| 51. |
- Hernández-Fisac, Inés, et al.
(författare)
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Oxo-4-methylpentanoic acid directs the metabolism of GABA into the Krebs cycle in rat pancreatic islets
- 2006
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 400:1, s. 81-89
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Tidskriftsartikel (refereegranskat)abstract
- OMP (oxo-4-methylpentanoic acid) stimulates by itself a biphasic secretion of insulin whereas L-leucine requires the presence of L-glutamine. L-Glutamine is predominantly converted into GABA (gamma-aminobutyric acid) in rat islets and L-leucine seems to promote its metabolism in the 'GABA shunt' [Fernandez-Pascual, Mukala-Nsengu-Tshibangu, Martin del Rio and Tamarit-Rodriguez (2004) Biochem. J. 379,721-729]. In the present study, we have investigated how 10mM OMP affects L-glutamine metabolism to uncover possible differences with L-leucine that might help to elucidate whether they share a common mechanism of stimulation of insulin secretion. In contrast with L-leucine, OMP alone stimulated a biphasic insulin secretion in rat perifused islets and decreased the islet content of GABA without modifying its extracellular release irrespective of the concentration of L-glutamine in the medium. GABA was transaminated to L-leucine whose intracellular concentration did not change because it was efficiently transported out of the islet cells. The L-[U-C-14]-Glutamine (at 0.5 and 10.0 mM) conversion to (CO2)-C-14 was enhanced by 10 mM OMP within 30% and 70% respectively. Gabaculine (250 mu M), a GABA transaminase inhibitor, suppressed OMP-induced oxygen consumption but not L-leucineor glucose-stimulated respiration. It also suppressed the OMP-induced decrease in islet GABA content and the OMP-induced increase in insulin release. These results support the view that OMP promotes islet metabolism in the 'GABA shunt' generating 2-oxo-glutarate, in the branched-chain a-amino acid transaminase reaction, which would in turn trigger GABA deamination by GABA transaminase. OMP, but not L-leucine, suppressed islet semialdehyde succinic acid reductase activity and this might shift the metabolic flux of the 'GABA shunt' from gamma-hydroxybutyrate to succinic acid production.
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| 52. |
- Hersleth, Hans-Petter, et al.
(författare)
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The crystal structure of peroxymyoglobin generated through cryoradiolytic reduction of myoglobin compound III during data collection
- 2008
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Ingår i: Biochemical Journal. - 0264-6021. ; 412, s. 257-264
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Tidskriftsartikel (refereegranskat)abstract
- Myoglobin has the ability to react with hydrogen peroxide, generating high-valent complexes similar to peroxidases (compounds I and II), and in the presence of excess hydrogen peroxide a third intermediate, compound III, with an oxymyoglobin-type structure is generated from compound II. The compound III is, however, easily one-electron reduced to peroxymyoglobin by synchrotron radiation during crystallograpic data collection. We have generated and solved the 1.30 angstrom (1 angstrom= 0.1 nin) resolution crystal structure of the peroxymyoglobin intermediate, which is isoelectric to compound 0 and has a Fe-O distance of 1.8 angstrom and O-O bond of 1.3 angstrom in accordance with a Fe-II-O-O- (or Fe-III-O-O2-) structure. The generation of the peroxy intermediate through reduction of compound III by X-rays shows the importance of using single-crystal microspectrophotometry when doing crystallography on metal loproteins. After having collected crystallographic data on a peroxy-generated myoglobin crystal, we were able (by a short annealing) to break the O-O bond leading to formation of compound II. These results indicate that the cryoradiolytic-generated peroxymyoglobin is biologically relevant through its conversion into compound II upon heating. Additionally, we have observed that the Xe1 site is occupied by a water molecule, which might be the leaving group in the compound II to compound III reaction.
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| 53. |
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| 54. |
- Hulkko, SM, et al.
(författare)
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The pro-apoptotic protein death-associated protein 3 (DAP3) interacts with the glucocorticoid receptor and affects the receptor function
- 2000
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Ingår i: The Biochemical journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 349349 Pt 3, s. 885-893
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Tidskriftsartikel (refereegranskat)abstract
- The yeast two-hybrid system was used to isolate cDNAs encoding proteins that interact with the glucocorticoid receptor (GR) ligand-binding domain in a ligand-dependent manner. One isolated cDNA encoded a fragment of death-associated protein 3 (DAP3), which has been implicated as a positive mediator of apoptosis. In vitro experiments showed that the full-length DAP3 also interacted with GR. The main interaction domain was mapped to the N-terminal region of DAP3 that had previously been shown to function in a dominant-negative fashion, protecting cells from apoptosis. Co-transfection experiments in COS-7 cells showed that DAP3 had a stimulatory effect on the ligand-induced transcriptional activation by GR and also increased the steroid-sensitivity. Furthermore, DAP3 formed a complex with several other nuclear receptors and some basic helix–loop–helix/Per–Arnt–Sim proteins, as well as with heat-shock protein 90 (hsp90) (Arnt is the aryl-hydrocarbon-receptor nuclear translocator, and Per and Sim are the Drosophila proteins Period and Single-minded). The results suggest that DAP3 could have an important role in GR action, possibly by modulating the cytoplasmic GR–hsp90 complex. Since glucocorticoids can induce apoptosis, the pro-apoptotic DAP3 protein may be involved in this function of GR.
