Light-dependent chemical modification of thylakoid membrane proteins with carboxyl-directed reagents

Spinach chloroplast thylakoid membranes were chemically modified with membrane penetrating reagents reactive toward protein carboxyl groups, a carbodiimide and the nucleophiles [¹⁴C]glycine ethyl ester or [³H]serotonin. The reagents, being weak bases, were accumulated within the inner aqueous space in the light, due to the low pH inside. Both the accumulation and the low pH stimulating effect on the carbodiimide activation step contributed to a greater labeling in the light compared to dark, and uncouplers inhibited most of the light-dependent increase. Hence, it is likely that the proteins showing the light-dependent, uncoupler-sensitive labeling have those parts located within the inner aqueous space or within the membrane itself. While many membrane proteins which separated on sodium dodecyl sulfate-polyacrylamide gels (12.5–25% gradient) showed some increased labeling in the light, the most conspicuous were the four polypeptides of the chlorophyll $\text{a}\text{b}$ light-harvesting complex. The light-harvesting complex was purified from dark- and light-treated, labeled membranes. The resultant preparation showed about a sixfold, light-dependent, uncoupler-sensitive labeling increase compared to dark conditions. Polypeptides near 6 and 8 kdalton showed light-dependent, uncoupler-resistent increases in carboxyl group modification, which could be due to localized acidic conditions near sites of proton release.     

Regulation of Pi,uptake by Acer pseudoplatanus cells

In view of the importance of P_i in the control of cell metabolism, it was of interest to study the mechanism and regulation of P_i uptake by Acer pseudoplatanus cells grown as cell suspensions. At low external P_i concentrations up to 10 mm, sycamore cells incorporate phosphate against a concentration gradient, by a process which is energy dependent. Under these conditions the intracellular P_i concentration is maintained constant (2–3 mm). On the contrary at high external P_i concentrations, higher than that which counterpoises the cytoplasmic P_i concentration (approximately 10 mm), P_i enters the cell by slow diffusion and the intracellular P_i concentration increases continuously as the extracellular P_i concentration increases from 15 to 50 mm. When sycamore cells are transferred to a phosphate-deficient medium, growth slows down considerably and ceases after 4–5 days. During this time, intracellular P_i concentration falls from 3 to 0.1 mm and phosphate esters from 8 to 2 mm. Phosphate starvation stimulates the uptake indicating that phosphate uptake depends on the intracellular phosphate and/or cytoplasmic ester-P pool. P_i uptake by P_i-starved cells is strongly dependent on the pH of the medium.     

Inactivation of Glutamine Synthetase by Ammonia Shock in the Gram-Positive Bacterium Streptomyces cattleya

In cultures of the gram-positive bacterium Streptomyces cattleya, a rapid inactivation of glutamine synthetase was seen after ammonia shock. pH activity curves for ammonia-shocked and control cultures are shown. A peak of glutamine synthetase activity was seen during fermentation for production of the antibiotic thienamycin.     

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.