ACTION SPECTRA OF PHOTOGEOTROPIC EQUILIBRIUM IN Phycomyces WILD TYPE AND THREE BEHAVIORAL MUTANTS

Photogeotropic equilibrium action spectra in the range from 301 to 740 nm were made for Phycomyces wild type and the three behavioral mutants C47 ( madA35 ), C109 ( madBlOl ) and LI (madCIIQ) , all of which have a raised phototropic threshold. In addition to two broad peaks at 365 and 455 nm, typical for flavins, the wild type action spectrum shows three novel peaks, which have not been observed previously. These peaks are located at 414, 491 and 650 nm. The 650 nm peak has a relative quantum efficiency of 3 × 10⁻⁸ compared to the peak at 414 nm. The wavelength dependent shapes of the fluence-response curves of the bending angle and the aiming error angle indicate more than one receptor pigment for phototropism. The shape of the action spectrum of C47 is basically unaltered in comparison to wild type. C109 and LI show substantial differences from the wild type. In the near UV two small peaks at 334 and 365 nm appear; the 414 and 491 nm peaks present in wild type and C47 are missing and two new peaks at 529 nm (not well resolved in C109) and 567 nm are found. None of the three mad mutations affects the 650 nm peak. A model of the sensory transduction chain is presented, which incorporates these and other known features.     

ACTION SPECTRA FOR PHOTOTROPIC BALANCE IN Phycomyces blakesleeanus: DEPENDENCE ON REFERENCE WAVELENGTH and INTENSITY RANGE

Abstract— Action spectra for phototropic balance of Phycomyces blakesleeanus sporangiophores were measured for various reference wavelengths and intensity ranges. Balance action spectra were made at fluence rates of 10^-4 W m^-2 with reference wavelengths of 450 nm, 394 nm, 507 nm, and broadband blue light. For broad-blue light and 450 nm light as references, typical flavin-like action spectra were found with a ma jor peak at 455 nm, a secondary peak at 477 nm, and a minor peak at 383 nm; these peaks are wider for broad blue than for 450 nm light. With the 394 nm reference, there is a major peak at 455 nm, a secondary peak at 477 nm and a minor peak at 394 nm. An action spectrum with 507 nm reference has a major peak at 455 nm and a minor peak at 383 nm, but no peak at 477 nm. A balance action spectrum was made with 450 nm reference light near threshold intensity (2 times 10^-8 W m^-2); there, the 386 nm peak is greatly reduced, while the 455 nm peak is enhanced. The intensity dependence of the 386 nm peak was studied in detail for reference light of 450 nm. We found that the relative quantum efficiency of the 386 nm light increases with the logarithm of the 450 nm fluence rate; in the high intensity range (0.3 W m^-2) the relative quantum efficiency of the 386 nm light is 1.3 and approaches zero at 10^-9 W m^-2. These findings indicate that P. blakesleeanus phototropism is mediated by multiple interacting pigments or by a photochromic photoreceptor.     

A FLAVOPROTEIN IN Phycomyces blakesleeanus WITH SHORT FLUORESCENCE LIFETIME

Abstract— The photoreceptor system for the blue-light-induced phototropism in the fungus Phycomyces blakesleeanus includes one or more flavin chromophores, probably located in the plasma membrane of the sporangiophore. From a plasma-membrane fraction of short synchronous sporangiophores, we have enriched, by column chromatography, specific proteins with covalently bound flavins. These flavoproteins were analyzed by a fluorescence lifetime assay. Flavoproteins with fluorescence lifetimes significantly less than 5 ns have been predicted to be involved in blue-light reception. We studied samples from a wild type strain and from the night-blind mutant LI. (genotype madC). For the enriched flavoprotein samples, we found predominant fluorescent components with lifetimes of 1.5 ns (74%) for wild type and 1.8 ns (83%) for LI. According to this assay, one or both of these flavoproteins are likely components of the blue-light photoreceptor system in P. blakesleeanus.     