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| 55. |
- Igamberdiev, A U, et al.
(författare)
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Implications of adenylate kinase-governed equilibrium of adenylates on contents of free magnesium in plant cells and compartments
- 2001
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 360, s. 225-231
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Tidskriftsartikel (refereegranskat)abstract
- On the basis of the equilibrium of adenylate kinase (AK; EC 2.7.4.3). which interconverts MgATP and free AMP with MgADP and free ADP, an approach has been worked out to calculate concentrations of free magnesium (Mg2+), based on concentrations of total ATP, ADP and AMP in plant tissues and in individual subcellular compartments. Based on reported total adenylate contents, [Mg2+] in plant tissues and organelles varies significantly depending on light and dark regimes, plant age and developmental stage. In steady-state conditions, [Mg2+] in chloroplasts is similar in light and darkness (in the millimolar range), whereas in the cytosol it is very low in the light and increases to about 0.4 mM in darkness. During the dark-to-light transition (photosynthetic induction), the [Mg2+] in chloroplasts falls to low values (0.2 mM or less), corresponding to a delay in photosynthetic oxygen evolution. This delay is considered to result from lower activities of Mg-dependent enzymes in the Calvin cycle. In mitochondria, the changes in [Mg2+] are similar but smoother. On the other hand, when the transition from light to darkness is considered, an initial increase in [Mg2+] occurs in both chloroplasts and mitochondria, which may be of importance for the control of key regulatory enzymes (e.g. mitochondrial malic enzyme and pyruvate dehydrogenase complex) and for processes connected with light-enhanced dark respiration. A rationale is presented for a possible role of [MgATP]/[MgADP] ratio (rather than [ATP(total)]/[ADP(total)]) as an important component of metabolic cellular control. It is postulated that assays of total adenylates may provide an accurate measure of [Mg2+] in plant tissues/cells and subcellular compartments, given that the adenylates are equilibrated by AK.
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| 56. |
- Jacobson, A, et al.
(författare)
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Expression of human hyaluronan synthases in response to external stimuli.
- 2000
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 348 Pt 1, s. 29-35
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Tidskriftsartikel (refereegranskat)abstract
- In the present study we have investigated the expression of mRNAs for hyaluronan synthase isoforms (HAS1, HAS2 and HAS3) in different cells in response to various stimuli. Human mesothelial cells, which synthesize large amounts of hyaluronan, express mRNAs encoding all three HAS isoforms, whereas their transformed counterparts, mesothelioma cells, which produce only minute amounts of hyaluronan, express only HAS3 mRNA. Human lung fibroblasts and the glioma cell line U-118 MG express only the HAS2 and HAS3 genes. The expression of the transcripts was higher in subconfluent than in confluent cultures and was well correlated with the production of hyaluronan by the cells. Stimulation of mesothelial cells with platelet-derived growth factor-BB induced an up-regulation of mRNA for HAS2 to a maximum after 6 h of stimulation; HAS1 and HAS3 genes were only induced slightly. Transforming growth factor-beta1 reduced HAS2 mRNA slightly, and hydrocortisone reduced it strongly, within 6 h of stimulation in mesothelial cell cultures but did not significantly affect the expression of mRNAs for HAS1 and HAS3. Induction of HAS1 and HAS2 protein levels in response to the stimuli above correlated with HAS transcript levels. Thus the expression of the three HAS isoforms is more prominent in growing cells than in resting cells and is differentially regulated by various stimuli suggesting distinct functional roles of the three proteins.
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| 57. |
- Johansson, Fredrik I, et al.
(författare)
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Oxidation and reduction of pyridine nucleotides in alamethicin-permeabilized plant mitochondria
- 2004
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Ingår i: Biochemical Journal. - 0264-6021. ; 380:1, s. 193-202
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Tidskriftsartikel (refereegranskat)abstract
- The inner mitochondrial membrane is selectively permeable, which limits the transport of solutes and metabolites across the membrane. This constitutes a problem when intramitochondrial enzymes are studied. The channel-forming antibiotic AlaM (alamethicin) was used as a potentially less invasive method to permearbilize mitochondria and study the highly branched electron-transport chain in potato tuber (Solanum tuberosum) and pea leaf (Pisum sativum) mitochondria. We show that AlaM permeabilized the inner membrane of plant mitochondria to NAD(P)H, allowing the quantification of internal NAD(P)H dehydrogenarses as well as matrix enzymes in situ. AlaM was found to inhibit the electron-tran sport chain at the external Ca2+-dependent rotenone-insensitive NADH dehydrogenase and around complexes III and IV. Nevertheless, under optimal conditions, especially complex I-mediated NADH oxidation in AlaM-treated mitochondria was much higher than what has been previously measured by other techniques. Our results also show a difference in substrate specificities for complex I in mitochondria as compared with inside-out submitochondrial particles. AlaM facilitated the passage of cofactors to and from the mitochondrial matrix and allowed the determination of NAD(+) requirements of malate oxidation in situ. In summary, we conclude that AlaM provides the best method for quantifying NADH dehydrogenase activities and that AlaM will prove to be an important method to study enzymes under conditions that resemble their native environment not only in plant mitochondria but also in other membrane-enclosed compartments, such as intact cells, chloroplasts and peroxisomes.
