Photoreactions of cytochrome b6 in systems using resolved chloroplast electron-transfer complexes

Photoreactions of cytochrome b₆ have been studied using resolved chloroplast electron-transfer complexes. In the presence of Photosystem (PS) II and the cytochrome b₆-f complex, photoreduction of the cytochrome can be observed. No soluble components are required for this reaction. Cytochrome b₆ photoreduction was found to be inhibited by quinone analogs, which inhibit at the Rieske iron-sulfur center of the cytochrome complex, by the addition of ascorbate and by depletion of the Rieske center and bound plastoquinone from the cytochrome complex. Photoreduction of cytochrome b₆ can also be demonstrated in the presence of the cytochrome complex and PS I. This photoreduction requires plastocyanin and a low-potential electron donor, such as durohydroquinone. Cytochrome b₆ photoreduction in the presence of PS I is inhibited by quinone analogs which interact with the Rieske iron-sulfur center. These results are discussed in terms of a Q-cycle mechanism in which plastosemiquinone serves as the reductant for cytochrome b₆ via an oxidant-induced reductive pathway.     

On the conformation of reconstituted ferredoxin:NADP oxidoreductase in the thylakoid membrane. Studies via triplet lifetime and rotational diffusion with eosin isothiocyanate as label

Eosin isothiocyanate was covalently bound to isolated ferredoxin-NADP⁺ reductase under protection of the NADP-binding domain. The bound label did not impair the functional reconstitution of the enzyme into depleted thylakoid membranes. Laser spectrophotometric experiments were carried out on thylakoids which were reconstituted with labeled ferredoxin-NADP⁺ reductase. Bound eosin isothiocyanate was used as a spectroscopic probe for conformational changes of ferredoxin-NADP⁺ reductase in either of two ways: We studied the rotational diffusion of labeled ferredoxin-NADP⁺ reductase in the membrane by the photoselection technique, and we studied the triplet lifetime of bound eosin, which measures polypeptide chain flexibility (via access of oxygen) around the binding site. The latter technique was complemented by measurements of the librational motion of bound dye. We observed: (1) When ferredoxin is absent, ferredoxin-NADP⁺ reductase undergoes very rapid rotational diffusion in the thylakoid membrane (correlation time less than 1 μs at 10°C). This is drastically slowed down (40 μs) upon addition of water-soluble ferredoxin. We propose that ferredoxin mediates the formation of a ternary complex with ferredoxin-NADP⁺ reductase and the Photosystem I complex. According to our data, this complex would live longer than required for the photoreduction of ferredoxin-NADP⁺ reductase by Photosystem I via ferredoxin. (2) Under the given incubation conditions, the binding sites for eosin isothiocyanate were located in the FAD domain of ferredoxin-NADP⁺ reductase. We found increased chain flexibility in this domain upon addition of NADP. This suggests induced fit for the binding of NADP and allosteric control of the FAD domain by the remote NADP domain. (3) Acidification of the internal phase of thylakoids decreased the chain flexibility in the FAD domain. This is of particular interest, since ferredoxin-NADP⁺ reductase is a peripheral external membrane protein. It suggests the existence of a binding protein for the oxidoreductase which spans the membrane and senses the internal pH     

Base-catalyzed oligomerization of levoglucosenone

Heating levoglucosenone in aqueous triethylamine gives a dimer and two trimers in yields of 8, 18, and 56%, respectively. These compounds have been isolated crystalline, and their structures and stereochemistry have been investigated by ¹³C- and ¹H-n.m.r. and other spectroscopic methods. These data indicate that the dimer is apparently formed by Michael addition to provide a C-3-C-4 linkage. Similar reactions provide a non-olefinic, C-3-C-4-linked, cyclic trimer and an olefinic, cyclic trimer containing two C-3-C-4 linkages and one C-2-C-3 linkage.     