ACTION SPECTRA OF PHYTOCHROME IN VIVO*

Abstract— A method is described to determine spectral properties of phytochrome in vivo. For photochrome in 7-day-old dark-grown Cucurbita pepo L. seedlings the mole fraction of the far-red-absorbing form (P_fr) present at photoequilibrium at 664 nm was found to be 0.76 ± 0.02 in vivo. Based on reflectance measurements, the photon fluence rate just below the surface of the cotyledons was calculated. Local rates of photoconversion for known local fluence rates were measured across cotyledons after non-saturating irradiations with wavelengths between 544 and 781 nm and in situ molar photoconversion coefficients were obtained. In contrast to purified oat phytochrome, the in situ molar photoconversion coefficients for P_fr show a strong shoulder between 660 and 700 nm. The maximum of P_fr absorption is at 726 nm. An isosbestic point of phytochrome is found at 686 nm. The mole fraction of P_fr present at photoequilibrium with 686 nm light is 0.58. The ratio of photoconversion quantum yields (that for P_r→ P_fr divided by that for P_fr→ P_r) is 1.38 ± 0.06.     

ACTION SPECTRA OF PHTHALOCYANINES WITH RESPECT TO PHOTOSENSITIZATION OF CELLS

Human carcinoma cells (NHIK 3025 cells) and Chinese hamster cells (V79 cells) were incubated with AlPcS1, AlPcS2 and AlPcS4, phthalocyanines with different lipophilicity but with similar photochemical properties when in monomeric solutions. The absorption- and fluorescence spectra of the dyes in the cells were recorded as well as their action spectra with respect to sensitizing cells to photoinactivation. These spectra show that under the present conditions AlPcS1 is strongly aggregated in both cell lines; AlPcS2 is aggregated in V79 cells but much less so in NHIK 3025 cells. A main finding is that the shapes of the action spectra are similar to that of the fluorescence excitation spectra, but not to the absorption spectra, indicating that the photosensitizing effects of the dyes are mainly due to their monomeric fraction in the cells. AlPcS2 and AlPcS4 localize intracellularly mainly in lysosomes while AlPcS1 was found to be more diffusely distributed in cells. As measured per quantum of fluorescence emitted, AlPcS1 and AlPcS2 are more efficient sensitizers than AlPcS4. The difference in efficiency between AlPcS2 and AlPcS4 is supposedly due to a different localization pattern on the suborganelle level.     

INVARIANT PROPERTIES OF ABSORPTION PROFILES IN SPORANGIOPHORES OF Phycomyces blakesleeanus UNDER BALANCING BILATERAL ILLUMINATION

Abstract— Absorption spectra and spectra of the refractive indices were separately measured for cytoplasm and vacuole of Phycomyces blakesleeanus in the wavelengths domains 300 to 750 nm and 250 to 750 nm respectively. These data were then used to simulate absorption profiles for different orientations of the photoreceptor for measured action spectra for phototropic balance. The photoreceptor was assumed to be a flavin. Using the reference wavelengths 394, 450 and 507 nm as an example it is shown that the absorption profiles have, independent of the photoreceptor orientation, invariant properties. Under bilateral balancing illumination the symmetry of the absorption profiles is conserved and the total amount of energy absorbed at proximal and distal sides is an invariant under a symmetry transformation front to the rear side.     

PROTEIN-RNA CROSSLINKS IN RIBOSOMES: ULTRAVIOLET ACTION SPECTRA. COMPARISON WITH INACTIVATION ACTION SPECTRUM

Abstract— RNA-protein crosslinking by UV of different wavelengths was studied in 70S E. coli ribosomes by three techniques: sucrose gradient centrifugation in the presence of sodium dodecyl sulfate (SDS), RNA solubilization in LiCI-urea concentrated solutions and RNA adsorption on nitrocellulose filters in the presence of SDS.
The centrifugational technique shows that the crosslinking reaction occurs in two steps, the first one corresponding to the fixation of a few protein molecules on 16 or 23 s RNAs and the second one corresponding to extensive RNA-protein crosslinking so that most protein molecules are no longer released by SDS from 30S and 50S subunits.
The initial rates for the first step of crosslinking were evaluated by the solubilization and adsorption techniques at 7 (or 6) wavelengths of irradiation between 223 and 290 mm. The action spectrum for RNA solubilization in LiCl-urea is perturbed at 223 nm by the breakage of protein chains. The action spectrum for retention on nitrocellulose filters seems to be exempt of this defect. It corresponds at high wavelengths to a nucleic chromophore and at low wavelengths to a proteic one. This means that RNA-protein crosslinking may occur through RNA and protein excitation. The similarity between the action spectrum for RNA retention on nitrocellulose filters and the action spectrum for inactivation of ribosomal synthesis activity suggests that RNA-protein crosslinking may be responsible for inactivation of ribosomes by UV.     

