| 1. |
- Valerio, M, et al.
(författare)
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Tissue-specificity of the regulation of ATP hydrolysis by isolated plant-mitochondria
- 1993
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Ingår i: FEBS Letters. - 0014-5793 .- 1873-3468. ; 318:2, s. 113-117
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Tidskriftsartikel (refereegranskat)abstract
- Pea leaf mitochondria had a high ATP hydrolase activity following the collapse of the membrane potential by addition of valinomycin in state 4. In mitochondria isolated from potato tubers such ATP hydrolase activity was not observed. Pea leaf mitochondria also had a DELTApH, in contrast to what was previously found for potato tuber mitochondria. This DELTApH could, however, not explain the different results on ATP hydrolysis since this activity was also observed in the presence of nigericin. The results suggest a tissue-specific regulation of ATP hydrolysis in resting organs (potato tubers) as compared to active organs (leaves).
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| 2. |
- Davydov, Albert, 1969-
(författare)
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Electron paramagnetic resonance and biochemical studies of red-ox properties of the diferric/radical center in mouse and Mycobacterium tuberculosis ribonucleotide reductase R2 proteins
- 1999
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Ribonucleotide reductase (RNR) catalyses the reduction of all four ribonucleotides to the corresponding deoxyribonucleotides that are used by the cells for DNA synthesis. The active enzyme from mouse, Mycobacterium tuberculosis (M. tuberculosis) and aerobically grown E. coli consists of two nonidentical subunits, proteins R1 and R2, both required for enzymatic activity. The R1 protein contains the regulatory sites and the binding site for the ribonucleotide substrates whereas the R2 subunit contains a stable tyrosyl radical and an antiferromagnetically coupled _-oxo-bridged diferric center. According to the proposed reaction mechanism the radical properties of the R2 protein are transferred to the active site of the R1 protein upon binding of the substrate molecule to the R1. This transfer is proposed to propagate via a conserved chain of hydrogen-bonded amino acids within the R1 and R2 proteins. Once generated the diferric/radical center may exist in different red-ox states: active (with Fe(III)Fe(III) center and tyrosyl radical), met (Fe(III)Fe(III) center without tyrosyl radical), mixed-valent (Fe(II)Fe(III) center) and fully reduced (Fe(II)Fe(II) center). Admission of oxygen to the fully reduced form of an R2 protein (a so-called regeneration reaction) results in a spontaneous formation of the active form. The regeneration reaction may be one of the possible ways employed by the cell for the generation of the active enzyme and therefore the study of this reaction is important for understanding the enzyme functionality.The aim of this thesis is to study the red-ox transitions of the diferric/radical center in mouse and Mycobacterium tuberculosis R2 proteins. Despite the significant similarities in the structure, the red-ox properties of the diferric/radical centers in mouse and M. tuberculosis R2 proteins are significantly different. The diferric/radical center in mouse R2 was found to be much more accessible for the external reductants than the centers of E. coli and M. tuberculosis R2 proteins. A higher accessibility of the diferric/radical center in mouse R2 protein can be explained by the presence of an open channel from the surface of the protein to the diferric/radical center. The tyrosyl radical in mouse R2 protein strongly interacts with the diferric center. Removing the tyrosyl radical in the active mouse R2 protein results in irreversible structural changes of the diferric cluster leading to an inactivation of the protein. Therefore the met form of mouse R2 protein can not be stabilized. Unlike mouse R2, the tyrosyl radical of M. tuberculosis R2 protein exhibits extremely weak magnetic interaction with the diferric center. The met form of M. tuberculosis R2 can be easily obtained and stabilized by the treatment of the active enzyme with hydroxyurea. The results of the chemical reduction of the diferric/radical center in the native mouse R2 protein as well as in two mutants (D266A and Y370W) in a proposed electron transfer pathway indicate that in all cases the second order rate constants in the mutants are comparable or faster than in native protein suggesting that the proposed radical transfer pathway is not important for the chemical reduction to proceed.Studying the oxidation of the fully reduced mouse R2 protein by different non-oxygen oxidants, we have demonstrated that the transitions between Fe(III)Fe(III), Fe(II)Fe(II) and Fe(II)Fe(III) states of this protein are fully reversible. The possibility to form a proper binuclear iron center without using molecular oxygen as an oxidant suggests that the _-oxo-bridge in mouse R2 protein does not necessary need molecular oxygen to be formed. Application of the low temperature reduction to mouse and M. tuberculosis R2 proteins demonstrated a presence of two structurally different diferric clusters giving rise to two distinct mixed-valent EPR signals. Whereas the shape of the mixed-valent EPR signal generated by _-irradiation at 77 K in mouse R2 protein is significantly affected by the presence of the tyrosyl radical, we did not observe any effect of the tyrosyl radical presence on the shape of mixed - valent signal generated in M. tuberculosis R2.
