Biologie - Open Access LMU - Teil 01/02
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The fluorescence decay time of the biliverdin IX7 chromophore present in biliproteins isolated from Pieris brassicae is determined to be 44 ± 3 ps. This value suggests a cyclic helical chromophore structure. The vibrational frequencies determined by CARS-spectroscopy are compared with those of model compounds. The data confirm that the chromophore in the protein-bound state adopts a cyclic-helical, flexible conformation.
The temperature dependence of spectral holes burnt into a phycocyanin-doped ethylene glycol/water glass is investigated in the temperature range between 1.5 and 15 K. The data are well described by a power law with an exponent of 1.16 ± 0.1. Chromoproteins thus behave very much the same as glasses doped with small impurity molecules.
The identification and localization of a marker protein for the intermembrane space between the outer and inner chloroplast envelopes is described. This 64-kDa protein is very rapidly labeled by [γ-32P]ATP at very low (30 nM) ATP concentrations and the phosphoryl group exhibits a high turnover rate. It was possible to establish the presence of the 64-kDa protein in this plastid compartment by using different chloroplast envelope separation and isolation techniques. In addition comparison of labeling kinetics by intact and hypotonically lysed pea chloroplasts support the localization of the 64-kDa protein in the intermembrane space. The 64-kDa protein was present and could be labeled in mixed envelope membranes isolated from hypotonically lysed plastids. Mixed envelope membranes incorporated high amounts of 32P from [γ-32P]ATP into the 64-kDa protein, whereas separated outer and inner envelope membranes did not show significant phosphorylation of this protein. Water/Triton X-114 phase partitioning demonstrated that the 64-kDa protein is a hydrophilic polypeptide. These findings suggest that the 64-kDa protein is a soluble protein trapped in the space between the inner and outer envelope membranes. After sonication of mixed envelope membranes, the 64-kDa protein was no longer present in the membrane fraction, but could be found in the supernatant after a 110000 × g centrifugation.
Calculations of excitation transfer rates among the chromophores of C-phycocyanin using the Förster inductive resonance transfer mechanism have been carried out using the new coordinates for the position and orientation of the chromophores (Schirmer, T., Bode, W. and Huber, R. (1987) J. Mol. Biol. 196, 677–695). Several of the rate constants are significantly altered from the results of our calculations using the previously published coordinates (Sauer, K., Scheer, H. and Sauer, P. (1987) Photochem. Photobiol. 46, 427–440). In particular, for the (αβ)3-trimers of Mastigocladus laminosus or for the (αβ)3-trimers or the (αβ)6-hexamers of Agmenellum quadruplicatum, the new calculations predict excited state relaxation components with exponential time constants shorter than 1 ps. In fact, some of the interchromophore interactions are so strong that exciton coupling is probably the relevant mechanism of interaction. The largest exciton energy is calculated to be about 56 cm−1, for the interaction between the adjacent α84 and β84 chromophores of neighboring monomer units within the (αβ)3-trimers or (αβ)6-hexamers. An energy transfer model invoking a combination of pairwise exciton formation followed by slower Förster transfer steps is described.
Reaction centers from Rhodobacter sphaeroides have been modified by treatment with sodium borohydride similar to the original procedure [Ditson et al., Biochim. Biophys. Acta 766, 623 (1984)], and investigated spectroscopically and by gel electrophoresis. (1) Low temperature (1.2 K) absorption, fluorescence, absorption- and fluorescence-detected ODMR, and microwave-induced singlet-triplet absorption difference spectra (MIA) suggest that the treatment produces a spectroscopically homogeneous preparation with one of the ‘additional’ bacteriochlorophylls being removed. The modification does not alter the zero field splitting parameters of the primary donor triplet (TP870). (2) From the circular dichroism and Raman resonance spectra in the1500–1800 cm-1 region, the removed pigment is assigned to BchlM, e.g. the "extra" Bchl on the "inactive" M-branch. (3) A strong coupling among all pigment molecules is deduced from the circular dichroism spectra, because pronounced band-shifts and/or intensity changes occur in the spectral components assigned to all pigments. This is supported by distinct differences among the MIA spectra of untreated and modified reaction centers, as well as by Raman resonance. (4) The modification is accompanied by partial proteolytic cleavage of the M-subunit. The preparation is thus spectroscopically homogeneous, but biochemically heterogenous.
