PHOTOREVERSIBLE Ca2+-DEPENDENT AGGREGATION OF PURIFIED PHYTOCHROME FROM ETIOLATED PEA AND RYE SEEDLINGS

The aggregation of phytochrome purified from etiolated pea ( Pisum satirum cv. Alaska) and rye ( Secale cereale cv. Cougar) tissues was investigated by centrifugation and turbidimetry. Purified pea phytochrome (A₆₆₉/A₂₈₀= 0.88), if irradiated with red light, became precipitable in the presence of CaCl₂. The precipitation upon red-light irradiation was optimal at a Ca^2- or Mg²⁺ concentration of 10–20 m M , was greater at increased phytochrome concentration or lower pH values, and was inhibited by 0.1 M KG. The precipitated phytochrome slowly became soluble after far-red light exposure.
Turbidity of pea phytochrome solutions after red-light irradiation also increased rapidly in the presence of either Ca²⁺ or Mg²⁺. Far-red light exposure after the red light cancelled the turbidity increase. Rye phytochrome showed less turbidity increase than pea phytochrome and occurred only in the presence of Ca²⁺. Partially degraded pea phytochrome produced by endogenous proteases in the extract did not show the turbidity increase. Undegraded pea phytochrome also associated with microsomal fractions under conditions similar to those described above, but the partially degraded phytochrome did not.     

FLUORESCENCE QUANTUM YIELDS OF 124-kDa PHYTOCHROME FROM OAT UPON EXCITATION WITHIN DIFFERENT ABSORPTION BANDS

Abstract— A fluorescence quantum yield (emission at650–850 nm) of π= (2.3 ± 0.3)10⁻³ was measured for the red-absorbing form (Pr) of 124-kDa phytochrome from etiolated oat seedlings ( Avena sativa ) upon excitation in the Soret band at Λ^exc= 380 nm. The small difference between this value and the previously determined quantum yield with Λ^exc= 640 nm, π= (3.5 ± 0.4)10⁻³is attributed to a blue-absorbing emitter responsible for the "anomalous" or "blue" emission of the chromoprotein in the region from ca. 400 to 550 nm. The absorption of Pr at 380 nm is consequently somewhat lower than that measured directly from the spectrum. Processes from upper excited states of the Pr phytochromobilin-derived chromophore other than rapid relaxation to the emitting state are not important. A quantum yield of Φ ' 1.2 times 10⁻³ is estimated for the blue fluorescence. The proportion of the blue emitters relative to Pr appears to be relatively high.     

COMPARATIVE PHOTOCHEMICAL ANALYSIS OF HIGHLY PURIFIED 124 KILODALTON OAT and RYE PHYTOCHROMES in vitro

Abstract A direct comparison of the photochemical interconversions between red (P_r-) and far-red (P_fr-) absorbing forms of highly-purified 124 kDa oat and rye phytochromes under identical experimental conditions was performed. In two different buffer systems at 5°C, the quantum yields for the P_r to P_tr and P_fr to P_r phototransformations under constant red and far-red illumination, φ _r and φ_fr respectively, were determined to be 0.152-0.154 and 0.060-0.065 for oat preparations and 0.172-0.174 and 0.074-0.078 for rye preparations. These values as well as the wavelength dependence of the photoequilibrium produced under continuous illumination throughout the visible and near-ultraviolet spectrum were based on the absorption spectra of the two phytochrome preparations and revised molar absorption coefficients. The molar absorption coefficients were estimated by quantitative amino acid analysis and shown to be identical for the two monocot phytochromes (i.e. 132 mM ⁻¹ cm⁻¹ at the red absorption maximum for the P_r form). Because these measurements were performed under identical experimental conditions, including buffer, temperature, light fluence rate, and instrumentation, the differences observed must reflect structural features inherent to the two different monocotyledonous phytochromes.     

