| 1. |
- Bardales, José R., et al.
(författare)
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CK2-mediated phosphorylation of a type II regulatory subunit of cAMP-dependent protein kinase from the mollusk Mytilus galloprovincialis
- 2007
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 461:1, s. 130-137
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Tidskriftsartikel (refereegranskat)abstract
- Two isoforms of regulatory (R) subunit of cAMP-dependent protein kinase (PKA), named R(myt1) and R(myt2), were identified so far in the sea mussel Mytilus galloprovincialis. Out of them, only R(myt2) was phosphorylated in vitro by casein kinase 2 (CK2) using GTP as phosphate donor. CK2 catalytic subunit (CK2alpha) itself was sufficient to phosphorylate R(myt2), but phosphorylation was enhanced by the presence of the regulatory subunit CK2beta. Even in the absence of CK2, R(myt2) was phosphorylated to a certain extent when it was incubated with GTP. This basal phosphorylation was partially abolished by the known inhibitors apigenin and emodin, which suggests the presence of a residual amount of endogenous CK2 in the preparation of purified R subunit. CK2-mediated phosphorylation significantly decreases the ability of R(myt2) to inhibit PKA catalytic (C) subunit activity in the absence of cAMP. On the other hand, the sequence of several peptides obtained from the tryptic digestion of R(myt2) showed that mussel protein contains the signature sequence common to all PKA family members, within the "phosphate binding cassette" (PBC) A and B. Moreover, the degree of identity between the sequences of peptides from R(myt2), as a whole, and those from type II R subunits was 68-75%, but the global identity percentage with type I R subunits was only about 30%, so that R(myt2) can be classified as a type II R subunit.
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| 2. |
- Basile, Maria, et al.
(författare)
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Paralemmin interacts with D3 dopamine receptors : implications for membrane localization and cAMP signaling
- 2006
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier. - 0003-9861 .- 1096-0384. ; 446:1, s. 60-68
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Tidskriftsartikel (refereegranskat)abstract
- Paralemmin is a novel lipid-anchored protein, which is highly expressed in neuronal plasma membranes. In this study, we demonstrate that paralemmin specifically interacts with the third intracellular loop of the D3 dopamine receptor. Utilizing co-immunoprecipitation and glutathione-S-transferase (GST) pulldown strategies, we demonstrate that paralemmin interacts exclusively with D3, but not D2 or D4 dopamine receptors or β-adrenergic receptors. Immunocytochemistry demonstrated co-localization of paralemmin and D3 receptor in vivo in hippocampus and cerebellum and in vitro in glial and neuronal cultures. Deletion mutational analysis indicates that amino acids 154–230 of paralemmin strongly interacted with amino acids 211–227 and 281–330 of the third intracellular loop of D3 receptor. The consequences of these interactions were investigated by co-expression in HEK293 cells. Cell surface biotinylation experiments demonstrate that paralemmin decreased D3 receptor concentration at the plasma membrane. Consistent with this observation, paralemmin expression decreased dopamine-stimulated adenylate cyclase activity. However, paralemmin also decreased basal, isoproterenol and forskolin-stimulated adenylate cyclase activity, suggesting a more general cellular function for paralemmin. Taken together, paralemmin has been implicated as a potent modulator of cellular cAMP signaling within the brain.
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| 3. |
- Burén, Jonas, et al.
(författare)
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Insulin action and signalling in fat and muscle from dexamethasone-treated rats
- 2008
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 474:1, s. 91-101
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Tidskriftsartikel (refereegranskat)abstract
- Glucocorticoids initiate whole body insulin resistance and the aim of the present study was to investigate effects of dexamethasone on protein expression and insulin signalling in muscle and fat tissue. Rats were injected with dexamethasone (1mg/kg/day, i.p.) or placebo for 11 days before insulin sensitivity was evaluated in vitro in soleus and epitrochlearis muscles and in isolated epididymal adipocytes. Dexamethasone treatment reduced insulin-stimulated glucose uptake and glycogen synthesis by 30-70% in epitrochlearis and soleus, and insulin-stimulated glucose uptake by approximately 40% in adipocytes. 8-bromo-cAMP-stimulated lipolysis was approximately 2-fold higher in adipocytes from dexamethasone-treated rats and insulin was less effective to inhibit cAMP-stimulated lipolysis. A main finding was that dexamethasone decreased expression of PKB and insulin-stimulated Ser(473) and Thr(308) phosphorylation in both muscles and adipocytes. Expression of GSK-3 was not influenced by dexamethasone treatment in muscles or adipocytes and insulin-stimulated GSK-3beta Ser(9) phosphorylation was reduced in muscles only. A novel finding was that glycogen synthase (GS) Ser(7) phosphorylation was higher in both muscles from dexamethasone-treated rats. GS expression decreased (by 50%) in adipocytes only. Basal and insulin-stimulated GS Ser(641) and GS Ser(645,649,653,657) phosphorylation was elevated in epitrochlearis and soleus muscles and GS fractional activity was reduced correspondingly. In conclusion, dexamethasone treatment (1) decreases PKB expression and insulin-stimulated phosphorylation in both muscles and adipocytes, and (2) increases GS phosphorylation (reduces GS fractional activity) in muscles and decreases GS expression in adipocytes. We suggest PKB and GS as major targets for dexamethasone-induced insulin resistance.
