Isolation, characterization and expression of a human brain mitochondrial glutaminase cDNA.
Various cDNAs that encode overlapping portions of the full-length human brain glutaminase (GA) cDNA were cloned and sequenced. The overall nucleotide sequence of hGA has a very high degree of identity with that of the rat kidney-type GA cDNA (77.4%) and the known portion of the cDNA that encodes the 5.0-kb porcine GA mRNA (81.1%). The identity is even more remarkable at the amino acid level, particularly in the C-terminal half where the three proteins share a 99.7% sequence identity. The hGA cDNA encodes a 73,427-Da protein that contains an N-terminal mitochondrial targeting signal and retains the primary proteolytic cleavage site characterized for the cytosolic precursor of the rat renal mitochondrial glutaminase. The entire coding region was assembled through the use of unique restriction sites and cloned into a baculovirus. Sf9 cells infected with the recombinant virus express high levels of properly processed and active glutaminase. Thus, expression of the isolated hGA cDNA should provide a means to purify large amounts of the mitochondrial glutaminase, a protein that catalyzes a key reaction in the metabolism of glutamine and the synthesis of important excitatory and inhibitory neurotransmitters.
Holcomb
T
T
Department of Biochemistry and Molecular Biology, Colorado State University, 316 MRB Bldg., Ft. Collins, CO 80523-1870, USA.
Taylor
L
L
Trohkimoinen
J
J
Curthoys
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NP
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D015723:Gene Library; D005972:Glutaminase / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D008928:Mitochondria / Q000737:chemistry*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera / Q000166:cytology; D014162:Transfection
D018076:DNA, Complementary; D005972:Glutaminase