The Experts below are selected from a list of 300 Experts worldwide ranked by ideXlab platform
Gerhard Kopperschläger - One of the best experts on this subject based on the ideXlab platform.
-
Photoaffinity labeling and photoaffinity cross-linking of Phosphofructokinase-1 from Saccharomyces cerevisiae by 8-azidoadeninenucleotides.
Archives of biochemistry and biophysics, 2001Co-Authors: Maren Knoche, Katrin Mönnich, Hans-jochen Schäfer, Gerhard KopperschlägerAbstract:Abstract Phosphofructokinase-1 from Saccharomyces cerevisiae is composed of four α- and four β-subunits, each of them carrying catalytic and regulatory bindings sites for MgATP. In this paper, various photoaffinity labels, such as 8-azidoadenosine 5′-triphosphate, 8-azido-1,N6-ethenoadenosine 5′-triphosphate, and 8-N3-3′(2′)-O-biotinyl-8-azidoadenosine 5′-triphosphate have been used to study their interaction with the enzyme in the dark and during irradiation. All nucleotidetriphosphates function as phosphate donor forming fructose 1,6-bisphosphate from fructose 6-phosphate. However, the kinetic analysis revealed distinctly differences between them. Photolabeling causes a decrease in enzyme activity to a similar extent, and ATP acts as competitive effector to inactivation. Three bifunctional diazidodiadeninedinucleotides (8-diN3AP4A, monoϵ-8-diN3AP4A, and diϵ-8-diN3AP4A) were applied for studying the spatial arrangement of the nucleotide binding sites. No cross-linking of the subunits was obtained by irradiation of the enzyme with 8-diN3AP4A. Photolabeling with diϵ-8-diN3AP4A resulted in the formation of two α-β cross-links with different mobilities in the SDS–polyacrylamide gel electrophoresis, while monoϵ-8-diN3AP4A yielded only one α-β cross-link. Because an interfacial location of the catalytic sites between two subunits is less likely, we suggest that the formation of cross-linked subunits may be the result of specific interactions of the bifunctional photolabels with regulatory sites at the interface of both subunits.
-
denaturation of Phosphofructokinase 1 from saccharomyces cerevisiae by guanidinium chloride and reconstitution of the unfolded subunits to their catalytically active form
Biochemistry, 2000Co-Authors: Jorg Bar, Ralph Golbik, Gerhard Hubner, Gerhard KopperschlägerAbstract:Unfolding and refolding of heterooctameric Phosphofructokinase-1 from Saccharomyces cerevisiae were investigated by application of kinetic, hydrodynamic, and spectroscopic methods and by use of guanidinium chloride (GdmCl) as denaturant. Inactivation of the enzyme starts at about 0.3 M GdmCl and undergoes a sharp unfolding transition in a narrow range of the denaturant concentration. The inactivation is accompanied by a dissociation of the enzyme into dimers (at 0.6 M GdmCl), which could be detected by changes of the circular dichroism and intrinsic fluorescence. Protein aggregates were observed from 0.7 to 1.5 M GdmCl that unfold at higher denaturant concentrations. Refolding of chemically denatured Phosphofructokinase proceeds as a stepwise process via the generation of elements of secondary structure, the formation of assembly-competent monomers that associate to heterodimers and the assembly of dimers to heterotetramers and heterooctamers. The assembly reactions seem to be rate-limiting. Recovery of t...
-
Purification, molecular and kinetic characterization of Phosphofructokinase-1 from the yeast Schizosaccharomyces pombe: evidence for an unusual subunit composition.
