Hideo Mizunuma

Identification of genetic mutations in Japanese patients with fructose-1,6-bisphosphatase deficiency

Fructose-1,6-bisphosphatase (FBPase) deficiency is an autosomal recessive inherited disorder and may cause sudden unexpected infant death. We reported the first case of molecular diagnosis of FBPase deficiency, using cultured monocytes as a source for FBPase mRNA. In the present study, we confirmed the presence of the same genetic mutation in this patient by amplifying genomic DNA. Molecular analysis was also performed to diagnose another 12 Japanese patients with FBPase deficiency. Four mutations responsible for FBPase deficiency were identified in 10 patients from 8 unrelated families among a total of 13 patients from 11 unrelated families; no mutation was found in the remaining 3 patients from 3 unrelated families. The identified mutations included the mutation reported earlier, with an insertion of one G residue at base 961 in exon 7 (960/961insG) (10 alleles, including 2 alleles in the Japanese family from our previous report [46% of the 22 mutant alleles]), and three novel mutations--a G-->A transition at base 490 in exon 4 (G164S) (3 alleles [14%]), a C-->A transversion at base 530 in exon 4 (A177D) (1 allele [4%]), and a G-->T transversion at base 88 in exon 1 (E30X) (2 alleles [9%]). FBPase proteins with G164S or A177D mutations were enzymatically inactive when purified from E. coli. Another new mutation, a T-->C transition at base 974 in exon 7 (V325A), was found in the same allele with the G164S mutation in one family (one allele) but was not responsible for FBPase deficiency. Our results indicate that the insertion of one G residue at base 961 was associated with a preferential disease-causing alternation in 13 Japanese patients. Our results also indicate accurate carrier detection in eight families (73%) of 11 Japanese patients with FBPase deficiency, in whom mutations in both alleles were identified.

Three-step purification method and characterization of the bovine brain 90-kDa heat shock protein.

A protein that cross-reacted with antibody against the 90-kDa heat shock protein (HSP90) of a mouse lymphoma cell line was purified from bovine brain by three steps. Fifty milligrams of the 90-kDa protein was recovered from 350 g of the brain cortex. The sedimentation coefficient and Stokes radius of the purified protein were 6.0 s and 6.7 nm, respectively. The molecular weight was calculated to be 170,000. The molecule was composed of two identical 90-kDa subunits. A partial amino acid sequence (23 residues) of this protein was homologous (96%) to human HSP90 (the sequence of 174-196). These facts led to the identification of the 90-kDa brain protein with HSP90. In bovine tissues, the brain contained this protein at a remarkably high concentration. The brain HSP90 was separable from glucocorticoid receptor by heparin-agarose and DNA-cellulose columns. It is concluded that HSP90 is present in brain cytosol and mostly as free molecules. Immunohistochemical studies showed that the protein was localized in nerve excitable cells. It was not found in nuclei but in cytosol.

Purification and Properties of Mouse Liver Fructose 1,6-Bisphosphatase

A simple procedure has been developed for the purification of mouse liver and kidney fructose-1,6-bisphosphatase. In addition to the conventional method, including substrate elution from phosphocellulose, Blue Sepharose column chromatography made the purification procedure highly reproducible. The enzyme from rabbit liver was also purified by this method with a small modification. The isolated preparation was electrophoretically homogeneous. The mouse liver enzyme was identical with the kidney enzyme, and different from the rabbit liver enzyme electrophoretically. The structural properties and the amino acid composition were similar to those of this enzyme from other mammalian livers; the molecular weight was 143,000, subunit size was 37,500, S20, w was 7.0, and partial specific volume was 0.74. Cysteine and methionine residues amounted to 5-6 mol per subunit. Tryptophan was not detected. The Km value for fructose-1,6-bisphosphate was 1.3 microM. The Ki value for AMP was 19 microM. EDTA strongly activated the activity of the mouse liver enzyme at neutral pH. A partial proteolytic digestion of the mouse liver enzyme decreased the activity at neutral pH, and increased it at alkaline pH.

Activation of Na+-K+-adenosine triphosphatase by spermine

Abstract Spermine activated Na+-K+-ATPase when the concentrations of K+ and ATP were low, whereas it inhibited K+-dependent and ouabain-inhibitable monophosphatase. The activating effect of sperimine was not due to the substitution for K+ or Na+. Excess K+ inhibited Na+-K+-ATPase partially, and reduced the spermine activation. When 1 mM ATP was used, spermine at higher concentrations inhibited Na+-K+-ATPase, and did not activate at all. It is suggested that the K+-sites essential to Na+-K+-ATPase and the K+-phosphatase co-exist at different places of the enzyme.

Effect of Mn2+ on fructose 2,6-bisphosphate inhibition of mouse liver, intestinal, and muscle fructose-1,6-bisphosphatases.

