Light-harvesting systems of brown algae and diatoms. Isolation and characterization of chlorophyll ac and chlorophyll afucoxanthin pigment-protein complexes

The present study examined the protein associations and energy transfer characteristics of chlorophyll c and fucoxanthin which are the major light-harvesting pigments in the brown and diatomaceous algae. It was demonstrated that sodium dodecyl sulfate (SDS)-solubilized photosynthetic membranes of these species when subjected to SDS polyacrylamide gel electrophoresis yielded three spectrally distinct pigment-protein complexes. The slowest migrating zone was identical to complex I, the SDS-altered form of the P-700 chlorophyll a-protein. The zone of intermediate mobility contained chlorophyll c and chlorophyll a in a molar ratio of 2 : 1, possessed no fucoxanthin, and showed efficient energy transfer from chlorophyll c to chlorophyll a. The fastest migrating pigment-protein zone contained fucoxanthin and chlorophyll a, possessed no chlorophyll c, and showed efficient energy transfer from fucoxanthin to chlorophyll a. It is demonstrated that the chlorophyll $\text{a}\text{c-}\text{protein}$ and the chlorophyll $\text{a}\text{fucoxanthin-protein}$ complexes are common to the brown algae and diatoms examined, and likely share similar roles in the photosynthetic units of these species.     

Glyphosine,a plant growth regulator,affects chloroplast membrane proteins

Glyphosine (N,N-bis(phosphonomethyl)glycine) is known to increase sucrose levels in sugarcane and to cause chlorosis in maize and other plants. It has been suggested (Crofts, S.M., Arntzen, C.J., Vanderhoef, L.N. and Zettinger, C.S. (1974) Biochim. Biophys. Acta 335, 211–217) that its primary mode of action is to inhibit the synthesis of plastid rRNA. Growth of Lemna gibba L. G-3 on 5 · 10^?4M glyphosine causes the plants to produce fronds lacking chlorophyll. The plastids in these white fronds contain only a few internal membrane structures, some of which are stacked. Sodium dodecyl sulfate polyacrylamide gel electrophoresis shows an accumulation of substantial amounts of both the large and small subunits of ribulosebisphosphate carboxylase by the white fronds. The membrane fraction from these fronds contains only traces of the light-harvesting chlorophyll $\text{a}\text{b}$ apoprotein in comparison to control plants. In vivo labeling and immunoprecipitation show that the large subunit of ribulose-bisphosphate carboxylase is actively synthesized by the white fronds. However, labeling of the chlorophyll $\text{a}\text{b}$ apoprotein and a 32000 dalton protein in the membrane fraction is extremely low compared to control plants. We conclude that in Lemna, glyphosine differentially affects the synthesis and/or processing of soluble proteins and some membrane chloroplast proteins, and could be useful in understanding the biogenesis of chloroplast membranes.     

The detergent and salt effect on the light-harvesting chlorophyll ab complex from green plants

The light-harvesting accessory pigment-protein complex (LHC) with a chlorophyll (Chl) $\text{a}\text{b}$ ratio of 1.2 was isolated by treating pea chloroplasts with Triton X-100. The LHC was used to investigate the action of ionic (sodium dodecyl sulfate) and non-ionic (Triton X-100) detergents. By optical methods (absorption and fluorescence spectra, measurements of fluorescence yield, ?, and lifetime, τ) two successive stages of the process were demonstrated, namely (1) interaction between detergent monomers and proteins and (2) solubilization of pigments into detergent micelles, which is facilitated by the presence of salts. The concentration ranges, characteristic of these stages, differ by 1.5–2 orders of magnitude for SDS, but slightly overlap for Triton X-100. At the second stage, certain changes occur in LHC absorption and fluorescence spectra. Several stable states of the LHC were established: (1) an aggregated state formed in the presence of 10 mM MgSO₄ with τ ≈ 0.6 ns; (2) the dialyzed LHC with τ ≈ 0.9 ns; (3) the states of the LHC in detergent solution with τ ≈ 2.3, 2.9, 3.4 ns; (4) a 30 kilodalton monomer obtained by SDS-polyacrylamide gel electrophoresis with τ ≈ 4.1 ns. The fluorescence parameters of the LHC states were compared with those of Chl a in detergent micelles (for the micelles τ = 5.6–6.0 ns. The $\text{τ}\text{?}$ ratio (the criterion for emission heterogeneity) for the LHC in the absence of a detergent was shown to be higher at least by a factor of 3.5 than that for Chl a in the presence of a detergent. Successive additions of the detergent to the LHC cause gradual decrease in the $\text{τ}\text{?}$ ratio, and for the LHC monomer it reaches practically the same value as for Chl a in detergent micelles. The results are discussed on the basis of the data obtained previously. It is suggested that in vivo LHCs do not form such aggregates as in water solution without a detergent.     