Yeast (Chichester England), 2000Co-Authors: R. Reuter, Jorg Bar, Manfred Naumann, Dieter Haferburg, Gerhard KopperschlägerAbstract:Phosphofructokinase-1 (Pfk-1) from Schizosaccharomyces pombe was purified by 54-fold enrichment to homogeneity elaborating the following steps: (a) Disruption of the cells with glass beads; (b) fractionated precipitation with polyethylene glycol 6000; (c) affinity chromatography on Cibacron-Blue F3G-A-Sephadex G 100; (d) ion exchange chromatography on Resource Q. The native enzyme exhibits a mass of 790±30 kDa, as detected by sedimentation equilibrium measurements. The apparent sedimentation coefficient was found to be s20,c=20.2±0.3 S. No significant dependence of the s-value on the protein concentration was observed in the range 0.07–0.7 mg/ml. Polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate and MALDI-TOF spectra showed that the enzyme is composed of subunits of identical size of 100±5 kDa, forming an octameric structure. The N-terminus of the enzyme was found to be blocked. Sequences of tryptic and chymotryptic peptides of the subunit coincide with the proposed amino acid sequence as deduced from the gene from the EMBL library. The Pfk-1 coding sequence of S. pombe was transformed into a Pfk-1 double deletion mutants of Saccharomyces cerevisiae resulting in glucose-positive cells with enzyme activity in the crude cell extract. The kinetic analysis revealed less cooperativity to fructose 6-phosphate (nH=1.6) and less inhibition by ATP as compared to the enzyme from baker's yeast. Fructose 2,6-bisphosphate (in micromolar range) and AMP (in millimolar range) were found to overcome ATP inhibition and to increase the affinity to fructose 6-phosphate. Copyright © 2000 John Wiley & Sons, Ltd.
-
EPITOPE MAPPING OF A MONOCLONAL ANTIBODY DIRECTED AGAINST THE ALPHA -SUBUNIT OF Phosphofructokinase-1 FROM SACCHAROMYCES CEREVISIAE BY SCREENING PHAGE DISPLAY LIBRARIES
Journal of Molecular Recognition, 1999Co-Authors: Margrit Hollborn, Jürgen Kirchberger, Gerd Birkenmeier, Gerhard KopperschlägerAbstract:Phosphofructokinase-1 from Saccharomyces cerevisiae is composed of two types of subunits, α and β. Subunit-specific monoclonal antibodies were raised to elucidate structural and functional properties of both subunits. One monoclonal antibody, α-F3, binds to an epitope either at the C-terminal or at the N-terminal part of the α-polypeptide chain. By screening a heptapeptide library with this monoclonal antibody, a set of heptapeptides was selected, which contained the consensus sequences D–A–F and D–S–F. Two heptapeptides with these motifs were synthesized in order assess their capacity to inhibit the binding of antibody α-F3 to native Phosphofructokinase-1. The peptide G–I–K–D–A–F–L inhibited the binding more strongly (IC50 = 1.5 µM) than the peptide A–P–W–H–D–S–F (IC50 = 33.3 µM). Sequence matching revealed the presence of the D–A–F motif in the polypeptide chain of Phosphofructokinase-1 at amino acid position 172–174. As a control, the nonapeptide A–P–T–S–K–D–A–F–L which corresponds to the sequence of the putative epitope was tested in the inhibition assay. In view of the high inhibitory capacity (IC50 = 0.3 µM) it was concluded that this nonapeptide represents the continuous epitope of Phosphofructokinase-1 that is recognized by antibody α-F3. Copyright © 1999 John Wiley & Sons, Ltd.
-
Phosphofructokinase-1 from Saccharomyces cerevisiae: analysis of molecular structure and function by electron microscopy and self-catalysed affinity labelling.
International journal of biological macromolecules, 1999Co-Authors: J. Kricke, Gerhard Kopperschläger, F. Mayer, T KriegelAbstract:Abstract Conventional and cryoelectron microscopy portray native octameric yeast Phosphofructokinase-1 (PFK) as consisting of two identical heterotetrameric tetrahedron-like moieties being rotated relative to each other. Immunoelectron microscopy employing subunit-specific IgG identifies α -type subunits in the contact zone of the two tetrahedrons, while β -chains are recognized exclusively at the tips of the octamer. The chemical reaction of Phosphofructokinase with analogues of fructose 6-phosphate followed by autocatalytic phosphoryl transfer from [ γ - 32 P]-ATP results in a specific labelling of the α -subunit. AMP and fructose 2,6-bisphosphate affect labelling by stimulating the binding of substrate analogue; AMP additionally promotes phosphoryl transfer. No stimulation of labelling is observed with proteolytically modified tetrameric 12-S Phosphofructokinase.
