Energy parameters of Acetabularia cell membrane]

The energy parameters of the mechanism involved in the action of Acetabularia cell membrane were studied. The resistance of the membrane is found to be near 3 kohm per sq. cm when resting and at the peak of excitation it is much lower (100 ohm/cm(2). The power of the mechanism regulating the action of the membrane during the peak. The total power of the membrane system of Acetabularia is 8 microwatt per sp. cm. reaches 6.5 microwatt/cm(2). The energy consumed by the regulatory mechanism during the action potential is minus 290 plus or minus 20 erg/cm(2) and the total energy dissipation is 20 erg/cm(2). The values of the energy consumed by the cell during the action potential are compared with its energy resource.     

Changes in membrane potential and resistance caused by transient increase of potassium conductance in the unicellular green alga Eremosphaera viridis

The electrophysiological membrane parameters of the unicellular green alga Eremosphaera viridis were determined using an improved computer-supported single-microelectrode technique. These cells developed an average membrane potential of-150 mV in the light and a specific resistance of 1 Ω m² with an external potassium concentration of 1.1 mM and pH 5.5. In the dark, many cells showed a less polarized potential of 30–40 mV and a smaller membrane resistance. At potassium concentrations in the external medium higher than 1 mM, the membrane potential strongly depends on the external potassium content apart from a small electrogenic component. At concentrations lower than 1 mM K⁺, a dependence of the membrane potential upon external potassium concentrations could not be verified. Inserting the internal ion activities in the Goldmann equation shows that, in this range, the proton conductance seems to be predominant over the potassium conductance. Transient changes in the membrane potential and in the membrane resistance were observed after switching off the light, after addition of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea or N,N′-dicyclohexylcarbodiimide, after a sudden decrease in temperature, and after current pulses. These changes resemble the action potentials (AP) found in other plant cells (Chara, Acetabularia). On average, the AP has a delay period of 5.1 s and a duration of 43.8 s showing a sudden decrease and a slower regeneration. The voltage peak during an AP followed exactly the Nernst potential of potassium over a range of external potassium concentrations from 5 μM to 0.2 M. This is true for depolarization or hyperpolarization, depending on the external K⁺-concentration. Tetraethylammonium-hydrogensulphate, a rather specific inhibitor of K⁺ channels in nervous cells, suppressed the AP. The correlation of the appearance of the AP with a short-term opening of potassium channels in the membrane of Eremosphaera is discussed.     

Adaptation in the Ventral Eye of Limulus is Functionally Independent of the Photochemical Cycle, Membrane Potential, and Membrane Resistance

The early receptor potential (ERP), membrane potential, membrane resistance, and sensitivity were measured during light and/or dark adaptation in the ventral eye of Limulus. After a bright flash, the ERP amplitude recovered with a time constant of 100 ms, whereas the sensitivity recovered with an initial time constant of 20 s. When a strong adapting light was turned off, the recovery of membrane potential and of membrane resistance had time-courses similar to each other, and both recovered more rapidly than the sensitivity. The receptor depolarization was compared during dark adaptation after strong illumination and during light adaptation with weaker illumination; at equal sensitivities the cell was more depolarized during light adaptation than during dark adaptation. Finally, the waveforms of responses to flashes were compared during dark adaptation after strong illumination and during light adaptation with weaker illumination. At equal sensitivities (equal amplitude responses for identical flashes), the responses during light adaptation had faster time-courses than the responses during dark adaptation. Thus neither the photochemical cycle nor the membrane potential nor the membrane resistance is related to sensitivity changes during dark adaptation in the photoreceptors of the ventral eye. By elimination, these results imply that there are (unknown) intermediate process(es) responsible for adaptation interposed between the photochemical cycle and the electrical properties of the photoreceptor.     

