Date of Award

8-20-2008

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Lambeth, David O.

Abstract

Nucleoside diphosphate kinases (NDP kinases) are a family of enzymes that transfer the gamma-phosphate group from a NTP (nucleoside triphosphate) to a NDP (nucleoside diphosphate). The reaction follows a ping-pong mechanism in which the enzyme is transiently phosphorylated on a conserved histidine residue. In the cell, activity appears to be ubiquitous as it is associated with the cytosol, nucleus, mitochondria and plasma membrane. NDP kinase enzymatic activity has been demonstrated in a variety of tissues. The classical role proposed for NDP kinase in mitochondrial matrix is the transfer of a phosphoryl group from GTP (produced in the TCA cycle) to ADP producing ATP. The matrix ATP is transported from mitochondria to the cytosol via the adenine nucleotide translocator. Cytosolic NDP kinase is then thought to use ATP to synthesize other NTPs required for cellular functions. Thus far, eight human nm23 genes encoding homologous NDP kinases have been identified. Nm23-H1 to H4 encodes NDP kinase isoforms 1 to 4, all of which have demonstrated NDP kinase activity, show a 58 to 88% homology to each other, and form hexamers. Nm23-H5 to H8 encode NDP kinases 5 to 8 and show more divergence as they are 25 to 45% homologous to group one and each other. The goal of this project was to investigate NDP kinase isoforms associated with mitochondria. Prior to this dissertation, evidence suggested that isoforms two, four, and six were associated with mitochondria. The relative amounts of isoforms four and six were determined in several tissues. Mitochondria from rats were isolated by differential centrifugation and submitochondrial localization of each isoform was determined by subfractionation followed by enzymatic assays and Western blots. Subcellular localization of isoform 6 was also determined using cultured cells with GFP fusion proteins. The relative amounts of each isoform were determined by Western blot. Evidence indicates that isoform two is associated with the outer surface of the outer mitochondrial membrane. Isoforms four and six appear to be located in the matrix. In addition, isoforms four and six appear to be expressed complimentary to each other in the tissues tested.

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