Fructose 2,6-bisphosphate inhibited all three fructose-1,6-bisphosphatases from the liver, intestine, and muscle of the mouse. The sensitivity of the liver enzyme to the inhibitor was significantly diminished when Mg2+ was replaced by Mn2+ as the activating cation. Inhibition of the liver enzyme by fructose 2,6-bisphosphate decreased as the concentration of the metal activator, Mn2+ or Mg2+, increased. The respective I50 values obtained by extrapolation of metal ion concentrations to zero were 40 microM with Mn2+ and 0.25 microM with Mg2+. The extent of desensitization to either fructose 2,6-bisphosphate or AMP inhibition by Mn2+ decreased in the order of the liver, intestine, and muscle enzyme. Only in the case of the liver enzyme was the substrate cooperativity induced by fructose 2,6-bisphosphate in the presence of Mg2+. In all three isoenzymes from the mouse, fructose 2,6-bisphosphate greatly potentiated the AMP inhibition of the enzyme in the presence of either Mg2+ or Mn2+. The liver enzyme with Mn2+ in addition to Mg2+ was still active in the presence of less than 1 microM fructose 2,6-bisphosphate, even though AMP was present at 100-200 microM.

Evidence for the intestinal type of fructose 1,6-bisphosphatase in mouse, rat, and golden hamster

Abstract The intestinal type of fructose 1,6-bisphosphatase was studied in six mammalian species. The immunotitration with antiserum against the mouse intestinal enzyme and measurement of isoelectric points confirmed that mouse intestinal fructose 1,6-bisphosphatase is a new isoenzyme different from the liver or muscle enzymes. In the rat and golden hamster both the Ki value for AMP and the isoelectric point of the intestinal enzyme were also clearly different from those of the muscle and liver enzymes of these species. These animals, as well as the mouse, seem to have the specific intestinal type of fructose 1,6-bisphosphatase. The levels of the liver enzyme activity in the animal species studied were nearly proportional to the magnitudes of their respective Ki values of the enzyme for AMP. On the other hand, the intestinal enzyme seems to be grouped into two classes among the species studied, that is, one having higher Ki with a higher level of activity in the tissue (golden hamster, guinea pig, and rabbit) and another having lower Ki values with a lower level of activity (mouse, rat, and dog). This grouping of animals was not consistent with that by the isoenyzme type in the tissue, that is, by whether the specific intestinal type is present or not.

Monocytes, not lymphocytes, show increased fructose-1,6-diphosphatase activity during culture

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Mouse thymoma cell line expresses a gluconeogenic enzyme, fructose 1,6-bisphosphatase

Fructose 1,6-bisphosphatase was observed in a thymic lymphoma cell line, WEH17.1 (11.5 +/- 0.8 munits/mg cytosol protein). Only a trace amount of the enzyme activity was observed in normal thymus tissue. The WEH17.1 enzyme had a pH optimum at around 7.5. The AMP-concentration giving 50% inhibition of the activity was about 73 microM. That of the crude mouse liver enzyme was 35 microM. The antibodies against the liver and intestinal enzymes cross-reacted with the WEH17.1 enzyme with a lower affinity than the liver enzyme. Immunoblot showed that the subunit molecular weight of the WEH17.1 enzyme was the same as that of the liver enzyme.

[60] Fructose-1,6-bisphosphatase from mouse and rabbit intestinal mucosa

Publisher Summary This chapter describes an assay method and the purification procedure for fructose-l,6-bisphosphatase from mouse and rabbit intestinal mucosa. The purified enzyme from intestinal mucosa has similar properties to those of the known liver, kidney, and muscle fructose-l,6-bisphosphatases, such as molecular and subunit molecular weights, adenosine monophosphate (AMP) inhibition, pH optimum, and the metal requirement. The production of fructose 6-phosphate is measured spectrophotometrically by following the reduction of NADP+ with glucose-6-phosphate isomerase and glucose-6-phosphate dehydrogenase. Purification of the enzyme from mouse small intestinal mucosa involves extraction, heat treatment, phosphocellulose treatment, phosphocellulose chromatography, and Blue Sepharose treatment. Intestinal homogenates contain a proteolytic activity that catalyzes the conversion of fructose-1,6-bisphosphatase to a form having the increased activity at alkaline pH. The heat treatment inactivates the proteolytic activity and there is no evidence that the enzyme is modified after this step. Rabbit intestinal enzyme is well adsorbed on Blue Sepharose gel and is completely eluted at high purity from the gel with the low concentration of AMP, while mouse intestinal enzyme is poorly adsorbed on Blue Sepharose gel around pH 6 under the present conditions.

Induction of fructose 1,6-bisphosphatase in HL-60 leukemia cells by retinoic acid.

The expression of the fructose 1,6‐bisphosphatase gene in HL‐60 cells was induced by retinoic acid. The levels of mRNA, enzyme activity and enzyme protein in the cell line began to rapidly increase after culturing with retinoic acid for 72 h. Retinoic acid dosedependently increased the enzyme activity with maximal stimulation at 1 μM. The responses of the fructose 1,6‐bisphosphatase gene expression by retinoic acid were markedly slower than those of the enzyme expression by 1α,25‐ dihydroxyvitamin D3. When HL‐60 cells were cultured in the presence of both retinoic acid and 1α,25‐dihydroxyvitamin D3, the effects of the two agents on enzyme activity, protein and mRNA were additive.