H+ -pumping rhodopsin from the marine alga Acetabularia

An opsin-encoding cDNA was cloned from the marine alga Acetabularia acetabulum. The cDNA was expressed in Xenopus oocytes into functional Acetabularia rhodopsin (AR) mediating H+ carried outward photocurrents of up to 1.2 microA with an action spectrum maximum at 518 nm (AR518). AR is the first ion-pumping rhodopsin found in a plant organism. Steady-state photocurrents of AR are always positive and rise sigmoidally from negative to positive transmembrane voltages. Numerous kinetic details (amplitudes and time constants), including voltage-dependent recovery of the dark state after light-off, are documented with respect to their sensitivities to light, internal and external pH, and the transmembrane voltage. The results are analyzed by enzyme kinetic formalisms using a simplified version of the known photocycle of bacteriorhodopsin (BR). Blue-light causes a shunt of the photocycle under H+ reuptake from the extracellular side. Similarities and differences of AR with BR are pointed out. This detailed electrophysiological characterization highlights voltage dependencies in catalytic membrane processes of this eukaryotic, H+ -pumping rhodopsin and of microbial-type rhodopsins in general.     

Intracellular axial current in Chara corallina reflects the altered kinetics of ions in cytoplasm under the influence of light

Recent experiments demonstrate that the concentration of Ca2+ in cytoplasm of Chara corallina internodal cells plays important role in electrical excitation of the plasma membrane. The concentration of free Ca2+ in the cytoplasm -[Ca2+]c is also sensitive to visible light. Both phenomena were simultaneously studied by noninvasive measuring action potential (AP) and magnetic field with a superconducting quantum interference device magnetometer in very close vicinity of electrically excited internodal C. corallina cells. A temporal shift in the depolarization maximum, which progressively occurred after transferring cells from the dark into the light, can be explained by the extended Othmer model. Assuming that the change in membrane voltage during the depolarization part of AP is the direct consequence of an activation of [Ca2+]c sensitive Cl- channels, the model simulations compare well with the experimental data. We can say that we have an example of electrically elicited AP that is of biochemical nature. Electric and magnetic measurements are in good agreement.     

Day/night variations in membrane bound leucyl-2-naphthylamide hydrolysing activity in rat anterior hypothalamus, pituitary and retina

It is well known that the suprachiasmatic nucleus of the anterior hypothalamus acts as pacemaker regulating circadian rhythms in mammals. The daily variations of neuropeptides as well as their receptors depend on this system (Moore 1983). Aminopeptidases play an important role in regulating the activity of brain peptides (Bauer 1982). In this study we investigated membrane bound leucyl-2-naphthylamide hydrolysing activity in the anterior hypothalamus, pituitary and retina of adult male rats at six time points of a 12:12h light:dark schedule (light from 7:00 to 19:00 h), in order to analyse its day/night variation. The fluorometric assay evidenced significant differences between the three regions: in the anterior hypothalamus being higher during the dark period compared with the light period and in the pituitary higher during the light period compared with the dark period. In the retina the levels of this activity showed a higher heterogeneity during the day. Day - night differences in membrane bound leucyl-2-naphthylamide hydrolysing activity may reflect differences in its susceptible endogenous substrates.     

Some aspects of glycoprotein metabolism in Acetabularia: Spatio‐temporal activity of β‐galactosidase and physiological effects of tunicamycin

Abstract

β‐galactosidase has been chosen as an indicator of glycoprotein metabolism in Acetabularia, an unicellular and uninucleate green alga. This catabolic enzyme was quantified by fluorecence spectrometry. It was found at all developmental stages, but the activity levels differed, peaking at the end of the growth phase, at the time of cap morphogenesis initiation, β‐galactosidase activity is also subjected to periodic modulation, displaying a bimodal rhythm with a prominent peak at 16 h. The distribution of the enzyme was examined by cytochemistry, using a substrate analogue (X‐gal). It is present both in the cytoplasm and in the cell wall. No apico‐basal gradient was detectable. The physiological role of glycoproteins was assessed with tunicamycin, an inhibitor of N‐linked glycoprotein synthesis. Two pulses of 3 or 4 h of inhibitor (10 μg ml‐1) always inhibited growth, but more severely during the light period. One pulse may inhibit growth during the light period and stimulate it during the dark one; it may also have little effect, in both periods. Cap formation is inhibited between time 0 and 7. During the dark or subjective dark period, it is often stimulated or not affected. The same results were obtained in constant light. Cap formation is also inhibited in anucleate algae treated during the light period.     