ACTION SPECTRUM FOR SUBLIMINAL LIGHT CONTROL OF ADAPTATION IN Phycomyces PHOTOTROPISM

Abstract -Adaptation processes enable phototropism and other blue light responses of Phycomyces to operate over a 10-decade range of Ruencc rate. Phototropic latency, used routinely to monitor the kinetics of sensitivity recovery after a step down in fluence rate, can be shortened by application of dim light for 35 min during the early part of the latency period. This light is termed subliminal , because it does not elicit phototropism under these experimental conditions; rather, it exerts its influence on the underlying adaptation kinetics. Fluence rate-response data for this latency reduction, obtained at 17 wavelengths of subliminal light from 347 to 742 nm, showed a variety of shapes that could be fit by zero, one, or two sigmoidal components, plus a constant term. At most wavelengths, the fluence-rate threshold for latency reduction by subliminal light tended to be well below the absolute threshold for phototropism, indicating that this effect is highly sensitive. An action spectrum for the sensitivity of the subliminal light effect, derived from the fluence rate-response curves, shows major peaks around 400 and 500 nm and a broad band from 570 to 670 nm, followed by a steep absorption edge. The sensitivity in the near ultraviolet region is relatively very low. The magnitude of the latency reduction also depends strongly on wavelength with a maximum at about 450 nm. The Huence-rate response data and the action spectrum–which is markedly different from that for phototropism and other blue-light responses of Phycornyces – indicate the participation of multiple pigments, or pigment states, in the photocontrol of adaptation.     

RESONANCE RAMAN SPECTRA OF BACTERIORHODOPSIN MUTANTS WITH SUBSTITUTIONS AT ASP-85, ASP-96, AND ARG-82

Detergent solubilized bacteriorhodopsin (BR) proteins which contain alterations made by site-directed mutagenesis (Asp-96----Asn, D96N; Asp-85----Asn, D85N; and Arg-82----Gln, R82Q) have been studied with resonance Raman spectroscopy. Raman spectra of the light-adapted (BRLA) and M species in D96N are identical to those of native BR, indicating that this residue is not located near the chromophore. The BRLA states of D85N and especially R82Q contain more of the 13-cis, C = N syn (BR555) species under ambient illumination compared to solubilized native BR. Replacement of Asp-85 with Asn causes a 25 nm red-shift of the absorption maximum and a frequency decrease in both the ethylenic (-7 cm-1) and the Schiff base C = NH+ (-3 cm-1) stretching modes of BRLA. These changes indicate that Asp-85 is located close to the protonated retinal Schiff base. The BRLA spectrum of R82Q exhibits a slight perturbation of the C = NH+ band, but its M spectrum is unperturbed. The Raman spectra and the absorption properties of D85N and R82Q suggest that the protein counterion environment involves the residues Asp-85-, Arg-82+ and presumably Asp-212-. These data are consistent with a model where the strength of the protein-chromophore interaction and hence the absorption maximum depends on the overall charge of the Schiff base counterion environment.     

ACTION SPECTRA FOR ACCUMULATION OF INORGANIC CARBON IN THE CYANOBACTERIUM, Anabaena variabilis

Abstract— Action spectra for accumulation of inorganic carbon were obtained for Anabaena variabilis , strainM–2, in the presence and absence of photosynthetic CO₂ fixation. The action spectrum for inorganic carbon accumulation in the presence of CO₂ fixation showed a peak around 684 nm, corresponding to chlorophyll a absorption in PS 1, while that for CO₂ fixation showed a peak around 630 nm, corresponding to phycocyanin absorption in PS 2. The action spectra obtained in the presence of iodoacetamide or diuron, which inhibit CO₂ fixation, showed two peaks, one at about 684 nm and the other at 630 nm, with the 630 nm peak height 80 to 90% of the 684 nm peak. These results indicate that inorganic carbon transport in A. variabilis can be driven with near equal efficiency by energy derived from absorption in photosystem 1 alone and with energy transferred to PS 1 after absorption by PS 2.     

