Nodulation (root nodule formation) in legume roots is initiated by the induction of cell divisions and formation of root nodule primordia in the plant root cortex, usually in front of the... Show moreNodulation (root nodule formation) in legume roots is initiated by the induction of cell divisions and formation of root nodule primordia in the plant root cortex, usually in front of the protoxylem ridges of the central root cylinder. We isolated a factor from the central cylinder (stele) of pea roots which enhances hormone-induced cell proliferation in root cortex explants at positions similar to those of nodule primordia. The factor was identified as uridine. Uridine may act as a morphogen in plant roots at picomolar concentrations. Show less
The biochemical isolation of pure and active proteins or chlorophyll protein complexes has been crucial for elucidating the mechanism of photosynthetic energy conversion. Most of the proteins... Show moreThe biochemical isolation of pure and active proteins or chlorophyll protein complexes has been crucial for elucidating the mechanism of photosynthetic energy conversion. Most of the proteins involved in this process are embedded in the photosynthetic membrane. The isolation of such hydrophobic integral membrane proteins is not trivial, and involves the use of detergents often combined with various time-consuming isolation procedures. We have applied the new procedure of perfusion chromatography for the rapid isolation of photosynthetic membrane proteins. Perfusion chromatography combines a highly reactive surface per bed volume with extremely high elution flow rates. We present an overview of this chromatographic method and show the rapid isolation of reaction centres from plant Photosystems I and II and photosynthetic purple bacteria, as well as the fractionation of the chlorophyll a/b-binding proteins of Photosystem I (LHC I). The isolation times have been drastically reduced compared to earlier approaches. The pronounced reduction in time for separation of photosynthetic complexes is convenient and permits purification of proteins in a more native state, including the maintainance of ligands and the possibility to isolate proteins trapped in intermediate metabolic or structural states. Show less
The lowest tripler state, T-0, of all-trans-tetradecahexaenal has been investigated by means of electron-spin-echo spectroscopy in a magnetic field and in zero field. The directions of the... Show moreThe lowest tripler state, T-0, of all-trans-tetradecahexaenal has been investigated by means of electron-spin-echo spectroscopy in a magnetic field and in zero field. The directions of the principal axes and the eigenvalues of the fine-structure tensor have been determined. The tripler state corresponds to a pi pi* excitation. In zero field, T-0 is populated predominantly through the upper spin sublevel which is found to decay fastest. Comparison of the relative energies and the decay rates of the sublevels of T-0 with those obtained previously for shorter polyenals reveals that both the zero-field splitting and the logarithm of the decay rates are inversely proportional to the total number of double bonds. Show less
The effect of redox-inactive cationic and anionic paramagnetic chromium(III) complexes on the H-1 NMR spectrum of the reduced type 1 blue copper protein amicyanin, AmCu1, from Thiobacillus versutus... Show moreThe effect of redox-inactive cationic and anionic paramagnetic chromium(III) complexes on the H-1 NMR spectrum of the reduced type 1 blue copper protein amicyanin, AmCu1, from Thiobacillus versutus has been studied as a means of defining sites for association at the protein surface. With [Cr(CN)(6)](3-) two sites are detected, one at the adjacent hydrophobic patch close to the exposed imidazole of the co-ordinated His-96, and the other at Phe-92 which has Lys-59, Lys-60, Arg-69 and Arg-100 in close proximity and is adjacent to the active site-co-ordinated Cys-93. In contrast, the cationic complexes [Cr(NH3)(6)](3+) and [Cr(en)(3)](3+) (en = ethane-2,2-diamine) cause no significant line broadening and no preferred sites for association are detected. Kinetic stopped-flow studies on the competitive inhibition by [Cr(CN)(6)](3-) of the [Fe(CN)(6)](3-) oxidation of AmCu1 indicate that [Fe(CN)(6)](3-) reacts at two sites, one of which is inhibited by [Cr(CN)(6)](3-) and the other is unaffected by [Cr(CN)(6)](3-). It is suggested that the first of these corresponds to reaction at the Phe-92 site, contributing 25% to the reaction, and the second to reaction at His-96. Therefore, in its reaction with [Fe(CN)(6)](3-) amicyanin has adjacent and remote binding sites. Show less