Development and plasticity of mitochondria and electrical properties of the cell membrane in blowfly photoreceptors

Blowfly photoreceptors are highly energy demanding sensory systems. Their information processing efficiency is enabled by the high temporal resolution of the cell membrane, requiring heavy metabolic support by the mitochondria. We studied the developmental changes of the mitochondrial apparatus and electrical properties of the photoreceptor membrane in the white eyed Calliphora vicina Chalky. Using in vivo microspectrophotometry and Western blot analysis, we found an age-dependent increase in the concentration of mitochondrial pigments. The maximal change occurred during the first week. The age-related changes were smaller in dark-bred than in light-bred flies. The mitochondrial pigment content increased after the switch from dark to light rearing and decreased after the switch from light to dark rearing. The electrical parameters of the photoreceptors were investigated with intracellular recordings. The resting membrane resistance and time constant decreased significantly after eclosion. The decrease was again most significant during the first week of adult life, paralleled with changes in the Na/K pump-dependent hyperpolarizing afterpotential. We conclude that the photoreceptor mitochondria exhibit remarkable ontogenetic and phenotypic plasticity, because the quantity of mitochondrial pigments tightly follows the development of the cell membrane as well as the energy demands of the photoreceptors under different rearing conditions.     

Synthesis of Chloroplast and Cytosol Ribosomes in Regenerating Acetabularia Cells

The incorporation of ³H-uridine into 80S cytosol and into 70S chloroplast ribosomes has been studied in Acetabularia in order to find out whether or not the nuclei undergo changes in the capability of rRNA formation during differentiation. To reinforce the differentiation process regenerating Acetabularia cells were chosen. Regeneration was induced by amputation of the apical part of the cell. The incorporation into cytosol ribosomes increased as early as 24 h after amputation. The stimulation reaches a maximum three to four days after amputation and does not depend on light at least for three days. It can be prevented, however, by addition of cycloheximide to the culture medium. The stimulation pattern of the uridine incorporation into chloroplast ribosomes is similar to the cytosol ribosomes with the exception that it is delayed by two days. This process is dependent on light and is not inhibited by cycloheximide. The response to amputation seems to be independent of chloroplast energy production as small and large fragments are stimulated to the same degree.     

Aspects of clock resetting in flowering of xanthium

Flowering is induced in Xanthium strumarium by a single dark period exceeding about 8.3 hours in length (the critical night). To study the mechanism which measures this dark period, plants were placed in growth chambers for about 2 days under constant light and temperature, given a phasing dark period terminated by an intervening light period (1 min to several hrs in duration), and finally a test dark period long enough normally to induce flowering. In some experiments, light interruptions during the test dark period were given to establish the time of maximum sensitivity.

If the phasing dark period was less than 5 hours long, its termination by a light flash only broadened the subsequent time of maximum sensitivity to a light flash, but the critical night was delayed. In causing the delay, the end of the intervening light period was acting like the dusk signal which initiated time measurement at the beginning of the phasing dark period.

If the phasing dark period was 6 hours or longer, time of maximum sensitivity during the subsequent test dark period was shifted by as much as 10 to 14 hours. In this case the light terminating the phasing dark period acted as a rephaser or a dawn signal.

Following a 7.5-hour phasing dark period, intervening light periods of 1 minute to 5 hours did not shift the subsequent time of maximum sensitivity, but with intervening light periods longer than 5 hours, termination of the light acts clearly like a dusk signal. The clock appears to be suspended during intervening light periods longer than 5 to 15 hours. It is restarted by a dusk signal. There is an anomaly with intervening light periods of 10 to 13 hours, following which time of maximum sensitivity is actually less than the usual 8 hours after dusk.

Ability of the clock in Xanthium to be rephased, suspended, restarted, or delayed, depending always upon conditions of the experiment, is characteristic of an oscillating timer and may confer upon this plant its ability to respond to a single inductive cycle. It is suggested that phytochrome may influence only the phase of the clock and not other aspects of flowering such as synthesis of flowering hormone.

