Search PubMed⌕ Search

Biomedical subjects

H S Sul

Publications and source records attributed to H S Sul.

At least 55 records · Page 3Linked to original sources
PubMed1991

Isolation and characterization of the transcriptionally regulated mouse liver (B-type) phosphofructokinase gene and its promoter.

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.

Animals↗
PubMed1989

Phosphofructokinase isozyme expression during myoblast differentiation.

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.

Animals↗
PubMed1989

Structure of mouse fatty acid synthase mRNA. Identification of the two NADPH binding sites.

Overlapping cDNA clones corresponding to 3.3 kb covering the carboxy-half and 3' non-coding regions of the single 8.2 kb mouse fatty acid synthase mRNA were isolated and sequenced. The sequence coded for 838 amino acid residues, followed by termination codon TAG, 771 nucleotides of 3' untranslated sequence and a poly A tail. For the first time, the two putative components of the NADPH binding sites of fatty acid synthase were identified, thereby making it possible to assign the enoyl reductase and beta-ketoacyl reductase domains of the multifunctional fatty acid synthase. Overall, the deduced amino acid sequence provides the domains for enoyl reductase, beta-ketoacyl reductase, acyl carrier protein and thioesterase of the mouse fatty acid synthase.

Amino Acid Sequence↗
PubMed1989

Hormonal regulation of mouse fatty acid synthase gene transcription in liver.

Blot analysis of poly(A+) RNA showed a rapid and dramatic insulin induction of fatty acid synthase (FAS) mRNA in diabetic mouse liver. Insulin administration to diabetic mice increased the mRNA level 4-fold within 1 h, and a maximum of 19-fold was reached in 6 h. The nutritional induction of FAS mRNA by fasting/refeeding was abolished by streptozotocin-induced diabetes and also by dibutyryl cAMP administered during refeeding in normal animals, demonstrating the roles of insulin and cAMP during nutritional manipulation. Run-on transcription analysis with isolated nuclei showed that the transcription rate of the FAS gene increased 3.5-fold after 30 min, reached a maximum of 7-fold after 2 h of insulin administration in diabetic animals, and was maintained at the maximum level to 6 h. The cAMP, administered to previously fasted normal mice during refeeding of a high carbohydrate diet, abolished the increased transcription of the FAS gene caused by nutritional manipulation. These results indicate positive control by insulin and negative control by dibutyryl cAMP of FAS gene transcription. Furthermore, the effect of insulin on the FAS mRNA level was abolished by cycloheximide administration. This indicates that ongoing protein synthesis is necessary for the transcriptional activation of the FAS gene by insulin.

Animals↗
PubMed1988

Liver (B-type) phosphofructokinase mRNA. Cloning, structure, and expression.

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.

Amino Acid Sequence↗
PubMed1988

Cloning and expression of mouse fatty acid synthase and other specific mRNAs. Developmental and hormonal regulation in 3T3-L1 cells.

Mouse liver mRNA enriched in sequence coding for fatty acid synthase by sucrose density gradient centrifugation was used as template for cDNA synthesis. Double-stranded cDNA sequences were inserted into pBR322 and lambda gt10 and cloned. Clones containing putative cDNA sequences for fatty acid synthase were identified by differential hybridization with [32P] cDNAs synthesized from sucrose gradient-purified liver mRNA from mice fasted or fasted and refed a high carbohydrate diet. Thirteen out of 45 differentially expressed clones were found to contain sequences complementary to fatty acid synthase mRNA. Northern blot analysis revealed that, unlike in avian and rat tissues, a single 8.2-kilobase (kb) mRNA codes for fatty acid synthase in mice. In addition to the fatty acid synthase cDNA clones, cDNA clones to two specific mRNAs of 5.1 and 7.2 kb were selected to study nutritional, hormonal, and developmental regulation at the level of mRNA abundance in mouse liver and in 3T3-L1 cells. The induction of fatty acid synthase in the livers of previously fasted mice fed a high carbohydrate diet was controlled pretranslationally by modulation of the fatty acid synthase mRNA content. The level of the two mRNAs with sizes of 5.1 and 7.2 kb were also elevated dramatically in the liver of mice fasted and refed a high carbohydrate diet. A detectable, but very low level of fatty acid synthase mRNA was found in 3T3-L1 preadipocytes. During the differentiation to adipocytes, both the rate of synthesis and relative mRNA level for fatty acid synthase increased in a parallel fashion to a maximum of 17-fold. The levels of 5.1- and 7.2-kb mRNAs, coding for proteins possibly involved in lipogenesis, increased 45- and 25-fold, respectively, during differentiation of 3T3-L1 adipocytes. Treatment of mature 3T3-L1 adipocytes with insulin elicited a 3-fold increase in both rate of synthesis and mRNA content of fatty acid synthase, while treatment with dibutyryl cAMP caused a 60% decrease in fatty acid synthase mRNA and an 80% decrease in the rate of the enzyme synthesis, indicating pretranslational control of fatty acid synthase expression by the lipogenic and lipolytic hormones. Similarly, insulin caused a 2- to 3-fold increase in both 7.2- and 5.1-kb mRNAs and dibutyryl cAMP decreased the levels of 7.2- and 5.1-kb mRNAs to 10 and 20% of control levels, respectively.