We present a combined fluorescence and hole-burning study of the biliprotein C-phycocyanin. Sharp zero-phonon holes compare with a broad structureless fluorescence. This finding is rationalized in terms of the special level structure in this pigment, the fast energy-transfer processes and a lack of correlation of the energies of the emissive states.
Highly frequency selective photochemistry at cryogenic temperatures is used to gain information on the excited state dynamics of large biomolecular aggregates, the phycobilisomes from the blue–green alga (cyanobacterium) Masticogladus laminosus. In particular, we show that in spite of the well organized structure of these aggregates disorder on a microscopic level dominates the optical spectra. The hole burning reaction in the resonantly excited chromophores is most probably due to a conformational change in the neighborhood of the chromophore. From the widths of the holes energy transfer times between different pigments on the order of 16 ps are determined. These transfer times are independent of the excitation energy. The Journal of Chemical Physics is copyrighted by The American Institute of Physics.
The glyoxylic-acid-induced fluorescence technique was applied to demonstrate patterns of catecholaminergic innervation within the auditory brainstem of echolocating bats and the house mouse. In the cochlear nucleus of the rufous horseshoe bat (Rhinolophus rouxi) and the mustache bat (Pteronotus parnelli), species-specific catecholaminergic innervation patterns are found that contrast with the relatively homogeneous innervation in the rodent. In both bats the subnuclei of the cochlear nucleus receive a differentially dense supply of catecholaminergic fibers, and within the subnuclei, the catecholamine innervation densities can be correlated with the tonotopic frequency representation. The areas devoted to the high-frequency echolocation calls are less densely innervated than those regions which are responsive to lower frequencies. Apart from this common scheme, there are noteworthy distinctions between the two bats which correlate with specialized cytoarchitectural features of the cochlear nucleus. The marginal cell group, located medially to the anteroventral cochlear nucleus of Pteronotus, receives the densest supply of catecholaminergic fibers of all auditory nuclei. This plexus is formed by a morphologically distinct population of catecholaminergic fibers.
A silver impregnation method is described which shows motor and sensory nerves and their endings in formalin-fixed mammalian muscles. The method works with the same reliability on flattened muscle pieces as well as on frozen sections. Large nerve bundles, myelinated and non-myelinated single axons, and terminals impregnated by this method stand out black against a light brown background.
The light-induced radical cation of the primary electron donor P960+• in photosynthetic reaction centers from Rhodopseudomonas viridis has been investigated by ESR, ENDOR and TRIPLE techniques. Both the comparison with the cation radical of monomeric bacteriochlorophyll b (BChl b) and with molecular-orbital calculations performed on P960+• using the results of an X-ray structure analysis, consistently show an asymmetric distribution of the unpaired electron over the two BChl b molecules which constitute P960+•. The possible relevance of this result for the primary electron transfer step in the reaction center is briefly discussed.
A guanosine 5-triphosphate (GTP)-dependent protein kinase was detected in preparations of outer chloroplast envelope membranes of pea (Pisum sativum L.) chloroplasts. The protein-kinase activity was capable of phosphorylating several envelope-membrane proteins. The major phosphorylated products were 23- and 32.5-kilo-dalton proteins of the outer envelope membrane. Several other envelope proteins were labeled to a lesser extent. Following acid hydrolysis of the labeled proteins, most of the label was detected as phosphoserine with only minor amounts detected as phosphothreonine. Several criteria were used to distinguish the GTP-dependent protein kinase from an ATP-dependent kinase also present in the outer envelope membrane. The ATP-dependent kinase phosphorylated a very different set of envelope-membrane proteins. Heparin inhibited the GTP-dependent kinase but had little effect upon the ATP-dependent enzyme. The GTP-dependent enzyme accepted phosvitin as an external protein substrate whereas the ATP-dependent enzyme did not. The outer membrane of the chloroplast envelope also contained a phosphotransferase capable of transferring labeled phosphate from [-32P]GTP to ADP to yield (-32P]ATP. Consequently, addition of ADP to a GTP-dependent protein-kinase assay resulted in a switch in the pattern of labeled products from that seen with GTP to that typically seen with ATP.