     

Changes in ascorbic acid content and ascorbate peroxidase activity during the development of Acetabularia mediterranea

Abstract. The level of peroxidase activity utilizing ascorbic acid changes during the development of the green alga, Acetabularia mediterranea. During development almost parallel levels of peroxidase activity and ascorbic acid content are detectable: both steadily decrease as algae progress from very young, slowly growing cells to the rapid growth stage and then to cells exhibiting differentiation into primordium and cap. Changes in the levels of the enzyme and its substrate in the cytoplasm and periplasm were demonstrated using biochemical and cytochemical procedures. Concomitant with these developmental changes, we also observed changes in the stage-specific patterns of ascorbic acid concentration: growing algae exhibit a pronounced negative apicobasal gradient of ascorbic acid. Acetabularia cultivated at 1,200 lux (the normal intensity in a 12-h-light/12-h-dark cycle) and at 700 lux (intensity at which growth is reduced, and cap formation is delayed) were also compared. The higher light intensity induced a moderate decrease in the ascorbic acid content without noticeable changes in the compartmental distribution in the cytoplasm and periplasm, and an increase in the level of periplasmic peroxidase activity with little change in the total peroxidase activity. Catalase was found to be present at very low levels and is unlikely to play a role in H₂O₂ catabolism. Possible roles for ascorbic acid and peroxidase in the development of Acetabularia are discussed.     

Light-Induced Translocation of RGS9-1 and Gβ5L in Mouse Rod Photoreceptors

The transducin GTPase-accelerating protein complex, which determines the photoresponse duration of photoreceptors, is composed of RGS9-1, Gβ5L and R9AP. Here we report that RGS9-1 and Gβ5L change their distribution in rods during light/dark adaptation. Upon prolonged dark adaptation, RGS9-1 and Gβ5L are primarily located in rod inner segments. But very dim-light exposure quickly translocates them to the outer segments. In contrast, their anchor protein R9AP remains in the outer segment at all times. In the dark, Gβ5L''s interaction with R9AP decreases significantly and RGS9-1 is phosphorylated at S⁴⁷⁵ to a significant degree. Dim light exposure leads to quick de-phosphorylation of RGS9-1. Furthermore, after prolonged dark adaptation, RGS9-1 and transducin Gα are located in different cellular compartments. These results suggest a previously unappreciated mechanism by which prolonged dark adaptation leads to increased light sensitivity in rods by dissociating RGS9-1 from R9AP and redistributing it to rod inner segments.     

Circadian rhythms in enzymatic activity of rat liver arginase and glucose 6-phosphate dehydrogenase

The circadian rhythms of glucose 6-phosphate dehydrogenase (G6PD) and of arginase activities and total protein content have been studied in the livers of 24-h fasted rats. Both G6PD and arginase activities reach a maximum at night and a minimum during the light period. On the other hand, the total protein level was maximal during the light period while it decreased to its lowest level during the dark period. These results are in agreement with the existence of a lipogenesis-lipolysis circadian rhythm in the rat, since the higher G6PD activity at night provides the necessary NADPH for lipid biosynthesis. The increase in arginase activity is also in agreement with an increase in amino-acid catabolism, probably as a source of energy and metabolic intermediates.     

Photocontrol of urease activity in spiropyran collagen membrane.

1. Collagen fibrils were modified with beta-1-[3,3-dimethyl-6'-nitrospiro-(indoline-2,2'-2H-benzopyran)] propionic anhydride. 2. Urease (urea amidohydrolase, EC 3.5.1.5) was immobilized in spiropyran collagen membrane. The activity of the urease-spiropyran collagen membrane was found to increase in the dark and then decrease with visible light irradiation. 3. The optimum pH of the urease-spiropyran collagen membrane under visible light was lowered in the dark. 4. The apparent Michaelis constant (K'm) of the urease-spiropyran collagen membrane in the dark was almost the same as that under visible light. The apparent maximum velocity was increased in the dark. 5. The diffusion coefficient of urea through the spiropyran collagen membrane in the dark was 1.4 times that under visible light. However, the increase of the diffusion rate was not responsible for the activity increase of the urease-spiropyran collagen membrane.     

