Studies on the activity and activation of rat liver microsomal glutathione transferase, in particular with a substrate analogue series

A number of potential substrates for the microsomal glutathione transferase have been investigated. Out of 11 epoxides tested, only two, i.e. androstenoxide and benzo(a)pyrene-4,5-oxide, were found to be substrates. Upon treatment of the enzyme with N-ethylmaleimide, its activity toward only certain substrates is increased. It appeared upon inspection of the bimolecular rate constants from the corresponding nonenzymatic reactions that the substrates for which the activity is increased are the more reactive ones. This hypothesis was investigated further using a series of para-substituted 1-chloro-2-nitrobenzene derivatives as substrates. Activation was seen only with the more reactive nitro-, aldehyde-, and acetaldehyde-substituted compounds and not with the amide and chloroanalogues, thus demonstrating the predicted effect with a related series of compounds. Interestingly, kcat values are increased 7-20-fold by N-ethylmaleimide treatment, whereas the corresponding kcat/Km value is increased only for the p-nitro derivative. Effective molarity and rate enhancement values were found to increase with decreasing reactivity of the substrate, attaining maximal values of 10(5) M and 10(8), respectively. It is concluded that the glutathione transferases are quite effective catalysts with their less reactive substrates. Hammett rho values for the kcat values of unactivated and activated enzyme were 0.49 and 2.0, respectively. The latter value is close to those found for cytosolic glutathione transferases, indicating that activation changes the catalytic mechanism so that it more closely resembles that of the soluble enzymes. The rho values for kcat/Km values were 3 and 3.5 for the unactivated and activated enzyme, respectively, values close to those observed for the nonenzymatic bimolecular rate constants and thereby demonstrating that these reactions have similar properties. The high coefficients of correlation between resonance sigma- values and all of these parameters demonstrate a strong dependence on substrate electrophilicity, as expected for nucleophilic aromatic substitution.     

Diel and seasonal locomotor activity patterns in Arctic charr, Salvelinm alpinus (L.)

One-year-old Arctic charr, Sulvelinus alpinus (L.), of the Hornavan strain were tested from February 1985 to January 1986 in an attempt to get an increased understanding of the annual rheotactic behaviour as well as the die1 and seasonal locomotor activity pattern. An annular stream tank equipped with photocells was used to measure the direction of swimming movements as well as the number of passings. From February to late May the locomotor activity was low but increased in July and peaked in September. After November the locomotor activity was again at low winter levels. During the activity peak from July to November the majority ofall movements was directed against the current while no preference for direction was noted during the rest of the year. The high level of swimming movements directed against the current in late summer and autumn may be related to an innate habitat change. From February until June, the charr exhibited a bimodal diurnal activity pattern. In July activity was evenly spread over the whole 24- hour period and in August and September activity was again mainly diurnal with a bimodal pattern. In October and November the activity was mainly nocturnal and in December and January activity was concentrated in the short light period. Both annual and die1 activity are discussed in relation to earlier findings in general locomotor activity in Arctic charr and other salmonids.     

Crystallographic refinement and structure of ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum at 1.7 A resolution

The amino acid sequence of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum has been fitted to the electron density maps. The resulting protein model has been refined to a nominal resolution of 1.7 A using the constrained-restrained least-squares refinement program of Sussman and the restrained least-squares refinement program of Hendrickson & Konnert. The crystallographic refinement, based on 76,452 reflections with F greater than sigma (F) in the resolution range 5.5 to 1.7 A resulted in a crystallographic R-factor of 18.0%. The asymmetric unit contains one dimeric ribulose-1,5-biphosphate carboxylase molecule, consisting of 869 amino acid residues and 736 water molecules. The geometry of the refined model is close to ideal, with root-mean-square deviations of 0.018 A in bond lengths and 2.7 degrees in bond angles. Two loop regions, comprising residues 54 to 63 and 324 to 335, and the last ten amino acid residues at the C terminus are disordered in our crystals. The expected trimodal distribution is obtained for the side-chain chi 1-angles with a marked preference for staggered conformation. The hydrogen-bonding pattern in the N-terminal beta-sheet and the parallel sheet in the beta/alpha-barrel is described. A number of hydrogen bonds and salt bridges are involved in domain-domain and subunit-subunit interactions. The subunit-subunit interface in the dimer covers an area of 2800 A2. Considerable deviations from the local 2-fold symmetry are found at both the N terminus (residues 2 to 5) and the C terminus (residues 422 to 457). Furthermore, loop 8 in the beta/alpha-barrel domain has a different conformation in the two subunits. A number of amino acid side-chains have different conformations in the two subunits. Most of these residues are located at the surface of the protein. An analysis of the individual temperature factors indicates a high mobility of the C-terminal region and for some of the loops at the active site. The positions and B-factors for 736 solvent sites have been refined (average B: 45.9 A2). Most of the solvent molecules are bound as clusters to the protein. The active site of the enzyme, especially the environment of the activator Lys191 in the non-activated enzyme is described. Crystallographic refinement at 1.7 A resolution clearly revealed the presence of a cis-proline at the active site. This residue is part of the highly conserved region Lys166-Pro167-Lys168.     

