Photocontrol of immobilized trypsin activity

Trypsin was coupled on an agarose gel which was modified with a spiropyran compound. The trypsin–spiropyran (agarose) gel showed reverse photochromism. The activity of the trypsin–spiropyran gel in the dark was 12% of that of native trypsin, and it was higher than that under visible light. The apparent Michaelis constant of the trypsin–spiropyran gel in the dark was larger than that under visible light. On the other hand, the maximum velocity in the dark was higher than that under visible light. The optimum pH of the trypsin–spiropyran gel in the dark was the same as that under visible light. Immobilized trypsin was stable in the pH range from 3 to 9. The trypsin–spiropyran gel was more stable against heat than the native trypsin.     

Amino acid esterification by alpha-chymotrypsin immobilized in spiropyran membrane.

α-Chymotrypsin was immobilized in a collagen membrane modified with a spiropyran compound. The immobilized chymotrypsin was used for the esterification of N-acetyl-l-tyrosine (AT). N-Acetyl-l-tyrosine ethyl ester (ATEE) was synthesized from AT and ethanol by immobilized chymotrypsin under visible light. The optimum pH for the esterification was 7. An increase of the chymotrypsin content in the spiropyran-collagen membrane increased the rate and the yield of ATEE. The yield of ATEE reached 40% under visible light. Initially, ATEE was synthesized in the dark. However, the ATEE synthesized was gradually hydrolyzed in the dark. The amount of ATEE in the reaction mixture increased with irradiation by visible light and decreased in the dark. Therefore, the esterification of N-acetyl-l-tyrosine was controlled by light irradiation.     

Photo control of enzyme activity of alpha-amylase.

Photo control of enzyme activity was performed by attaching a photochromic spiropyran compound to α-amylase. Modified α-amylase exhibited reverse photochromism in water: a colored form in the dark and a colorless form under light irradiation, which indicated that bound spiropyran possessed a hydrophilic structure (an open-ring form) in the dark and a hydrophobic structure (a closed-ring form) under light irradiation. The activity of modified α-amylase under light irradiation was extremely retarded as compared with that determined in the dark. The photo-induced change of the activity reversibly occurred in accordance with the photochromism of bound spiropyran. The mechanism of the photo control is discussed.     

Photocontrol of affinity chromatography: Purification of asparaginase by photosensitive AHA-gel

An agarose gel modified with N-(ω-aminohexytl)-L -aspartic acid (AHA) and spiropyran compound (AHA–spiropyran gel) was prepared and the photocontrolled binding and releasing of asparaginase were investigated with the AHA–spiropyran gel. Asparaginase was bound on the AHA–spiropyran gel under visible light and was released in the dark. The optimum conditions for photocontrolled binding and releasing of asparaginase were a 0.05M phosphate buffer concentration and pH 7.0. Seventy-five percent of the bound asparaginase was released from the AHA–spiropyran gel column in the dark. Ninetyfold purification of asparaginase was performed with the AHA–spiropyran gel Column.     

Photocontrol of affinity chromatography. I. Trypsin purification by photosensitive soybean trypsin inhibitor (STI) gel

Soybean trypsin inhibitor (STI) was immobilized on the agarose gel modified with spiropyran compound (spiropyran gel), and photocontrolled binding and releasing of trypsin was examined. The STI-spiropyran gel showed reverse photochromism. Trypsin was bound on the STI-spiropyran gel in the dark and released with visible light irradiation. The optimum conditions for photocontrolled binding and releasing of trypsin were pH 6.6 and the buffer concentration of 0.05 m. Approximately 60–80% of bound trypsin was released with visible light irradiation. The activity of released trypsin was the same as that of native trypsin. Approximately 21-fold purification of trypsin was performed with the STI-spiropyran gel column.     

Photocontrol of urease-collagen membrane activity.

(1) Urease (EC 3.5.1.5.) was modified with beta-1-[3,3-dimethyl-6'-nitrospiro-(indoline-2,2'-2H-benzopyrene)] propionic anhydride. Three amino acid residues of urease were modified by the anhydride at a molar ratio of 2000. (2) The activity of modified urease was decreased with ultraviolet irradiation and then restored to the initial activity with visible light irradiation. (3) Modified urease was used to prepare a urease-collagen membrane. The apparent Michaelis constant (Km) of the modified urease-collagen membrane ultraviolet light was identical to that of the membrane under visible light. (4) The optimum pH of the modified urease-collagen membrane was displaced toward lower pH values with ultraviolet irradiation. At higher ionic strength, the pH activity curve of the membrane was displaced toward higher pH values. (5) The thermostability of urease was increased with its modification.     

