Lithium Dodecyl Sulfate-polyacrylamide Gel Electrophoresis Research Articles

Expression, purification, immunogenicity and protective efficacy of a recombinant nucleoside hydrolase from Leishmania donovani, a vaccine candidate for preventing cutaneous leishmaniasis

The nucleoside hydrolase gene from Leishmania donovani was cloned and expressed in Escherichia coli as a full length 36-kDa protein (LdNH36). Following lysis and extraction, the protein was purified by anion exchange and gel filtration chromatography. The purified protein had a molecular mass of approximately 36-kDa and was confirmed to be >99% pure. Using a nucleoside hydrolase assay, the protein was found to exhibit a Km of 741 ± 246 μM. Protein integrity was confirmed by lithium dodecyl sulfate polyacrylamide gel electrophoresis (LDS-PAGE), mass spectrometry (MS), and enzymatic assay. Analysis of antibody levels from immunized mice indicated that LdNH36 alone or in a stable emulsion with the Toll-like receptor-4 ligand glucopyranosyl lipid adjuvant (GLA-SE) as immunostimulant induced high levels of antigen-specific IgG antibodies. The cellular immune response indicated a Th1 response in mice immunized with LdNH36, but only when formulated with GLA-SE. Mice immunized with the LdNH36 antigen in combination with the GLA-SE adjuvant and challenged with Leishmania mexicana showed significant reductions (>20 fold) in parasite burden, confirming the protective efficacy of this vaccine candidate.

Surface modification of permalloy (Ni80Fe20) nanoparticles for biomedical applications

We report a simple and novel method for surface biofunctionalization onto recently reported Ni80Fe20 permalloy nanoparticles (~71 nm) and the immobilization of a model protein, IgG from human serum. The strategy of protein immobilization involved attachment of histidine-tagged streptavidin to the Ni80Fe20 nanoparticles via a non-covalent ligand binding followed by biotinylated human IgG binding on the nanoparticle surface using the specific high affinity avidin–biotin interaction. The biofunctionalization of Ni80Fe20 permalloy nanoparticles was confirmed by Fourier Transform InfraRed (FTIR) spectroscopy and protein denaturing gel electrophoresis (lithium dodecyl sulfate-polyacrylamide gel electrophoresis, LDS-PAGE). This protocol for surface functionalization of the novel nanometer-sized Ni80Fe20 permalloy particles with biological molecules could open diverse applications in disease diagnostics and drug delivery.

Proteomic Analysis of Wheat Flour Allergens

Wheat can cause severe IgE-mediated systematic reactions, but knowledge on relevant wheat allergens at the molecular level is scanty. The aim of the present study was to achieve a more detailed and comprehensive characterization of the wheat allergens involved in food allergy to wheat using proteomic strategies, referred to as "allergenomics". Whole flour proteins were separated by two-dimensional gel electrophoresis with isoelectric focusing and lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, IgE-binding proteins were detected by immunoblotting with sera of patients with a food allergy to wheat. After tryptic digestion, the peptides of IgE-binding proteins were analyzed by matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry. In this study, we identified four previously reported wheat allergens or their sequentially homologous proteins [serpin, alpha-amylase inhibitor, gamma-gliadin, and low molecular weight (LMW) glutenin] by a database search. As a result of the high resolution of two-dimensional gel electrophoresis, nine subunits of LMW glutenins were identified as the most predominant IgE-binding antigens. The two-dimensional allergen map can be beneficial in many ways. It could be used, for example, for precise diagnosis of wheat-allergic patients and assessment of wheat allergens in food. Additionally, we compared allergenomics to conventional biochemical methods and evaluated the usefulness of a proteomic strategy for identifying putative allergens to wheat allergy.

Isolation, size estimates, and spectral heterogeneity of an oligomeric series of light-harvesting 1 complexes from Rhodobacter sphaeroides.

