Glycation as a post-translational modification of myosin structure and function; Role of Glutathione; Glycation and aging
Open Access
- Author:
- Ramamurthy, Bhagavathi
- Graduate Program:
- Physiology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- November 08, 2002
- Committee Members:
- Peter Farrell, Committee Member
Sally Johnson, Committee Member
James Harold Marden, Committee Member
Charles H Lang, Committee Member
Lars Larsson, Committee Chair/Co-Chair - Keywords:
- aging
glutathione
myosin
glycation - Abstract:
- The aims of the studies conducted in this thesis were to investigate the effect of the post-translational modification glycation on myosin function in order to better understand the mechanisms underlying the aging-related slowing of skeletal muscle. Non-enzymatic glycosylation (glycation) has been recognized as an important post-translational modification underlying alterations of structure and function of extracellular proteins. The effect of glycation on intracellular proteins is, on the other hand, less well known in spite of the vital importance of intracellular proteins for cell, tissue, and organ function. The aim of this study was to explore the effects of glycation on the structure and function of skeletal muscle myosin. Myosin was incubated for up to 30 minutes with glucose and subsequently tested for structural and functional modifications by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and a single fiber in vitro motility assay, respectively. MALDI spectra revealed glycation-related structural alterations as evidenced by disappearance of specific Lys-C proteolysis products and appearance of higher mass peaks that are attributed to crosslinking by glucose. This change was paralleled by a significant reduction in in vitro motility speed, suggesting a structure-related decline in myosin mechanics in response to glucose exposure. There is complete reversal of motility speed after reaction with the Schiff base-cleaving agent hydroxylamine hydrochloride. Glutathione (GSH) is an abundant tripeptide in skeletal muscle and is known for its antioxidant function and ability to protect cells from toxicity of xenobiotics. Earlier studies have suggested that abundant thiol compounds may inhibit the process of glycation. In order to understand the effect of GSH on glycated myosin function, we used a single fibre in vitro motility assay. Myosin function responded to glucose exposure in a dose-dependent manner, i.e., motility speeds were reduced by 10, 34, and 90% of pre-incubation values after 30 minutes exposure to 1, 3 and 6 mM glucose concentration, respectively. The 30-minute 6 mM glucose incubation was followed by a 20-minute 10 mM GSH incubation. After glucose exposure (0.10 ± 0.07 ?m/s, n=3), GSH treatment restored (p<0.001) motility (0.98 ± 0.06 ?m/s, n=3) close to pre-incubation levels (1.12 ± 0.06 ?m/s, n=3). Antibodies against Advanced Glycation Endproducts (AGEs) were used to quantify the amount of AGEs in SOL and EDL muscle cross sections from male and female Wistar rats of 5 different age-groups (3-6 months, 11-12 months, 16-19 months, 20-25 months and 27-30 months). Confocal microscopy was used to detect the primary anti-AGE-RNAse antibody. Intracellular AGEs showed a significant increase (p<0.001) in both SOL and EDL muscles of males and females across age groups. However, sarcolemmal AGE formation was significant (p<0.05) across age-groups only in the fast-twitch EDL muscle of male rats. Immunoblotting showed myosin along with 2 other proteins of ~57 kD, to be glycated in all age groups of both muscle types in both the sexes. Cross sections were double labelled for Type I and Type IIA fiber types in EDL and SOL muscles respectively. The increase in AGEs was irrespective of fiber type in both SOL and EDL muscles.