Adipose Tissue↗
PubMed1987

Structure and expression of murine malic enzyme mRNA. Differentiation-dependent accumulation of two forms of malic enzyme mRNA in 3T3-L1 cells.

Many murine cells express two mRNAs with markedly different sizes (2.0 and 3.1 kilobases (kb)) that hybridize with cDNA probes for cytosolic malic enzyme ((S)-malate NADP+ oxidoreductase (oxaloacetate-decarboxylating, EC 1.1.1.40). A series of overlapping cDNA clones corresponding to 3129 nucleotides of malic enzyme mRNA was isolated and sequenced to determine the relationship between the two mRNAs and establish the primary structure of mouse malic enzyme. The larger mRNA has an open reading frame of 1716 nucleotides followed by a 3' untranslated region of 1348 nucleotides. The sequence of an exceptionally G/C-rich (88%) portion (65 nucleotides) of the 5' noncoding region was also established. An uncommon poly A addition signal (AUUAAA) is used during the processing of the 3.1-kb mRNA. The 2.0-kb mRNA results from the utilization of another poly A addition signal that truncates the 3' noncoding sequence by approximately 1 kb. The mRNA coding sequence indicates that the malic enzyme subunit contains 572 amino acid residues and has a Mr of 64,000. Two putative components of an NADP-binding domain are located between residues 100 and 165. During the differentiation of 3T3-L1 preadipocytes into adipocytes both the rate of synthesis and relative mRNA concentration for malic enzyme and another lipogenic enzyme, ATP-citrate lyase, are coordinately increased 5-7-fold. However, as preadipocytes approach confluence, the mRNA levels for both lipogenic enzymes transiently increase 3-4-fold, whereas the rates of synthesis of the two proteins are only slightly elevated. Thus, lipogenic enzyme expression is controlled at a pretranslational level during adipogenesis, but the accumulation of the same enzymes may also be subject to translational control in the fibroblast-like preadipocytes. In contrast, mRNA coding for a third enzyme required for lipogenesis, glycerol-3-phosphate dehydrogenase, is not detected in 3T3-L1 preadipocytes, but rapidly accumulates during adipocyte development.

Adipose Tissue↗
PubMed1984

Coordinate regulation of the biosynthesis of ATP-citrate lyase and malic enzyme during adipocyte differentiation. Studies on 3T3-L1 cells.

The biosynthesis and degradation of two lipogenic enzymes were studied during the differentiation of 3T3-L1 preadipocytes into adipocytes. The activity and mass of malic enzyme, rose by an order of magnitude during adipocyte development and the enzyme accounted for 0.3% of the cytosol protein in mature fat cells. Similarly, the activity and amount of ATP-citrate lyase increased approximately 7-fold during the adipose conversion. The relative rates of synthesis of the two enzymes were less than or equal to 0.02% in preadipocytes, but increased sharply as the cells began to differentiate. Maximal steady state rates of malic enzyme and ATP-citrate lyase synthesis in 3T3-L1 adipocytes were 13- and 8-fold higher, respectively, than the basal rates in preadipocytes. In contrast, the half-lives of malic enzyme (67 h) and ATP-citrate lyase (47 h) were not altered during adipocyte development. Thus, accelerated rates of enzyme synthesis account for the differentiation-dependent accumulation of the two lipogenic enzymes. Increased rates of malic enzyme, ATP-citrate lyase, and fatty acid synthetase biosynthesis are expressed in a highly coordinated manner during adipocyte differentiation and are associated with parallel elevations in the levels of translatable mRNAs for these enzymes.

ATP Citrate (pro-S)-Lyase↗
PubMed1984

Cloning of cDNA sequences for murine ATP-citrate lyase. Construction of recombinant plasmids using an immunopurified mRNA template and evidence for the nutritional regulation of ATP-citrate lyase mRNA content in mouse liver.

