PHOTOREVERSIBLE PROTON DISSOCIATION AND ASSOCIATION IN PEA PHYTOCHROME AND ITS CHROMOPEPTIDES

Abstract— Effect of red-light irradiation on the medium pH at 10d?C was measured and compared among unbuffered solutions of the 121-kDa native pea (Pisum sativum cv. Alaska) phytochrome and its 114- and 62-kDa fragments in a red-light-absorbing form (P_r), all of which converted to far-red-light-absorbing form (Pf_r) on red-light irradiation. Red-light irradiation induced alkalinization in the solutions of the phytochrome and the fragments in the pH range 6.6-7.2 and 6.2-7.8, respectively. The amount of protons taken up by the 121-kDa phytochrome was less than one half of that of the 114-kDa fragment. Red-light irradiation induced acidification in the solutions of the 114- and the 62-kDa fragments above pH 7.8. In the solutions of the 121-kDa phytochrome, however, the irradiation induced no pH change at pH 7.2-8.2, and only a slight acidification at pH 8.2-8.7, which may be ascribed to a small amount of contamination from the 114-kDa fragment. All these red-light-induced pH changes were reversible following exposure to far-red light. The 7-kDa polypeptide(s) of the native 121-kDa phytochrome, which is lacking in the 114-kDa fragment, thus, prohibited proton transfer between phytochrome and the medium. A red-light-induced pH change was also measured in unbuffered solutions of the 39-kDa fragment of the phytochrome and of the 114-kDa fragment in the presence of 0.8 mM soyasaponin I. The 39-kDa fragment showed partially photoreversible conversion between a spectral form having an absorption maximum at 659 nm (P₆₅₉) and a bleached form, P_***. The 114-kDa fragment in the presence of the saponin showed a photoreversible conversion between P65V and Pb,. Exposure of P₆₅₉ from the 39-kDa fragment and from the 114-kDa fragment in the presence of the saponin to red light, caused acidification of the medium in the pH range 6.8-8.8 and 7.2-9.0, respectively, but no change at pH 6.2-6.8 and 6.4-7.2, respectively. The acidification of the latter was reversible following a far-red-light irradiation, but that of the former was only partially photoreversible. Proton uptake of phytochrome was inhibited by tryptic degradation to the 39-kDa fragment and also by the presence of the saponin. Only proton release was observed during the photoconversion from P₆₅₉ and P_***hl. It is suggested that a phytochrome molecule has possible site(s) for both proton release and for uptake and that the proton release reaction may be correlated to the photoconversion process(es) prior to the bleached intermediate (I_***) of phytochrome.     

PHOTOREVERSIBLE CHANGES IN pH OF PEA PHYTOCHROME SOLUTIONS

Abstract— Photoinduced pH changes in unbuffered solutions of undegraded pea phytochrome were studied at 10_oC by using a glass electrode. Red light irradiation caused alkalinization of the solutions in the pH range 5.2–xs7.5 and acidification in pH 7.5–8.9. The pH changes were fully reversed by a subsequent irradiation with far-red light. The red and far-red light effects were repeatedly reversible. The solution of tryptic peptide of phytochrome (mol. wt 60000) showed similar photoreversible pH changes.     

THE ELECTRICAL RESPONSE OF CEPHALOPOD VISUAL CELL MEMBRANE FRAGMENTS

Abstract— Microvilli membrane fragments of squid ( Todarodes pacificus ) visual cells were adsorbed onto an oil surface in an aqueous medium. An electrical conductance increase was observed on illuminating the adsorbed film, and photopotential responses were recorded in a salt free medium of pH below 6, or in a saline of pH over 6. These electrical photoresponses are discussed in relation to the late receptor potential of squid retina.     

THE DISTANCE BETWEEN THE PHYTOCHROME CHROMOPHORE AND THE N-TERMINAL CHAIN DECREASES DURING PHOTOTRANSFORMATION. A NOVEL FLUORESCENCE ENERGY TRANSFER METHOD USING LABELED ANTIBODY FRAGMENTS

A novel antibody-fluorescence method has been developed to elucidate the chromophore topography in phytochrome as it undergoes a photochromic transformation. Förster energy transfer from N-terminal bound, fluorescently labeled Oat-25 Fab antibody fragments to the phytochrome chromophore was measured. The results suggest that the chromophore moves relative to the N-terminus upon the Pr → Pfr phototransformation. This conclusion is consistent with previous models which have proposed a reorientation and an interaction of the Pfr chromophore with the N-terminus. The method described appears to be the first study of a Forster energy transfer measurement using a donor-label attached to a Fab fragment of a photosensor protein.     