Organization of chlorophyll-protein complexes of Photosystem I in Chlamydomonas reinhardii

The polypeptide pattern, chlorophyll-protein complexes, fluorescence emission spectra and light intensity required for saturation of electron flow via Photosystem (PS) II and PS I in a pale-green photoautotrophic mutant, y-lp, were compared to those of the parent strain, Chlamydomonas reinhardii y-1 cells. The mutant exhibits a 686 nm fluorescence yield at 25°C and 77 K 2–6-fold higher than that of the parent strain cells, and is deficient in thylakoid polypeptides 14, 17.2, 18 and 22 according to the nomenclature of Chua (Chua, N.-H. (1980) Methods Enzymol. 60C, 434–446). All chlorophyll-protein complexes ascribed to PS II and the CP I complex were present in both type of cells. However, a chlorophyll-protein complex CP Ia containing — in the parent strain — the 66–68 kDa polypeptides of CP I and the four above-mentioned polypeptides, was absent in the mutant. It was previously reported that a chlorophyll-protein complex, CP O, obtained from C. reinhardii contains five polypeptides, namely, 14, 15, 17.2, 18 and 22 (Wollman, F.A. and Bennoun, P. (1982) Biochim. Biophys. Acta 680, 352–360). A CP O-like complex was present also in the mutant y-lp cells but it contains only one polypeptide, 15. Energy transfer from PS II to PS I was not impaired in the mutant, although a 4-fold higher light intensity was required for the saturation of PS I electron flow in the y-lp cells as compared with the parent strain. No difference was found in the light saturation curves for PS II activity between the mutant and parent strain cells. Based on these and additional data (Gershoni, J.M., Shochat, S., Malkin, S. and Ohad, I. (1982) Plant Physiol. 70, 637–644), it is concluded that the chlorophyll-protein complexes of PS I in Chlamydomonas comprise a reaction center-core antenna complex containing the 66–68 kDa polypeptides (CP I), a connecting antenna consisting of four polypeptides (14, 17.2, 18 and 22), and a light-harvesting antenna containing one polypeptide, 15. These appear to be organized as a complex, CP Ia. The interconnecting antenna is deficient in the y-lp mutant and thus the CP Ia complex is unstable and energy is not transferred from CP O to CP I. The effective cross-section of PS I antenna is thus reduced and a high fluorescence is emitted at 686 nm.     

A carotenoid-protein from cyanobacteria

Easily solubilized carotenoid-containing proteins have been found in aqueous extracts from three genera of cyanobacteria. The three proteins have been purified, and the absorption spectra have been determined to be virtually identical with absorption maxima at 495 and 465 nm. During the purification the orange protein spontaneously changed to a red protein with a single, broad absorption maximum at 505 nm. The orange protein showed a molecular weight of 47 000 on gel filtration while that of the red protein was 26 700. Sodium dodecyl sulfate polacrylamide gel electrophoresis indicated a single polypeptide of M_r 16 000 in both the red and orange forms, but this method removed the chromophore from the proteins. The main carotenoid component of the complex was determined to be 3′-hydroxy-4-keto-ββ-carotenoid or 3′-hydroxyechinenone. The number of carotenoid molecules per molecule of orange protein of molecular weight 47 000 was between 20 and 40. The stoichiometry of carotenoid to protein seemed reasonably constant.     

