Concentration of liver and kidney fructose-1,6-diphosphatase determined by specific radioimmunoassay

Mazzotta, Mary Y.; Veneziale, Carlo M.

doi:10.1016/0005-2744(80)90051-0

Cited by 18 publications

(11 citation statements)

References 15 publications

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“…Since Fru-1,6-P2ase activity is elevated in diabetes and decreased by insulin treatment (12,13), it was of interest to determine whether these changes are caused by changes in mRNA abundance. Fig.…”

Section: Resultsmentioning

confidence: 99%

“…However, insulin acted more rapidly to reduce PEPCK mRNA levels, which were reduced to control levels after only 6 hr of insulin treatment (lane 8). The changes in Fru-1,6-P2ase mRNA were coincident with changes in the enzyme's activity seen in extracts from livers of diabetic and insulin-treated diabetic rats (data not shown) (12,13).…”

Section: Resultsmentioning

confidence: 99%

“…Phosphorylation of the yeast enzyme, on the other hand, at an amino-terminal extension (11) results in a marked inhibition of activity (6). Liver Fru-1,6-P2ase activity has been reported to increase during diabetes and starvation (12,13), suggesting that the enzyme is subject to long-term regulation at the levels of transcription and/or translation.…”

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confidence: 99%

“…The labeled probe was hybridized with the membranes at 42°C for 12-24 hr, after which the membranes were washed with four 500-ml washes of 15 mM NaCI/1.5 mM sodium citrate, pH 7.4/0.1% NaDodSO4 (30 min per wash) at 50°C. The membranes were then exposed to x-ray film for [12][13][14][15][16][17][18][19][20][21][22][23][24] hr with an intensifying screen at -70°C.…”

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cDNA sequence of rat liver fructose-1,6-bisphosphatase and evidence for down-regulation of its mRNA by insulin.

el-Maghrabi

Pilkis²,

Marker³

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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A coding-length clone of rat liver fructose-1,6-bisphosphatase (EC 3.1.3.11) was isolated by immunological screening of a cDNA library in Agtll. Its identity was verified by comparing the deduced amino acid sequence with that obtained by direct sequencing of a complete set of CNBr and proteolytic peptides from the purified protein. The enzyme subunit is composed of 362 amino acids and has N-acetylvaline as the amino-terminal residue. The cDNA, 1255 base pairs (bp) long, consisted of 1086 bp of coding region, 15 bp of 5' untranslated sequence, and 154 bp at the 3' untranslated end. The 3' untranslated sequence contained a polyadenylylation signal (AATAAA) followed after 30 bp by a stretch of 7 adenines at the end of the clone. The deduced amino acid sequence was identical to the primary sequence of the protein and confirmed the alignment of five nonoverlapping peptides. It also confirmed the 27-residue extension, unique to the rat liver subunit, ending with a carboxyl-terminal phenylalanine. RNA blot analyses using the radiolabeled liver cDNA as a probe revealed a single band of fructose-1,6-bisphosphatase mRNA, 1.4 kilobases long, in liver and kidney but not in nongluconeogenic tissues. Fructose-1,6-bisphosphatase mRNA was increased 10-fold in livers from diabetic rats and was reduced to control levels after 24 hr of insulin treatment, suggesting that the changes in enzyme activity observed in diabetes and after insulin treatment are due to alterations in mRNA abundance.The hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate, catalyzed by fructose-1,6-bisphosphatase (Fru-1,6-P2ase; EC 3.1.3.11), is an essential step in the gluconeogenic pathway (1) and is under both acute and long-term hormonal control. Acute hormonal regulation occurs primarily by modulation of the potent inhibitor fructose 2,6-bisphosphate (Fru-2,6-P2) (2, 3), which has been postulated to bind either to the active site (2, 4) or to a separate allosteric site (3, 5). Most of the studies on allosteric regulation of Fru-1,6-P2ase by Fru-2,6-P2 have been carried out on the rat liver enzyme, but identification ofthe Fru -2,6-P2 binding site has been hampered both by lack of primary sequence and x-ray crystallographic information and by lack of a cDNA clone with which to do site-directed mutagenesis studies. Yeast (6) and rat liver (7) Fru-1,6-P2ase have also been reported to be regulated by cAMP-dependent phosphorylation. Phosphorylation of the rat liver enzyme at a site(s) located on a carboxyl-terminal extension unique to this form (8, 9)t has been reported to increase enzyme activity (7, 10), but the changes have not been universally observed (8). Phosphorylation of the yeast enzyme, on the other hand, at an amino-terminal extension (11) results in a marked inhibition of activity (6). Liver Fru-1,6-P2ase activity has been reported to increase during diabetes and starvation (12, 13), suggesting that the enzyme is subject to long-term regulation at the levels of transcription and/or translation.Since the DNA encoding mammalian ...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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cDNA sequence of rat liver fructose-1,6-bisphosphatase and evidence for down-regulation of its mRNA by insulin.