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.     

Primary structure of subunit B of the 11S globulin of cotton seeds of variety 108-F. VI. Reconstruction of the polypeptide chain

Subunit B of the 11S globulin of cotton seeds, the polypeptide chain of which contains about 190 amino acid residues, has been reconstructed on the basis of large fragments from three types of tryptic hydrolyses.Institute of the Chemistry of Plant Substances, Academy of Science of the Uzbek SSR, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 230–233, March–April, 1984.     

Method optimization in capillary zone electrophoretic analysis of hGH tryptic digest fragments

The mobile phase composition was optimized for the separation of tryptic digest fragments of human growth hormone by capillary zone electrophoresis. The effect of pH (pH 2.4, 6.1, 8.1 and 10.4) was evaluated since pH determines the relative charge of species, the prime contributor to selectivity; pH 8.1 was selected for the optimization studies. Tricine (buffer), sodium chloride (ionic strength adjustor), and morpholine (mobile phase additive) concentrations were systematically varied a pH 8.1. All three exhibited major effects on the electroosmotic flow velocity and current, and minor effects on selectivity. Tricine was the most crucial for good resolution, although addition of morpholine helped to resolve closely eluting species. The optimum separation conditions were found to be pH 8.1 with 0.1 M tricine, 0.02 M morpholine and no salt.     

Effect of thiocyanate counterion condensation on poly(allylamine hydrochloride) chains on the buildup and permeability of polystyrenesulfonate/polyallylamine polyelectrolyte multilayers

In this study, we investigate the buildup of PEI-(PSS-PAH)(n) polyelectrolyte multilayers at pH 7.4 in the presence of either NaCl or NaSCN as a supporting electrolyte. It appears that in the presence of increasing thiocyanate concentrations (from 0.1 to 0.5 M), the thickness increment, obtained from optical waveguide lightmode spectroscopy experiments, increases whereas it stays practically constant for increasing sodium chloride concentrations (between 0.1 and 0.5 M). The hydration of the films differs also markedly between both electrolyte solutions. The differences in the construction of the polyelectrolyte multilayers in the presence of both supporting electrolytes are rationalized in terms of strong SCN(-) condensation on the PAH chains. The occurrence of this ion condensation is indirectly demonstrated by means of zeta potential measurements and directly demonstrated by means of attenuated total internal reflection infrared spectroscopy on the multilayer films. Moreover when the films are built up in the presence of SCN(-), these ions are only slowly exchanged by the Cl(-) ions introduced in the bulk. Conversely the thick films obtained from 0.5 M NaSCN solutions do not deswell when the buffer solution is replaced by a 0.5 M NaCl containing buffer. The permeability of the films constructed in the presence of both sodium salts is also studied by means of cyclic voltametry and is found to be markedly different in the case of films made from five bilayers at 0.5 M salt concentration. This difference is due to the different morphology and porosity of the films constructed in the presence of 0.5 M NaCl and 0.5 M NaSCN.     

ANALYSIS OF PHOTOTRANSFORMATION INTERMEDIATES IN THE PATHWAY FROM THE RED-ABSORBING TO THE FAR-RED-ABSORBING FORM OF AVENA PHYTOCHROME BY A MULTICHANNEL TRANSIENT SPECTRUM ANALYZER

