Identification of the overtone of the Fe-CO stretching mode in heme proteins: a probe of the heme active site.

Two CO-isotope sensitive lines have been detected in the overtone region of the resonance Raman spectra of CO-bound hemeproteins. One line is assigned as the overtone of the Fe-CO stretching mode and is located in the 1000- to 1070-cm-1 region. The other line is found in the 1180- to 1210-cm-1 region and is assigned as a combination between a porphyrin mode, nu 7, and the Fe-CO stretching mode. The high intensities of these lines, which in the terminal oxidase class of proteins are of the same order as those of the fundamental stretching mode, indicate that the mechanism of enhancement for modes involving the Fe-CO moiety is different from that for the modes of the porphyrin macrocycle and call for reexamination of Raman theory of porphyrins as applied to axial ligands. The anharmonicity of the electronic potential function was evaluated, revealing that in the terminal oxidases the anharmonicity is greater than in the other heme proteins that were examined, suggesting a distinctive interaction of the bound CO with its distal environment in this family. Furthermore, the anharmonicity correlates with the frequency of the C-O stretching mode, demonstrating that both of these parameters are sensitive to the Fe-CO bond energy. The overtone and combination lines involving the bound CO promise to be additional probes of heme protein structural properties.

Green fluorescent protein as a vital marker and reporter of gene expression in Drosophila.

We have used the green fluorescent protein (GFP) from the jellyfish Aequorea victoria as a vital marker/reporter in Drosophila melanogaster. Transgenic flies were generated in which GFP was expressed under the transcriptional control of the yeast upstream activating sequence that is recognized by GAL4. These flies were crossed to several GAL4 enhancer trap lines, and expression of GFP was monitored in a variety of tissues during development using confocal microscopy. Here, we show that GFP could be detected in freshly dissected ovaries, imaginal discs, and the larval nervous system without prior fixation or the addition of substrates or antibodies. We also show that expression of GFP could be monitored in intact living embryos and larvae and in cultured egg chambers, allowing us to visualize dynamic changes in gene expression during real time.

Quantitative measurement of intraorganelle pH in the endosomal-lysosomal pathway in neurons by using ratiometric imaging with pyranine.

Organelle acidification is an essential element of the endosomal-lysosomal pathway, but our understanding of the mechanisms underlying progression through this pathway has been hindered by the absence of adequate methods for quantifying intraorganelle pH. To address this problem in neurons, we developed a direct quantitative method for accurately determining the pH of endocytic organelles in live cells. In this report, we demonstrate that the ratiometric fluorescent pH indicator 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) is the most advantageous available probe for such pH measurements. To measure intraorganelle pH, cells were labeled by endocytic uptake of HPTS, the ratio of fluorescence emission intensities at excitation wavelengths of 450 nm and 405 nm (F450/405) was calculated for each organelle, and ratios were converted to pH values by using standard curves for F450/405 vs. pH. Proper calibration is critical for accurate measurement of pH values: standard curves generated in vitro yielded artifactually low organelle pH values. Calibration was unaffected by the use of culture medium buffered with various buffers or different cell types. By using this technique, we show that both acidic and neutral endocytically derived organelles exist in the axons of sympathetic neurons in different steady-state proportions than in the cell body. Furthermore, we demonstrate that these axonal organelles have a bimodal pH distribution, indicating a rapid acidification step in their maturation that reduces the average pH of a fraction of the organelles by 2 pH units while leaving few organelles of intermediate pH at steady state. Finally, we demonstrate a spatial gradient or organelle pH along axons, with the relative frequency of acidic organelles increasing with proximity to the cell body.

Development of a K(+)-channel probe and its use for identification of an intracellular plant membrane K+ channel.

Polyclonal antibodies were generated against a 9-amino acid, synthetic peptide corresponding to the selectivity filter in the pore region of K(+)-channel proteins. The sequence of amino acids in the ion-conducting pore region of K+ channels is the only highly conserved region of members of this protein family. The objectives of the present work were (i) to determine whether the anti-channel pore peptide antibody was immunoreactive with known K(+)-channel proteins and (ii) to demonstrate the usefulness of the antibody by employing it to identify a newly discovered K(+)-channel protein. Anti-channel pore peptide was immunoreactive with various K(+)-channel subtypes native to a number of different species. Immunoblot analysis demonstrated affinity of the antibody for the drk1, maxi-K, and KAT1 K(+)-channel proteins. Studies also suggested that the anti-channel pore peptide antibody did not immunoreact with membrane proteins other than K+ channels. The anti-channel pore peptide antibody was used to establish the identity of a 62-kDa chloroplast inner envelope polypeptide as a putative component of a K(+)-channel protein. It was concluded that an antibody generated against the conserved pore region/selectivity filter of K+ channels has broad but selective affinity for this class of proteins. This K(+)-channel probe may be a useful tool for identification of K(+)-channel proteins in native membranes.