Molecular cloning, cDNA sequence, and bacterial expression of human glutamine:fructose-6-phosphate amidotransferase.
Glutamine:fructose-6-phosphate amidotransferase (GFAT) has recently been shown to be an insulin-regulated enzyme that plays a key role in the induction of insulin resistance in cultured cells. As a first step in understanding the molecular regulation of this enzyme the human form of this enzyme has been cloned and the functional protein has been expressed in Escherichia coli. A 3.1-kilobase cDNA was isolated which contains the complete coding region of 681 amino acids. Expression of the cDNA in E. coli produced a protein of approximately 77 kDa and increased GFAT activity 4.5-fold over endogenous bacterial levels. Recombinant GFAT activity was inhibited 51% by UDP-GlcNAc whereas bacterial GFAT activity was insensitive to inhibition by UDP-GlcNAc. On the basis of these results we conclude that: 1) functional human GFAT protein was expressed, and 2) the cloned human cDNA encodes both the catalytic and regulatory domains of GFAT since the recombinant GFAT was sensitive to UDP-GlcNAc. Overall, the development of cloned GFAT molecular probes should provide new insights into the development of insulin resistance by allowing quantitation of GFAT mRNA levels in pathophysiological states such as non-insulin-dependent diabetes mellitus and obesity.
McKnight
G L
GL
ZymoGenetics, Inc., Seattle, Washington 98105.
Mudri
S L
SL
Mathewes
S L
SL
Traxinger
R R
RR
Marshall
S
S
Sheppard
P O
PO
O'Hara
P J
PJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D004247:DNA; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)