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| 3. |
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| 4. |
- Sehlstedt, Ulrica, 1969-
(författare)
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A biophysical study of nucleic acid interactions with analogues and drugs
- 1998
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- work presented in this thesis concerns studies on the physicochemical nature of interactions between nucleic acids and small ligands. The outcome of such studies can yield insights at a molecular level into the physiological mechanisms of action of biologically active nucleic-acid binding molecules. The thesis work includes investigations of a number of such low molecular weight compounds designed for nucleic acid sequence probing or therapeutic use. The interactions have been characterised by means of various optical spectroscopic techniques - including linear dichroism, circular dichroism and fluorescence - as well as nuclear magnetic resonance spectroscopy.The fluorescent dye 4',6-diamidino-2-phenylindole (DAPI) is known to adopt different DNA binding modes in regions containing consecutive AT base-pairs as compared to those consisting of long sequences of GC base-pairs. In mixed sequence DNA, DAPI exhibits a pronounced preference to bind in the minor groove of AT rich regions. To verify whether the variation in ligand mode of binding could be attributed to the exocyclic N2 group of guanine, guanines in the polynucleotide [poly(dG-dC)]2 were substituted for the 2-desamino analogue inosine, denoted I. Comparison of the spectral characteristics of DAPI in complex with either of the polymeric nucleic acid host structures revealed a clear difference in binding geometries; the spectroscopic properties of the IC complex closely resemble those of the AT complex, in which DAPI is inserted edgewisely along the minor groove. These results are rationalised in terms of steric hindrance and decreased electronegative attraction caused by the amino group protruding into the minor groove of B-type GC tracts.Studies of complexes between DNA and a series of cobalt porphyrins and their unmetallated analogues revealed contact energy transfer from the DNA bases and binding orientation angles nearly parallel to the planes of the DNA base-pairs, indicative of intercalation, for the non-metal porphyrins. The metalloporphyrins, on the other hand, are suggested to bind in a partially melted region of DNA. This hypothesis is supported by cleavage reactions in which a break in one of the DNA strands is induced by a single activation event on the cobalt porphyrins.The DNA interaction properties for a series of quinoxaline derivatives with a positively charged side chain were examined with respect to variation of the size of the molecular heterocyclic ring system. Derivatives with three, four, and five rings were included in the investigations. All but the tricyclic compound are found to bind in an intercalative mode irrespective of DNA base sequence. For the tricyclic derivative it is suggested that the binding involves a competitive equilibrium between intercalation preferred by the ring system, and minor groove binding favoured by the side chain.The second part of this thesis focuses on the interactions between nucleic acids and two compounds with the potential of being used in the novel therapeutic antigene/antisense strategies, either by means of its own action (PNA, peptide nucleic acid) or as an antigene enhancer (9-OH-B220).The binding of the biologically active quinoxaline derivative 9-OH-B220 to double and triple helices of synthetic DNA and RNA was characterised. The drug is found to adopt an intercalative binding geometry in all complexes except when the RNA triplex serves as a host structure. In the latter case, the spectroscopic properties are indicative of a binding of the drug chromophore in the wide and shallow minor groove of the RNA triplex polymer. The drug is also found to enhance the thermal stability of each nucleic acid structure, with the DNA triplex stabilising capacity being extraordinary; when the DNA triplex is formed in a 0.1 M NaCl buffer, its triplex-to-duplex equilibrium is shifted towards higher temperature by 52.5oC upon drug association. The results indicate that 9-OH-B220 has the potential of being used both as a partner in an antigene strategy and as an antiretroviral agent.An NMR study of the base-pair breathing dynamics of hybrid duplexes formed between DNA and the nucleic acid analogue PNA revealed intriguing kinetic features of these complexes. PNA strand bases open and close with unusually high rates. The bases in the complementary DNA strands are influenced by this fast kinetics in different ways; while the DNA strand guanines are virtually unaffected, the thymine imino protons become hypersensitive to exchange catalysis. Hence, we conclude that base-pair opening is an asymmetric process in these hybrid duplexes. A model compatible with experimental data, in which a longitudinal breathing motion within the backbones is a pre-equilibrium state to that of lateral base opening, is presented and discussed. The results are of importance for efficient development of new DNA modulating drugs.
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| 5. |
- Ternström, Tomas, et al.