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| 4. |
- Chen, Mingzhi, et al.
(författare)
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Predicting protein folding cores by empirical potential functions
- 2009
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Ingår i: Archives of Biochemistry and Biophysics. - New York : Elsevier. - 0003-9861 .- 1096-0384. ; 483:1, s. 16-22
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Tidskriftsartikel (refereegranskat)abstract
- Theoretical and in vitro experiments suggest that protein-folding cores form early in the process of folding, and that proteins may have evolved to optimize both folding speed and native-state stability. In our previous work (Chen et al., Structure, 14, 1401 (2006)), we developed a set of empirical potential functions and used them to analyze interaction energies among secondary-structure elements in two β-sandwich proteins. Our work on this group of proteins demonstrated that the predicted folding core also harbors residues that form native-like interactions early in the folding reaction. In the current work, we have tested our empirical potential functions on structurally-different proteins for which the folding cores have been revealed by protein hydrogen-deuterium exchange experiments. Using a set of 29 unrelated proteins, which have been extensively studied in the literature, we demonstrate that the average prediction result from our method is significantly better than predictions based on other computational methods. Our study is an important step towards the ultimate goal of understanding the correlation between folding cores and native structures.
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| 5. |
- Cristea, Mirela, et al.
(författare)
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Expression of manganese lipoxygenase in Pichia pastoris and site-directed mutagenesis of putative manganese ligands
- 2005
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 434:1, s. 201-211
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Tidskriftsartikel (refereegranskat)abstract
- Manganese lipoxygenase is secreted by the fungus Gaeumannomyces graminis. We expressed the enzyme in Pichia pastoris, which secreted approximately 30 mg Mn-lipoxygenase/L culture medium in fermentor. The recombinant lipoxygenase was N- and O-glycosylated (80-100 kDa), contained approximately 1 mol Mn/mol protein, and had similar kinetic properties (K(m) approximately 7.1 microM alpha-linolenic acid and V(max) 18 nmol/min/microg) as the native Mn-lipoxygenase. Mn-lipoxygenase could be quantitatively converted, presumably by secreted Pichia proteases, to a smaller protein (approximately 67 kDa) with retention of lipoxygenase activity (K(m) approximately 6.4 microM alpha-linolenic acid and V(max) approximately 12 nmol/min/microg). Putative manganese ligands were investigated by site-directed mutagenesis. The iron ligands of soybean lipoxygenase-1 are two His residues in the sequence HWLNTH, one His residue and a distant Asn residue in the sequence HAAVNFGQ, and the C-terminal Ile residue. The homologous sequences of Mn-lipoxygenase are H274VLFH278 and H462HVMN466QGS, respectively, and the C-terminal amino acid is Val-602. The His274Gln, His278Glu, His462Glu, and the Val-602 deletion mutants of Mn-lipoxygenase were inactive, and had lost >95% of the manganese content. His-463, Asn-466, and Gln-467 did not appear to be critical for Mn-lipoxygenase activity, as His463Gln, Asn466Gln, Asn466Leu, and Gln467Asn mutants metabolized alpha-linolenic acid to 11- and 13-hydroperoxylinolenic acids. We conclude that His-274, His-278, His-462, and Val-602 likely coordinate manganese.
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| 6. |
- Eriksson, Sandra, et al.