Purification and characterization of peptic fragments derived from the carboxyl-terminal half of human albumin

Utilizing a combination of conventional and affinity-chromatographic procedures, we have purified four fragments of human albumin that were generated by controlled limited proteolysis with pepsin [0.3 mM albumin; 37°C; 10 min; pH 3.51; 4.2 mM octanoate; pepsin/albumin, 1:1000 (w/w)]. These fragments have a molecular weight range of 9200-17,000 Da. Amino acid compositions, N- and C-terminal sequences, molecular weights, and other internal markers were used to determine the location of these fragments within the parent molecule. All of the fragments were shown to be derived from the C-terminal half of human albumin. The presence of multiple pepsin-sensitive bonds near the C terminus of each fragment complicated the assignment of specific residue numbers to each fragment. Two pairs of similar peptides were identified: (A) those corresponding to a single-loop structure (residues 309–380 and 309–387) and (B) those containing multiple loops and intraloop cleavages [residues 309–(491–495) with 408–423 deleted]. Purification of these fragments without disulfide bond reduction confirms portions of the loop structure of human albumin and demonstrates increased susceptibility of two specific regions of the C-terminal half of the molecule to peptic digestion.     

Photoresponses of the copepod Mesocyclops edax

The predatory copepod Mesocyclops edax is an important componentof many zooplankton communities where it typically makes extensivedid vertical migrations. To describe the effect of light onadults we measured their photoresponses in the laboratory. Theresponse spectrum is characterized by a wide plateau of greatestsensitivity from about 480 – 580 nm. These animals areadapted to perceive light during the day since their regionof maximum sensitivity overlaps the spectral region of highestquantal intensity underwater (575 – 700 nm). The thresholdintensity for positive phototaxis by dark adapted animals wasabout 5 x 10^–1 Wm^–2 at 540 nm, and they were positivelyphototactic up to an intensity of 5 x 10^–1 Wm^–2.Above this intensity phototaxis is no longer observed. Light-adaptedanimals were less sensitive than dark-adapted, but their generalpattern of response to light intensity did not differ. Thereis no rhythm in phototaxis. Their photoresponses may providea mechanism for controlling vertical migration so as to minimizeexposure to planktivorous fish. ¹Contribution No. 1375-AEL from UM-CEES, Appalachian EnvironmentalLaboratory.     

Enriched populations of rat retinal ganglion cells: Studies using a cell-type specific surface marker

Cells dissociated from adult and neonatal rat retinas were separated by density gradient centrifugation. Previous work had shown that rat retinal cells labelled by an immunofluorescence assay for the Thy-1 antigen were chiefly or exclusively ganglion cells, and so the proportion of Thy-1 positive cells in the density gradient fractions was used as an index of the enrichment of ganglion cells. The proportion of Thy-1 positive neonatal cells was increased from about 0.4% in the initial dissociate to about 8% in the most enriched fraction of a Percoll step gradient. Amongst adult cells the initial 0.7% Thy-1 positive cells were increased to roughly 2% in the best fraction of a metrizamide step gradient.

The presence of relatively large numbers of Thy-1 positive cells in other fractions suggested that it would be difficult to further increase the proportion of rat ganglion cells by methods based on their sedimentation properties. These results demonstrate the importance of cell-type specific markers in attempts to purify cells from the central nervous system.     

Mating strategy shifts in male green treefrogs (Hyla cinerea): An experimental study

Four field experiments were designed to study calling and satellite mating strategies in the green treefrog, Hyla cinerea. (1) The calling male was removed from 19 satellite associations and 11 of the 19 satellite males began calling. (2) After the calling male was removed from 10 satellite associations, a speaker broadcast synthetic mating calls. All of the satellite males oriented to the speaker. (3) Synthetic mating calls were played back to 14 calling males. Eleven males stopped calling and oriented to the speaker during at least one trial. (4) Sequences of synthetic mating calls and synthetic encounter calls were broadcast to 12 calling males. Nine males became satellites when the speaker emitted mating calls; none did so when encounter calls were presented.