Fiona E Karet - One of the best experts on this subject based on the ideXlab platform.
-
Human H(+) ATPase a4 subunit mutations causing renal tubular acidosis reveal a role for interaction with Phosphofructokinase-1
American journal of physiology. Renal physiology, 2008Co-Authors: Katherine G. Blake-palmer, Simon H. Chang, Aiwu Zhou, Sara L Sorrell, Babak Javid, Katherine Bowers, Seema Qamar, Fiona E KaretAbstract:The vacuolar-type ATPase (H+ATPase) is a ubiquitously expressed multisubunit pump whose regulation is poorly understood. Its membrane-integral a-subunit is involved in proton translocation and in h...
-
human h atpase a4 subunit mutations causing renal tubular acidosis reveal a role for interaction with Phosphofructokinase 1
American Journal of Physiology-renal Physiology, 2008Co-Authors: Katherine G Blakepalmer, Simon H. Chang, Aiwu Zhou, Sara L Sorrell, Babak Javid, Katherine Bowers, Seema Qamar, Fiona E KaretAbstract:The vacuolar-type ATPase (H+ATPase) is a ubiquitously expressed multisubunit pump whose regulation is poorly understood. Its membrane-integral a-subunit is involved in proton translocation and in h...
-
the a subunit of the v type h atpase interacts with Phosphofructokinase 1 in humans
Journal of Biological Chemistry, 2003Co-Authors: Aiwu Zhou, Rafia S Allamki, Fiona E KaretAbstract:Abstract V-type or H+-ATPases are a family of ATP-dependent proton pumps that move protons across the plasma membrane at specialized sites such as kidney epithelial cells and osteoclasts as well as acidifying intracellular compartments. The 100-kDa polytopic a-subunit of this group of ATPases is suggested to play an important role in coupling the two functions of the pump, ATP hydrolysis and proton transport. In man, different a-subunit isoforms are encoded by four genes. ATP6V0A4 encodes a4, which is expressed apically in α-intercalated cells in both human and mouse kidney. We sought binding partners for the C terminus of a4 in order to address its potential role in the H+-ATPase complex. Random peptide phage display analysis revealed a consensus motif (WLELRP) with almost complete homology to part of the enzyme Phosphofructokinase 1 (PFK-1). Activity of this enzyme is the rate-limiting step in glycolysis. Specificity of a4 binding to this peptide was confirmed by enzyme-linked immunosorbent assay. Protein-protein interaction was further demonstrated by co-immunoprecipitation of a4 with PFK-1 from solubilized human kidney membrane proteins. An in vitro bead-bound PFK-1 pull-down assay showed that this interaction was also true for the ubiquitously expressed a1 subunit. Finally, PFK-1 co-immunolocalized with a4 in α-intercalated cells in the collecting ducts of human kidney. These findings indicate a direct link between V-type H+-ATPases and glycolysis via the C-terminal region of the a-subunit of the pump and suggest a novel regulatory mechanism between H+-ATPase function and energy supply. This interaction between the a-subunit and PFK-1 also provides new evidence that the C terminus of this subunit lies cytoplasmically in vivo.
Francisco Sobrino - One of the best experts on this subject based on the ideXlab platform.
-
Streptozotocin-induced diabetes increases fructose 2,6-biphosphate levels and glucose metabolism in thymus lymphocytes.
Life sciences, 1996Co-Authors: Victoria R. Moreno-aurioles, Rocio Montaño, Manuel Conde, Regla Bustos, Francisco SobrinoAbstract:Abstract Acute effect of streptozotocin-induced diabetes on several parameters of glucose metabolism was investigated in thymus lymphocytes (thymocytes). The cells from diabetics rats accumulated in vitro about 2-fold more fructose 2, 6-bisphosphate (Fru-2, 6-P2) in the presence of increasing glucose concentration than cells from normal rats. An increased production of lactate was also observed. Phosphofructokinase-1 (PFK-1) and Phosphofructokinase-2 (PFK-2) activities were enhanced in cells from diabetic rats compared with those from normal rats. [U-14C]glucose incorporation into glycogen was also increased in cells from diabetic rats and the 14C02 liberation was lesser than in cells from normal animals. From these data it may be concluded that the response of thymocytes to streptozotocin-induced diabetes is similar to that observed in other extrahepatic tissues.