Photoresponsive polypeptides: Photochromism and conformation of poly (L-glutamic acid) containing spiropyran units

Spirobenzopyran units were bound to the side chains of poly (L -glutamic acid) and partially methylated poly(L -glutamate)s. The modified polymers were found to exhibit “reverse photochromism” in hexafluoro-2-propanol (HFP), so the samples kept in the dark were characterized by an intense absorption band in the visible range of the spectrum, which was completely erased upon exposure to sunlight or irradiation at 500–550 nm. The CD spectra showed that the macromolecules adopted a random coil conformation in the dark, whereas the bleached solutions after exposure to light displayed the typical CD pattern of the α-helix. The back reaction in the dark was accompanied by the progressive decrease of the helix content and recovery of the original disordered conformation. The photoinduced conformational changes resulted in large and reversible viscosity variations. When spiropyran side chains were converted to “spiropyran salts” of trifluoroacetic acid, the system was still photochromic, but the macromolecules were disordered both in the dark and light conditions. However, when appropriate amounts of methanol were added as a cosolvent to the HFP solutions, the system responded to light, giving reversible variations of the α-helix content. Irradiation at appropriate solvent compositions allowed modulation of the extent of the photoresponse. © 1993 John Wiley & Sons, Inc.     

Light and dark active phosphodiesterase regulation in salamander rods

We studied the activation of 3',5'-cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE) by using a cell-permeant enzyme inhibitor. Rods of Ambystoma tigrinum held in a suction electrode were jumped into a stream of 3-isobutyl-1-methylxanthine (IBMX), 0.01-1 mM. Initial transient light-sensitive currents fit the notion that dark and light-activated forms of PDE contributed independently to metabolic activity and were equivalently inhibited by IBMX (apparent Ki 30 microns). Inhibition developed within 50 ms, producing a step decrease of enzyme velocity, which could be offset by activation with flashes or steps of light. The dark PDE activity was equivalent to light activation of enzyme by 1,000 isomerization rod-1s-1, sufficient to hydrolyze the free cGMP pool (1/e) in 0.6 s. Steady light activated PDE in linear proportion to isomerization rate, the range from darkness to current saturation amounting to a 10-fold increase. The conditions for simultaneous onset of inhibitor and illumination to produce no net change of membrane current defined the apparent lifetime of light-activated PDE, TPDE* = 0.9 s, which was independent of both background illumination and current over the range 0-3 x 10(5) isomerization s-1, from 50 to 0 pA. Adaptation was a function of current rather than isomerization: jumps with different proportions of IBMX concentration to steady light intensity produced equal currents, and followed the same course of adaptation in maintained light, despite a 10-fold difference of illumination. Judged from the delay between IBMX- and light-induced currents, the dominant feedback regulatory site comes after PDE on the signal path. The dark active PDE affects the hydrolytic flux and cytoplasmic diffusion of cGMP, as well as the proportional range of the cGMP activity signal in response to light.     

Role of Light in the Appearance of Glutamine Synthetase in Leaves of Pennisetum glaucum

Leaves of Pennisetum [Pennisetum glaucum (L) HHB 67] seedlings contained two isozymes of glutamine synthetase (GS, EC 6.3.1.2): cytosolic GS1 and chloroplastic GS2. Leaves of seedlings grown in light for seven days contained about twofold higher GS activity than etiolated leaves. In both light and dark grown seedlings, total GS, GS1 and GS2 activity declined with plant age with more pronounced effect in leaves of etiolated seedlings, and GS2 declined at a much faster rate than GS1. Exposure of etiolated seedlings to light markedly enhanced GS1 and GS2 activity. This increase in activity was not affected by cycloheximide, precluding light dependent de novo synthesis of the enzyme. Treatment of etiolated seedlings with photosynthetic inhibitor, dichlorophenyl dimethyl urea (DCMU) inhibited light dependent appearance of GS. Exogenous supply of sucrose to dark grown seedlings greatly increased the GS activity in dark. These results suggest that light-mediated stimulation in activity of GS in Pennisetum leaves is dependent on photosynthetic reaction.     

