Aldolase and fructose bisphosphatase: key enzymes in the control of gluconeogenesis and glycolysis.
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High-throughput screening of cells expressing active catalytic antibody clones by flow cytometry is described. A fluorogenic retro-aldol retro-Michael substrate was designed and synthesized with incorporation of a chloromethyl moiety for intracellular retention. Hybridoma or transfected mammalian cells expressing catalytic antibody molecules could be rapidly isolated.
We studied 28 Polish hereditary fructose intolerant (HFI) patients (26 unrelated) by direct sequencing of the ALDOB coding region/splice sites. Eight different mutations were found including two novel ones (each found in two unrelated individuals): c.250delC (frameshift) and c.522 C > G (p.Y174X). The most frequent mutation c.448 G > C (p.A150P, 67% of chromosomes) was screened for in a group of 1049 randomly selected unrelated individuals. Eight (1:131) carriers were found allowing to estimate the HFI prevalence in Poland as 1:31,000.
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Coexistence of two different HNF1-like factors (AlF-A1 and AlF-A2) in rat liver is described. The two factors had similar molecular weight and the same sequence-specificity in DNA-binding. One of these factors AlF-A1 was retained on wheat germ agglutinin-sepharose column and eluted from it with N-acetylglucosamine, while AlF-A2 was not, suggesting that AlF-A1 is glycosylated. Using rat brain nuclear extract, both factors stimulated transcription in vitro, but their stimulatory effects differed from each other.
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