Phototransformation of the red-absorbing form of phytochrome (Pr) to the far-red-absorbing form (Pfr) was followed with a custom-built transient spectrum analyzer. Large phytochrome, which consisted of approximately 120000-dalton monomers, was immunopurified or conventionally purified from etiolated oat (Avena sativa L., cv. Garry) shoots. Phototransformation was initiated by exciting Pr with a 115-mJ, 600-ns half-width, 655-nm laser pulse. Absorption spectra were recorded on a microsecond time scale at predetermined times after the flash. It has been reported earlier that flash excitation of large oat Pr produces a transformation intermediate with maximum absorbance near 700 nm in a difference spectrum and that this intermediate decays by two kinetically distinct reactions. Difference spectra for these two reactions are indistinguishable. Both show bleaching centered at 690 nm with no detectable associated absorbance increase between 570 and 830 nm. Subsequent appearance of absorbance at 724 nm, which presumably but not necessarily represents the appearance of Pfr, had earlier been shown to occur by two kinetically distinct reactions for large oat phytochrome. Data presented here indicate in addition the occurrence of a third, slower reaction. Difference spectra for the two faster reactions are indistinguishable, both with maxima near 728 nm and minima near 650 nm. The difference spectrum for the slowest component, however, was qualitatively different exhibiting a maximum near 722 nm with no corresponding minimum. About 15-20% of the absorbance increase at 724 nm occurred by this slowest reaction, which exhibited a half-life of 3 s at 25°C and a Q₁₀ of 1.2 for immunopurified and 1.5 for conventionally purified phytochrome. The percentage occurring by this reaction was independent of temperature over the range studied (1-25dEC). For immunopurified phytochrome the enthalpy of activation, Gibbs free energy of activation, and entropy of activation of this slowest reaction were found to be about lOkJ-mol^-1, 75kJ.mol^-1, and -220 J.mol^-1 K^-1, respectively, and for conventionally purified phytochrome 25kJ.mol^-1, 75kJ.mol^-1and —170 J.mol^-1 K^-1, respectively. The thermodynamic characteristics of this reaction indicate that it may involve a significant ordering of the protein moiety as it transforms to Pfr.     

STUDIES ON THE PROTEIN CONFORMATION OF PHYTOCHROME

Abstract— The extinction coefficients for large rye phytochrome were found to be: Fluorescence and circular dichroism spectra of large- and small-molecular-weight rye phytochrome give no evidence for a protein conformational change on phototransformation of phytochrome. The large molecule has a fluorescence emission peak at 331 nm when excited at 290 nm, and an excitation peak for this emission at 288 nm. The circular dichroism spectra indicate that large rye phytochrome has about 17–20% a-helix content, 30%β-structure and 50% random coil, and that the small rye phytochrome has about 10–13%α-helix content. The ultraviolet difference spectra for large and small rye phytochrome are similar and differ from the difference spectrum of the small oat phytochrome in the relative size of the 296–298 nm peak. The difference spectra may reflect changes in chromophore absorbance and in the environment of amino acid residues near the chromophore, particularly of tyrosine, and perhaps of tryptophan and cysteine.     

Analysis of aggregates of human immunoglobulin G using size-exclusion chromatography,static and dynamic light scattering

Large aggregates (Mr: 10(6)-10(7) g/mol) of human immunoglobulins are present in extremely small concentrations in IgG preparations (<0.1%). Traces of large protein aggregates cannot be determined by conventional size-exclusion chromatography (SEC) using UV detection due to limitations in sensitivity. The conventional analysis of IgG by SEC is limited to dimers and oligomers. Using light scattering it is possible to determine significant differences concerning the aggregate composition and the extent of protein aggregation in samples of different process steps. Two different pilot preparations were analyzed by SEC with UV and static light scattering detection and compared to dynamic light scattering in the batch mode. The change of large aggregates could be monitored and data were corroborated by dynamic light scattering.     

Capillary zone electrophoresis of alpha 1-acid glycoprotein fragments from trypsin and endoglycosidase digestions

Capillary zone electrophoresis with fused-silica tubes having hydrophilic coating on the inner walls was evaluated in the separation of peptide and glycopeptide fragments from trypsin digestion of alpha 1-acid glycoprotein. Submapping of glycosylated and nonglycosylated tryptic fragments of the glycoprotein by capillary electrophoresis was facilitated by selective isolation of the glycopeptides on concanavalin A silica-based stationary phases prior to the electrophoretic run. In addition, the electrophoretic map and submaps of the whole tryptic digest and its concanavalin A fractions, respectively, allowed the elucidation of the microheterogeneity of the glycoprotein. Also, capillary zone electrophoresis proved suitable for the mapping of the oligosaccharide chains cleaved from the glycoproteins by endoglycosidase digestion. The oligosaccharides cleaved from human and bovine alpha 1-acid glycoprotein were analyzed after derivatization with 2-aminopyridine, which allowed their sensitive detection by on column UV absorption. The separation was best achieved when 0.1 M phosphate solution, pH 5.0, containing 50 mM tetrabutylammonium bromide was used as the running electrolyte. The effect of the organic salt on separation was attributed to ion-pair formation and/or hydrophobic interaction.     