(författare)
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Development, evaluation and application of tripeptidyl-peptidase II sequence signatures
- 2009
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 484:1, s. 39-45
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Tidskriftsartikel (refereegranskat)abstract
- Tripeptidyl-peptidase II (TPP II) is a cytosolic peptidase that has been implicated in fat formation and cancer, apparently independent of the enzymatic activity. In search for alternative functional regions, conserved motifs were identified and eleven signatures were constructed. Seven of the signatures covered previously investigated residues, whereas the functional importance of the other motifs is unknown. This provides directions for future investigations of alternative activities of TPP II. The obtained signatures provide an efficient bioinformatic tool for the identification of TPP II homologues. Hence, a TPP II sequence homologue from fission yeast, Schizosaccharomyces pombe, was identified and demonstrated to encode the TPP II-like protein previously reported as multicorn. Furthermore, an homologous protein was found in the prokaryote Blastopirellula marina, albeit the TPP II function was apparently not conserved. This gene is probably the result of a rare gene transfer from eukaryote to prokaryote.
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| 7. |
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| 8. |
- Goldstone, J. V., et al.
(författare)
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Cytochrome P450 1D1 : A novel CYP1A-related gene that is not transcriptionally activated by PCB126 or TCDD
- 2009
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Ingår i: Archives of Biochemistry and Biophysics. - Amsterdam : Elsevier inc.. - 0003-9861 .- 1096-0384. ; 482:1-2, s. 7-16
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Tidskriftsartikel (refereegranskat)abstract
- Enzymes in the cytochrome P450 1 family oxidize many common environmental toxicants. We identified a new CYP1, termed CYP1D1, in zebrafish. Phylogenetically, CYP1D1 is paralogous to CYP1A and the two share 45% amino acid identity and similar gene structure. In adult zebrafish, CYP1D1 is most highlyexpressed in liver and is relatively highly expressed in brain. CYP1D1 transcript levels were higher at 9 h post-fertilization than at later developmental times. Treatment of zebrafish with potent aryl hydrocarbon receptor (AHR) agonists (3,3',4,4',5-pentachlorobiphenyl or 2,3,7,8-tetrachlorodibenzo-p-dioxin) did not induce CYP1D1 transcript expression. Morpholino oligonucleotide knockdown of AHR2, which mediates induction of other CYP1s, did not affect CYP1D1 expression. Zebrafish CYP1D1 heterologously expressed in yeast exhibited ethoxyresorufin- and methoxyresorufin-O-dealkylase activities. Antibodies against a CYP1D1 peptide specifically detected a single electrophoretically-resolved protein band in zebrafish liver microsomes, distinct from CYP1A. CYP1D1 in zebrafish is a CYP1A-like gene that could have metabolic functions targeting endogenous compounds.
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| 9. |
- Josephy, David, et al.
(författare)
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Single-nucleotide polymorphic variants of human glutathione transferase T1-1 differ in stability and functional properties
- 2009
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 490:1, s. 24-29
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Tidskriftsartikel (refereegranskat)abstract
- We have previously expressed hexa-histidine-tagged human glutathione transferase GST T1-1 at very high levels in an Escherichia coli lacZ mutagenicity assay strain. Ethylene dibromide (EDB), which is activated by GST T1-1, produces a potent response in the mutation assay. We have now constructed and expressed two SNP variants of wild-type GST T1-1:D141N and E173K. The EDB activation activities of both variant enzymes, as measured by the lacZ mutagenicity assay, are greatly reduced The D141N variant behaved similarly to the wild-type enzyme, in terms of expression level and specific activities for conjugation of glutathione with 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP), ethylene diiodide (EDI), and 4-nitrobenzyl chloride (NBCl), and for peroxidative detoxication of cumene hydroperoxide (CuOOH). In contrast, variant E173K is poorly expressed, has no detectable activity with EPNP, NBCl, or CuOOH, and has EDI activity much lower than that of the wild-type enzyme. The circular dichroism (CD) thermal denaturation profiles of the wild-type protein and variant D141N show a sharp two-state transition between native and denatured states. Variant E173K showed a very different profile, consistent with improper or incomplete protein folding. Our results show that SNP variants can give rise to GSTT1-1 proteins with significantly altered properties. (C) 2009 Elsevier Inc. All rights reserved.
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| 10. |
- Kim, Maria V, et al.
(författare)
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Structure and properties of K141E mutant of small heat shock protein HSP22 (HspB8, H11) that is expressed in human neuromuscular disorders.