Mouse liver mRNA that was enriched in sequences coding for ATP-citrate lyase by polysome immunoadsorption was used as a template for cDNA synthesis. Double-stranded cDNA sequences were inserted into the plasmid pBR322 and cloned in Escherichia coli RR1. Twenty-seven plasmids containing putative cDNA sequences for ATP-citrate lyase were identified by differential hybridization with single-stranded 32P-cDNAs synthesized from immunopurified mRNA, sucrose gradient-purified ATP-citrate lyase mRNA, and mRNA isolated from the livers of mice that were nutritionally induced or de-induced for ATP-citrate lyase biosynthesis. A subgroup of five recombinant plasmids was characterized further in hybridization-selection experiments. Each of these plasmids positively selected ATP-citrate lyase mRNA as determined by in vitro translation and specific immunoprecipitation. The length of ATP-citrate lyase mRNA was estimated to be 4900 bases in a Northern blot analysis. A 32P-cDNA probe derived from a 1500-base pair insert was used to investigate the basis for the 20-30-fold induction of ATP-citrate lyase that occurs when starved animals are fed a high carbohydrate/low fat diet. Dot-blot hybridization analysis disclosed that the relative content of liver ATP-citrate lyase mRNA increased 25-fold after 15 h of refeeding, indicating that the synthesis of the lipogenic enzyme is controlled at a pretranslational level in the nutritional paradigm.

ATP Citrate (pro-S)-Lyase↗
PubMed1984

Cloning of cDNA sequences for murine malic enzyme and the identification of aberrantly large malic enzyme mRNA in MOD-1 null mice.

Polysomes containing cytosolic malic enzyme mRNA and malic enzyme nascent chains were complexed with specific antibodies and purified by chromatography on protein A-Sepharose. When poly(A+) mRNA derived from the immunoselected polysomes was translated in vitro, full length malic enzyme (subunit Mr = 58,000) accounted for a significant fraction (approximately 20%) of the polypeptides synthesized. Double-stranded cDNA, synthesized using partially purified malic enzyme mRNA as a template, was inserted into pBR 322 and cloned. Twenty-five candidate malic enzyme cDNA clones were identified by differential hybridization. Four clones were studied further and each of these was shown to have malic enzyme cDNA sequences by hybrid-selected translation and specific immunoprecipitation. Plasmid pME1, which contains a 1400-base pair insert, hybridized to two mouse liver malic enzyme mRNAs with lengths of 2300 and 3500 bases. Similar analyses were performed on liver mRNAs isolated from MOD-1 mutant mice which lack cytosolic malic enzyme activity. These Northern blots disclosed a pair of aberrantly large malic enzyme mRNAs with lengths of 2800 and 4000 bases. Furthermore, anti-malic enzyme antibodies exclusively precipitated a polypeptide translation product with a Mr of 77,000 when MOD-1 mRNA was used to direct in vitro protein synthesis. Thus, it is possible that MOD-1 malic enzyme mRNA contains an additional polypeptide coding sequence. The translation of such a sequence might disrupt enzyme function and/or markedly decrease enzyme stability. The malic enzyme cDNA probe was also employed to demonstrate that the induction of malic enzyme in the livers of previously starved mice that were fed a high carbohydrate, fat-free diet was controlled pretranslationally by a parallel modulation of the malic enzyme mRNA concentration.

Animals↗
PubMed1982

Cardiac phosphorylase kinase. Deactivation by selective dephosphorylation of alpha' and beta subunits.

We have documented previously that although the phosphorylation of the beta subunit of cardiac phosphorylase kinase plays a major role in activation of the enzyme, enzyme activity is also modulated by either cAMP-dependent or cAMP-independent phosphorylation of the alpha' subunit (Sul, H. S., Cooper R. H., Whitehouse S., and Walsh D. A. (1982) J. Biol. Chem. 257, 3484-3490). This paper reports that deactivation of 32P-labeled cardiac phosphorylase kinase by protein phosphatase occurs concomitantly with the dephosphorylation of either the alpha' or beta subunits. However, enzyme activation attributable to alpha' subunit phosphorylation is still apparent after dephosphorylation of the beta subunit and, similarly, that caused by beta subunit phosphorylation is maintained after alpha' subunit dephosphorylation. These data support out previously stated conclusion that both alpha' and beta subunit phosphorylation regulates the activity of cardiac phosphorylase kinase.