Internal and external membrane proteins of the cyanobacterium, Synechococcus cedrorum

The protein composition and architecture of the photosynthetic membranes from the cyanobacterium, Synechococcus cedrorum, were analyzed with the aid of site-specific labels. Using membranes labeled with ³⁵S, about 50 membrane proteins can be detected by sodium dodecyl sulfate acrylamide gel electrophoresis. Approximately half of the proteins are accessible to modification by the impermeant probe, lactoperoxidase, indicating that they have surface-exposed domains. At least six of these external proteins can be removed by EDTA washing; the correspondence in molecular weights between five of these EDTA-extractable proteins and those of typical chloroplast coupling factor preparations may indicate that they are subunits of a membrane-bound ATPase. The photoactive, lipophilic compound, [¹²⁵I]iodonaphthyl azide, was used to label protein domains in contact with the lipid bilayer. Iodonaphthyl azide modification led to a labeling pattern significantly different from that seen with lactoperoxidase. In particular, proteins in the 13 000–20 000 dalton range that were labeled poorly or not at all by lactoperoxidase were heavily modified by iodonaphthyl azide.Photosystem I and II particles, extracted from the membrane by digitonin treatment, were iodinated by lactoperoxidase after isolation. The PS I particles acted as a relatively tight complex, with most of the proteins remaining inaccessible to surface modification. The PS II particles, on the other hand, responded as a more open structure, with most of the subunits yielding to lactoperoxidase iodination. Similar studies on a highly fluorescent, temperature-sensitive mutant of S. cedrorum revealed a different organization of the PS II complex. This mutant, when grown at 40°C, inserts a 51 kdalton polypeptide in place of a 53 kdalton protein. This protein also replaces the 53 kdalton species in the PS II complex of the mutant after 40°C growth. The structure of this complex is altered in that more sites become accessible to lactoperoxidase. This is particularly true of the 51 kdalton protein, which is barely labeled in wild-type PS II complexes.     

Studies on P-700 photo-oxidation and reduction in Photosystem I subchloroplast particles

The photochemical oxidation and reduction of P-700 were studied in digitonin- and in sodium dodecyl sulphate (SDS)-Photosystem I (PS I) particles in the presence of ascorbate. In digitonin-PS I particles, reduction of P-700⁺ occurs by the bound iron-sulphur protein (P-430) and by ascorbate. The relative contribution of these back reactions depends on the length of the exposure to light and on the temperature and pH of the reaction medium. Experiments performed under anaerobic conditions demonstrate that some endogenous component may serve as the electron acceptor of P-430^?. The rate of the latter reaction is also dependent upon the temperature and pH of the sample. At pH 9 and lower temperatures the rate of this reaction is so much reduced that the reduction of P-700⁺ by ascorbate, which increases rapidly at high pH, can be observed even during illumination. The effects of secondary electron acceptors and of the presence of SDS on the absorption changes due to P-700 are also reported. Low concentrations of SDS are shown to retard the back reaction of P-700⁺ with P-430^?. Studies with SDS-PS I particles (CP_I) confirm the absence of the iron-sulphur centres in this preparation. Three larger P-700-chlorophylla-protein complexes prepared by mild electrophoresis in the presence of SDS plus Triton X-100, however, still contain P-430.     

Titles of related papers in other sections

Electrophoretic analysis by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis showed that the light-harvesting chlorophyll $\text{a}\text{b}\text{-}\text{protein}$ complex of barley thylakoids contains only one polypeptide of apparent molecular weight 26 000. The barley mutant, deficient in chlorophyll b and this light-harvesting complex, lacks this polypeptide.The addition of a nonionic detergent, Triton X-100, to the sodium dodecyl solubilization buffer prior to SDS polyacrylamide tube gel electrophoresis, allowed separation of a relatively stable complex, characterized as an oligomeric form of the light-harvesting complex. The oligomer also contained a polypeptide with an apparent molecular weight of 26 000. The absorption and fluorescence spectral properties of the oligomer are similar to those of the monomer. It is suggested that the oligomer of the light-harvesting chlorophyll $\text{a}\text{b}\text{-}\text{protein}$ is closer to the in vivo form rather than the monomer.     