An ATP-dependent protein kinase was partially purified from isolated outer envelope membranes of pea (Pisum sativum L., Progress No. 9) chloroplasts. The purified kinase had a molecular weight of 70 kilodaltons, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was of the cyclic nucleotide and Ca2+, calmodulin-independent type. The purification involved the detergent solubilization of purified outer envelopes by 0.5% cholate and 1% octylglycoside, followed by centrifugation on a linear 6 to 25% sucrose gradient. Active enzyme fractions were further purified by affinity chromatography on histone III-S Sepharose 4B and ion exchange chromatography on diethylaminoethyl cellulose. The protein kinase eluted at 100 millimolar and 50 millimolar NaCl, respectively. The protein kinase was essentially pure as judged by Western blot analysis. The enzyme has a KM of 450 micromolar for ATP and a Vmax of 25 picomoles of 32P incorporated into histone III-S per minute per microgram. Inhibition by ADP is competitive (Ki 150 micromolar).
The Q4 class I gene has been shown to participate in gene conversion events within the mouse major histocompatibility complex. Its complete genomic nucleotide sequence has been determined. The 5' half of Q4 resembles H-2 genes more strongly than other Q genes. Its 3' end, in contrast, is Q-like and contains a translational stop signal in exon 5 which predicts a polypeptide with an incomplete membrane spanning segment. The presence of two inverted B1 repeats suggests that part of the Q4 gene may be mobile within the genome. Gene transfer experiments have shown that the Q4 gene encodes a ß2-microglobulin associated polypeptide of Mr 41 000. A similar protein was found in activated mouse spleen cells. The Q4 polypeptide was found to be secreted both by spleen cells and by transfected fibroblasts and was not detectable on the cell surface. Antibody binding and twodimensional gel electrophoresis indicate that the Q4 molecule is identical to a mouse class I polypeptide, Qb-1, which has been previously described.
The Edinger-Westphal nucleus in goldfish was identified by retrograde labeling from the ciliary ganglion. In the same animals a few neurons near this nucleus (perinuclear Edinger-Westphal neurons) were labeled by a different retrograde tracer injected into the cerebellum. No double-labeled cells were found. Similar results were obtained in kelp bass, except that in this species no cerebellar-projecting perinuclear neurons were observed. Cerebellar-projecting Edinger-Westphal neurons have previously been described in some mammals, but not in other vertebrates. Therefore the homology of cerebellar-projecting cells of the Edinger-Westphal region in mammals and teleost fishes is doubtful.
Injection of horseradish peroxidase into the secondary gustatory nucleus of the green sunfish, Lepomis cyanellus, resulted in retrogradely filled neurons bilaterally in the viscerosensory column of the brainstem and in anterograde transport revealing ipsilateral terminal fields in the preglomerular tertiary gustatory nucleus, the nucleus of the torus lateralis and the central and periventricular nucleus of the inferior lobe. Thus, the glomerular nucleus of percomorph teleosts is not a tertiary gustatory center. It is proposed that the term ‘nucleus glomerulosus’ be reserved for the nucleus involved with vision and that the preglomerular subdivision involved in gustation be termed ‘nucleus gustatorius tertius’.
Excitation energy transfer in C-phycocyanin is modeled using the Forster inductive resonance mechanism. Detailed calculations are carried out using coordinates and orientations of the chromophores derived from X-ray crystallographic studies of C-phycocyanin from two different species (Schirmer et al, J. Mol. Biol. 184, 257–277 (1985) and ibid., 188, 651-677 (1986)). Spectral overlap integrals are estimated from absorption and fluorescence spectra of C-phycocyanin of Mastigocladus laminosus and its separated subunits. Calculations are carried out for the β-subunit, αβ-monomer, (αβ)3-trimer and (αβ)0-hexamer species with the following chromophore assignments: β155 = 's’(sensitizer), β84 =‘f (fluorescer) and α84 =‘m’(intermediate):]:. The calculations show that excitation transfer relaxation occurs to 3=98% within 200 ps in nearly every case; however, the rates increase as much as 10-fold for the higher aggregates. Comparison with experimental data on fluorescence decay and depolarization kinetics from the literature shows qualitative agreement with these calculations. We conclude that Forster transfer is sufficient to account for all of the observed fluorescence properties of C-phycocyanin in aggregation states up to the hexamer and in the absence of linker polypeptides.