LIGHT-INDUCED CAROTENE SYNTHESIS IN MUTANTS OF PHYCOMYCES WITH ABNORMAL PHOTOTROPISM

Mutants of Phycomyces. abnormal in their phototropic responses (the mad mutants), have been tested for their responses in light-induced carotene synthesis (LICS). The amount of carotene synthesized at any given fluence is significantly lower in the madA, madB and madD mutants than in the wild type. The amount of carotene is not lower in other mad mutants ( madC, madE, madF and madG ). The double mutant mad A madB and the triple mutant mad A madB madC show stronger effects. The wild type strain, as well as those carrying a single mad mutation ( madA and madB ) or those carrying two or three mad mutations ( madA madB. madA madB madC ) show closely similar sensitivity to LICS. This contrasts with phototropism and photoinitiation of sporangiophores which are sensitive to extremely small signals in the wild type and in which the madA mutation decreases the sensitivity by nearly a factor of 10⁴ and madB mutation by a factor of 10⁵. It appears that LICS does not share the signal amplification mechanisms characteristic of the other two responses.     

BIOSYNTHESIS OF PTERIDINES IN Neurospora crassa,Phycomyces blakesleeanus AND Euglena gracilis: DETECTION AND CHARACTERIZATION OF BIOSYNTHETIC ENZYMES*,†

Abstract— Occurrence, biosynthesis and some functions of tetrahydrobiopterin (H₄biopterin) in animals are well known. The biochemistry of H₄biopterin in other organisms than animals was hitherto not widely investigated. Recently H₄biopterin was found in the phytoflagellate Euglena gracilis, in the zygomycete Phycomyces blakesleeanus and in the ascomycete Neurospora crassa. In Euglena, Neurospora and Phycomyces the enzymatic activities of GTP cyclohydrolase I, 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase are detectable and the biosynthesis follows the same steps as were shown for animals. The biosynthetic enzymes, however, show a much lower sensitivity to those inhibitors that act on vertebrate enzymes. 2,4-Diamino-6-hydroxypyrimidine as inhibitor of GTP cyclohydrolase I and N-acetylserotonin or N-methoxyacetylserotonin as inhibitors of sepiapterin reductase can decrease pteridine biosynthesis significantly, in vitro and in vivo. The apparent K_mvalues are in general higher when compared with the respective animal enzymes. In Neurospora, the conversion of GTP to dihydroneopterin triphosphate was closely associated with subsequent production of 6-hydroxymethyl-7, 8-dihydropterin due to the high activity of dihydroneopterin aldolase, different from all other tested organisms. Investigations involving inhibition of pteridine synthesis might be a useful tool for evaluating the hypothesis that pteridines in fungi and plants are co-chromophores of various blue light-dependent, flavin-containing phototrcptors.     

ACTION SPECTRA FOR HEMATOPORPHYRIN DERIVATIVE AND PHOTOFRIN II WITH RESPECT TO SENSITIZATION OF HUMAN CELLS IN VITRO TO PHOTOINACTIVATION

Abstract— Exposure of Raji cells to haematoporphyrin derivative (HPD) and red light caused marked cytotoxicity. This was completely inhibited under anaerobic conditions. By using sodium dithionite in aqueous solutions, precise and graded oxygen concentrations could be achieved. Cytotoxicity was directly proportional to the oxygen concentration of the medium until a maximum was reached at a pO₂ of 90 mm Hg. Sodium dithionite did not affect the viability of test cells and did not alter the chromatographic profile of HPD. Dithionite did not interfere with the uptake of HPD by cells. Dependency of phototoxicity upon aerobic conditions suggests that the cytotoxic agent is derived from oxygen and is consistent with the hypothesis that singlet oxygen and/or oxygen-derived free radicals play an important role in photochemotherapy with HPD.     