Large-scale Generation of Affinity-purified Recombinant Phytochrome Chromopeptide

Abstract— Two different yeast expression systems, Pichia pastoris and Hansenula polymorpha, are compared for their capability to express in functional form the 65 kDa N-ter-minal portion of oat phytochrome A (phyA, spanning amino acids 1-595). The front half of phytochrome was selected for this investigation because it exhibits a greater stability than the full-length protein, and it harbors full spectroscopic and kinetic properties of phytochrome, allowing an exact proof of the functional integrity of the recombinant material. In the comparison between the two expression systems used, special emphasis was given to optimizing the yield of the expression and to improving the quality of the expressed material with respect to the proportion of functional protein. From identical volumes of cell culture, H. polymorpha synthesized between 8- and 10-fold more functional protein than P. pastoris. Following the observation by Wu and Lagarias (Proc. Natl. Acad. Sci. USA 93, 8989-8994,1996) that P. pastoris endogenously produces the chromophore of phytochrome, phytochromobilin (PpHB) in significant amounts that leads to formation of spectrally active phytochrome during expression, the invention of an alternative high-yield expression system was strongly demanded. A Histag was attached to the C-terminus of the recombinant protein, which allows for a convenient and efficient purification and selects the full-length proteins over translationally truncated peptides. Fully reconstituted chromo-proteins showed an A660A280 ratio of 1.2, indicating the high degree of reconstitutable apoprotein obtained by this procedure. The assembly between apoprotein and the chromophore phycocyanobilin when followed time-resolved yielded a time constant (obs) of 35 s. The λ_max values of the red-(Pr) and the far red-absorbing (Pfr) forms of phytochrome (665 and 729 nm) of the recombinant 65 kDa chromopeptide, reconstituted with PcjiB are nearly identical to those of native full-length oat phytochrome. The kinetic parameters of the affinity-purified 65 kDa phytochrome chromoprotein for the Pr I700 Pfr conversion are compared to those of the recombinant 65 kDa chromoprotein, lacking the His-tag and to wild-type oat phytochrome. Referring to wild-type phytochrome allows determination of whether the recombinant material has lost spectral properties during the purification procedure. The decay of the primary intermediate (I₇₀₀) occurs with nearly the same time constant for the His-tagged chromoprotein and for the reference (110 and 90 mUs, respectively). The formation of the Pfr form was fitted with three exponentials in both the His-tagged and the reference chromoprotein with the middle component being slightly smaller and the longest component being remarkably larger for the His-tagged protein (1.5, 10 and 300 ms) than for the reference (1.4, 18 and 96 ms). This selective slowing down of the long kinetic component in the millisecond time range may be indicative of stronger interactions between protein domains involving the C-terminus that in the His-tagged form exhibits increased polarity.     

A STUDY OF THE FLASH INDUCED 518 NM ABSORBANCE CHANGE KINETICS UNDER ANAEROBIC CONDITIONS

The preillumination induced acceleration of the flash-induced 518 nm absorbance change (ΔA₅₁₈) decay was studied in lettuce leaves and chloroplasts. In leaves, the acceleration was inhibited by DCMU or reversibly by removal of oxygen. In chloroplasts with added ADP and phosphate and/or reconstructed electron transport, the acceleration was also inhibited by DCMU or the lack of O₂.
Anaerobic inhibition of ΔA₅₁₈ decay acceleration was no longer observed when hydroxylamine replaced water as electron donor to PSII. Anaerobiosis was also shown to reversibly inhibit the initial rate of FeCN reduction in chloroplasts. These results suggest the mechanism of anaerobic inhibition of ΔA₅₁₈ decay acceleration to be associated with the O₂ evolving system.     

DETECTION AND QUANTITATION OF THREE PHYTOCHROMES IN UNIMBIBED SEEDS OF: Avena sativa L.

Three phytochrome apoproteins in unimbibed seeds of Avena saliva L. were identified with monoclonal antibodies directed to, and specific for, three oat phytochromes with monomeric molecular masses of 125, 124 and 123 kDa [Wang et al., 1991, Planta 184, 96–104]. All three phytochromes were readily detected in embryo-containing portions. Only trace amounts were found in endosperm tissue. Phytochrome photoreversibility was detected after concentration and partial purification of embryo extracts by fractionation with ammonium sulfate, indicating that at least one of these seed phytochromes had its chromophore prosthetic group bound to it. Immunoblot analyses were performed to quantitate each of the three phytochromes in unimbibed seeds. Quantitation of phytochromes in detergent-free extracts led to serious underestimates of phytochrome contents in the unimbibed seeds. In contrast, more than 93% of each of the three phytochromes in the unimbibed seeds was extracted when a modified sodium dodecyl sulfate sample buffer was used as the extraction medium. In such extracts, we measured per embryo 1.40 ± 0.12. 1.60 ± 0.05 and 6.13 ± 0.31 ng of 125–, 124– and 123-kDa phytochrome, respectively.     