ORIGIN OF THE RED SHIFTED ABSORPTION IN PHYCOCYANIN 632 FROM Mastigocladus laminosus

A phycocyanin (PC) with γ_max= 632 nm (PC632) was isolated from extracts of the cyanobacterium Mastigocladus laminosus. The complex contained three polypeptides migrating in SDS-PAGE around 22 kDa. The N-termini of the three polypeptides are identical. They are homologous to rod-core linker polypeptides from this and another cyanobacterium and are identical to one of them. Reconstitution of PC (γ_max=614–620 nm) with the 22 kDa polypeptide produced again the red-shifted PC632. Phycobilisomes isolated at pH 6 or 7 contain, if any, only traces of 22 kDa polypeptides. Origin and functions of the polypeptides are discussed, they are most likely proteolytic fragments of allophycocyanin-PC rod-core linkers that contain the PC-binding domain.     

甲基丙烯酸2-羟乙酯水溶液聚合与硫氰化钠的效应

<正> 甲基丙烯酸2-羟乙酯(HEMA)是一带有羟基的功能性单体,它可以在自由基聚合引发剂如过氧化苯甲酰、偶氮二异丁腈、过氧化二碳酸二异丙酯或氧化还原引发体系过硫酸盐-乙酸二甲胺基乙酯的作用下进行自由基聚合,我们已报道带有羟基的单体如HEMA、甲基丙烯酸羟丙酯(HPMA)及甲基丙烯酯3-甲氧基-2-羟丙酯(MHPMA)可用     

Molecular dynamics simulations of polyelectrolyte-polyampholyte complexes. Effect of solvent quality and salt concentration

Complexation between polyelectrolyte and polyampholyte chains in poor solvent conditions for the polyelectrolyte backbone has been studied by molecular dynamics simulations. In a poor solvent a polyelectrolyte forms a necklace-like structure consisting of polymeric globules (beads) connected by strings of monomers. The simulation results can be explained by assuming the existence of two different mechanisms leading to the necklace formation. In the case of weak electrostatic interactions, the necklace formation is driven by optimization of short-range monomer-monomer attraction and electrostatic repulsion between charged monomers on the polymer backbone. In the case of strong electrostatic interactions, the necklace structure appears as a result of counterion condensation. While the short-range attractions between monomers are still important, the correlation-induced attraction between condensed counterions and charged monomers and electrostatic repulsion between uncompensated charges provide significant contribution to optimization of the necklace structure. Upon forming a complex with both random and diblock polyampholytes, a polyelectrolyte chain changes its necklace conformation by forming one huge bead. The collapse of the polyelectrolyte chain occurs due to the neutralization of the polyelectrolyte charge by polyampholytes. In the case of the random polyampholyte, the more positively charged sections of the chain mix with negatively charged polyelectrolyte forming the globular bead while more negatively charged chain sections form loops surrounding the collapsed core of the aggregate. In the case of diblock polyampholyte, the positively charged block, a part of the negatively charged block, and a polyelectrolyte chain form a core of the aggregate with a substantial section of the negatively charged block sticking out from the collapsed core of the aggregate. In both cases the core of the aggregate has a layered structure that is characterized by the variations in the excess of concentration of monomers belonging to polyampholyte and polyelectrolyte chains throughout the core radius. These structures appear as a result of optimization of the net electrostatic energy of the complex and short-range attractive interactions between monomers of the polyelectrolyte chain.     

PHOTOCONTROL OF PHYTOCHROME DESTRUCTION AND BINDING IN DICOTYLEDONOUS vs MONOCOTYLE-DONOUS SEEDLINGS. THE INFLUENCE OF WAVELENGTH AND IRRADIANCE

Abstract— The irradiance and wavelength dependence of phytochrome destruction in vivo was analysed in etiolated cotyledons of Cucurbita pepo L. and etiolated seedlings of Amaranthus caudatus L. In contrast to grass seedlings, the rate of P _tot destruction could only be saturated by light sources that establish relatively high P _fr levels (about 50% of total phytochrome, corresponding to the photostationary state established by 693 nm light). To explain the irradiance dependence of P _tot destruction in dicots at irradiances above 0.1 Wm^-2, where the light reaction is at least one order of magnitude faster than P _fr destruction, we suggest there is a fast intercalary dark reaction between photoreaction and destruction. This dark reaction is probably—as in grass seedlings—the binding of P _fr to a receptor site. We conclude that the differences between dicots and grass seedlings with respect to the phytochrome system are of a quantitative rather than a qualitative nature.     