Isolation and characterization of a major chlorophyll ac2 light-harvesting protein from a Chroomonas species (Cryptophyceae)

Three chlorophyll-protein complexes of a Chroomonas species (Cryptophyceae) have been separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The two bands at 100 and 42 kDa are Complex I (CP I) and Complex IV (CP IV), the ubiquitous chlorophyll a-proteins associated with Photosystems I and II, respectively. The third 55 kDa band, which had two peptide subunits (24 and 20 kDa), contained both chlorophyll a and chlorophyll c₂ in a molar ratio of 1.4 chlorophyll a to 1 chlorophyll c₂ ($\text{chlorophyll}\text{a}\text{chlorophyll}\text{c}{}_2$ ratio in whole cells = 4). A chlorophyll $\text{a}\text{c}{}_2$ fraction with similar spectral and electrophoretic properties was isolated by digitonin-sucrose density gradient centrifugation. This fraction had no photochemical activity and contained only a single carotenoid species with absorbance maxima in methanol at 424, 448 and 476 nm. Efficient energy transfer from chlorophyll c₂ to chlorophyll a occurred in the complex.     

Ca2+-dependent cross-linking processes in human platelets

When platelet cytoplasmic Ca²⁺ is increased by the ionophore A23187 in the presence of the protease inhibitor leupeptin, there is the coincident appearance of a cross-linked polymer and the partial disappearance of monomeric protein and glycoprotein units. In the absence of leupeptin only 30% of the polymer was formed. The disappearance of monomeric protein bands, as detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis, is prevented by histamine, which as a pseudodonor amine is a known inhibitor of transglutaminase-catalyzed cross-linking. [¹⁴C]Histamine, at a tracer concentration, is incorporated into the polymer as well as into myosin, glycoproteins IIB and III, actin and tropomyosin. The lose of monomeric protein bands is mostly due to their conversion into polymers. Control measurements show that leupeptin effectively inhibited platelet Ca²⁺-dependent proteases. The cross-linking processes bringing about the observed increase in polymer formation are thus the result of a Ca²⁺-dependent platelet transglutaminase activity. The latter is located in the platelet cytosol and has been identified as platelet factor XIII on the basis of its specific cross-linking of fibrin. Platelet factor XIII, upon activation, may function physiologically to couple membrane proteins to cytoplasmic structural proteins. Thus, a new concept is proposed for the stabilization of platelet membranes and platelets as they form the hemostatic plug.     

Analysis of absorption spectra changes induced by temperature lowering on phycobilisomes,thylakoids and chlorophyll-protein complexes

Using fourth derivative analysis, differences between room and low temperature absorption spectra were studied. The positions of most absorption bands of the water-soluble, accessory pigment complex, the phycobilisome, remained unchanged after cooling. The stability of the wavelength positions of chlorophyll a forms in vivo as a function of temperature (Gulyaev, B.A. and Litvin, F.F. (1967) Biofizika 12, 845–854) was generally confirmed. The wavelength positions of all chlorophyll a forms in the P-700 chlorophyll a protein complex were unchanged when the preparations were cooled to ?196°C. Likewise, with other chlorophyll-containing materials: the light-harvesting chlorophyll $\text{a}\text{b}$ protein complex and the thylakoids of higher plants, algae, and cyanobacteria, the wavelength positions of most chlorophyll a forms were stable upon cooling. An exception was a 680 nm chlorophyll a band which was generally split at low temperature into two bands with the materials investigated. An interpretation of the multiplicity of chlorophyll spectral forms and the spectral changes induced by cooling for these forms is given using exciton theory and the energy-coupling variation of chlorophyll a molecules.     

Resolutions and identification of the core deoxynucleoproteins of the simian virus 40

Low molecular weight basic core proteins of SV₄₀ are resolved by Tris-Acetate-SDS polyacrylamide gel electrophoresis into a minimum of four polypeptides. These are the electrophoretic counterparts of the evolutionarily conserved histones F3, F2b, F2a2, and F2a1. Host African green monkey kidney cells contain an active protease activity which readily cleaves histone F3 during nuclear isolation in hypotonic buffers.     