(författare)
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From Snapshot to Movie: φ Analysis of Protein Folding Transition States Taken One Step Further
- 1999
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Ingår i: Proceedings of the National Academy of Sciences. - 1091-6490. ; 96:26, s. 14854-14859
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Tidskriftsartikel (refereegranskat)abstract
- Kinetic anomalies in protein folding can result from changes of the kinetic ground states (D, I, and N), changes of the protein folding transition state, or both. The 102-residue protein U1A has a symmetrically curved chevron plot which seems to result mainly from changes of the transition state. At low concentrations of denaturant the transition state occurs early in the folding reaction, whereas at high denaturant concentration it moves close to the native structure. In this study we use this movement to follow continuously the formation and growth of U1A's folding nucleus by φ analysis. Although U1A's transition state structure is generally delocalized and displays a typical nucleation-condensation pattern, we can still resolve a sequence of folding events. However, these events are sufficiently coupled to start almost simultaneously throughout the transition state structure.
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| 6. |
- Sonesson, Anders
(författare)
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Plasma membrane/cytoskeleton interactions in plants
- 1997
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The way in which the cortical cytoskeleton associates to the plasma membrane (PM) must be elucidated to understand the structural dynamics of many processes in the plant cell. In the present study, isolated cauliflower (Brassica oleracea L.) PM vesicles were used to characterize membrane/cytoskeleton interactions. Both actin and tubulin were shown to copurify with PM vesicles and this copurification was specific to the PM. In order to expose the PM-associated cytoskeleton, Brij 58 was used to form sealed, inside-out vesicles (cytoplasmic side out). Both actin and tubulin colocalized with Brij 58-treated PM vesicles in sucrose-gradient centrifugation, indicating a lack of disruption of the actin/PM and tubulin/PM associations following inside-out vesicle formation. In order to characterize the PM links to actin and tubulin, inside-out PM vesicles were washed in a range of media. Both actin and tubulin co-sedimented with the PM vesicles in the presence of 50 mM DTT, 10 mM CaCl2 and 2 M NaCl (separately). Actin, but not tubulin, was completely released in the presence of 0.6 M KI, and both proteins were released in either 6 M urea or at pH>11.4. The association of actin to the PM was sensitive to extensive dialysis against a low ionic-strength medium. The presence of a pool of a- and b-tubulin with hydrophobic properties, "hydrophobic tubulin", was shown by Triton X-114 fractionation. This hydrophobicity was restricted to PM-associated tubulin and was not seen in soluble tubulin. Hydrophobic tubulin constituted at least 15% of the recovered PM-associated tubulin. Washes in the presence of high concentrations of salt or calcium did not release the hydrophobic tubulin, while high pH (“11.0) did release this fraction. The hydrophobicity of a fraction of the recovered tubulin could reflect a direct or indirect interaction of this tubulin with the PM lipid bilayer or to an integral membrane protein. Hydrophobic tubulin was associated in a salt-sensitive manner to detergent-resistant proteins, likely to be of peripheral/cytoskeletal origin. It is possible that the presence of hydrophobic tubulin in isolated PM reflects the anchoring of the cortical tubulin cytoskeleton to the PM. Further elucidation of the cytoskeleton anchoring mechanism, based on the present study will lead to a better understanding of the structural dynamics of many cellular events.
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| 7. |
- Evenäs, Johan, et al.
(författare)
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NMR studies of the E140Q mutant of the carboxy-terminal domain of calmodulin reveal global conformational exchange in the Ca2+-saturated state
- 1997
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 36:12, s. 3448-3457
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Tidskriftsartikel (refereegranskat)abstract
- In the present investigation, the Ca2+ activation of the C-terminal domain of bovine calmodulin and the effects of replacing the bidentate Ca2+-coordinating glutamic acid residue in the 12th and last position of loop IV with a glutamine are studied by NMR spectroscopy. The mutation E140Q results in sequential Ca2+ binding in this domain and has far-reaching effects on the structure of (Ca2+)2 TR2C, thereby providing further evidence for the critical role of this glutamic acid residue for the Ca2+- induced conformational change of regulatory EF-hand proteins. Analyses of the NOESY spectra of the mutant under Ca2+-saturated conditions, such that 97% of the protein is in the (Ca2+)2 form, revealed two sets of mutually exclusive NOEs. One set of NOEs is found to be consistent with the closed structure observed in the apo state of the C-terminal domain of the wild- type protein, while the other set supports the open structure observed in the Ca2+-saturated state. In addition, several residues in the hydrophobic core exhibit broadened resonances. We conclude that the (Ca2+)2 form of the mutant experiences a global conformational exchange between states similar to the closed and open conformations of the C-terminal domain of wild-type calmodulin. A population of 65 ± 15% of the open conformation and an exchange rate of (1-7) x 104 s-1 were estimated from the NMR data and the chemical shifts of the wild-type protein. From a Ca2- titration of the 15N-labeled mutant, the macroscopic binding constants (log(K1) = 4.9 ± 0.3 and log(K2) = 3.15 ± 0.10] and the inherent chemical shifts of the intermediate (Ca2+)1 form of the mutant were determined using NMR. Valuable information was also provided on the mechanism of the Ca2+ activation and the roles of the structural elements in the two Ca2+- binding events. Comparison with the wild-type protein indicates that the (Ca2+)1 conformation of the mutant is essentially closed but that some rearrangement of the empty loop IV toward the Ca2+-bound form has occurred.