-
Streptozotocin-Induced Diabetes Increases Fructose 2,6-Bisphosphate Levels and Glucose Metabolism in Rat Macrophages
Biochemical medicine and metabolic biology, 1993Co-Authors: R. Bustos, Victoria R. Moreno-aurioles, Rocio Montaño, Manuel Conde, Francisco SobrinoAbstract:The acute effects of streptozotocin-induced diabetes on several parameters of glucose metabolism were investigated in rat peritoneal macrophages. These cells accumulated in vitro about twofold more fructose 2,6-bisphosphate in the presence of increasing glucose concentration than cells from normal rats, and an increased production of lactate was observed. Phosphofructokinase-1, Phosphofructokinase-2, hexokinase, and pyruvate kinase activities were increased in cells from diabetic rats compared with those from normal rats. Transport of 2-deoxy-D-glucose was increased in cells from diabetic rats. [U-14C]Glucose incorporation into glycogen was also increased in cells from diabetics and the 14CO2 liberation was less than in cells from normal animals. Moreover, macrophages from diabetics did not possess a more active pentose phosphate pathway (measure with [1-14C]glucose oxidation) nor a greater production of superoxide anion (index of activation of macrophages) than in cells from normal animals.
-
Glucocorticoids inhibit fructose 2,6-bisphosphate synthesis in rat thymocytes. Opposite effect of cycloheximide
Biochimica et biophysica acta, 1991Co-Authors: V.r. Moreno-aurioles, Francisco SobrinoAbstract:The content of fructose 2,6-bisphosphate (Fru(2,6)P2) and lactate production in triamcinolone acetonide-treated rats thymocytes was studied. The effect in vitro of corticosterone and dexamethasone on normal thymocytes was also examined. Glucocorticoids produced a marked decrease in Fru(2,5)P2 content and lactate production. The largest effect was observed with triamcinolone acetonide (7.5 mg per kg body weight), which after 20 h of treatment produced over 90% of inhibition. This change was accompanied by the decrease of both Phosphofructokinase-1 and -2 activities and ATP levels, without modifications of hexoses phosphate content. The inhibitory actions of glucocorticoids were abolished by cycloheximide, an inhibitor of protein synthesis. Furthermore this drug, by itself, increased Fru(2,6)P2 content by more than 50% compared with the controls.
Aiwu Zhou - One of the best experts on this subject based on the ideXlab platform.
-
Human H(+) ATPase a4 subunit mutations causing renal tubular acidosis reveal a role for interaction with Phosphofructokinase-1
American journal of physiology. Renal physiology, 2008Co-Authors: Katherine G. Blake-palmer, Simon H. Chang, Aiwu Zhou, Sara L Sorrell, Babak Javid, Katherine Bowers, Seema Qamar, Fiona E KaretAbstract:The vacuolar-type ATPase (H+ATPase) is a ubiquitously expressed multisubunit pump whose regulation is poorly understood. Its membrane-integral a-subunit is involved in proton translocation and in h...
-
human h atpase a4 subunit mutations causing renal tubular acidosis reveal a role for interaction with Phosphofructokinase 1
American Journal of Physiology-renal Physiology, 2008Co-Authors: Katherine G Blakepalmer, Simon H. Chang, Aiwu Zhou, Sara L Sorrell, Babak Javid, Katherine Bowers, Seema Qamar, Fiona E KaretAbstract:The vacuolar-type ATPase (H+ATPase) is a ubiquitously expressed multisubunit pump whose regulation is poorly understood. Its membrane-integral a-subunit is involved in proton translocation and in h...