el-Maghrabi

Pilkis²,

Marker³

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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“…12ng of phosphofructokinase) with increasing amounts of unlabelled enzyme (0-320ng) and a constant amount of antiserum sufficient to precipitate 40-50% of the labelled antigen. The final volume was made to 1.0ml with egg albumin buffer [0.1 M-sodium phosphate buffer (pH 7.5)11% ovalbumin/0.02% NaN3] (Mazzotta & Veneziale, 1980). All reaction mixtures were produced in duplicate and maintained at 4°C for 48 h. The second-stage incubation mixture consisted of 50gu1 of a diluted (1: 50) goat normal serum and 50pl of rabbit anti-goat serum in each reaction mixture.…”

Section: Radioiodination Ofphosphofructokinasementioning

confidence: 99%

Quantification of liver and kidney phosphofructokinase by radioimmunoassay in fed, starved and alloxan-diabetic rats

Donofrio¹,

Thompson²,

Reinhart³

et al. 1984

Biochemical Journal

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A newly developed specific radioimmunoassay was used to quantify phosphofructokinase protein directly and independently of assayable activity in liver and kidney cytosol of normal fed, starved and alloxan-diabetic rats. In the fed state, liver phosphofructokinase concentration was 0.096 pm and the kidney enzyme was 0.086 pM (jumol/kg of tissue). In the starved state (24h), liver and kidney phosphofructokinase concentrations decreased by 30%. Prolonged starvation up to 72h did not further decrease enzyme concentration. In liver, total enzyme content during starvation declined by more than 50%, secondary also to a decrease in liver weight. In the alloxan-diabetic rats, there was a 22% decrease in enzyme protein concentration in liver and kidney. Total enzyme content per liver actually decreased much more (46%), because diabetes also resulted in a decrease in liver size. In conjunction with assayable activity measurements, the results of the radioimmunoassay allowed us to calculate the apparent specific activity of the enzyme. The specific activity of the kidney enzyme was 2-3 times that of the liver. Little or no change in specific activity of the liver or kidney enzyme occurred as a result of starvation or chemically induced diabetes. Tissue enzyme concentrations of phosphofructokinase unequivocally reconcile the ultimate results of changing rates of synthesis and degradation and are useful data in the design of spectrophotometric, kinetic, aggregation-disaggregation and other studies.

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Mechanism of Action of Fructose 1,6‐Bisphosphatase

Benkovic

DeMaine

1982

Advances in Enzymology - And Related Areas of Molecular Biology

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Concentration of liver and kidney fructose-1,6-diphosphatase determined by specific radioimmunoassay

Cited by 18 publications

References 15 publications

cDNA sequence of rat liver fructose-1,6-bisphosphatase and evidence for down-regulation of its mRNA by insulin.

cDNA sequence of rat liver fructose-1,6-bisphosphatase and evidence for down-regulation of its mRNA by insulin.

Quantification of liver and kidney phosphofructokinase by radioimmunoassay in fed, starved and alloxan-diabetic rats

Mechanism of Action of Fructose 1,6‐Bisphosphatase

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