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| 8. |
- Walse, B, et al.
(författare)
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Structure of a cyclic peptide with a catalytic triad, determined by computer simulation and NMR spectroscopy
- 1996
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Ingår i: Journal of Computer-Aided Molecular Design. - 0920-654X. ; 10:1, s. 11-22
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Tidskriftsartikel (refereegranskat)abstract
- We report the design of a cyclic, eight-residue peptide that possesses the catalytic triad residues of the serine proteases. A manually built model has been relaxed by 0.3 ns of molecular dynamics simulation at room temperature, during which no major changes occurred in the peptide. The molecule has been synthesised and purified. Two-dimensional NMR spectroscopy provided 35 distance and 7 torsion angle constraints, which were used to determine the three-dimensional structure. The experimental conformation agrees with the predicted one at the beta-turn, but deviates in the arrangement of the disulphide bridge that closes the backbone to a ring. A 1.2 ns simulation at 600 K provided extended sampling of conformation space. The disulphide bridge reoriented into the experimental arrangement, producing a minimum backbone rmsd from the experimental conformation of 0.8 A. At a later stage in the simulation, a transition at Ser3 produced more pronounced high-temperature behaviour. The peptide hydrolyses p-nitrophenyl acetate about nine times faster than free histidine.
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| 9. |
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| 10. |
- Domar, Ulla, 1938-
(författare)
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Human intestinal alkaline phosphatase : tissue expression and serum levels
- 1992
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Human alkaline phosphatase (ALP) comprises four isozymes, viz liver/bone/ kidney or tissue unspecific (AP), intestinal (LAP), placental (PLAP) and germ cell or PLAP-like alkaline phosphatase, with their main expression in specific tissues as indicated by their names. The isozymes are coded by different genes, but they are closely related, with more than 50% amino acid sequence homologies. Their biological function is unclear. In certain malignant and benign diseases, serum elevations of one or more of the isozymes occur, which is of diagnostic importance. In this study, the special expression of the intestinal isozyme in human tissues and sera, in normal as well as in pathological conditions, has been investigated by use of isozyme specific monoclonal antibodies.Monoclonal antibodies against the AP, IAP and PLAP isozymes were prepared, and specific assays developed, based on these monoclonal antibodies and the catalytic activity of the isozymes. By use of these assays the basal levels of all three isozymes were examined in selected normal organs. The isozymes were found to be expressed in measurable amounts in all the examined organs.IAP was immunohistochemically localized to the epithelial cells of membranes lining the ducts and tubules of the kidney, liver, pancreas and small intestine.Normal human serum contained all three isozymes. The AP isozyme constituted about 90% of the total ALP activity, the IAP isozyme less than 10% and the PLAP isozyme about 1%. Considerable interindividual variations of the serum IAP activity were observed. The serum activities of the IAP isozyme were related to the individual ABO blood group and secretor status. Non-secretors had low levels of IAP activity amounting to about one tenth of the activity in sera from blood group B or 0 secretors, while blood group A secretors had serum IAP activities in the same order as non-secretors. High individual day to day variations were observed.Fat absorption caused serum IAP to increase significantly for all persons, but it was rapidly cleared from the blood. We found that the release of IAP into the blood was linked to lipid absorption, but removal from the blood was not linked to lipoprotein clearance.Certain tumors of the testis expressed elevated levels of all three ALP isozymes. The highest activitiy of LAP was observed in one yolk sac tumor, in agreement with the endodermal origin of this tumor. In seminoma tissue the AP and PLAP isozymes were significantly, and IAP moderately elevated.Cirrhosis of the liver caused significantly increased serum levels of IAP besides the AP isozyme. In inflammatory diseases of the small intestine, normal serum IAP activities were observed.
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