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.     

EXPRESSION OF OAT phyA cDNA IN THE MOSS Ceratodon purpureus

The possibility of transforming Ceratodon purpureus protoplasts by PEG-mediated direct DNA uptake was tested. Transformation with a plasmid carrying a kanamycin-resistance gene resulted in kanamycin-resistant colonies of C. purpureus protonemata. A full-length cDNA clone coding for oat phyA phytochrome was isolated. The clone HM4.1 which is 3.7-kb long exhibits about 99% nucleotide sequence identity to the known phytochrome clone AP3. The expression of HM4.1 in C. purpureus protonemata was tested. A construct with the 35S-promotor and the structural gene of HM4.1 was cotransformed with the plasmid containing the kanamycin-resistance. Kanamycin-resistant colonies were tested for the presence of HM4.1 sequences in a genomic Southern experiment. Two out of 19 kanamycin-resistant colonies reacted positively with a HM4.1 specific probe. The expression of phyA in the positive colonies was examined with monoclonal antibodies specific for oat phytochrome. The Western blot experiment with protein extracts of the two positive colonies grown in the dark revealed clear signals at 124-kDa which were not detected in control plants. These data demonstrate the possibility of expressing oat phyA-apoprotein in C. purpureus protonemata. The transgenic moss protonemata did not show phenotypical alterations in response to the foreign phytochrome polypeptide; it is not known at the moment if the tetrapyrole chromophore is attached to the oat polypeptide in the protonemata or not.     

Molecular mechanisms of polypeptide aggregation in human diseases

Protein aggregation is implicated in a plethora of neurodegenerative diseases. The proteins found to aggregate in these diseases are unrelated in their native structures and amino acid sequences, but form similar insoluble fibrils with characteristic cross-beta sheet morphologies called amyloid in the aggregated state. While both the mechanism of aggregation and the structure of the aggregates are not fully understood at the molecular level, recent studies provide strong support for the idea that protein aggregation into highly stable, insoluble amyloid structures is a general property of the polypeptide chain. For proteins with a unique native state, it is known that aggregation occurs under conditions that promote native-state destabilization in vitro and in vivo. Taken together, the results of several important recent investigations suggest three broad molecular frameworks that may underlie the conversion of normally soluble peptides and proteins into insoluble amyloid fibrils: (1) edge-strand hydrogen bonding, (2) domain-swapping, and (3) self-association of amyloidogenic fragments. We argue that these underlying scenarios are not mutually exclusive and may be protein-dependent - i.e., a protein with a high content of hinge-regions may aggregate via a runaway domain-swap, whereas a protein with a high content of amyloidogenic fragments may aggregate primarily by the self-association of these fragments. These different scenarios provide frameworks to understand the molecular mechanism of polypeptide aggregation.     

PHYTOCHROME CONTROL OF THE DEVELOPMENT OF PHOTOPHOSPHORYLATION

In the cotyledons of the mustard (Sinapis ah L.) seedling the development of the capacity for photophosphorylation is strongly influenced by pretreatment of the seedling with red light pulses. The red light acts through phytochrome. After a red light pretreatment the capacity for photophosphorylation increases linearly with the chlorophyll content, at least up to 30 min after the onset of continuous white light. It is proposed that the reaction chain required for photophosphorylation is completed under the influence of phytochrome even in the absence of chlorophyll. As soon as chlorophyll becomes available photophosphorylation functions instantaneously. Without a red light pretreatment there is a lag of more than 15 min before photophosphorylation becomes detectable after the onset of continuous white light even though chlorophyll a is available. Although phytochrome strongly influences the rate of chlorophyll accumulation as well it is improbable that the control by phytochrome of development of photophosphorylation and of chlorophyll accumulation are causally connected.