PHYTOCHROME IN GREEN-TISSUE: PARTIAL PURIFICATION and CHARACTERIZATION OF THE 118-KILODALTON PHYTOCHROME SPECIES FROM LIGHT-GROWN Avena sativa L.*

The predominant, immunochemically-detectable phytochrome polypeptide rapidly extracted directly into boiling sodium dodecyl sulfate-containing buffer from fresh or freeze-dried green Avena tissue has an apparent molecular mass of 118 kilodaltons (kDa). This result indicates that the 118-kDa phytochrome species obtained from green Avena by extraction and rapid processing under non-denaturing conditions in previous studies was not derived by partial proteolysis of a larger polypeptide present in the cell. Additional data do, however, demonstrate the presence in green tissue homogenates of proteolytic activity that can cause a = 6-kDa reduction in apparent molecular mass and a blue-shift in the P_fr absorbance maximum of phytochrome during handling. This proteolytic activity contrasts with that previously encountered in etiolated tissue in that it is not inhibited by phenylmethylsulfonyl fluoride, but is inhibited by iodoacetamide and leupeptin. This result indicates that the activity is associated with a thiol-like protease. A partial purification procedure that incorporates the use of iodacetamide and a novel chromatographic step is described for green-tissue phytochrome. This procedure provides 50% recovery with a 90-fold enrichment of phytochrome relative to the initial extract in which the chromoprotein is 0.003% of the total soluble protein. The final fraction is apparently free of proteolytic activity. Immunoblot analysis of this fraction demonstrates that the predominant immunoreactive band has a monomeric molecular mass of 118 kDa. Comigration of this band with a band exhibiting zinc-induced fluorescence on blots of the partially purified preparations verifies that the 118-kDa species is the principal tetrapyrrole-bearing polypeptide present. Spectral properties of the final fraction are identical to those published for crude green-tissue extracts, indicating the stability of the molecule's spectral properties throughout the procedure. Size exclusion chromatography under nondenaturing conditions shows that the 118-kDa phytochrome species from green tissue comigrates with the dimeric, etiolated-tissue molecule, and is therefore suggestive of similar quaternary structure. Together these data reinforce previous conclusions that the predominant phytochrome molecule present in the living cells of green tissue is resolvable as a 118-kDa species, distinct from the well-characterized 124-kDa molecule from etiolated tissue (Tokuhisa et al., 1985, Planta 164, 321–332), and indicate that the partial purification protocol described here sustains the green-tissue phytochrome in its native state throughout the procedure.     

EVIDENCE FOR A SEQUENTIAL PATHWAY FROM Pr TO Pfr OF THE PHOTOTRANSFORMATION OF 124-kDa OAT PHYTOCHROME

Abstract. Phototransformation kinetics of 124-kDa oat phytochrome at 298 K after a red (660-nm) laser flash excitation were recorded at different wavelengths. The kinetics of the dark relaxation processes for lumi-R to P_fr can be satisfactorily described by only 3 rate constants: k = 28000 s^-1 370 s^-1 and 20 s^-1. The first rate constant is due to the decay of lumi-R to meta -Ra. The latter two rate constants correspond to processes establishing the far-red (>700 nm) absorption band. No meta -Rb could be detected. From the wavelength dependency of the amplitudes of these two rates, parallel pathways in the formation of P_fr could be excluded. A unique sequential pathway for the dark relaxation leading to P_fr seems to be an intrinsic property of 124-kDa phytochrome, however. Assuming a sequential pathway, molar extinction coefficients for intermediates have been calculated. These values agree with molar extinction coefficients obtained from low-temperature spectra. The process with a rate constant of 370 s^-1 corresponds to absorbance changes for the formation of meta -Rc from meta -Ra and the rate constant of 20 s^-1 describes the absorbance changes due to the transformation of meta -Rc to P_fr.     