Biogenesis of Thylakoid Membranes in Chlamydomonas reinhardtii y1 (A Kinetic Study of Initial Greening)

Initiation of thylakoid membrane assembly was examined in degreened cells of Chlamydomonas reinhardtii y1 cells depleted of thylakoid membranes and photosynthetic activity by growth in the dark for 3 to 4 d. Photoreductive activities of photosystem II (PSII) and photosystem I (PSI) increased with no apparent lag when degreened cells were exposed to light at 38[deg]C. However, fluorescence transients induced by actinic light, which reflect the functional state of PSII, changed only slightly during the first 2 h of greening. When these cells were treated with 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) or saturating light, fluorescence increased commensurate with the cellular content of chlorophyll. In similar experiments with greening cells of C. reinhardtii CC-2341 (ac-u-g-2.3), a PSI-minus strain, fluorescence increased with chlorophyll without treatment with DCMU. These data suggested that fluorescence of initial PSII centers in greening y1 cells was quenched by activity of PSI. Continuous monitoring of fluorescence in the presence or absence of DCMU showed that assembly of quenched PSII centers occurred within seconds after exposure of y1 cells to light. These results are consistent with initial assembly of PSI and PSII within localized domains, where their proximity allows efficient energy coupling.     

Visible light during mycelial growth and conidiation of Metarhizium robertsii produces conidia with increased stress tolerance

Light conditions during mycelial growth are known to influence fungi in many ways. The effect of visible-light exposure during mycelial growth was investigated on conidial tolerance to UVB irradiation and wet heat of Metarhizium robertsii, an insect-pathogenic fungus. Two nutrient media and two light regimens were compared. Conidia were produced on (A) potato dextrose agar plus yeast extract medium (PDAY) (A1) under dark conditions or (A2) under continuous visible light (provided by two fluorescent lamps with intensity 5.4 W m(-2)). For comparison, the fungus was also produced on (B) minimal medium (MM) under continuous-dark incubation, which is known to produce conidia with increased tolerance to heat and UVB radiation. The UVB tolerances of conidia produced on PDAY under continuous visible light were twofold higher than conidia produced on PDAY medium under dark conditions, and this elevated UVB tolerance was similar to that of conidia produced on MM in the dark. The heat tolerance of conidia produced under continuous light was, however, similar to that of conidia produced on MM or PDAY in the dark. Conidial yield on PDAY medium was equivalent when the fungus was grown either under continuous-dark or under continuous-light conditions.     

Aromatic L-Amino Acid Decarboxylase Is Modulated by D1 Dopamine Receptors in Rat Retina

Aromatic L-amino acid decarboxylase (AAAD) activity of rat retina increases when animals are placed in a lighted environment from the dark. The increase of activity can be inhibited by administering the selective dopamine D1 receptor agonist SKF 38393, but not the selective D2 agonist quinpirole, or apomorphine. Conversely, in the dark, enzyme activity can be enhanced by administering the selective D1 antagonist SCH 23390 or haloperidol, but not the selective D2 antagonist (-)-sulpiride. Furthermore, in animals exposed to room light for 3 h, the D1 agonist SKF 38393 reduced retinal AAAD activity, and this effect was prevented by prior administration of SCH 23390. In contrast, quinpirole had little or no effect when administered to animals in the light. Kinetic analysis indicated that the apparent Vmax for the enzyme increases with little change in the apparent Km for the substrate 3,4-dihydroxyphenylalanine or the cofactor pyridoxal-5'-phosphate. We suggest that dopamine released in the dark tonically occupies D1 receptors and suppresses AAAD activity. When the room light is turned on, D1 receptors are vacated and selective D1 agonists can either prevent the rise of AAAD or reverse light-enhanced AAAD activity.     

Decay of membrane potential under phosphorylating conditions in chloroplasts with in vivo activated ATPase

(1) The relation between the membrane potential and phosphorylation was studied in chloroplasts rapidly prepared from illuminated spinach leaves (light chloroplasts) and from dark-adapted leaves (dark chloroplasts). Light chloroplasts had a higher ATP hydrolysis activity than dark chloroplasts. (2) In the presence of ADP or ATP, a rapidly decaying phase of the field-indicating 518 nm absorbance change with a half-time of 15 ms became apparent in addition to the slow phase with a half-time of more than 300 ms in either type of chloroplast. Under these conditions, light chloroplasts showed a larger rapid phase than dark chloroplasts. (3) The rapid phase was suppressed by dicyclohexylcarbodiimide and was assumed to reflect the dissipation of membrane potential due to proton movements inside the CF₁-CF₀ ATP synthetase. (4) A model for the proton movement in ATP synthetase is proposed.     