- 2006
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 454:1
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Tidskriftsartikel (refereegranskat)abstract
- Some properties of the K141E mutant of human HSP22 that is expressed in distal hereditary motor neuropathy were investigated. This mutation slightly decreased intrinsic fluorescence of HSP22 and induced changes in the far UV CD spectra that correlate with increase of disordered structure. Destabilized K141E mutant was more susceptible to trypsinolysis than the wild type protein. Mutation K141E did not significantly affect the hydrophobic properties measured by bis-ANS binding and did not affect the quaternary structure of HSP22. With insulin as a substrate the chaperone-like activity of K141E mutant and the wild type protein were similar. However with alcohol dehydrogenase and rhodanese the chaperone-like activity of K141E mutant was remarkably lower than the corresponding activity of the wild type protein. It is concluded that K141E mutation induces destabilization of HSP22 structure and probably by this means diminish the chaperone-like activity of HSP22 with certain protein substrates.
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| 11. |
- Kurtovic, Sanela, et al.
(författare)
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Colorimetric endpoint assay for enzyme-catalyzed iodide ion release for high-throughput screening in microtiter plates
- 2007
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 464:2, s. 284-287
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Tidskriftsartikel (refereegranskat)abstract
- Efforts are being made to engineer enzymes with enhanced activities against haloalkanes, a toxicologically important class of compounds widely used and frequently occurring in the environment. Here we describe a facile, inexpensive, and robust method for the screening of libraries of mutated enzymes with iodoalkane substrates. Iodide formed in the enzymatic reaction is oxidized to iodine, which in the presence of starch gives blue color that can be measured at 610 nm or scored with the human eye. The assay can be performed with enzymes in crude cell lysates in 96-wells microtiter plates. Expression clones of several glutathione transferases showed diverse activities with different iodoalkanes, and a mutant library of human glutathione transferase A1-1 expressed variants with enhanced substrate selectivities. © 2007 Elsevier Inc. All rights reserved.
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| 12. |
- Kurz, Tino, 1974-, et al.
(författare)
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Autophagy, ageing and apoptosis : The role of oxidative stress and lysosomal iron
- 2007
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 462:2, s. 220-230
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Tidskriftsartikel (refereegranskat)abstract
- As an outcome of normal autophagic degradation of ferruginous materials, such as ferritin and mitochondrial metalloproteins, the lysosomal compartment is rich in labile iron and, therefore, sensitive to the mild oxidative stress that cells naturally experience because of their constant production of hydrogen peroxide. Diffusion of hydrogen peroxide into the lysosomes results in Fenton-type reactions with the formation of hydroxyl radicals and ensuing peroxidation of lysosomal contents with formation of lipofuscin that amasses in long-lived postmitotic cells. Lipofuscin is a non-degradable polymeric substance that forms at a rate that is inversely related to the average lifespan across species and is built up of aldehyde-linked protein residues. The normal accumulation of lipofuscin in lysosomes seems to reduce autophagic capacity of senescent postmitotic cells-probably because lipofuscin-loaded lysosomes continue to receive newly formed lysosomal enzymes, which results in lack of such enzymes for autophagy. The result is an insufficient and declining rate of autophagic turnover of worn-out and damaged cellular components that consequently accumulate in a way that upsets normal metabolism. In the event of a more substantial oxidative stress, enhanced formation of hydroxyl radicals within lysosomes jeopardizes the membrane stability of particularly iron-rich lysosomes, specifically of autophagolysosomes that have recently participated in the degradation of iron-rich materials. For some time, the rupture of a limited number of lysosomes has been recognized as an early upstream event in many cases of apoptosis, particularly oxidative stress-induced apoptosis, while necrosis results from a major lysosomal break. Consequently, the regulation of the lysosomal content of redox-active iron seems to be essential for the survival of cells both in the short- and the long-term. © 2007 Elsevier Inc. All rights reserved.
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| 13. |
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| 14. |
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| 15. |
- Petzold, Katja, et al.