A series of light-harvesting 1 (LH1) complexes was isolated by lithium dodecyl sulfate-polyacrylamide gel electrophoresis at 4 degrees C from Rhodobacter sphaeroides M21, which lacks the peripheral light-harvesting 2 (LH2) complex. This ladder of LH1 bands was also demonstrated in the wild type, partially superimposed upon a smaller number of LH2 complexes. An assessment of electrophoretic mobility vs acrylamide concentration, in which the reaction center LM particle and annular LH1 and LH2 complexes were used as standards of known structure, indicated that the LH1 gel bands 2 to 10 represent regular oligomers of an alpha beta heterodimeric unit, that vary in size from (alpha beta)(2-3) to (alpha beta)(10-11). The isolated LH1 complexes exhibited oligomeric state dependent optical properties, characterized by red shifts in near-IR absorption and emission maxima at 77 K of approximately 6 nm as aggregate sizes increased from approximately 3 to 7-8 alpha beta-heterodimers, accompanied by shifts in highly polarized fluorescence from the blue to the red side of the absorption band. This has been explained by the oligomerization of heterodimers to form a curvilinear array of excitonically coupled chromophores, with the anisotropic long-wavelength component, designated originally as B896, corresponding to low energy excitonic transitions arising from interactions within inhomogeneous BChl clusters [Westerhuis et al. (1999) J. Phys. Chem. B 103, 7733-7742]. Differences in electrophoretic profiles of LH1 bands between strains M21 and M2192, an LH1-only strain that also lacks PufX, further suggested that the more rapidly migrating bands represent arced fragments of the curvilinear array of LH1 complexes thought to exist as a large closed circular structure only in the latter strain. The electrophoretic banding pattern also indicated that the LH1 complex may be located at the peripheries of dimeric intramembrane particle arrays seen in freeze-fracture replicas of tubular M21 membranes; the possible role for the PufX protein in the assembly of these structures is discussed.

Cloning and expression of two novel hemin binding protein genes from Treponema denticola.

Treponema denticola does not appear to produce siderophores, so it must acquire iron by other pathways. Indeed, T. denticola has been shown to have an iron-regulated 44-kDa outer membrane protein (HbpA) with hemin binding ability. To characterize the HbpA protein, its gene was cloned from genomic DNA libraries of T. denticola. Sequence analysis of the hbpA open reading frame indicated that it encoded a 42.8-kDa protein with a 23-amino-acid signal peptide. HbpA has no significant homology to any proteins in the databases. Southern blot analysis demonstrated that hbpA is present in several T. denticola ATCC strains and clinical isolates, but not in Treponema pectinovorum, Treponema socranskii, or Escherichia coli. HbpA, expressed as a recombinant protein in E. coli and purified by antibody affinity chromatography, has hemin binding activity as determined by lithium dodecyl sulfate-polyacrylamide gel electrophoresis with tetramethylbenzidine staining. Northern blot analysis showed that there were two hbpA-containing transcripts, of approximately 1.3 and 2.6 kb, and that the RNA levels were low-iron induced. Interestingly, the 2.6-kb mRNA also encoded a second protein with significant homology to hbpA. This downstream gene, called hbpB, was cloned and sequenced and its product was expressed as a fusion protein in E. coli. The hbpB gene product is 49% identical to HbpA and binds hemin. Thus, T. denticola has two novel hemin binding proteins which may be part of a previously unrecognized iron acquisition pathway.

Presence of Na+-stimulated P-type ATPase in the membrane of a facultatively anaerobic alkaliphile, Exiguobacterium aurantiacum.

It was found that a facultatively anaerobic alkaliphile, Exiguobacterium aurantiacum, possesses a membrane-bound ATPase, which was activated specifically by Na+. The Na+-stimulated ATPase activity reached a maximum value at 200 mM NaCl. In the presence of 200 mM NaCl, the activity was drastically reduced by vanadate, a potent inhibitor of P-type ATPase, with a half-maximal inhibition at 1 microM. Incubation of the membranes with [gamma-32P]ATP followed by acidic lithium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the existence of two phosphorylated intermediates with apparent molecular masses of 60 and 100 kDa. Only phosphorylation of the 100-kDa polypeptide was inhibited by vanadate. The membrane extract containing Na+-stimulated ATPase, when reconstituted into soybean phospholipid vesicles, exhibited 22Na+ transport by the addition of ATP, which was inhibited by vanadate and gramicidin. It is likely that the Na+-stimulated ATPase belongs to P-type and is involved in Na+ transport.