Tacrolimus-Induced Neurotoxicity and Nephrotoxicity Is Ameliorated by Administration in the Dark Phase in Rats

1. Tacrolimus, a potent immunosuppressant, induces impaired renal function and neurological complications. We investigated the influence of dosing time on the neurotoxicity, nephrotoxicity, and immunosuppressive effect of tacrolimus in rats. 2. The repeated injection of tacrolimus in the light phase (8:00) produced a significantly greater increase than that in the dark phase (20:00) in the duration of harmine-induced tremors and in the blood urea nitrogen (BUN) concentration in rats. An immunosuppressive effect of tacrolimus on the xenotransplantation of mouse-to-rat skin grafts was apparent in the dark phase but not in the light phase. 3. The dosing time-dependent pharmacokinetic results were not observed when tacrolimus concentrations in rat whole blood were measured after a single or repeated injection in the light or dark phase. 4. These findings suggest that treatment in the active phase of the diurnal cycle ameliorates neurotoxicity and nephrotoxicity while maintaining the immunosuppressive effect of tacrolimus. The present findings have important implications for therapeutic approaches to avoid tacrolimus-induced neurotoxicity and nephrotoxicity.     

NaCl胁迫对互花米草细胞膜和光响应曲线特征参数的影响

以大米草的互花米草为材料,研究了不同盐浓度对其细胞膜透性、丙二醛(MDA)含量和光响应曲线的特征参数的变化情况。结果表明:盐浓度低于300mmol·L-1时,互花米草细胞膜透性和MDA含量较对照组无显著差异;其较高的最大光合速率(>30μmol·m-2·s-1),表观量子效率(>0.05mol·mol-1Photons)以及较低的暗呼吸速率(<1.5μmolCO2·m-2·s-1)和光补偿点(<20μmol·m-2·s-1)为其有机物质积累、竞争、建立种群并扩散提供条件。盐浓度高于500mmol·L-1时,互花米草膜透性和MDA含量显著上升,最大光合速率(Amax)及表观量子效率(Q)显著下降,暗呼吸速率(Rday)和光补偿点(LCP)上升。表明细胞膜和光合作用有关酶受到迫害,抑制了其正常生长。盐胁迫下互花米草光合速率降低,但蒸腾速率的显著下降提高了单叶水分利用效率,从而部分缓解了渗透势变化对细胞的迫害,为其生存和生长提供条件。     

光照或黑暗条件下野火病菌侵染对烟草光合机构的影响

烟草野火病菌(Pst)是一种兼性营养型的细菌致病菌,它可以引起烟草发生褐色病斑,名为野火病.近年来Pst受到很多关注,然而大多数对Pst的研究主要集中在寄主和非寄主植物对Pst侵染的防御机制和产自于野火病菌的野火毒素上,Pst侵染对烟草叶片光合性能的影响及其机理尚未见报道.研究Pst侵染后对光系统Ⅱ(PSⅡ)的影响不仅可以帮助阐明烟草-Pst 相互作用的机制,还可以从生理角度加深对细菌致病菌病害的了解.本研究采用叶绿素荧光快速诱导动力学曲线分析、类囊体膜蛋白Western分析、活性氧(ROS)和叶绿素含量测定等方法,探讨光照(200 μmol·m^-2·s^-1)或黑暗条件下Pst侵染对烟草光系统Ⅱ的影响.结果表明: 与未处理相比,Pst侵染3 d后在光照和黑暗条件下叶片侵染区域叶绿素含量均显著下降,出现萎黄病变,注射区域呈现出明显的野火病特征.光照和黑暗条件下,侵染3 d后烟草叶片过氧化氢含量明显升高,光照条件下要比黑暗条件下升高比例更大.Pst侵染3 d后,光照和黑暗条件下烟草叶片注射区域叶绿素荧光动力学曲线中K点和J点的相对可变荧光W_K和V_J逐渐增大,叶片最大光化学效率(F_v/F_m)和单位面积有活性反应中心的数目(RC/CSm)均显著下降.此外,相对于光照条件,Pst侵染后在黑暗条件下W_K和V_J的升高程度更大,说明对K点和J点的抑制程度更严重.Pst侵染3 d后,在光照和黑暗条件下放氧复合体(OEC)的核心组分PsaO、光系统Ⅱ反应中心核心蛋白D1蛋白均发生明显的降解,且在黑暗条件下降解更为严重.表明Pst侵染后,在光照和黑暗条件下均会使光合电子传递链Q_A到Q_B的电子传递受到限制,放氧复合体受到伤害,烟草叶片光系统Ⅱ供体侧、受体侧、反应中心的数目和活性均受到伤害,光系统Ⅱ发生光抑制或类似光抑制的伤害,且在黑暗条件下对光系统Ⅱ的伤害程度比光照条件下更为严重.     

