Glyoxylase II (Glo II, E.C. 3.1.2.6) catalyzes the hydrolysis of S-D-Lactoylglutathione (SLG) to D-Lactate and glutathione. This is the rate limiting step in the conversion of methylglyoxal to D-Lactate. The purpose of the present study was to determine whether or not a relationship exists between some naturally occuring metabolites and in vivo modulation of Glo II. We have observed a non-competitive inhibition (~ 45%) of Glo II in crude preparation of rat liver by GTP (0.3 mM). A factor (apparently protein),devoid of Glo II,when reconstituted with the purified Glo II, enhanced Glo II activity. This coordinate activation and inhibition of Glo II suggest a mechanism whereby SLG levels can be modulated in vivo.

The glyoxalase system mediates the conversion of methylglyoxal, a toxic ketoaldehyde, to D-lactic acid. The system is composed of two enzymes, glyoxalase I (Glo-I) and glyoxalase II (Glo-II), and exhibits an absolute requirement for a catalytic quantity of glutathione (GSH). Glo-I catalyzes the isomerization of a hemithioacetal, formed non-enzymatically from methylglyoxal and GSH, to the corresponding a -D-hydroxyacid thioester, s-D-lactoylglutathione (SLG). Glo-II catalyzes the irreversible breakdown of SLG to D-lactate and GSH. We have observed that ATP or GTP significantly inhibits the Glo-II activity of tissue homogenates from various sources. We have developed a rapid, one step chromatography procedure to purify Glo-II such that the purified enzyme remains "sensitive" to inhibition by ATP or GTP (Glo-II-s). Studies indicate that inhibition of Glo-II-s by nucleotides is restricted to ATP, GTP, ADP, and GDP, with ATP appearing most effective. Kinetics studies have shown that ATP acts as a partial non-competitive inhibitor of Glo-II-s activity, and further suggest that two kinetically distinguishable forms of the enzyme exist. The sensitivity of pure Glo-II-s to nucleotide inhibition is slowly lost on storage even at -80° C. This loss is accelerated at higher temperatures or in the presence of ATP. Kinetics studies on the resultant "insensitive" enzyme (Glo-II-i) show that a significant reduction of the affinity of the enzyme for the substrate, SLG, occurs and further suggest that only one form of the enzyme is kinetically distinguishable after "de-sensitization". Tryptophan fluorescence studies of the two enzyme preparations suggest that a subtle conformational change in the enzyme has occurred during de-sensitization. We have also observed that Glo-II-i is "resensitized" to nucleotide inhibition after incubation in the presence of a reagent that reduces disulfide bonds. The resensitized enzyme exhibits an increased KM value similar to that of the original Glo-II-s. Kinetics studies show that ATP or GTP again act as partial non-competitive inhibitors of the resensitized enzyme and suggest that only one form of the enzyme is present. The physiological significance of the two enzyme forms is discussed.

Glyoxalase 11, the second enzyme of the glyoxalase system, hydrolyzes S-D-lactoylglutathione (SLG) to regenerate glutathione (GSH) and liberate free D-lactate. It was found that GTP binds with Gil from rat liver and inhibits Gil activity. Preincubation experiments showed that the binding is relatively tight, since more than 15 minutes are required to release GTP from the complex following dilution. Inhibition kinetics studies indicate that GTP is a "partially competitive inhibitor"; Thus, it would appear that the binding sites for substrate (SLG) and inhibitor (GTP) are different, but spatially close. Glyoxalase 11 binds to a GTP affinity medium, and with polyacrylamide gel electrophoresis, Gil has a higher relative mobility when GTP is present (ATP has no effect). The functional consequences of GTP binding with a specific site on Gil are still unclear. It is speculated that Gil may interact with tubulin by serving as a dissociable GTP carrier, delivering GTP to the tubulinGTP binding site, and thus facilitating tubulin polymerization.

It is the purpose of this investigation to determine the variation of glyoxalase activity in liver of normal mice and in the liver of mice bearing a lymphosarcoma and to compare the glyoxalase activity of the lymphosarcoma with values previously reported in the literature for other tumor types. Further, if there is indeed a variation in liver glyoxalase activity between normal and tumor-bearing mice, it will be compared to the variation in the activity of two other enzymes present in liver tissue in relatively high concentration.

In this research, a model system for studying the effects of the toxic metabolite, methylglyoxal, was created using Chinese Hamster Ovary (CHO) cells which produce tissue plasminogen activator (t-PA). The human gene for glyoxalase I was subcloned into an inducible mammalian expression vector. This vector was then used to create three stable CHO integrants, two control and one putative glyoxalase I producing cell lines. The CHO clones were characterized for the production of glyoxalase I using both SDS-PAGE gels and glyoxalase activity assays. In addition, the cell lines were evaluated to determine the levels of free methylglyoxal produced. The putative glyoxalase producer showed higher levels of glyoxalase I activity than the parent cell line and produced a unique protein band at the correct molecular weight. They also had a significantly lower level of free methylglyoxal than either of the two control cell lines. These cells can now be used as a tool to determine the specific effect of methylglyoxal on the yield and quality of tissue plasminogen activator produced. / Graduation date: 2000

Thesis (M.S.)--Miami University, Dept. of Chemistry and Biochemistry, 2003. / Title from first page of PDF document. Document formatted into pages; contains xii, 83 p. : ill. Includes bibliographical references.

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In patients with diabetes, hyperglycemia leads to functional impairment of endothelial cells (ECs) and microangiopathy. Inflammation and endothelial dysfunction (ED) have been associated with the development of several cardiovascular complications. Concentration of methylglyoxal (MG) - a highly reactive aldehyde is increased in diabetes. In a non-pathological state, MG is detoxified by the enzymes glyoxalase-1 (GLO1) and glyoxalase 2 in presence of glutathione. This thesis examines the role of MG accumulation in ECs and bone marrow cells (BMCs), with the consequences it has for their function. To this end, a transgenic mouse model was used in which the human enzyme GLO1 is overexpressed in the vasculature By using a GLO1 overexpressing mouse model studies described here examined the contribution of MG-induced inflammation in vivo to cardiovascular complications of diabetes, namely diabetic heart failure and peripheral vascular disease. This study confirmed that accumulation of MG leads to inflammation and cell death, and further explained how MG affects the role of ECs in development of the heart failure and BMCs in the revascularization. Overexpression of GLO1 in the vasculature diminished MG-induced inflammation, reduced EC death and delayed and limited the loss of cardiac function in streptozotocin (STZ)-induced diabetic mice (Chapter 2). The in vitro part of this study showed that MG and tumor necrosis factor (TNF- have a synergistic effect on cell death (Chapter 3). Overexpressing the GLO1 in BMCs only, restored neovascularization in ischaemic tissue of mice with STZ-induced diabetes (Chapter 4). Taken together, the results of this thesis suggest that hyperglycemia increased MG leads to endothelial inflammation, EC death and decreased angiogenic potential of BMCs. Furthermore, this MG-induced inflammation and reduced cell function observed, identifies a potential target for therapy of the cardiovascular complications seen in diabetes.

A new potent competitive inhibitor of glyoxalase II, S-carbobenzoxglutathione (CBG) (Ki=0.065mM) was synthesized. The homogenous enzyme was obtained by a simple two-step CBG-affinity column chromatographic procedure.

It was the purpose of this study to purify glyoxalase I and then characterize its physical and kinetic properties. A comparison of the enzyme from the liver of normal and tumor-bearing mice and from the tumor itself was also to be made.