Crystal structure of the complex of ribulose-1,5-bisphosphate carboxylase and a transition state analogue, 2-carboxy-D-arabinitol 1,5-bisphosphate

The crystal structure of the binary complex of nonactivated ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum and a transition state analogue, 2-carboxy-D-arabinitol 1,5-bisphosphate has been determined to 2.6 A resolution with x-ray crystallographic methods. The transition state analogue binds in a rather extended conformation at the active site. The orientation of the transition state analogue within the active site could be determined from the electron density maps. The P1 phosphate group of the analogue binds at a site built up of residues from loops 5 and 6 of the alpha/beta-barrel. The phosphate group interacts with the side chains of the conserved residues Arg-288, His-321, and Ser-368 and with main chain nitrogens from residues Thr-322 and Gly-323. The second phosphate group of the transition state analogue binds at the opposite side of the barrel close to loops 1 and 8. Significant differences for the positions and interactions of the P2 phosphate group with the enzyme are found in the two subunits of the dimer. The different mode of binding for this phosphate group in the two subunits is interpreted as a consequence of different conformations of the polypeptide chain observed in loops 6 and 8. The P2 phosphate group interacts with the sidechains of Lys-166 and Lys-329. Loop 6, which is disordered in the nonactivated, nonliganded enzyme is considerably more ordered in one of the subunits, probably due to the interaction of the side chain of Lys-329 with the P2 phosphate group. Almost all oxygen atoms are hydrogen bonded to groups on the enzyme. The carboxyl group forms hydrogen bonds to the side chain of the conserved Asn-111. The binding of the transition state analogue to the nonactivated enzyme is different from the binding of the analogue to activated spinach ribulose-bisphosphate carboxylase.     

Stereoselectivity in β-cyclodextrin complexation of 1,4-dihydropyridine derivatives

Structure–interaction relationships, stereoselectivity, and solubility enhancement in inclusion compexation of β-cyclodextrins (CDs) with some racemic and enantiomerically pure 1,4-dihydropyridine derivatives (DHPs) were investigated. 1:1 and 1:2 (mole ratio) complexes were prepared and characterized by X-ray powder diffraction, differential scanning calorimetry (DSC), MS-FAB spectrometry, ¹H-NMR spectroscopy, water and phase solubility. The solubility studies have revealed different complexation equilibria for optically pure DHP enantiomers, and corresponding racemic mixtures in water solutions. By means of ¹H-NMR chemical shift measurements, the inclusion of aromatic fragments of racemic and enantiomerically pure DHP molecules within the cavities of different CDs was elucidated. Considerable stereoselectivity in complexation interactions was observed. The results indicate the potential use of cyclodextrins as chiral selectors for enantiomeric resolution of 1,4-DHP calcium antagonists. © 1993 Wiley-Liss, Inc.     

Characterization of a newin vitro model for studies of reepithelialization in human partial thickness wounds

Summary Reepithelialization of artificial partial thickness wounds made in biopsies of human skin was determined after 3, 5, or 7 d of incubation, submerged or elevated to the air-liquid interface. The biopsies were reepithelialized within 5–7 d, with a more complete epidermal healing in wounds exposed to air. Both types of wounds showed similar time-course in deposition of basement membrane components, as detected by immunofluorescence labeling. Laminin and collagen type VII were deposited underneath the migrating tips, whereas collagen type IV was detected after reepithelialization. Markers of terminal differentiation showed a pattern close to normal in the air-liquid incubated wounds after reepithelialization. Involucrin was detected in the suprabasal regions of the migrating epidermis and thereafter in the upper half of neo-epidermis in the air-liquid incubated wound. Filaggrin could not be detected in the submerged wounds at any time during healing, whereas wounds exposed to air showed a well-differentiated epidermis by Day 7. Tritiated thymidine-incorporation indicated proliferation of epidermal and dermal cells during reepithelialization and a maintained viability, as shown by cultivation of endothelial- and fibroblast-like cells obtained from the dermis 7 d after wounding. Reepithelialization in this humanin vitro model is supported by a matrix close to normal with the possibility of extracellular influences and cell-cell interactions and, in addition, the technique is simple and reproducible. Therefore, we suggest this model for studies of regeneration in culture and as a complement toin vivo studies on epidermal healing.     

Male-specific cell migration into the developing gonad

Background: The gene Sry acts as a developmental switch, initiating a pathway of gene activity that leads to the differentiation of testis rather than ovary from the indifferent gonad (genital ridge) in mammalian embryos. The early events following Sry expression include rapid changes in the topographical organization of cells in the XY gonad. To investigate the contribution of mesonephric cells to this process, gonads from wild-type mice (CD1), and mesonephroi from a transgenic strain ubiquitously expressing β-galactosidase (ROSA26), were grafted together in vitro. After culture, organs were fixed and stained for β-galactosidase activity to identify cells contributed from the mesonephros to the male or female gonad.Results: Migration of mesonephric cells occurred into XY but not XX gonads from 11.5–16.5 days post coitum (dpc). Somatic cells contributed from the mesonephros were distinguished by their histological location and by available cell-specific markers. Some of the migrating cells were endothelial; a second population occupied positions circumscribing areas of condensing Sertoli cells; and a third population lay in close apposition to endothelial cells.Conclusions: Migration from the mesonephros to the gonad is male specific at this stage of development and depends on an active signal that requires the presence of a Y chromosome in the gonad. The signals that trigger migration operate over considerable distances and behave as chemoattractants. We suggest that migration of cells into the bipotential gonad may have a critical role in initiating the divergence of development towards the testis pathway.