Cloning and expression in Escherichia coli of a rat hepatoma cell cDNA coding for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

Crepin, Karine M.; Darville, Martine I.; Hue, Louis; Rousseau, Gg.

doi:10.1016/0261-5614(90)90102-x

Cited by 3 publications

(5 citation statements)

References 19 publications

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“…1). We then used an antiserum (anti‐L) raised against the unique N‐terminal sequence of the L isozyme [24]. This antiserum revealed the expected 55 000‐ M r band with purified liver enzyme ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Immunoblots were carried out with an anti‐L serum, which was raised in rabbits immunized with a synthetic peptide corresponding to the unique N‐terminal sequence of the L isozyme of PFK‐2 [24]. This antiserum did not immunoprecipitate PFK‐2 activity; however, it was active in immunoblots and specifically recognized its cognate antigen [24]. The BCL‐2 antiserum, which recognizes all PFK‐2 isozymes, was prepared as described [25].…”

Section: Methodsmentioning

confidence: 99%

“…Total RNA was extracted from rat epididymal fat pads by the guanidinium thiocyanate method [24] or from about 10 6 cultured cells with the ‘High pure RNA isolation kit’ from Boehringer‐Mannhein. The integrity of RNA and the absence of DNA was verified by gel staining.…”

Section: Methodsmentioning

confidence: 99%

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Expression and regulation of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐ bisphosphatase isozymes in white adipose tissue

Bruni

Vandoolaeghe

Rousseau

et al. 1999

European Journal of Biochemistry

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The aim of this work was to identify the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) isozyme(s) present in white adipose tissue. Ion-exchange chromatography of PFK-2 from rat epididymal fat pads yielded an elution pattern compatible with the presence of both the L (liver) and M (muscle) isozymes. This was consistent with a study of the phosphorylation of the purified adipose tissue enzyme by cAMP-dependent protein kinase, by specific labelling of the preparation with [2-32 P]fructose 2,6-bisphosphate and by reaction with antibodies. Characterization of the PFK-2/FBPase-2 mRNAs showed that mature adipocytes express the mRNA that codes for the L isozyme and the two mRNAs that code for the M isozyme. Preadipocytes expressed mRNA that codes for the M isozyme. Incubation of rat epididymal fat pads with adrenaline stimulated glycolysis but decreased fructose 2,6-bisphosphate concentrations without significant inactivation of PFK-2. These results support previous findings showing that fructose 2,6-bisphosphate is not involved in the adrenaline-induced stimulation of glycolysis in white adipose tissue.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Expression and regulation of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐ bisphosphatase isozymes in white adipose tissue

Bruni

Vandoolaeghe

Rousseau

et al. 1999

European Journal of Biochemistry

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“…The amino acid sequence of the protein from rat liver (Lively eta/., 1988), and the entire nucleotide sequence of the cognate cDNA have been determined (Colosia et aL, 1987). The coding sequence of the liver gene can be expressed as a functional protein in Escherichia coil (Crepin et al, 1989;Tauler et aL, 1989).…”

Section: Introductionmentioning

confidence: 99%

Carbon metabolism in leaves of transgenic tobacco (Nicotiana tabacum L.) containing elevated fructose 2,6‐bisphosphate levels

et al. 1995

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SummaryThe aim of this work was to investigate the role of fructose 2,6-bisphosphete (Fru 2,6-P z) during photosynthesis. The level of Fru 2,6-P2 in tobacco plants was elevated by the introduction of a modified mammalian gene encoding 6-phosphofructo-2-kinase (6-PF-2-K). Estimates of the metabolite control coefficient (C) for Fru 2,6-P 2 levels in response to increased 6-PF-2-K activity, suggest that small increases in 6-PF-2-K activity have little effect upon steadystate Fru 2,6-P 2 levels (C = +0.08 for a 0-58% increase in 6-PF-2-K activity). However, larger changes resulted in dramatic rises in Fru 2,6-P2 levels (C = +3.35 for 206-268% increase in 6-PF-2-K activity). Transgenic plants contained Fru 2,6-Pz levels in the dark that ranged from 104 to 230% of the level in wild-type tobacco. Plants with altered levels of Fru 2,6-P 2 were used to determine the effects of this signal metabolite upon carbohydrate metabolism during the initial phase of the light period. Here we provide direct evidence that Fru 2,6-P 2 contributes to the regulation of carbon partitioning in tobacco leaves by inhibiting sucrose synthesis.

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“…the relative activities of the kinase and phosphatase. For example, the liver and the skeletal muscle enzymes are the same gene products except for the N-termini (Crepin et al, 1989;Kitamura et al, 1989). The muscle enzyme lacks 22 amino acids from the N-terminus, and the first 9 amino acids from the N-terminus are different from those of the liver enzyme.…”

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confidence: 99%

Reversible unfolding of fructose 6‐phosphate, 2‐kinase:fructose 2,6‐bisphosphatase

1994

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Reversible unfolding of rat testis fructose 6-phosphate,2-kinase:fructose 2,6-bisphosphatase in guanidine hydrochloride was monitored by following enzyme activities as well as by fluorescence methodologies (intensity, emission maximum, polarization, and quenching), using both intrinsic (tryptophan) and extrinsic (5((2-(iodoacety1)amino) ethy1)naphthalene-1-sulfonic acid) probes. The unfolding reaction is described minimally as a 4-state transition from folded dimer + partially unfolded dimer -+ monomer -+ unfolded monomer. The partially unfolded dimer had a high phosphatase/kinase ratio due to preferential unfolding of the kinase domain. The renaturation reaction proceeded by very rapid conversion (less than 1 s) of unfolded monomer to dimer, devoid of any enzyme activity, followed by slow (over 60 min) formation of the active enzyme. The recovery rates of the kinase and the phosphatase were similar. Thus, the refolding appeared to be a reversal of the unfolding pathway involving different forms of the transient dimeric intermediates. Fluorescence quenching studies using iodide and acrylamide showed that the tryptophans, including Trp-15 in the N-terminal peptide, were only slightly accessible to iodide but were much more accessible to acrylamide. Fructose 6-phosphate, but not ATP or fructose 2,6-bisphosphate, diminished the iodide quenching, but all these ligands inhibited the acrylamide quenching by 25%. These results suggested that the N-terminal peptide (containing a tryptophan) was not exposed on the protein surface and may play an important role in shielding other tryptophans from solvent.

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Cloning and expression in Escherichia coli of a rat hepatoma cell cDNA coding for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

Cited by 3 publications

References 19 publications

Expression and regulation of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐ bisphosphatase isozymes in white adipose tissue

Expression and regulation of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐ bisphosphatase isozymes in white adipose tissue

Carbon metabolism in leaves of transgenic tobacco (Nicotiana tabacum L.) containing elevated fructose 2,6‐bisphosphate levels

Reversible unfolding of fructose 6‐phosphate, 2‐kinase:fructose 2,6‐bisphosphatase

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Cloning and expression in Escherichia coli of a rat hepatoma cell cDNA coding for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

Cited by 3 publications

References 19 publications

Expression and regulation of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐ bisphosphatase isozymes in white adipose tissue

Expression and regulation of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐ bisphosphatase isozymes in white adipose tissue

Carbon metabolism in leaves of transgenic tobacco (Nicotiana tabacum L.) containing elevated fructose 2,6‐bisphosphate levels

Reversible unfolding of fructose 6‐phosphate, 2‐kinase:fructose 2,6‐bisphosphatase

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Expression and regulation of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐ bisphosphatase isozymes in white adipose tissue

Expression and regulation of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐ bisphosphatase isozymes in white adipose tissue