Cation-mediated regulation of excitation energy distribution in chloroplasts lacking organized Photosystem II complexes

Despite the total loss of Photosystem II activity, thylakoids isolated from the green nuclear maize mutant hcf1-3 contain normal amounts of the light-harvesting chlorophyll $\text{a}\text{b}$ pigment-protein complex (LHC). We interpret the spectroscopic and ultrastructural characteristics of these thylakoids to indicate that the LHC present in these membranes is not associated with Photosystem II reaction centers and thus exists in a ‘free’ state within the thylakoid membrane. In contrast, the LHC found in wild-type maize thylakoids shows the usual functional association with Photosystem II reaction centers. Several lines of evidence suggest that the free LHC found in thylakoids isolated from hcf1-3 is able to mediate cation-dependent changes in both thylakoid appression and energy distribution between the photosystems: (1) Thylakoids isolated from hcf1-3 and wild-type seedlings exhibit a similar Mg²⁺-dependent increase in the short/long wavelength fluorescence emission peak ratio at 77 K. This Mg²⁺ effect is lost following incubation of thylakoids isolated from either source with low concentrations of trypsin. Such treatment results in the partial proteolysis of the LHC in both membrane types. (2) Thylakoids isolated from both hcf1-3 and wild-type seedlings show a similar Mg²⁺ dependence for the enhancement of the maximal yield of room temperature fluorescence and light scattering; both Mg²⁺ effects are abolished by brief incubation of the thylakoids with low concentrations of trypsin (3) Mg²⁺ acts to reduce the relative quantum efficiency of Photosystem I-dependent electron transport at limiting 650 nm light in thylakoids isolated from hcf1-3. (4) The pattern of digitonin fractionation of thylakoid membranes, which is dependent upon structural membrane interactions and upon LHC in the thylakoids, is similar in thylakoids isolated from both hcf1-3 and wild-type seedlings. We conclude that the surface-exposed segment of the LHC, but not the LHC-Photosystem II core association, is necessary for the cation-dependent changes in both thylakoid appression and energy distribution between the two photosystems, and that the LHC itself is able to transfer excitation energy directly to Photosystem I in a Mg²⁺-dependent fashion in the absence of Photosystem II reaction centers. The latter phenomenon is equivalent to a cation-induced change in the absorptive cross-section of Photosystem I.     

Ouabain-insensitivity of highly active Na+, K+-dependent adenosinetriphosphatase from rat kidney.

Highly purified preparations of Na⁺+K⁺-dependent adenosinetriphosphatase were isolated from rat kidney by two different procedures. The I₅₀ values for ouabain inhibition of the rat kidney enzyme at various stages of purification were determined to be essentially the same for all fractions tested (0.7 to 1.0 × 10^?4M). These results suggest that the marked insensitivity of the rat enzyme to inhibition by cardiac glycosides is due to the primary structure of the enzyme, and not to some other component in the tissue.     

Cis- and trans-diamminedichloroplatinum(II) binding products different tertiary structural changes on SV40 DNA

The proteases secreted into culture medium by MCF-7 breast cancer cells produced both plasminogen-dependent and -independent proteolysis, as shown by casein-polyacrylamide gel electrophoresis. All of these proteases except the largest (M_r 120,000) were retained on a benzamidine-Sepharose affinity column, a characteristic of trypsinlike proteases. Among the proteases which activated plasminogen, all except a major protease of M_r 59,000 were antigenically similar to urokinase. These urokinaselike proteases (M_r 65,000 to 25,000) were isolated on a antiurokinase-Sepharose affinity column. The findings indicate that in a stable cell line derived from a human breast cancer there are two distinct types of plasminogen activators, opening the possibility that these activator types may be modulated in separate ways.     

The effect of ammonium nitrate on the synthesis of nitrogenase and the concentration of leghemoglobin in pea root nodules induced by Rhizobium leguminosarum

The effects of NH₄NO₃ on the development of root nodules of Pisum sativum after infection with Rhizobium leguminosarum (strain PRE) and on the nitrogenase activity of the bacteriods in the nodule tissue were studied. The addition of NH₄NO₃ decreased the nitrogenase activity measured on intact nodules. This reduction of nitrogen fixation did not result from a reduced number of bacteroids or a decreased amount of bacteroid proteins per gram of nodule. The synthesis of nitrogenase, measured as the relative amount of incorporation of [³⁵S]sulfate into the components I and II of nitrogenase was similarly not affected.The addition of NH₄NO₃ decreased the amount of leghemoglobin in the nodules and there was a quantitative correlation between the leghemoglobin content and the nitrogen-fixing capacity of the nodules. The conclusion is that the decrease of nitrogen-fixing capacity is caused by a decrease of the leghemoglobin content of the root nodules and not by repression of the nitrogenase synthesis.