Fluorescence Analysis of Oat phyA Deletion Mutants Expressed in Tobacco Suggests that the N-Terminal Domain Determines the Photochemical and Spectroscopic Distinctions between phyA' and phyA"†

Abstract— In vivo low-temperature (85 K) fluorescence spectroscopy has defined two phytochrome A (phyA) subpopulations, designated phyA' and phyA", in etiolated seedlings (V. A. Sineshchekov, J. Photochem. Photobiol. 28, 53–55, 1995). Phytochrome A' is the more abundant but light-labile species characterized by longer wavelength emission/absorption maxima (687/673 nm) and by a higher extent of the photoconversion of its red-absorbing form (Pr) into photoproduct (lumi-R) at 85 K (γ₁≈ 0.5). Phytochrome A" is the minor but relatively light-stable species, characterized by shorter wavelength maxima (682/668 nm) and by a lower γ₁ (<0.05). To help define domains within phyA responsible for these differences, the low-temperature spectral properties of transgenic tobacco expressing full-length (FL) oat phyA and C-and N-terminally truncated versions (CD [Δ786–1129] and NA [Δ7–69], respectively) were compared. Oat phytochrome expression was more pronounced than that of tobacco in the basal section of etiolated seedlings following 2 h irradiation with white light. Seedlings expressing FL and CD phyA had spectral properties for phyA' and phyA" that were indistinguishable from that of wild-type tobacco. Conversely, expression of NA phyA generated an abundant phy species that behaved like phyA". From this we conclude that the N-terminal domain of phyA is involved in determining the photochemical and spectroscopic distinctions between the native phyA' and phyA" species.     

124-kDa PHYTOCHROME IN MODEL MEMBRANE SYSTEMS: STUDIES OF THE Ii700 INTERMEDIATES WITH THE PROTEIN COVALENTLY BOUND TO PREFORMED LIPOSOMES

Abstract— We have described the covalent binding of 124-kDa oat phytochrome to large unilamellar liposomes composed of either dioleoyl phosphatidylcholine or dipalmkoyl phosphatidylcholine or soybean lecithin, without affecting the photochromic properties of the protein. These phytochrome-liposome systems have now been studied by laser flash photolysis. The liposomes, independent of their membrane rigidity (liquid-crystal vs gel-like phase), do not influence the ratio and reactivity of the two primary photoproducts, Iⁱ₇₀₀- of the red absorbing form of phytochrome, P_l Thus, the lifetimes of the Iⁱ₇₀₀ intermediates and the activation parameters associated with Iⁱ₇₀₀Iⁱ_bl are the same as those measured for nonbound phytochrome in buffer solution. The temperature increase from about 273 K. to 297 K lowers the population of the shorter-lived Iⁱ₇₀₀ intermediate to the same extent both in the liposome-P_l and in nonbound P_l, whereas it does not affect the relative population of the Iⁱ₇₀₀ intermediates from non-bound P_l in the presence of 25% ethylene glycol added to the buffer solution (ionic strength 0.17).     

SPECTRAL CHARACTERIZATION OF LIGHT-REDUCIBLE CYTOCHROME IN A PLASMA MEMBRANE-ENRICHED FRACTION AND IN OTHER MEMBRANES FROM CAULIFLOWER INFLORESCENCES

Abstract— Low temperature spectroscopy has been used to characterize microsomal fractions obtained from cauliflower inflorescences ( Brasska oleracea L.) by differential centrifugation and partition in an aqueous polymer two-phase system. The plasma membrane-enriched fraction (U₃) was found to contain one dominant b -cytochrome, which could be reduced both by blue light and by dithionite. An action spectrum of the blue light-induced absorbance change [LIAC, Δ(A₄₃₀—A₄₁₀)] associated with the reversible reduction of this b -type cytochrome indicated that the primary light-receptor was a flavin-like compound. Another microsomal fraction (L₃) containing membranes from mitochondria, endoplasmic reticulum and other organelles also contained light-reducible cytochrome. One of these could be identified as cytochrome c oxidase, and another may be identical to cytochrome b ₅ of the endoplasmic reticulum.     