Adenosine Triphosphate↗
PubMed1981

Phosphorylation and activation of the cardiac isoenzyme of phosphorylase kinase by the cAMP-dependent protein kinase.

The cAMP-dependent protein kinase catalyzes the phosphorylation of the alpha- and beta-subunits of the cardiac isozyme of phosphorylase kinase. beta-Subunit phosphorylation achieves a maximum level of between 1 to 2 mol of phosphate/mol of phosphorylase kinase, a value less than the stoichiometric content of beta-subunits in the enzyme. This, less than stoichiometric incorporation, is not a result of the presence of endogenous phosphate in equivalent sites in the remaining beta-subunit moieties. Pretreatment of phosphorylase kinase with phosphoprotein phosphatase, under conditions proven to dephosphorylate such sites, does not modify the observed extent of beta-subunit phosphorylation. alpha'-Subunit phosphorylation is initiated at a slower rate than beta but achieves a higher maximum level of incorporation. alpha'-Subunit phosphorylation, but not the extent of beta-subunit phosphorylation, is stimulated by MnCl2 and partially inhibited by NaF; neither is effected by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The activation of cardiac phosphorylase kinase that occurs concomitantly with phosphorylation appears to be dependent upon phosphate incorporation into both the alpha- and beta-subunits. At low levels of activation a close correlation is observed between activation and either alpha-subunit phosphorylation, beta-subunit phosphorylation, or total phosphorylation. However, the cAMP-dependent catalyzed phosphorylation of alpha, at a time after which beta-subunit phosphorylation is already maximal, also results in activation of cardiac phosphorylase kinase.

Animals↗
PubMed1980

Purification and properties of the cardiac isoenzyme of phosphorylase kinase.

This study reports the purification of bovine cardiac phosphorylase kinase to near homogeneity (approximately 1240-fold), as based upon SDS gel electrophoresis and sucrose density gradient ultracentrifugation (co-migration of enzyme activity and protein with constant specific activity throughout the peak). The molecular weight of the enzyme following purification is identical with that in the soluble extract from beef hearts, and similar to that of the rabbit skeletal muscle isoenzyme (i.e. approximately 1.3 X 10(6)) based on co-migration in sucrose density gradient ultracentrifugation. The molecular weights of the subunits determined by SDS gel electrophoresis are: alpha', 134,000; beta, 125,000; gamma, 48,000. The subunit stoichiometry is determined to be alpha 1 beta 1.01 gamma 1.35 with a faint band co-migrating with purified bovine brain calmodulin. The enzyme displays little, if any, activity below pH 6.0, but activity increases markedly in the pH range of 6.8 to 8.2. After phosphorylation with pure cAMP-dependent protein kinase catalytic subunit and [gamma-32P]ATP (0.14 mM ATP, 4 mM magnesium acetate) in which the beta subunit was maximally phosphorylated (stoichiometry, 0.25 mol/mol; ratio of 32P in the alpha' and beta subunits, 1.90:1), enzyme activity was increased approximately 2-fold at pH 6.8, while at higher pH values, the effect of phosphorylation was less marked (20 to 50% increase at pH 8.2). Ca2+ is required for enzyme activity; ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) (0.5 mM) completely inhibited the activity of th nonphosphorylated and phosphorylated enzyme, and activity was restored by the addition of Ca2+ (Ka for Ca2+, 1.94 and 1.35 microM for the nonphosphorylated and phosphorylated enzyme, respectively.

Animals↗
PubMed1979

Comparison of the adenine nucleotide translocase in hepatomas and rat liver mitochondria.

Various biochemical properties of the adenine nucleotide translocase were compared with mitochondria prepared from control and host liver, and Morris hepatomas 7777, 7800 and 5123C. The transport of phosphoenolpyruvate on the adenine nucleotide translocase was found to be three to four times more active, and inhibition of the transporter by palmitoyl-CoA and atractylate considerably less in hepatoma the active transport of phosphoenolypyruvate was associated with a greater stimulation of calcium egress from the mitochondria matrix by the anion in the hepatoma. The diminished sensitivity of the adenine nucleotide translocase to palmitoyl-CoA in hepatoma mitochondria was associated with lower levels of long chain acyl-CoA esters in the whole tissue. A change in activation energy at 6 degrees C for the adenine nucleotide translocase was found in host liver mitochondria while no break point in the temperature curve was observed in hepatoma mitochondria. These results are most consistent with a change in the structure-function relationship of hepatoma mitochondria due to differences in lipid composition.

Acyl Coenzyme A↗