Cell adhesion to proteins separated by lithium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto a polyvinylidene difluoride membrane: a new cell-blotting technique

Cell blotting, although conceptually simple, has failed to achieve wide practical application. Described here is a new cell-blotting technique which involves cell adhesion to protein bands after separation by lithium dodecyl sulfate-polyacrylamide gel electrophoresis (LDS-PAGE) and blotting onto polyvinylidene difluoride (PVDF) membrane at 4°C. Cell bands adherent on PVDF are detected using hematoxylin, or propidium iodide (PI) staining followed by viewing under ultraviolet (UV) light. The technique allows quick microscopic visualization of adherent cells composing the bands, without requiring clearing of the membrane. Representative cell adhesion proteins from different sources, i.e., plant lectins (e.g., phytohemagglutinin, PHA; concanavalin A, ConA; and wheat germ agglutinin, WGA); extracellular matrix (ECM) proteins; and integral membrane proteins (e.g., recombinant soluble vascular cell adhesion molecule-1, rs VCAM-1) were tested for cell binding by the new cell-blotting technique using human lymphoid progenitor (NALM-6) and myeloid progenitor (KG1a) cell lines. Cell adhesion proteins retained their adhesion function in all cases tested. Specificity of cell binding onPVDF blot was demonstrated by inhibition of cell adhesion to WGA protein bands using an appropriate sugar, i.e., N-acetyl D-glucosamine. The cell blotting assay was comparable in sensitivity to Coomassie blue staining of protein bands. The ability to conduct protein extraction, separation and blotting at low temperature avoids thermal denaturation, thereby preserving the adhesion properties of the proteins. The electrophoretic/blotting system has unique detergent removal/protein renaturation properties and the ability to preserve functionally active adhesion protein complexes. The cell-blotting technique described is sufficiently robust for routine application in the investigation of novel cell adhesion proteins.

Utilization of haem from the haptoglobin-haemoglobin complex by Bacteroides fragilis

Possession of specialized iron acquisition systems is a prerequisite for the survival of pathogenic bacteria in their host. The purpose of this study was to determine whether Bacteroides fragilis, a clinically important Gram-negative anaerobic bacterium, possesses a specific haem-uptake system. Growth studies indicated that this microorganism can utilize haem from either haemoglobin or haptoglobin-haemoglobin as its sole source of iron. Iron-repressible haem-binding protein complexes ( HBP complexes), involved in the uptake of haem from haptoglobin-haemoglobin were detected by means of lithium dodecyl sulfate polyacrylamide gel electrophoresis ( LDS-PAGE). Four polypeptides of approximately 60, 58, 49 and 35 kDa, which are part of these HBP complexes, were identified as haem-binding proteins. A 44 kDa iron-repressible outer-membrane protein is needed for a functional HBP complex, but the exact role of this protein in the uptake of haem is still unknown.

Discovery of novel hematopoietic cell adhesion molecules from human bone marrow stromal cell membrane protein extracts by a new cell- blotting technique

In an attempt to define the role of cell adhesion molecules (CAMs) within the bone marrow (BM) microenvironment in normal hematopoiesis and in leukemia development, a novel cell-blotting technique that involved cell adhesion to protein bands after separation by lithium dodecyl sulfate-polyacrylamide gel electrophoresis (LDS-PAGE) and blotting onto polyvinylidene difluoride (PVDF) membrane has been developed. Human BM stromal cell membrane fractions have been prepared from Dexter-type cultures after cell lysis by sonification and differential centrifugations of the sonification contents. The 20,000 g pellets representing membrane fractions have been solubilized by 2% Triton X-100, 0.575% LDS, and 8 mol/L urea in sequential order. The protein extracts are fractionated by LDS-PAGE and screened for CAMs by the new cell-blotting technique. This led to identification of nine protein bands in lanes containing LDS extracts showing adhesion of KG1a (CD34+ progenitor myeloid) cells. Evidence that the BM proteins exhibiting KG1a cell adhesion are novel CAMs is based on the observations that these proteins, in comparison with known CAMs, specifically VCAM-1, CD54, and CD44, show (1) contrasting detergent- solubility properties, (2) different temperature requirement for mediating cell adhesion function, and (3) markedly distinct electrophoretic mobilities. The various cell types tested, notably KG1a, NALM-6, WIL-2, Ramos, HS-Sultan, K562, JY B lymphoblastoid cells, and T lymphoblasts, showed distinctive patterns of binding to different subsets of BM CAMs. These results demonstrate a new approach to studies of molecular mechanisms that may determine specificity of hematopoietic cellular localization within BM microenvironment and may play an important role in controlling hematopoiesis.