(författare)
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Folding of the αΙΙ-spectrin SH3 domain under physiological salt conditions
- 2008
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier. - 0003-9861 .- 1096-0384. ; 474:1, s. 39-47
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Tidskriftsartikel (refereegranskat)abstract
- The SH3 domain has often been used as a model for protein folding due to its typical two-state behaviour. However, recent experimental data at low pH as well as molecular dynamic simulations have indicated that the folding process of SH3 probably is more complicated, and may involve intermediate states. Using both kinetic and equilibrium measurements we have obtained evidence that under native-like conditions the folding of the spectrin SH3 domain does not follow a classic two-state behaviour. The curvature we observed in the Chevron plots is a strong indication of a non-linear activation energy relationship due to the presence of high-energy intermediates. In addition, circular dichroism measurements indicated that refolding after thermal denaturation did not follow the same pattern as thermal unfolding but rather implied less cooperativity and that the refolding transition increased with increasing protein concentration. Further, NMR experiments indicated that upon refolding the SH3 domain gave rise to more than one conformation. Therefore, our results suggest that the folding of the SH3 domain of II-spectrin does not follow a classical two-state process under high-salt conditions and neutral pH. Heterogeneous folding pathways, which can include folding intermediates as well as misfolded intermediates, might give a more reasonable insight into the folding behaviour of the II-spectrin SH3 domain.
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| 16. |
- Rodríguez, M., et al.
(författare)
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Towards biosensing of arteriosclerotic nanoplaque formation using femtosecond spectroscopy.
- 2007
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 460:1, s. 92-99
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Tidskriftsartikel (refereegranskat)abstract
- The ultrafast dynamics of proteoheparan sulfate (HS-PG) in Krebs blood substitute solution was measured using femtosecond transient absorption spectroscopy after UV excitation. Interacting with blood lipoproteins and Ca2+ ions, the proteoglycan HS-PG is the key component of the so-called nanoplaque, the earliest stage in atherogenesis. Since tryptophan (Trp) residues are the main optically active parts of HS-PG, analogous measurements were performed on bare Trp in Krebs solution. The comparison reveals distinct differences to main characteristics of the HS-PG broadband absorption spectra. Analyzing the Trp spectra, we show that the results from transient absorption spectroscopy resemble the time constants of the chromophore ultrafast solvation dynamics that have been found by another group using fluorescence up-conversion techniques. Yet, the broadband transient absorption provides more details about the molecular dynamics, including stimulated emission, excited state absorption and resonant energy transfer. Furthermore, the absorption long time dynamics upon adding Ca2+ to the HS-PG probe were investigated by transient absorption spectroscopy and by surface force and ellipsometry investigations. Notably, a Ca2+-induced conformational change responsible for arteriosclerotic nanoplaque formation was detected. Slight differences, which are only visible as broad spectral features in the sub-picosecond time scale, provide a first insight into the molecular formation of nanoplaques in blood vessels, which may yield a better understanding of the genesis of arteriosclerosis.
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| 17. |
- Sedlák, Erik, et al.
(författare)
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Effect of Hofmeister ions on protein thermal stability : roles of ion hydration and peptide groups?
- 2008
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier. - 0003-9861 .- 1096-0384. ; 479:1, s. 69-73
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Tidskriftsartikel (refereegranskat)abstract
- We have systematically explored the Hofmeister effects of cations and anions (0.3–1.75 M range) for acidic Desulfovibrio desulfuricans apoflavodoxin (net charge −19, pH 7) and basic horse heart cytochrome c (net charge +17, pH 4.5). The Hofmeister effect of the ions on protein thermal stability was assessed by the parameter dTtrs/d[ion] (Ttrs; thermal midpoint). We show that dTtrs/d[ion] correlates with ion partition coefficients between surface and bulk water and ion surface tension effects: this suggests direct interactions between ions and proteins. Surprisingly, the stability effects of the different ions on the two model proteins are similar, implying a major role of the peptide backbone, instead of charged groups, in mediation of the interactions. Upon assessing chemical/physical properties of the ions responsible for the Hofmeister effects on protein stability, ion charge density was identified as most important. Taken together, our study suggests key roles for ion hydration and the peptide group in facilitating interactions between Hofmeister ions and proteins.
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| 18. |
- Tang, Wanjin, et al.
(författare)
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Regulation of human CYP27A1 by estrogens and androgens in HepG2 and prostate cells
- 2007
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 462:1, s. 13-20
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Tidskriftsartikel (refereegranskat)abstract
- The regulation of the human CYP27A1 gene by estrogens and androgens was studied in human liver-derived HepG2 and prostate cells. Our results show that the promoter activity, enzymatic activity and mRNA levels of CYP27A1 in HepG2 cells are downregulated by estrogen in presence of ERα or ERβ. Similar effects by estrogen were found in RWPE-1 prostate cells. In contrast, estrogen markedly upregulated the transcriptional activity of CYP27A1 in LNCaP prostate cancer cells. 5α-Dihydrotestosterone and androgen receptor upregulated the transcriptional activity of CYP27A1 in HepG2 cells. Progressive deletion experiments indicate that the ERβ-mediated effects in HepG2 and LNCaP cells are conferred to the same region (−451/+42) whereas ERα-mediated effects on this promoter are more complex. The results indicate that the stimulating effect of androgen in HepG2 cells is conferred to a region upstream from –792 in the CYP27A1 promoter. In summary, we have identified the human CYP27A1 gene as a target for estrogens and androgens. The results imply that expression of CYP27A1 may be affected by endogenous sex hormones and pharmacological compounds with estrogenic or androgenic effects.