TRYPTOPHAN EMISSION FROM HUMAN HEMOGLOBIN AND ITS ISOLATED SUBUNITS

Abstract— The emission spectra of human adult hemoglobin A₀ and its isolated α and ß subunits were obtained using a highly sensitive photon-counting spectrofluorometer. The quantum yields of the emissions, relative to free tryptophan, were also measured as well as the excitation polarization spectra for hemoglobin A₀ and apohemoglobin. The fluorophore bis-ANS was utilized to probe for the presence of apoproteins in the hemoprotein preparations. The work suggests that tryptophan may be useful as an intrinsic probe to study dynamical processes in hemoglobin.     

ON THE PRIMARY PHOTOPROCESS OF 124-kdalton PHYTOCHROME

The photoreaction between P_τ and the first detectable intermediate, lumi-R, of 124-kdalton oat phytochrome has been investigated at low temperatures. The temperature dependence of the quantum yields of the photoreactions, P_τ to lumi-R and lumi-R to P_τ, has been determined. From measurements over a temperature range from 119 to 155 K, an activation barrier of 3.6 ± 0.5 kJ mol ₁ is found for the photoreaction of P_τ with 661-nm actinic light. A higher value (5.7 ± 0.7 kJ mol ^-1) is found for the photoreaction of lumi-R to P^τ. with 698-nm actinic light. Increased quantum yields are found in deuterated buffer solutions at low temperatures. The activation energies for deuterated phytochrome (3.2 ± 0.7 kJ mol^–1 for P_τ with 661-nm irradiation and 6.2 ± 1.2 kJ mol^-1 for lumi-R at 698-nm irradiation) are identical within the limits of error with those of protonated phytochrome. The lack of a deuterium effect for the activation energies favors the Z,E-isomerization rather than proton transfer or tautomerization for the chromophore photochemistry during P_τ⇄lumi-R conversion.     

Isolation and characterization of a new subunit of phycocyanin from Chroomonas placoidea

A new phycocyanin（PC） fluorescent subunit namedβ₂（18kDa） was isolated and characterized by both SDS-PAGE and isoelectric focusing（IEF） from a species of cryptophytic alga Chroomonas placoidea.PC was separated and purified by ammonium sulfate sedimentation followed by two steps of Sephadex G-100 chromatography.After denatured in 4 mol/L urea for 48 h,PC was divided into two fractions by passing through a Sephacryl S-100 chromatography column twice.The blue fraction（S-1） containedβsubunits with a maximal absorbance at 595 nm in visible light region.While the green fraction（S-2） enriched inαsubunits showed a characteristic long wavelength absorbance at 680-700 nm region and exhibited a relatively low molecular weight of 9.4（α₁） and 8.5 kDa（α₂）.Fraction S-1 also consisted of two different fluorescent subunits with molecular weight of 20.1 kDa（β₁） and 18 kDa （β₂） and differed from each other on isoelectric points of pH 5.7（ft） and 6.0（ft）,respectively.Further investigation of peptide sequence will help a lot in elucidating the new subunit ft that was smaller in size and more neutral than the known ft subunit,and may provide an alternative explanation in structure of cryptophytic phycobiliproteins.     

Stabilization of phytochrome intermediates by low temperature

Abstract— The photocon versions between the red-absorbing form (P_r) and the far-red absorbing form (P_fr) of phytochrome were examined at low temperatures. Partially purified preparations of the chromoprotein were examined in phosphate buffer and in 25 per cent buffer plus 75 per cent glycerol. Actinic irradiation of P, below – 150°C produces an intermediate with maximum absorbance near 695 nm, R₆₉₅. Actinic irradiation of R₆₉₅ converts it back to P. Above – 150°C R₆₉₅ decays to a low extinction form of phytochrome, R, which in turn decays to P_fr upon further warming. Light absorption by P_fr below – 150°C results in the formation of an intermediate form of phytochrome with maximum absorbance near 660 nm, FR₆₆₀. FR₆₆₀ decays upon warming to a lower extinction form, FR'. which in turn decays to P_r on continued warming. No evidence was obtained to suggest that any of the observed intermediate states are involved in more than one direction of phytochrome photocon version.     

