RESUMEN
We have isolated and characterized a mouse gene encoding liver (B-type) phosphofructokinase, a key regulatory enzyme in glycolysis. The gene spans approximately 21.5 kilobase pairs and consists of 22 exons. Compared with the muscle (A-type) phosphofructokinase gene, the sizes of the introns are different although exon lengths are highly conserved. Two transcription start sites 10 bases apart were determined by primer extension experiments. The immediate 5' sequence does not possess a TATA or CCAAT box but contains multiple GC boxes (positions -10, -43, -50, -62, and +28 in the 5'-untranslated region) which may be Sp1-binding sites. An unusual feature of 200 base stretches of CT repeats is present at position -480 to -693. In addition, direct repeats of CTCGAAGGAG are found at positions -447 and -478. DNase I footprinting showed five regions where liver nuclear proteins may interact. Two proximal 5'-flanking regions spanning -1 to -20 and -30 to -70, which contain GC boxes. Also protected was a region spanning -70 to -90, which contains an AP-1 like sequence (TCAGTCA). The consensus AP-1 sequence, however, did not inhibit footprinting, indicating involvement of a distinct protein. Two distal regions spanning from -450 to -470 and from -500 to -520 were also protected. The former is positioned between the direct repeats and the latter is at the start of the CT repeats. The rate of transcription of the liver phosphofructokinase gene, as measured by run-on assays, increased 5-fold in livers of previously starved mice fed a high carbohydrate diet compared to starved controls. Administration of dibutyryl cAMP blocked the increase in transcription caused by refeeding. Functional analysis of the promoter region of the gene will be necessary to elucidate the mechanisms of transcriptional regulation by fasting/refeeding and by cAMP. These results provide a useful system for the study of regulatory elements in liver phosphofructokinase gene transcription.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Hígado/enzimología , Fosfofructoquinasa-1/genética , Regiones Promotoras Genéticas , Transcripción Genética , Animales , Secuencia de Bases , AMP Cíclico/metabolismo , ADN , Desoxirribonucleasa I , Ingestión de Alimentos , Exones , Ayuno , Intrones , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mapeo RestrictivoRESUMEN
Isozyme expression of phosphofructokinase (PFK), the key regulatory enzyme for glycolysis, was studied during differentiation of mouse C2 myoblasts to myotubes. The total PFK activity increased 20-fold during in vitro myogenesis. The rate of synthesis, relative to the rate of total protein synthesis, measured by pulse labeling and immunoprecipitation was lowest for muscle PFK (PFK-A), 0.008% in myoblasts, while those for liver (PFK-B) and brain (PFK-C) PFK were 0.017 and 0.014%, respectively. The relative rate of PFK-A synthesis increased sharply (5-fold) at an initial period of differentiation (8 h) and reached maximum of 10-fold at 48 h, to make PFK-A the major isoform synthesized in myotubes. The relative rates of synthesis for both PFK-B and PFK-C did not change drastically, decreasing slightly at 8 h, but were restored to 1.5-2-fold of myoblasts. cDNA sequences coding for mouse muscle PFK were cloned and used along with those for mouse liver PFK, which we have previously cloned, to measure by Northern blot analysis under highly stringent conditions the steady-state mRNA concentrations for muscle and liver PFK during C2 differentiation. The hybridizable mRNA level for PFK-A increased gradually, reaching 13-fold at 48 h when 80% of cells was fused to myotubes. The PFK-A mRNA level at 96 h was 90-fold of that for myoblasts. In contrast, the mRNA level for PFK-B increased slightly during differentiation, showing a maximum of 4-fold at 96 h. These results indicate isozyme-specific control of muscle PFK gene expression during C2 myoblast differentiation.
Asunto(s)
Isoenzimas/metabolismo , Músculos/enzimología , Fosfofructoquinasa-1/metabolismo , Animales , Encéfalo/enzimología , Diferenciación Celular , Línea Celular , ADN/genética , Regulación de la Expresión Génica , Glucólisis , Hígado/enzimología , Ratones , Músculos/citología , ARN Mensajero/genéticaRESUMEN
Mouse liver mRNA enriched in sequences coding for liver phosphofructokinase by polysome immunoadsorption was used as a template for the synthesis of cDNA. The double-stranded cDNA was inserted into the expression vector lambda gt11 and cloned. Preliminary identification of clones containing cDNA sequences for phosphofructokinase was made by screening the library with anti-rat liver phosphofructokinase serum and horseradish peroxidase-conjugated goat anti-rabbit IgG as second antibody. Subsequently, by selecting antibodies specific to fusion proteins expressed by putative clones and by reacting with Western blots of mouse liver proteins several clones were positively identified as containing liver phosphofructokinase sequences. A cDNA clone corresponding to 2708 nucleotides of liver phosphofructokinase mRNA was further characterized and sequenced. The liver phosphofructokinase mRNA has an open reading frame of 2343 nucleotides followed by a 3'-untranslated region of 303 nucleotides. The G/C-rich (76%) portion of the 5'-untranslated region precedes a characteristic translational start site of CCGCC(AUG). The mRNA coding sequence indicates that the liver phosphofructokinase subunit is composed of 780 amino acid residues and has a Mr of 85,000. Comparison of the deduced amino acid sequence of mouse liver phosphofructokinase with the known rabbit muscle phosphofructokinase shows 68% homology. The N-half of the liver phosphofructokinase has conserved substrate binding sites for ATP and fructose-6-P. The 25 C-terminal residues, which contain the ATP inhibitory site, are the least homologous (20%) but contain a putative phosphorylation site (Arg-Arg-X-X-Ser). The liver phosphofructokinase mRNA is under nutritional and hormonal regulation. The liver phosphofructokinase mRNA level increased 4-fold when previously starved mice were refed a high carbohydrate, fat-free diet. This increase in mRNA level was blocked by 50% by the administration of dibutyryl cAMP. The induction of liver phosphofructokinase mRNA by fasting/refeeding was also diminished in streptozotocin diabetic mice.
Asunto(s)
Hígado/enzimología , Fosfofructoquinasa-1/genética , ARN Mensajero/genética , Secuencia de Aminoácidos , Animales , Bacteriófago lambda/genética , Secuencia de Bases , Clonación Molecular , ADN/genética , ADN Recombinante , Técnicas para Inmunoenzimas , Ratones , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Conejos , Ratas , Homología de Secuencia de Ácido NucleicoRESUMEN
1. The aim of these studies was to investigate a mitochondrial basis for changes in gluconeogenesis during hibernation. 2. State 3 respiration rates in liver mitochondria from hibernating ground squirrels were reduced by 62-66%. The limiting reaction appeared to be electron transport, particularly in respiratory complex III. 3. The mitochondrial ATP + ADP + AMP content was reduced by 29% during hibernation; cellular adenine nucleotide content was unchanged. 4. Pyruvate carboxylation in intact mitochondria was decreased 75% during hibernation, although total pyruvate carboxylase activity was not lower. 5. Rates of gluconeogenesis in intact hepatocytes isolated from hibernators were lower than in cells from non-hibernators.