-
the a subunit of the v type h atpase interacts with Phosphofructokinase 1 in humans
Journal of Biological Chemistry, 2003Co-Authors: Aiwu Zhou, Rafia S Allamki, Fiona E KaretAbstract:Abstract V-type or H+-ATPases are a family of ATP-dependent proton pumps that move protons across the plasma membrane at specialized sites such as kidney epithelial cells and osteoclasts as well as acidifying intracellular compartments. The 100-kDa polytopic a-subunit of this group of ATPases is suggested to play an important role in coupling the two functions of the pump, ATP hydrolysis and proton transport. In man, different a-subunit isoforms are encoded by four genes. ATP6V0A4 encodes a4, which is expressed apically in α-intercalated cells in both human and mouse kidney. We sought binding partners for the C terminus of a4 in order to address its potential role in the H+-ATPase complex. Random peptide phage display analysis revealed a consensus motif (WLELRP) with almost complete homology to part of the enzyme Phosphofructokinase 1 (PFK-1). Activity of this enzyme is the rate-limiting step in glycolysis. Specificity of a4 binding to this peptide was confirmed by enzyme-linked immunosorbent assay. Protein-protein interaction was further demonstrated by co-immunoprecipitation of a4 with PFK-1 from solubilized human kidney membrane proteins. An in vitro bead-bound PFK-1 pull-down assay showed that this interaction was also true for the ubiquitously expressed a1 subunit. Finally, PFK-1 co-immunolocalized with a4 in α-intercalated cells in the collecting ducts of human kidney. These findings indicate a direct link between V-type H+-ATPases and glycolysis via the C-terminal region of the a-subunit of the pump and suggest a novel regulatory mechanism between H+-ATPase function and energy supply. This interaction between the a-subunit and PFK-1 also provides new evidence that the C terminus of this subunit lies cytoplasmically in vivo.
Simon H. Chang - One of the best experts on this subject based on the ideXlab platform.
-
Capillary electrophoresis-based assay of Phosphofructokinase-1.
Analytical biochemistry, 2014Co-Authors: Andrew Malina, Sherrisse K. Bryant, Simon H. Chang, Grover L. Waldrop, S. Douglass GilmanAbstract:Abstract An assay was developed for Phosphofructokinase-1 (PFK-1) using capillary electrophoresis (CE). In the glycolytic pathway, this enzyme catalyzes the rate-limiting step from fructose-6-phosphate and magnesium-bound adenosine triphosphate (Mg–ATP) to fructose-1,6-bisphosphate and magnesium-bound adenosine diphosphate (Mg–ADP). This enzyme has recently become a research target because of the importance of glycolysis in cancer and obesity. The CE assay for PFK-1 is based on the separation and detection by ultraviolet (UV) absorbance at 260 nm of Mg–ATP and Mg–ADP. The separation was enhanced by the addition of Mg 2+ to the separation buffer. Inhibition studies of PFK-1 by aurintricarboxylic acid and palmitoyl coenzyme A were also performed. An IC 50 value was determined for aurintricarboxylic acid, and this value matched values in the literature obtained using coupled spectrophotometric assays. This assay for PFK-1 directly monitors the enzyme-catalyzed reaction, and the CE separation reduces the potential of spectral interference by inhibitors.
-
human h atpase a4 subunit mutations causing renal tubular acidosis reveal a role for interaction with Phosphofructokinase 1
American Journal of Physiology-renal Physiology, 2008Co-Authors: Katherine G Blakepalmer, Simon H. Chang, Aiwu Zhou, Sara L Sorrell, Babak Javid, Katherine Bowers, Seema Qamar, Fiona E KaretAbstract:The vacuolar-type ATPase (H+ATPase) is a ubiquitously expressed multisubunit pump whose regulation is poorly understood. Its membrane-integral a-subunit is involved in proton translocation and in h...
-
Human H(+) ATPase a4 subunit mutations causing renal tubular acidosis reveal a role for interaction with Phosphofructokinase-1
American journal of physiology. Renal physiology, 2008Co-Authors: Katherine G. Blake-palmer, Simon H. Chang, Aiwu Zhou, Sara L Sorrell, Babak Javid, Katherine Bowers, Seema Qamar, Fiona E KaretAbstract:The vacuolar-type ATPase (H+ATPase) is a ubiquitously expressed multisubunit pump whose regulation is poorly understood. Its membrane-integral a-subunit is involved in proton translocation and in h...