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| 58. |
- Jonsson, Magnus, et al.
(författare)
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Semenogelins I and II bind zinc and regulate the activity of prostate-specific antigen
- 2005
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Ingår i: Biochemical Journal. - 0264-6021. ; 387:Part 2, s. 447-453
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Tidskriftsartikel (refereegranskat)abstract
- In semen. the gel proteins SgI and SgII (semenogelins I and II) are digested by PSA (prostate-specific antigen), resulting in liquefaction and release of motile spermatozoa. Semen contains a high concentration of Zn2+, which is known to inhibit the protease activity of PSA. We characterized the binding of Zn2+ to SgI and SgII and found evidence that these proteins are involved in regulating the activity of PSA. Intact SgI and SgII and synthetic semenogelin peptides were used in the experiments. Binding of Zn2+ was studied by radioligand blotting, titration with a zinc (II) fluorophore chelator and NMR analysis. A chromogenic substrate was used to measure the enzymatic activity of PSA. SgI and SgII bound Zn2+ with a stoichiometry of at least 10 cool (mol of protein)(-1) and with an average dissociation constant of approx. 5 mu M per site. Moreover, Zn2+-inhibited PSA was activated by exposure to SgI or SgII. Since both proteins have high affinity for Zn2+ and are the dominating proteins in semen, they probably represent the major Zn2+ binders in semen, one function of which may be to regulate the activity of PSA. The system is self-regulating, and PSA is maintained in an active state by its substrate.
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| 59. |
- Kalamajski, Sebastian, et al.
(författare)
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Asporin competes with decorin for collagen binding, binds calcium and promotes osteoblast collagen mineralization
- 2009
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Ingår i: Biochemical Journal. - 0264-6021. ; 423, s. 53-59
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Tidskriftsartikel (refereegranskat)abstract
- The interactions of the ECM (extracellular matrix) protein asporin with ECM components have previously not been investigated. Here, we show that asporin binds collagen type I. This binding is inhibited by recombinant asporin fragment LRR (leucine-rich repeat) 10-12 and by full-length decorin, but not by biglycan. We demonstrate that the polyaspartate domain binds calcium and regulates hydroxyapatite formation in vitro. In the presence of asporin, the number of collagen nodules, and mRNA of osteoblastic markers Osterix and Runx2 were increased. Moreover, decorin or the collagen-binding asporin fragment LRR 10-12 inhibited the pro-osteoblastic activity of full-length asporin. Our results suggest that asporin and decorin compete for binding to collagen and that the polyaspartate in asporin directly regulates collagen mineralization. Therefore asporin has a role in osteoblast-driven collagen biomineralization activity. We also show that asporin can be expressed in Escherichia coli (Rosettagami (TM)) with correctly positioned cysteine bridges, and a similar system can possibly be used for the expression of other SLRPs (small LRR proteoglycans/proteins).
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| 60. |
- Kallas, Åsa M., et al.
(författare)
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Enzymatic properties of native and deglycosylated hybrid aspen (Populus tremula x tremuloides) xyloglucan endotransglycosylase 16A expressed in Pichia pastoris
- 2005
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Ingår i: Biochemical Journal. - 0264-6021. ; 390, s. 105-113
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Tidskriftsartikel (refereegranskat)abstract
- The cDNA encoding a xyloglucan endotransglycosylase, PttXET16A, from hybrid aspen (Populus tremula x tremuloides) has been isolated from an expressed sequence tag library and expressed in the methylotrophic yeast Pichia pastoris. Sequence analysis indicated a high degree of similarity with other proteins in the XTH (xyloglucan transglycosylase/hydrolase) gene subfamily of GH16 (glycoside hydrolase family 16). In addition to the conserved GH16 catalytic sequence motif, PttXET16A contains a conserved N-glycosylation site situated proximal to the predicted catalytic residues. MS analysis indicated that the recombinant PttXET16A expressed in P. pastoris is heterogeneous due to the presence of variable N-glycosylation and incomplete cleavage of the a-factor secretion signal peptide. Removal of the N-glycan by endoglycosidase H treatment did not influence the catalytic activity significantly. Similarly, site-directed mutagenesis of Asn(93) to serine to remove the N-glycosylation site resulted in an enzyme which was comparable with the wild-type enzyme in specific activity and thermal stability but had clearly reduced solubility. Hydrolytic activity was detected neither in wild-type PttXET16A before or after enzymatic deglycosylation nor in PttXET16A N93S (Asn(93) -> Ser) mutant.
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