ribosylation site was located within the head domain of desmin that has been suggested to play an important role in filament formation;To better understand muscle ADP-ribosyltransferase structure and catalytic properties, significant amounts of pure enzyme are needed. To this regard, skeletal muscle arginine-specific mono(ADP-ribosyl) transferase has been expressed in E. coli. The enzyme appears in the inclusion bodies and a method using the combination of oxidized and reduced glutathione and a pair of refolding assistants, polyoxyethylene 10 lauryl ether (POE10L) and [beta]-cyclodextrin, was successfully used to yield the active enzyme.</p>
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dc.identifier.articleid 11909
dc.identifier.contextkey 6423339
dc.identifier.s3bucket isulib-bepress-aws-west
dc.identifier.submissionpath rtd/10910
dc.language.iso en
dc.source.bitstream archive/|||Fri Jan 14 18:30:55 UTC 2022
dc.subject.disciplines Animal Sciences
dc.subject.disciplines Cell Biology
dc.subject.disciplines Molecular Biology
dc.subject.disciplines Physiology
dc.subject.disciplines Veterinary Physiology
dc.subject.keywords Biochemistry and biophysics
dc.subject.keywords Biochemistry
dc.title The role(s) of arginine-specific mono(ADP-ribosyl)transferase in skeletal muscle cells
dc.type article
dc.type.genre dissertation
dspace.entity.type Publication
relation.isOrgUnitOfPublication faf0a6cb-16ca-421c-8f48-9fbbd7bc3747 dissertation Doctor of Philosophy
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