Date of Award

1-1-1993

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

Abstract

Prolonged hyperglycemia, as seen in diabetes, results in nonenzymatic glycation of proteins and DNA. Previous investigation by others has shown that many intracellular proteins are glycated, both, in vivo and in vitro. Nonetheless, only a few proteins have been analyzed for the effect of glycation on their function.Glucose and galactose were found to modify a large number of the lysyl residues in selected cytoskeletal proteins. These results led to the study of myosin and S-1 (subfragment-1 of myosin), first with FITC (fluorescein isothiocyanate; isomer #1) and later with comparatively less reactive sugars (sorbitol, glucose, fructose, and ribose).FITC caused a decrease in the actin-activated Mg$\sp{2+}$-ATPase of both S-1 and myosin. Measurements of actin affinity (an increase in the K$\sb{\rm m}$ for actin binding) for FITC-modified S-1 revealed that lysine residues are essential for the binding of actin to S-1. Intrinsic lysyl residues must also play a role in actin-activated myosin ATPase because, the V$\sb{\rm max}$ of the actin-activated myosin ATPase was decreased when myosin was derivatized with FITC. A role for S-1 lysine involvement in actin-S-1 interactions was also revealed when FITC modified S-1 was found less able to stimulate G-actin polymerization. SDS-PAGE of S-1 digested by trypsin, localized FITC to the actin-binding region at the 50-20kDa junction.The effect of sugars on myosin and S-1 was also studied. Fructose, a relatively reactive sugar, caused a time and concentration dependent decrease in the actin-activated Mg$\sp{2+}$-ATPase activity of myosin. Of significant interest, glucose, which is less reactive than fructose or ribose, decreased myosin actin-activated Mg$\sp{2+}$-ATPase activity more than the other sugars. In contrast to myosin, the actin-activated Mg$\sp{2+}$-ATPase activity of S-1 was affected most by ribose derivatization and least with glucose. The K$\sb{\rm m}$ for actin binding to S-1 was increased with sugar derivatization, indicating that the actin-binding site was modified by sugars. In addition, sugar derivatization lowered the ability of S-1 to cause G-actin polymerization.

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