Discovery of Novel Hematopoietic Cell Adhesion Molecules From Human Bone Marrow Stromal Cell Membrane Protein Extracts by a New Cell-Blotting Technique

AbstractIn an attempt to define the role of cell adhesion molecules (CAMs) within the bone marrow (BM) microenvironment in normal hematopoiesis and in leukemia development, a novel cell-blotting technique that involved cell adhesion to protein bands after separation by lithium dodecyl sulfatepolyacrylamide gel electrophoresis (LDS-PAGE) and blotting onto polyvinylidene difluoride (PVDF) membrane has been developed. Human BM stromal cell membrane fractions have been prepared from Dexter-type cultures after cell lysis by sonification and differential centrifugations of the sonification contents. The 20,000g pellets representing membrane fractions have been solubilized by 2% Triton X-100, 0.575% LDS, and 8 mol/L urea in sequential order. The protein extracts are fractionated by LDS-PAGE and screened for CAMs by the new cell-blotting technique. This led to identification of nine protein bands in lanes containing LDS extracts showing adhesion of KG1a (CD34+ progenitor myeloid) cells. Evidence that the BM proteins exhibiting KG1a cell adhesion are novel CAMs is based on the observations that these proteins, in comparison with known CAMs, specifically VCAM-1, CD54, and CD44, show (1) contrasting detergent-solubility properties, (2) different temperature requirement for mediating cell adhesion function, and (3) markedly distinct electrophoretic mobilities. The various cell types tested, notably KG1a, NALM-6, WIL-2, Ramos, HS-Sultan, K562, JY B lymphoblastoid cells, and T lymphoblasts, showed distinctive patterns of binding to different subsets of BM CAMs. These results demonstrate a new approach to studies of molecular mechanisms that may determine specificity of hematopoietic cellular localization within BM micro-environment and may play an important role in controlling hematopoiesis.

Human neutrophil cytochrome b contains multiple hemes. Evidence for heme associated with both subunits.

Human phagocyte cytochrome b is the terminal component of the microbicidal superoxide generating system. Although the primary structure of this protein has been determined, little is known about the placement of the heme prosthetic groups in this heterodimeric integral membrane protein. Analysis of the cytochrome using lithium dodecyl sulfate-polyacrylamide gel electrophoresis at 0 degree C followed by tetramethylbenzidine heme staining demonstrated the presence of heme in both the 91- and 22-kDa subunits identified by Western blot analysis using peptide specific antisera. Exposure of cytochrome b (purified or in isolated neutrophil plasma membranes) to Staphylococcal protease V8 or trypsin did not affect absorbance spectra. However, such treatment resulted in degradation of both subunits to smaller fragments, including characteristic immunoreactive 20-kDa fragments of both the large and small subunits of the cytochrome that retained one or both of the hemes. The spectral stability to proteolysis and size of the proteolytic heme-containing fragments generated explains previous reports which suggested that the heme resided in the small subunit. Our current results indicate that human neutrophil cytochrome b is a bi-heme or possibly tri-heme molecule with at least one heme residing in the large subunit and one shared between both subunits and that the heme-containing regions of the cytochrome probably lie within the membrane lipid bilayer. Such a multi-heme structure would be consistent with an electron transfer function for this cytochrome by providing an efficient mechanism for transferring electrons across the plasma membrane to the extracellular surface where oxygen could be reduced to create superoxide.