Comparison of the protein conformations between different forms (Pr and Pfr) of native (124 kDa) and degraded (118/114 kDa) phytochromes from Avena sativa

Abstract— Circular dichroic properties of native, 124 kDa phytochrome from etiolated Avena sativa seedlings have been examined and compared with those of degraded phytochrome (118/114 kDa). The CD spectrum of the P_r form of 124 kDa phytochrome does not differ significantly in the visible region from that of 118/114 kDa P_r. In contrast, the CD spectrum of the P_fr form of 124 kDa phytochrome differs from that of the 118/114 kDa species in the far-red, red and blue regions of the spectrum. This result confirms that the NH₂-terminal polypeptide segment has a critical role in chromophore-protein interaction in the P_fr but not in the P_r form. In the UV region, 124 kDa phytochrome exhibits a photoreversible difference between the CD spectra of P_r and P_fr, whereas no such difference is observed for 118/114 kDa preparations. These data suggest a possible photoreversible change in secondary structure of the 124 kDa phytochrome polypeptide that requires the presence of the 6/10 kDa NH₂-terminal domain to occur.     

A PHYTOCHROME STUDY USING TWO-LASER/TWO-COLOR FLASH PHOTOLYSIS: I700 IS A MANDATORY INTERMEDIATE IN THE PrPfr PHOTOTRANSFORMATION

Abstract— The phototransformation of native (124 kDa)oat phytochrome, P_r P_fr, Has been studied at 10C by two laser/ two-color flash photolysis. the overall P_rP_fr reaction yield did not vary with temperature within the range4–21C. Foloeing the excitation of P_r with a single 15 ns laser flash at 650nm, the formation of P_fr was quantitavely measured in a time-resolved experiment in the presence of a second 8 ns laser flash at 710 nm delayed from the initial flash. the second laser flash causes at 1.0 s after the initial laser flash a depletion of the uintermediate I₇₀₀ as welll as a reduction of the P_fr absorption at 730 nm. The depletion of I₇₀₀ correlates quantitavely with the reduction of P_fr formation. The absorpton spectra of I₇₀₀ and of the following intermendiate, I_bi, were calculated assuming that the amount of P_r, which is photoconverted by a single laser, equals the amount of P_fr formed.     

PHOTOLYSIS OF POLYRIBOBROMOURIDYLIC ACID

Abstract— Photolysis of polyribobromouridylic acid with 313 nm light at neutral pH caused extensive debromination and a loss of A₂₈₀ (280–nm absorbance) without comparable increase in A₂₆₀. At an exposure of 190μE/cm ² , strand breakage occurred on the average of one break every 170 BrU residues. Little if any pyrimidine hydrate was produced. Exhaustive RNase hydrolysis of photolysed polymer gave a mixture of mononucleotides and oligonucleotides. The mononucleotide fraction was found to be composed of unaltered BrUMP and contained little if any UMP. Irradiation of the polymer at alkaline pH caused little or no debromination or spectral change.     

ON THE NATURE OF THE INTERMEDIATE ELECTRON ACCEPTOR (A1) IN THE PHOTOSYSTEM I REACTION CENTER

Abstract— A sodium dodecyl sulfate-Photosystem I (PSI) complex has been prepared and characterized with respect to its electron acceptors. Component X and iron-sulfur centers A and B are absent from this preparation but the intermediate electron acceptor (A₁) is present. Flash-induced absorbance changes at 25°C show charge separation, followed by a back-reaction with a half-time of 5 µs. The spectrum of the flash-induced change from 350 to 550 nm indicates a contribution from the intermediate electron acceptor, A₁, as well as from P700⁺. EPR studies show that A₁ is associated with a free-radical signal having a g-value of 2.0025 and a linewidth of 12 gauss. A, would appear to be associated with a monomeric form of either Chi α or pheophytin a.     

A RAPID PROCEDURE FOR THE PURIFICATION OF 124 kDALTON PHYTOCHROME FROM AVENA

Abstract— A modification of previously published procedures is described which permits the preparation of high-purity 124 kDa phytochrome within 14 h. The last 5 h are used for chromatography on a molecular sieve column, so all the handling steps are completed within 9 h. The procedure is simpler and faster than previously published methods.