Linear dichroism and orientation of the phycomyces photopigment: I. Response and absorption studies. II. Fluorescence studies

NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document. The response of five strains of the fungus Phycomyces to linearly polarized light was studied. The sporangiophores of the strains, C2, C5, C9, C158, and NRRL 1555 (wildtype) differed primarily in optical attenuation. Their abilities to distinguish between longitudinally and transversely polarized blue light were found to be approximately the same. The angle (with respect to the transverse axis) at which the [...] vector of the polarized light must be oriented to give a maximum response during perpendicular incidence into the cell was measured. It was found to be 42° ± 3° for 280 nm light in the wild type strain. In the C158 strain this angle was 7° ± 3° at 456 nm and 7 ± 8° at 486 nm. The in vivo attenuation of polarized light as a function of the angle between the [...] vector and the cell axis was measured. The maximum transmission differences resulting from anisotropic attenuation were 4 ± 2% at 320 nm, 3 ± 2% at 456 nm, and 2 ± 1% at 486 nm. These results indicate that the polarized light effect in Phycomyces cannot arise from reflections at the cell surface, nor from attenuations due to internal screening or scattering and, therefore, must be due to the dichroism and orientation of the visual pigment. An attempt was made to detect the presence of the photopigment in cell wall and plasmalemma fractions using fluorescence kinetics and polarization. Excitation of these preparations with high intensity 488 nm laser light and monitoring fluorescence intensity with a microphotometer, a fluorescence decay and a periodic dependence of fluorescence intensity on the [...] vector angle of exciting light was found. The relevance of this fluorescence to the in vivo photosystem is questionable because of the high excitation intensity necessary to produce any detectable fluorescence

Formyl Met-Leu-Phe-Stimulated FPR1 Phosphorylation in Plate-Adherent Human Neutrophils: Enhanced Proteolysis but Lack of Inhibition by Platelet-Activating Factor

N-formyl-Met-Leu-Phe (fMLF) is a model PAMP/DAMP driving human PMN to sites of injury/infection utilizing the GPCR, FPR1. We examined a microtiter plate format for measurement of FPR1 phosphorylation in adherent PMN at high densities and found that a new phosphosensitive FPR1 fragment, 25K-FPR1, accumulates in SDS-PAGE extracts. 25K-FPR1 is fully inhibited by diisopropylfluorophosphate PMN pretreatment but is not physiologic, as its formation failed to be significantly perturbed by ATP depletion, time and temperature of adherence, or adherence mechanism. 25K-FPR1 was minimized by extracting fMLF-exposed PMN in lithium dodecylsulfate at 4°C prior to reduction/alkylation. After exposure of adherent PMN to a 5 log range of PAF before or after fMLF, unlike in suspension PMN, no inhibition of fMLF-induced FPR1 phosphorylation was observed. However, PAF induced the release of 40% of PMN lactate dehydrogenase, implying significant cell lysis. We infer that PAF-induced inhibition of fMLF-dependent FPR1 phosphorylation observed in suspension PMN does not occur in the unlysed adherent PMN. We speculate that although the conditions of the assay may induce PAF-stimulated necrosis, the cell densities on the plates may approach levels observed in inflamed tissues and provide for an explanation of PAF’s divergent effects on FPR1 phosphorylation as well as PMN function

Structural changes are induced in human neutrophil cytochrome b by NADPH oxidase activators, LDS, SDS, and arachidonate: intermolecular resonance energy transfer between trisulfopyrenyl-wheat germ agglutinin and cytochrome b558

AbstractAnionic amphiphiles such as sodium- and lithium dodecyl sulfate (SDS, LDS), or arachidonate (AA) initiate NADPH oxidase and proton channel activation in cell-free systems and intact neutrophils. To investigate whether these amphiphiles exert allosteric effects on cytochrome b, trisulfopyrenyl-labeled wheat germ agglutinin (Cascade Blue®–wheat germ agglutinin, CCB-WGA) was used as an extrinsic fluorescence donor for resonance energy transfer (RET) to the intrinsic heme acceptors of detergent-solubilized cytochrome b. In solution, cytochrome b complexed with the CCB-WGA causing a rapid, saturable, carbohydrate-dependent quenching of up to ∼55% of the steady-state fluorescence. Subsequent additions of SDS, LDS, or AA to typical cell-free oxidase assay concentrations completely relaxed the fluorescence quenching. The relaxation effects were specific, and not caused by dissociation of the CCB-WGA–cytochrome b complex or alterations in the spectral properties of the chromophores. In contrast, addition of the oxidase antagonist, arachidonate methyl ester, caused an opposite effect and was able to partially reverse the activator-induced relaxation. We conclude that the activators induce a cytochrome b conformation wherein the proximity or orientation between the hemes and the extrinsic CCB fluorescence donors has undergone a significant change. These events may be linked to NADPH oxidase assembly and activation or proton channel induction

APPROVAL

of a dissertation submitted b