Date of Award

1989

Publication Type

Doctoral Thesis

Degree Name

Ph.D.

Department

Chemistry and Biochemistry

Keywords

Chemistry, Biochemistry.

Rights

info:eu-repo/semantics/openAccess

Abstract

Oncomodulin is an oncodevelopmental Ca$\sp{2+}$ binding protein of the Troponin C superfamily. The reactivity of the cys-18 thiol of oncomodulin has been probed with 5,5$\sp\prime$-dithiobis(2-nitrobenzoate) (DTNB). The kinetics of the reaction indicate that the thiol is $\sim$10 times more accessible in the presence of Ca$\sp{2+}$ than in EGTA. In addition, oncomodulin has the ability to dimerize via intermolecular disulfide bond formation in vitro. The kinetics of dimerization indicate that the second order rate constant for the formation of dimer is $\sim$6 fold higher than that observed for the reaction of the free thiol with DTNB, perhaps an indication that intermolecular electrostatic attractions precede and facilitate dimerization. The disulfide linked dimer of oncomodulin appears to be more calmodulin like in structure than the monomer since the dimer displayed affinity for the amphiphilic peptide melittin in the same range as calmodulin. To this end, oncomodulin dimer was shown to activate two calmodulin dependent enzymes, bovine heart phosphodiesterase and bovine brain calcineurin, with activity constants of 63nM and 1nM, respectively, indicating that these enzymes have different domain requirements for activation. In addition, oncomodulin was shown to interact with glutathione reductase (GSSGRase) (from bovine intestinal mucosa and rat liver) in a calcium dependent manner. This is evidenced by the fact that glutathione reductase binds to oncomodulin Sepharose only in the presence of calcium and was eluted by the application of EGTA. The inclusion of increasing amounts of reduced oncomodulin in the GSSGRase assay resulted in inhibition of the enzyme with 50% inhibition occurring at $\sim$1E-5M. The closely related carp and rabbit parvalbumins as well as calmodulin had no effect on GSSGRase activity. This is the first report of a regulatory role for oncomodulin which is not shared by calmodulin. The kinetics obtained illustrate that reduced oncomodulin behaves as a noncompetitive inhibitor of GSSG utilization by GSSGRase. In addition, oxidized oncomodulin can act as a substrate for GSSGRase in vitro, as evidenced by SDS-PAGE analysis of the reaction products. These results suggest that oncomodulin monomer is involved in the maintenance of glutathione levels in tumour cells. Therefore, it appears from these studies that oncomodulin monomer and dimer are both important in the metabolism of neoplastic cells, each possessing different regulatory roles, some of which seem to be oncomodulin specific effects. Studies performed on the stability of oncomodulin dimer to 10mM GSH indicate that the dimer may not survive the reducing conditions present intracellularly. Hence, the functions of oncomodulin dimer may lie in the extracellular environment.Dept. of Chemistry and Biochemistry. Paper copy at Leddy Library: Theses & Major Papers - Basement, West Bldg. / Call Number: Thesis1989 .P355. Source: Dissertation Abstracts International, Volume: 50-03, Section: B, page: 0940. Thesis (Ph.D.)--University of Windsor (Canada), 1989.

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