Ketohexokinase: expression and localization of the principal fructose-metabolizing enzyme.
Ketohexokinase (KHK, also known as fructokinase) initiates the pathway through which most dietary fructose is metabolized. Very little is known about the cellular localization of this enzyme. Alternatively spliced KHK-C and KHK-A mRNAs are known, but the existence of the KHK-A protein isoform has not been demonstrated in vivo. Using antibodies to KHK for immunohistochemistry and Western blotting of rodent tissues, including those from mouse knockouts, coupled with RT-PCR assays, we determined the distribution of the splice variants. The highly expressed KHK-C isoform localized to hepatocytes in the liver and to the straight segment of the proximal renal tubule. In both tissues, cytoplasmic and nuclear staining was observed. The KHK-A mRNA isoform was observed exclusively in a range of other tissues, and by Western blotting, the presence of endogenous immunoreactive KHK-A protein was shown for the first time, proving that the KHK-A mRNA is translated into KHK-A protein in vivo, and supporting the suggestion that this evolutionarily conserved isoform is physiologically functional. However, the low levels of KHK-A expression prevented its immunohistochemical localization within these tissues. Our results highlight that the use of in vivo biological controls (tissues from knockout animals) is required to distinguish genuine KHK immunoreactivity from experimental artifact.
Diggle
Christine P
CP
Leeds Institute of Molecular Medicine, University of Leeds, St James's University Hospital, Beckett Street, Leeds LS9 7TF, United Kingdom.
Shires
Michael
M
Leitch
Derek
D
Brooke
David
D
Carr
Ian M
IM
Markham
Alex F
AF
Hayward
Bruce E
BE
Asipu
Aruna
A
Bonthron
David T
DT
D017398:Alternative Splicing; D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D005631:Fructokinases / Q000235:genetics / Q000378:metabolism*; D005632:Fructose / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D009928:Organ Specificity; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013045:Species Specificity
D007527:Isoenzymes; D011994:Recombinant Proteins; D005632:Fructose; D005631:Fructokinases; C021475:ketohexokinase