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| 19. |
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| 20. |
- Ålander, J., et al.
(författare)
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Microsomal glutathione transferase 1 exhibits one-third-of-the-sites-reactivity towards glutathione
- 2009
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 487:1, s. 42-48
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Tidskriftsartikel (refereegranskat)abstract
- The trimeric membrane protein microsomal glutathione transferase 1 (MGST1) possesses glutathione transferase and peroxidase activity. Previous data indicated one active site/trimer whereas structural data suggests three GSH-binding sites. Here we have determined ligand interactions of MGST1 by several techniques. Nanoelectrospray mass spectrometry of native MGST1 revealed binding of three GSH molecules/trimer and equilibrium dialysis showed three product molecules/trimer (K(d) = 320 +/- 50 mu M). All three product molecules Could be competed out with GSH. Reinvestigation of GSH-binding showed one high affinity site per trimer, consistent with earlier data. Using single turnover stopped flow kinetic measurements, Kd could be determined for a low affinity GSH-binding site (2.5 +/- 0.5 mu M). Thus we can reconcile previous observations and show here that MGST1 contains three active sites with different affinities for GSH and that only the high affinity site is catalytically competent.
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| 21. |
- Almeida, Joao, et al.
(författare)
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Characterization of major enzymes and genes involved in flavonoid and proanthocyanidin biosynthesis during fruit development in strawberry (Fragaria x ananassa)
- 2007
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861. ; 465:1, s. 61-71
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Tidskriftsartikel (refereegranskat)abstract
- The biosynthesis of flavonoids and proanthocyanidins was studied in cultivated strawberry (Fragaria x ananassa) by combining biochemical and molecular approaches. Chemical analyses showed that ripe strawberries accumulate high amounts of pelargonidin-derived anthocyanins, and a larger pool of 3',4'-hydroxylated proanthocyanidins. Activities and properties of major recombinant enzymes were demonstrated by means of in vitro assays, with special emphasis on specificity for the biologically relevant 4'- and 3',4'-hydroxylated compounds. Only leucoanthocyanidin reductase showed a strict specificity for the 3',4'-hydroxylated leucocyanidin, while other enzymes accepted either hydroxylated substrate with different relative activity rates. The structure of late flavonoid pathway genes, leading to the synthesis of major compounds in ripe fruits, was elucidated. Complex developmental and spatial expression patterns were shown for phenylpropanoid and flavonoid genes in fruits throughout ripening as well as in leaves, petals and roots. Presented results elucidate key steps in the biosynthesis of strawberry flavonoid end products. (c) 2007 Elsevier Inc. All rights reserved.
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| 22. |
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| 23. |
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| 24. |
- Hansson, Markus, et al.
(författare)
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Biosynthesis, processing, and sorting of human myeloperoxidase.
- 2006
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861. ; 445:2, s. 214-224
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Forskningsöversikt (refereegranskat)abstract
- Exclusively synthesized by normal neutrophil and monocyte precursor cells, myeloperoxidase (MPO) functions not only in host defense by mediating efficient microbial killing but also can contribute to progressive tissue damage in chronic inflammatory states Such as atherosclerosis. The biosynthetic precursor, apoproMPO, is processed slowly in the ER, undergoing cotranslational N-glycosylation, transient interactions with the molecular chaperones calreticulin and calnexin, and heme incorporation to generate enzymatically active proMPO that is competent for export into the Golgi. After exiting the Golgi the propeptide is removed prior to final proteolytic processing in azurophil granules, resulting in formation of a symmetric MPO homodimer linked by a disulfide bond. Some proMPO escapes granule targeting and becomes constitutively secreted to the extracellular environment. Although the precise mechanism is Unknown, the pro-segirient is required for normal processing and targeting. as propeptide-deleted MPO precursor is either degraded or constitutively secreted. Characterizing the molecular consequences of naturally Occurring mutations that cause inherited MPO deficiency provides unique insight into the structural determinants of MPO involved in biosynthesis, processing and targeting. (C) 2005 Elsevier Inc. All rights reserved.