Effects of Daylength on Endogenous Gibberellins in Solanum andigena III. Gibberellin Production by the Leaves

Using an agar diffusion technique, it was found that leaves from potato plants growing under long days produced more gibberellin-like substances than did leaves from plants growing under short days. Short day plants irradiated with red light during the long dark period and harvested during the ensuing light period, contained levels of gibberellin-like substances approximating those found in long day grown plants. Red irradiation during the long dark period also resulted in an increase in gibberellin production in short day plants. Four zones of gibberellin-like activity (A, B, C, D) were separated by thin layer chromatography in extracts from potato leaves. Red light treatment reduced the levels of peak D and brought about a concomitant increase in the levels of peak A.     

The effects of light on induction, time courses, and kinetic patterns of net nitrate uptake in barley

Barley seedlings ( Hordeum vulgare L.) were grown hydroponically with (induced) or without (uninduced) nitrate in a light/dark cycle with high photon flux density to determine the effects of light on time courses, induction and kinetics of net nitrate uptake. Nitrate uptake was induced by external nitrate in both light and dark and was prevented by 1 mol m^–3 p-fluorophenylalanine. In high light, nitrate uptake was about 2-fold higher than in low light. During time course experiments the uptake rates oscillated due to daily light–dark changes. Rates of nitrate uptake also increased at about 2200 h during continuous darkness. This increase coincided approximately with the time at which the dark period started during the previous culture of the plants, indicating that it was due to a mechanism associated with an endogenous diurnal rhythm. When calculating the kinetics of nitrate uptake, a model with two saturable systems, including a high-affinity system (HATS) and a low-affinity system (LATS), gave the best fit to data in all treatments. The apparent affinity of the HATS ranged from 7·7 to 12·2 mmol m^–3 in induced plants in all light conditions. The effect of light on the HATS was mainly an increase of apparent V _max in the step from low to high light. In uninduced plants the HATS operated at a very low activity which was strongly enhanced during induction. Interpretation of the calculated kinetics of the LATS was much more difficult on the basis of net uptake data. The apparent affinity of the LATS increased from 24·3 mol m^–3 in low light up to 0·17 mol m^–3 after acceleration in high light. These extreme changes in apparent affinity of the LATS could not be explained satisfactorily, and the nature of this system is also discussed with respect to the method used.     

光和暗条件下低温对黄瓜和水稻叶绿素蛋白质复合体的影响

本试验以黄瓜和水稻幼苗为材料,研究了光照和黑暗条件下低温对植物叶绿素蛋白质复合体的影响。SDS—PAGE电泳结果表明:5℃及12h 280μmol m~(-2)S~(-1)处理2d,Chl-蛋白质复合体的降解明显大于5℃暗低温处理;低温与光照对P700-CPa_1的影响大于LHCP。叶绿素荧光测定表明;5℃及12h 280μmol m~(-2)s~(-1)的处理对PSⅡ的影响亦大于暗低温处理。由此认为:低温与光对植物叶绿体的PSⅠ和PSⅡ都有明显的影响,其机理可能与常温下高光强引起的光抑制相类似;不同的是低温下中等光强就能引起光抑制。因此,在光照低温下往往加剧植物冷害的发生。     

光照和生长阶段对菖蒲根系泌氧的影响

以自然湖泊沉积物为研究基质,利用微型电机控制溶氧微电极实现纵向精确微位移,在照光与遮光条件下,对典型湿地植物菖蒲幼苗、成株根系根基部起总根长1/4处(根1/4)、根系中部(根1/2)、从根基部起总根长3/4处(根3/4)及根尖(根1)处根系微界面径向溶氧浓度变化进行原位精确测定。结果表明:无论有无光照,菖蒲幼苗、成株根系不同部位均存在从根表面至沉积物氧饱和度由高到低的氧扩散层,其厚度0.18—0.68 mm;根1/2、3/4、1处氧扩散能力菖蒲成株较幼苗显著增强(P<0.01),根1/4处二者则无显著差异(P>0.05);光照对菖蒲幼苗、成株根系不同部位氧扩散能力的影响存在差异,光照对菖蒲幼苗根1/2及菖蒲成株根1/2、根3/4处影响显著(照光组显著高于遮光组,P<0.01),而对菖蒲幼苗根1/4、根3/4、根1及菖蒲成株根1/4、根1处无显著影响(P>0.05);从根系泌氧空间差异上看,照光条件下菖蒲幼苗、成株分别表现为根1/2>根3/4≈根1≈根1/4(P<0.01,P>0.05)和根1/2>根3/4>根1>根1/4(P<0.01),遮光条件下菖蒲幼苗、成株分别表现为根1/2≈根3/4≈根1≈根1/4(P>0.05)和根1/2>根3/4≈根1>根1/4(P<0.01,P>0.05)。