Identification of novel adhesion proteins in mouse peritoneal macrophages

When mouse peritoneal macrophages were made to adhere firmly on glass surface and then removed by sequential treatment with hypotonic triethanolamine and Nonide P-40, a set of proteins were found to be left behind at the sites of adherent cells. Such glass-adherent proteins were detected as round or ellipsoidal patches of autofluorescence under a confocal laser microscope, and visualized ultrastructurally as aggregates to narrow threads of unique loop structures which were composed of linearly aligned particles of 22 ± 2 nm in diameter. Lithium dodecylsulfate-polyacrylamide gel electrophoresis of the glass-adherent proteins showed two major bands, 12 kDa and 14 kDa, which always co-existed in any different sample. The polyclonal antibody raised against these two proteins specifically stained the glass-adherent proteins in situ. The adhesion of macrophages to glass was significantly blocked with Fab fragments of the antibody. The in situ cross-linking experiment suggested that these two proteins might be closely associated with each other to form complexes. Hence, these proteins can be reasonably considered to be responsible for non-specific adhesion of macrophages to glass.

Detection of hemoprotein peroxidase activity on polyvinylidene difluoride membrane

The feasibility of detecting hemoproteins after electroblotting was examined. Hemorproteins were subjected to lithium dodecyl sulfate-polyacrylamide gel electrophoresis (LDS-PAGE) and then electroblotted and peroxidase activity was detected with 3,3′,5,5′-tetramethylbenzidine. The sensitivity and specificity of tetramethylbenzidine staining of LDS-PAGE gels was retained when proteins were electroblotted. Subsequent staining of the membrane with Coomassie blue R-250 revealed a protein pattern similar to that in the polyacrylamide gel. Thus electroblotting of hemoproteins does not affect resolution of the electrophoretic pattern and heme-associated peroxidase activity. Additionally, the ability to stain hemoproteins on polyvinylidene difluoride membranes offers the advantage of utilizing the same membrane for further biochemical and immunological characterizations.

Characterization of a P-type ATPase of the Archaebacterium Methanococcus voltae

The vanadate-sensitive ATPase of Methanococcus voltae has been purified by a procedure which includes, purification of the cytoplasmic membrane by sucrose gradient centrifugation, solubilization with Triton X-100, and DEAE-Sephadex and Sephacryl S-300 chromatography. While the DEAE-Sephadex step provided a preparation consisting of two polypeptides (74 and 52 kDa), the Sephacryl S-300 step yields a product with a subunit of 74 kDa. Incubation of either membranes or purified ATPase with [gamma-32P]ATP followed by acidic (pH 2.4) lithium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the vanadate-sensitive labeling of a 74-kDa acyl phosphate intermediate. These results indicate that the M. voltae ATPase is of the P-type.

Characterization and localization of phosphatidylglycerophosphate and phosphatidylserine synthases in Rhodobacter sphaeroides

Catalytic properties and membrane associations of the phosphatidylglycerophosphate (PGP) and phosphatidylserine (PS) synthases of Rhodobacter sphaeroides were examined to further characterize sites of phospholipid biosynthesis. In preparations of cytoplasmic membrane (CM) enriched in these activities, apparent Km values of PGP synthase were 90 microM for sn-glycerol-3-phosphate and 60 microM for CDP-diacylglycerol; the apparent Km of PS synthase for L-serine was near 165 microM. Both enzymes required Triton X-100 with optimal PS synthase activity at a detergent/CDP-diacylglycerol (mol/mol) ratio of 7.5:1.0, while for optimal PGP synthase, a range of 10-50:1.0 was observed. Unlike the enzyme in Escherichia coli and several other Gram-negative bacteria, the PS synthase activity had a specific requirement for magnesium and was tightly associated with membranes rather than ribosomes in crude cell extracts. Sedimentation studies suggested that the PGP synthase was distributed uniformly over the CM in both chemoheterotrophically and photoheterotrophically grown cells, while the PS synthase was confined mainly to a vesicular CM fraction. Solubilized PGP synthase activity migrated as a single band with a pI value near 5.5 in a chromato-focusing column and 5.8 on isoelectric focusing; in the latter procedure, the pI was shifted to 5.3 in the presence of CDP-diacylglycerol. The PGP synthase activity gave rise to a single polypeptide band in lithium dodecyl sulfate-polyacrylamide gel electrophoresis at 4 degrees C.