Effect of neonatal MSG treatment on day-night alkaline phosphatase activity in the rat duodenum

The day-night variation of food intake and alkaline phosphatase (AP) activity was studied in the duodenum of rats neonatally treated with monosodium glutamate (MSG) and saline-treated (control) rats. The animals were kept under light-dark conditions (light phase from 09:00 h to 21:00 h) with free access to food. AP activity was cytophotometrically analyzed in the brush-border of enterocytes separated from the tip, middle and cryptal part of the villi every 6 h over a 24-hour period. In comparison with the controls, MSG-treated rats consumed about 40% less food during the dark period and their 24-hour food intake was thus significantly lowered (P<0.001). On the other hand, the nocturnal feeding habit showed a similar pattern: food consumption was high during the night (65% vs. 75%) and the lowest consumption was found during the light phase (35% vs. 25%) in MSG-treated and control rats, respectively. In agreement with the rhythm of food intake, the highest AP activity was observed during the dark phase and was lowest during the light phase in both groups of animals. These significant day-night variations showed nearly the same pattern in the enterocytes of all observed parts along the villus axis. In comparison with the controls, a permanent increase of AP activity was observed in neonatal MSG-treated rats. This increase was more expressive during the dark phase of the day in the cryptal (P<0.001) and middle part of the villus (P<0.01). From the viewpoint of feeding, this enzyme in MSG-treated rats was enhanced in an inverse relation to the amount of food eaten i.e. despite sustained hypophagia the mean AP activity in the enterocytes along the villus axis was higher than in the control animals during all investigated periods. The present results suggest that the increased AP activity in MSG-treated rats is probably not a consequence of actual day-night eating perturbations but could be a component of a more general effect of MSG. This information contributes to better understanding of the function of intestinal AP and its relation to day-night feeding changes especially in connection with the MSG syndrome.     

Selective modification of coupling factor 1 in spinach chloroplast thylakoids by a fluorescent maleimide

N-(1-Anilinonaphthyl-4)maleimide (ANM) has been used to modify coupling factor 1 (CF1), the terminal coupling factor of photophosphorylation in chloroplasts. As with other monofunctional maleimides, incubation of thylakoids with ANM in the light, but not in the dark, causes energy transfer inhibition of photophosphorylation. In the dark, sites on both the gamma and epsilon subunits of CF1 are modified. The light-accessible site is also on the gamma subunit. Trypsin digestion of the enzyme after dithiothreitol activation reveals that the dark-and light-accessible sites on the gamma subunit are different amino acid residues. Fluorescence of ANM bound at the dark-and light-accessible sites has been measured after isolation of CF1 from thylakoids. The fluorescence emission maximum of ANM at the light-accessible site is blue-shifted and the quantum yield is increased 2-fold relative to ANM bound at dark-accessible sites. On the soluble enzyme, fluorescence polarization is high and equivalent for ANM bound at both dark-and light-accessible sites. Fluorescence energy transfer from a tryptophan in a hydrophilic region of the epsilon subunit to ANM bound to the epsilon subunit but not to the gamma subunit has been observed. The significance of these observations is discussed with respect to the structure of the gamma subunit and its role in conformational transitions within CF1 that occur during energization of the membrane.     

Rhabdom changes in the shrimp,Palaemonetes

The fine structure of the principal compound eye of the shrimp, Palaemonetes, was studied under conditions of light and dark adaptation. Ommatidium the situation in other decapod crustaceans. Light and dark adapted eyes differ in that the rhabdom changes its shape; morphological evidence suggests a possible sequence of events involving production, utilization, and degradation of photoreceptor membrane, a discontinuous process occurring only during changes from light to dark and dark to light. A hypothesis of membrane turnover is proposed.     

Control of the Photosynthetic Apparatus of Acetabularia mediterranea by Blue Light : Analysis by Light-Saturation Curves

During growth, Acetabularia mediterranea requires the action of blue light to maintain high rates of photosynthesis. In the present study, blue light-dependent alterations of the photosynthetic apparatus, which can be detected by analysis of light-saturation curves and by measurements of partial reactions of the photosynthetic electron transport chain, are described. Light-saturation curves of photosynthesis in vivo were measured with a new closed oxygen electrode system after culture of Acetabularia in continuous red or blue light. These curves were compared to those of 2,6-dichlorophenol-indophenol reduction by isolated chloroplast membranes. The analysis lead to the following statements: (a) only one reaction limits electron transport rates in vitro (dichlorophenol-indophenol reduction) at all light intensities irrespective of the light quality during growth, and (b) the limiting step is light driven and located in the reaction center of photosystem II. Presumably, this same reaction determines the flow of electrons under low light intensities in vivo in cells from white, blue, and red light. In addition to photosynthesis, the rates of dark respiration changed due to the action of blue light. Concomitantly, the light compensation point of apparent photosynthesis was shifted during monochromatic irradiations.