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| 25. |
- Horjales, Sofia, et al.
(författare)
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Translational machinery and protein folding: Evidence of conformational variants of the estrogen receptor alpha
- 2007
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861. ; 467:2, s. 139-143
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Tidskriftsartikel (refereegranskat)abstract
- As an approach to understand how translation may affect protein folding, we analyzed structural and functional properties of the human estrogen receptor alpha synthesized by different eukaryotic translation systems. A minimum of three conformations of the receptor were detected using limited proteolysis and a sterol ligand-binding assay. The receptor in vitro translated in rabbit reticulocyte lysate was rapidly degraded by protease, produced major bands of about 34 kDa and showed a high affinity for estradiol. In a wheat germ translation system, the receptor was more slowly digested. Two soluble co-existing conformations were evident by different degradation patterns and estradiol binding. Our data show that differences in the translation machinery may result in alternative conformations of the receptor with distinct sterol binding properties. These studies suggest that components of the cellular translation machinery itself might influence the protein folding pathways and the relative abundance of different receptor conformers. (C) 2007 Published by Elsevier Inc.
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| 26. |
- Ilari, Andrea, et al.
(författare)
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Crystal structure and ligand binding properties of the truncated hemoglobin from Geobacillus stearothermophilus
- 2007
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861. ; 457:1, s. 85-94
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Tidskriftsartikel (refereegranskat)abstract
- A novel truncated hemoglobin has been identified in the thermophilic bacterium Geobacillus stearothermophilus (Gs-trHb). The protein has been expressed in Escherichia coli, the 3D crystal structure (at 1.5 angstrom resolution) and the ligand binding properties have been determined. The distal heme pocket displays an array of hydrogen bonding donors to the iron-bound ligands, including Tyr-B10 on one side of the heme pocket and Trp-G8 indole nitrogen on the opposite side. At variance with the highly similar Bacillus subtilis hemoglobin, Gs-trHb is dimeric both in the crystal and in solution and displays several unique structural properties. In the crystal cell, the iron-bound ligand is not homogeneously distributed within each distal site such that oxygen and an acetate anion can be resolved with relative occupancies of 50% each. Accordingly, equilibrium titrations of the oxygenated derivative in solution with acetate anion yield a partially saturated ferric acetate adduct. Moreover, the asymmetric unit contains two subunits and sedimentation velocity ultracentrifugation data confirm that the protein is dimeric. (c) 2006 Elsevier Inc. All rights reserved.
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| 27. |
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| 28. |
- Picaud, S, et al.
(författare)
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Amorpha-4,11-diene synthase: Mechanism and stereochemistry of the enzymatic cyclization of farnesyl diphosphate
- 2006
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861. ; 448:1-2, s. 150-155
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Tidskriftsartikel (refereegranskat)abstract
- Recombinant amorpha-4,11-diene synthase from Artemisia annua, expressed in Escherichia coli, was incubated with the deuterium-labeled farnesyl diphosphates, (1R)-[1-H-2]FPP, (1S)-[1-H-2]FPP, and [1,1-H-2(2)]FPP. GC-MS analysis of amorpha-4,11-diene formed from the deuterated FPPs shows that the deuterium atoms are retained in the product. Furthermore, analysis of the MS-spectra obtained with the differently labeled substrate indicates that the H-1si-proton of FPP is transferred during the cyclization reaction to carbon 10 of amorphadiene while the H-1re-proton of FPP is retained on C-6 of the product. Proton NMR and COSY experiments proved that the original H-1si-proton of FPP is located at C-10 of amorpha-4,11-diene as a result of a 1,3-hydride shift following initial 1,6-ring closure. The results obtained support the previously suggested mechanism for the cyclization of farnesyl diphosphate by amorph-4,11-diene synthase involving isomerization of FPP to (R)-nerolidyl diphosphate (NPP), ionization of NPP, and C-1,C-6-ring closure to generate a bisabolyl cation, followed by a 1,3-hydride shift, 1,10-ring closure to generate the amorphane skeleton, and deprotonation at either C-12 or C-13 to afford the final product (1S,6R,7R,10R)-amorpha-4,11-diene. (c) 2005 Elsevier Inc. All rights reserved.
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