Cytochromes of plant plasma membranes. Characterization by absorbance difference spectrophotometry and redox titration

The cytochrome composition of plasma membranes (PM) obtained by phase partitioning of microsomal fractions from spinach leaves (Spinacea oleracea L. cv. Medania), cauliflower inflorescences (Brassica oleracea L.), sugar beer leaves (Beta vulgaris L.) and barley (Hordeum vulgare L. cv. Kristina) roots and leaves was characterized by absorbance difference spectrophotometry at different reducing conditions at 20 and – 196°C, by redox titration, and by heme staining of polypeptide bands after lithium dodecyl sulfate polyacrylamide gel electrophoresis (LDS‐PAGE). The location of the α‐bands in the difference spectra and the loss of heme after treatment with LDS indicated that predominantly cytochromes of the b‐type were present in all species tested. The total concentration of cytochrome was ca 0.35 nmol (mg protein)−1. The main component (ca 70% of total) was completely reduced by ascorbate and partly by NADH and had a midpoint potential of ca 150 mV. At – 196°C, ascorbate reduction revealed a symmetrical α‐band at ca 557 nm with PM from spinach leaves, cauliflower and sugar beet leaves, but with barley root and leaf PM ascorbate reduction resulted in an asymmetrical α‐band (shoulder at 552, maximum at 559 nm). In the dithionite‐reduced minus ascorbate‐reduced spectrum at –196°C a split α‐band (552 + 558 nm) was seen with PM from all species. This minor component had a midpoint potential of ca – 50 mV and is probably identical to cytochrome b5, the presence of which would explain the relatively high NADH‐cytochrome c reductase activities observed with plant PM. With PM from cauliflower, CO‐difference spectra indicated that cytochromes P‐420 and P‐450 were present at concentrations up to 0.06 and 0.03 nmol (mg protein)−1, respectively. Visualization of cytochromes by heme staining after LDS‐PAGE was complicated by endogenous peroxidase activity and by loss of heme during solubilisation. A presumptive b‐cytochrome (heme‐stained band at 94 kDa) was only detected with barley leaf PM.

Oligomerization states and associations of light-harvesting pigment-protein complexes of Rhodobacter sphaeroides as analyzed by lithium dodecyl sulfate-polyacrylamide gel electrophoresis

Oligomerization states and associations of light-harvesting pigment-protein complexes of Rhodobacter sphaeroides as analyzed by lithium dodecyl sulfate-polyacrylamide gel electrophoresis

Structural properties of the D1 and surrounding photosystem II polypeptides as revealed by their interaction with cross-linking reagents.

Treatment of Chlamydomonas reinhardtii thylakoids with cross-linking reagents including glutaraldehyde causes polymerization of all thylakoid polypeptides, but not of the reaction center II polypeptide D1 unless the thylakoids are presolubilized by octyl beta-D-glucoside (Adir, N., and Ohad, I. (1986) Biochim. Biophys. Acta 850, 264-274). The results presented here show that this is a general property of D1 as it can be demonstrated in thylakoids of cyanophytes, Dasicladaceae, green algae, and C3 and C4 plants. Solubilization of the membranes by ionic detergents, deoxycholate, lauryl sucrose, or dodecyl beta-D-maltoside is not effective in inducing cross-linking of the D1 polypeptides by glutaraldehyde. The most effective alkyl glucosides were those with 7-9 carbon alkyl chains. The same behavior toward glutaraldehyde was exhibited by the unprocessed D1 precursor and by the palmitoylated D1 protein. Based on the refractility of the D1 protein to cross-linking reagents, a procedure was developed for its isolation from cross-linked thylakoids by lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Isolated D1 retained its behavior toward cross-linking by glutaraldehyde and generated tryptic fragments similar to those obtained following trypsin treatment of intact thylakoids. Denaturation of isolated D1 protein by acetone facilitates cross-linking by glutaraldehyde and extensive degradation by trypsin. The photosystem II polypeptides are differentially cross-linked with increasing concentrations of glutaraldehyde, the most susceptible being the 28- and 23-kDa components of the light-harvesting chlorophyll a-b protein complex and the core complex 44- and 51-kDa polypeptides, and the least affected being the cytochrome b559, the D2 protein, and a 24-kDa component of the light-harvesting chlorophyll a-b protein complex. These results reflect the relative position and interaction of the photosystem II polypeptides within the complex and suggest that strong and specific hydrophobic interactions may be responsible for the tight and stable conformation of D1. This may be based mostly on the conserved amino acid sequences of D1 and possibly plays a role in the process of D1 integration and removal from the reaction center during its light-dependent turnover.