PHOTODYNAMIC ACTION OF MEROCYANINE 540 IN ARTIFICIAL BILAYERS AND NATURAL MEMBRANES: ACTION SPECTRA AND QUANTUM YIELDS

The action spectra and quantum yields for singlet oxygen (1O2) generation by merocyanine 540 (MC540) in liposomes and isolated erythrocyte membranes were obtained using electron spin resonance techniques. Oxygen consumption was measured by spin label oximetry in the presence of histidine for fully-saturated dimyristoylphosphatidylcholine vesicles, mono-unsaturated 1-palmitoyl-2-oleoylphosphatidylcholine vesicles and erythrocyte membranes. The quantum yield for the photogeneration of 1O2 by membrane-bound MC540 in aqueous buffer was determined to be 0.065 +/- 0.005, which is approx. 1/10 of the value determined for Rose Bengal under similar conditions. Using unilamellar liposomes and isolated erythrocyte membranes containing MC540 at different monomer/dimer ratios, we have observed that the action spectra of 1O2 generation closely overlap the absorption spectra of the monomeric dye in these systems. It is likely that factors which affect the monomer-dimer equilibrium of MC540 will influence the production of 1O2. These findings have important implications for the phototherapeutic efficacy of MC540.     

ACTION SPECTRA FOR LIGHT-INDUCED DE-EPOXIDATION AND EPOXIDATION OF XANTHOPHYLLS IN SPINACH LEAF*

Abstract— The action spectra for violaxanthin de-epoxidation and zeaxanthin epoxidation in New Zealand spinach leaf segments, Tetragonia expansa, were determined at equal incident quanta of 2·0 × 10¹⁵ quanta cm^-2 sec^-1. Precise action spectra were not obtained due to variable leaf activity. The de-epoxidation action spectrum had major peaks at approximately 480 and 648 nm. Blue light was slightly more effective than red light and little activity was observed beyond 700 nm. The epoxidation action spectrum showed major peaks at around 440 and 670 nm. Blue light was more effective than red light and light beyond 700 nm showed definite activity. The net result of de-epoxidation and epoxidation is a cyclic scheme, the violaxanthin cycle, which consumes O₂ and photoproducts. The action spectra indicate that the violaxanthin cycle is more active in blue than in red light and therefore could account for O₂ uptake stimulated by blue light. However, the violaxanthin cycle is not the pathway for O₂ uptake by photosynthetic system 1. It was suggested that the violaxanthin cycle may function as a pathway for the consumption of excess photoproducts generated in blue light or the conversion of these photo-products to other forms of energy.     

ACTION SPECTRA OF THE ANTILEUKEMIC and ANTIVIRAL ACTIVITIES OF MEROCYANINE 540

Action spectra of the antileukemic and antiviral activities of merocyanine 540 (MC540) were determined using L1210 leukemia cells and human Herpes simplex virus type 1. The major peak of both action spectra aligned closely with the absorption spectrum of membrane-bound dye monomer, and by implication, the action spectrum of 1O2 generation. These results are compatible with the notion that the antileukemic and antiviral activities of MC540 are primarily attributable to membrane-bound monomer and at least in part mediated by 1O2.     

ACTION SPECTRA FOR HYPHAL AGGREGATION,THE FIRST STAGE OF FRUITING,IN THE BASIDIOMYCETE Pleurotus ostreatus*

Abstract. Action spectra were determined for hyphal aggregation in Pleurotus ostreatus at wavelengths between 360 and 600 nm. The action spectrum for a 50% response had two maxima, one at 370 nm in the near-UV and the other a broad peak at 440–450 nm in the blue. Both were approximately of the same magnitude. A minimum was present at 400 nm and wavelengths greater than 530 nm invoked no response. Action spectra for a range of responses, 10–90%, were also determined which showed gradual changes in the peaks in the blue region. It was concluded that hyphal aggregation in P. ostreatus is under the control of a cryptochrome-like photoreceptor system.     

ALTERATIONS IN DNA IRRADIATED WITH ULTRAVIOLET RADIATION—I. THE FORMATION PROCESS OF CYCLOBUTYLPYRIMIDINE DIMERS: CROSS SECTIONS, ACTION SPECTRA and QUANTUM YIELDS

Abstract— We have determined the dimerization and monomerization cross sections of the Thy < > Thy (cyclobutyl dimer of thymine and thymine) and the Cyt < > Thy (cyclobutyl dimer of cytosine and thymine) dimers in Escherichia coti [³H]-DNA ([³H]-thymine labeled DNA) at five wavelengths in the range 240–300 nm. It may be concluded from the dimerization action spectra for the two dimers that the excitation of Thy (thymine) is mainly responsible for the photochemical dimerization reaction in both cases. The calculated quantum yields of dimerization and monomerization are also presented in this paper and several questions, raised by the results obtained at 300 nm, are discussed.