Danielsen, E.; Bauer, R.; Hemmingsen, L.; Bjerrum, M.J.; Butz, T.; Tröger, W.; ... ; Pouderoyen, G. van 1995
The present work uses Cd-111m-perturbed angular correlations of gamma-rays (PAC) to investigate the structure of the metal site of the His117Gly mutant of Pseudomonas aeruginosa azurin in aqueous... Show moreThe present work uses Cd-111m-perturbed angular correlations of gamma-rays (PAC) to investigate the structure of the metal site of the His117Gly mutant of Pseudomonas aeruginosa azurin in aqueous solution and the effect on the structure upon addition of the following exogenous ligands: imidazole, 4-methyl imidazole, 1-methyl imidazole, 2-methyl imidazole and histidine. The nuclear quadrupole interaction of cadmium bound to the mutant without addition of exogenous ligands shows a strong pH dependence with three different nuclear quadrupole interactions consistent with two pK(a) values at about 7.2 and 8.6 at 2 degrees C. Addition of the imidazole derivatives resulted in a significant change in the PAC spectrum showing that they coordinate. This is in accordance with observations by EPR for the same mutant with copper at the metal site [den Blaauwen, T. & Canters, G. W. (1993) J. Am. Chem. Soc. 115, 1121-1129]. However, whereas EPR and ultraviolet/visual absorption show that the characteristics of the wild-type copper protein are regained by addition of the imidazole derivatives with the exception of the possible bidentates (histidine and histamine), the comparison of the PAC results to model calculations shows that the cadmium ion must be fourfold coordinated in most cases, probably binding an additional water or hydroxide ligand. A fourfold coordination is in contrast to cadmium-substituted wild-type azurin where PAC data inferred a threefold coordination by a Cys and two His residues. Show less
A magnetophotoselection (MPS) study on the triplet state of the primary donor in the D1-D2-cytochrome b559 complex is performed at 6 K. For this purpose a novel double-modulation technique was... Show moreA magnetophotoselection (MPS) study on the triplet state of the primary donor in the D1-D2-cytochrome b559 complex is performed at 6 K. For this purpose a novel double-modulation technique was developed, resulting in a 2-fold better signal-to-noise ratio, compared to the conventional technique. The MPS data were found to depend on the excitation wavelength, The angles calculated from the MPS at the longest wavelength are representative for the a,transition moment of P680, phi(x) = 55 degrees +/- 1 degrees, phi(y) = 30 degrees +/- 4 degrees, phi(z) = 90 degrees +/- 12 degrees, where phi(i) is the angle with the ith triplet axis, and yield a positive sign for E. The angles found for P680 differ from those reported for monomeric chlorophyll a in solution (Vrieze, J.; Hoff, A. J. Chem. Phys. Lett. 1995, 237, 493). This difference is discussed in terms of the structure of P680. Assuming C-2 symmetry, with the 2-fold axis perpendicular to the membrane plane, a model is presented in which P680 is an excitonically-coupled dimer of chlorophyll a molecules, whose triplet state at 6 K resides on one of the constituent monomers. The angles of the triplet magnetic axes of this monomer with the membrane plane correspond to those determined in an EPR study on oriented samples (Van Mieghem, F. J. E.; Satoh, K.; Rutherford, A, W. Biochim. Biophys. Acta 1991, 1058, 379-385). The ($) over right arrow Q(y) transition moments of the two chlorophylls of P680 are close to antiparallel, and the ($) over right arrow Q(y)((-)) axis of the lower excitonic component lies in the membrane plane. The Chi porphyrin planes make an angle of 60 degrees, and their intercenter distance is estimated to be 11 Angstrom. Show less
Bloemberg, G.V.; Lagas, R.M.; Leeuwen, S. van; Marel, G.A. van der; Boom, J.H. van; Lugtenberg, E.J.J.; Spaink, H.P. 1995
We describe an insert for optical and magnetic-resonance experiments on single molecules in a solid matrix at liquid-helium temperatures. The experimental arrangement allows in situ adjustment of... Show moreWe describe an insert for optical and magnetic-resonance experiments on single molecules in a solid matrix at liquid-helium temperatures. The experimental arrangement allows in situ adjustment of the focusing lens and of the sample. A parabolic mirror serves to collect the fluorescence emission and to direct the light onto a photodetector. Microwaves can be irradiated through a coil around the sample while a superconducting magnet provides the possibility of a stationary magnetic field. (C) 1995 American Institute of Physics. Show less