Low-temperature fluorescence emission spectra and chlorophyll-protein complexes in mutants of Chlamydomonas reinhardtii: Evidence for a new chlorophyll- a-protein complex related to Photosystem I

With the aim of confirming the relationships between low-temperature emission bands of chlorophyll fluorescence of whole cells and chlorophyll-protein complexes as defined by their electrophoretic behaviour, a study was performed with the wild type of Chlamydomonas reinhardtii and eight mutants which lacked one or several chlorophyll-protein complexes and showed impaired photosynthetic functions. Chlorophyll-protein complexes from chloroplast fragments or Triton X-100-treated particles, which were enriched in both Photosystems I and II (PS I and PS II), were analyzed by lithium dodecylsulfate polyacrylamide gel electrophoresis at 4°C, using either lithium dodecylsulfate or n- octyl-β- d-glucopyranoside in the solubilization mixtures. Absorption and fluorescence emission spectra were measured at 77 K with whole cells and also, for fluorescence, with isolated chlorophyll-protein complexes. Fluorescence emissions in the 680–682 nm ( F 682), 686 nm ( F 686), 696 nm ( F 696), 703 nm ( F 703), 707 nm ( F 707) and 712–717 nm ( F 715) regions were observed with whole cells of the different strains. A comparative study of the chlorophyll-protein complexes, the fluorescence emissions and the photochemical activities shown by each strain confirmed the following correlations, which have been previously described or proposed in the literature for algae or higher plants: F 682 with CP II, the chlorophyll (Chl) a + b complex corresponding to the main light-harvesting antenna; F 686 and F 696 with CP IV and CP III, the Chl a complexes corresponding to the antenna and to the core of PS II respectively; F 707 with CP 0, the Chl a + b complex part of the PS I antenna; F 715 with CP I, the Chl a complex corresponding to the core of PS I. A new PS-I-related Chl a-protein complex, tentatively designated CP 0a, was also observed. This complex showed an apparent relative molecular mass slightly smaller than that of CP I. It was the only PS I-related complex in the double mutant Fl 5 Pg 27 which lacks Chl b, CP 0, CP I and CP II. It appeared to be correlated with F 703, which was observed with cells of the same double mutant. The emission spectrum of another mutant, Fl 50 which contained CP I and CP II but was deficient in CP 0 and CP 0a, showed a F 715 contribution significantly reduced, indicating that CP 0 and CP 0a play an essential part in energy transfer from CP II to CP I. The probable pathways of light energy transfer in C. reinhardtii were examined. It is proposed that CP 0a acts as a connecting antenna between CP 0 and CP I, the energy transfer from CP II to PS I occurring through the following sequence: CP II → CP 0 → CP 0a → CP I core antenna → PS I reaction center.

Synthesis of a Putative c-Type Cytochrome by Intact, Isolated Pea Chloroplast

In addition to chlorophyll-protein complexes, other proteins were labeled when isolated developing pea (Pisum sativum L.) chloroplasts were incubated with [(14)C]-5-aminolevulinic acid. The major labeled band (M(r) = 43 kilodaltons by lithium dodecyl sulfate-polyacrylamide gel electrophoresis) was labeled even in the presence of chloramphenicol. Heme-dependent peroxidase activity (as detected by the tetramethyl benzidine-H(2)O(2) stain) was not visibly associated with this band. The radioactive band was stable to heat, 5% HCl in acetone, and was absent if the incubation with [(14)C]-5-aminolevulinic acid was carried out in the presence of N-methyl protoporphyrin IX dimethyl ester (a specific inhibitor of ferrochelatase). Organic solvent extraction procedures for the enrichment of cytochrome f from chloroplast membranes also extracted this unknown labeled product. It was concluded that this labeled product was probably a c-type cytochrome; however, the possibility that it might be a protein containing a covalently linked linear tetrapyrrole was not ruled out.