Bacterial expression, purification, and characterization of rat kidney-type mitochondrial glutaminase.
The human gene that encodes the kidney-type glutaminase (KGA) spans 84-kb, contains 19 exons, and encodes two alternatively spliced mRNAs. Various segments of the rat KGA cDNA were PCR amplified and cloned into a bacterial expression vector to determine whether the N- and C- terminal ends of the glutaminase protein were essential for activity. A recombinant glutaminase, lacking the coding sequence contained in exon 1, was found to be fully active. In contrast, proteins that lacked sequences from exons 1 and 2 and exons 1-3 were inactive. An additional construct that corresponded to the sequence encoded by exons 2-14 also retained full activity. Both of the fully active, truncated proteins were purified to apparent homogeneity using an incorporated N-terminal His(6)-tag and Ni(2+)-affinity chromatography. The K(M) values for glutamine of the native and recombinant forms of glutaminase were nearly identical. However, the two truncated forms of the glutaminase exhibit the characteristic phosphate activation profile only when dialyzed into a buffer lacking phosphate. Dialysis versus 10mM Tris-phosphate was sufficient to form an active tetramer. Thus, the deleted N-terminal sequence may contribute to the phosphate-dependent oligomerization and activation of the native glutaminase.
Kenny
John
J
Department of Biochemistry and Molecular Biology, Colorado State University, Ft. Collins, CO 80523-1870, USA.
Bao
Yuhne
Y
Hamm
Brian
B
Taylor
Lynn
L
Toth
Ann
A
Wagers
Brian
B
Curthoys
Norman P
NP
D000818:Animals; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005091:Exons / Q000235:genetics; D017353:Gene Deletion; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D005972:Glutaminase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D006639:Histidine; D007544:Isopropyl Thiogalactoside / Q000031:analogs & derivatives / Q000494:pharmacology; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D009842:Oligopeptides / Q000235:genetics; D010710:Phosphates / Q000737:chemistry / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D013010:Sonication
D018076:DNA, Complementary; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D010710:Phosphates; D011994:Recombinant Proteins; D005973:Glutamine; D007544:Isopropyl Thiogalactoside; D006639:Histidine; D005969:Glutamate Dehydrogenase; D005972:Glutaminase