Isolation and characterization of a 23 kDa protein essential for photosynthetic oxygen evolution

A 23 kDa protein has recently been demonstrated to participate in photosynthetic oxygen evolution by reconstitution experiments on inside-out thylakoid vesicles (Åkerlund H-E, Jansson C and Andersson B (1982) Biochim Biophys Acta 681:1–10). Here we describe the isolation of the 23 kDa protein from a spinach chloroplast extract using ion-exchange chromatography. The protein was obtained in a yield of 25% and with less than 1% of contaminating proteins. The ability of the protein to stimulate oxygen evolution in inside-out thylakoids was preserved throughout the various fractionation steps. The isolated protein was highly water soluble and appeared as a monomer. Its isoelectric point was at pH=7.3. The amino acid composition showed a high content of polar amino acids, resulting in a polarity index of 49%. The isolated protein lacked metals and other prosthetic groups. Its function as a catalytic or regulating subunit in the oxygen evolving complex is discussed.Abbreviations kDa kiloDalton - PAGE polyacrylamide gel electrophoresis     

Identification of polypeptides associated with the 23 and 33 kDa proteins of photosynthetic oxygen evolution

An immunological approach was used for nearest-neighbor analyses for the 23 and 33 kDA proteins of the oxygen-evolving complex. Functional Photosystem II particles with a simple polypeptide composition were partly solubilized with detergent and incubated with monospecific antibodies against either the 23 or the 33 kDa protein. SDS-polyacrylamide gel electrophoresis revealed that the immunoprecipitates, apart from the antigenic proteins, also contained polypeptides at 24, 22 and 10 kDa. In contrast, polypeptides of the light-harvesting and Photosystem II core complexes showed very poor coprecipitation with the 23 and 33 kDa proteins. The 24, 22 and 10 kDa polypeptides were not precipitated by the antibodies if the 23 and 33 kDa proteins had been removed from the particles prior to solubilization. These observations demonstrate a close association between the 24, 22 and 10 kDa polypeptides and the 23 and 33 kDa proteins of the oxygen-evolving complex. None of these precipitated polypeptides contained any manganese. It is suggested that the 24, 22 and 10 kDa polypeptides are subunits of the oxygen-evolving complex and involved in the binding of the extrinsic 23 and 33 kDa proteins to the inner thylakoid surface.     

A protein essential for recovering oxygen evolution in cholate-treated chloroplasts

A novel method for the reconstitution of oxygen evolution in cholate-extracted spinach thylakoid membranes was established and a protein essential for the reconstitution was purified from cholate extracts. Purification of the protein was accomplished by chromatography on a DEAE-Sephacel column. This protein (M_r 17 000) was reinserted into vesicular membranes reconstituted from cholate-extracted thylakoids in the presence of 25% glycerol to reactivate oxygen evolution.     

Isolation and partial characterization of an opioid-like 88 kDa hibernation-related protein

Previous studies show that infusion of hibernating woodchuck albumin (HWA) induces hibernation in summer-active ground squirrels and results in profound behavioral and physiological depression in primates. These effects are reversed by the administration of opiate antagonists, suggesting that the putative hibernation induction trigger (HIT) may act through opioid receptors. We have demonstrated that both HIT-containing plasma and the synthetic α opioid -Ala²- -Leu⁵-enkephalin (DADLE), which mimics the activity of HIT in hibernators, extend tissue survival time of a multi-organ autoperfusion system by 3-fold. In this study we present the first data showing biological activity with a much more highly purified plasma fraction from hibernating woodchucks, identified as the hibernation-related factor (HRF). Both the HRF and DADLE show opiate-like contractile inhibition in the mouse vas deferens (Mvd) bioassay. We also have preliminary evidence in an isolated rabbit heart preparation indicating that the HRF and DADLE act similarly to restore left ventricular function following global myocardial ischemia. Furthermore, we have partially sequenced an α 1-glycoprotein-like 88 kDa hibernation-related protein (p88 HRP) present in this fraction, which may prove to be the blood-borne HIT molecule.     

Isolation and characterization of a 22 kDa protein with antifungal properties from maize seeds.

We have purified a 22 kDa protein from maize seeds to homogeneity by ammonium sulfate precipitation, chitin extraction and Mono-S column chromatography. The purified protein inhibited the growth of the agronomically important pathogens of potato wilt (Fusarium oxysporum) and tomato early blight (Alternaria solani). Sequence analysis of the purified protein showed that it has 52% homology with the sweet protein thaumatin (Edens, L., Hselinga, L., Klok, R., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. (1982) Gene 18, 1-12), 57% homology with the pathogenesis-related protein (Cornelissen, B. J. C., Huijsduijnen, R. A. M., and Bol, J. F. (1986) Nature 321, 531-532) and 99% homology with the 22 kDa trypsin/alpha-amylase inhibitor (Richardson, M., Valdes-Rodriguez, S., and Blanco-Labra, A. (1987) Nature 327, 432-434).     

Isolation, characterization and expression of a cDNA coding for a jasmonate-inducible protein of 37 kDa in barley leaves

In barley leaves, there is a dramatic alteration of gene expression upon treatment with jasmonates leading to the accumulation of newly formed proteins, designated as jasmonate-inducible proteins (JIPs). In the present study, a new jasmonate-inducible cDNA, designated pHvJS37, has been isolated by differential screening of a γgt10 cDNA library constructed from mRNA of jasmonate-treated barley leaf segments. The open reading frame (ORF) encodes a 39-9 kDa polypeptide which cross-reacts with antibodies raised against the in vivo JIP-37. The hydropathic plot suggests that the protein is mainly hydrophilic, containing two hydrophilic domains near the C-terminus. Database searches did not show any sequence homology of pHv.JS37 to known sequences. Southern analysis revealed at least two genes coding for JIP-37 which map to the distal portion of the long arm of chromosome 3 and are closely related to genes coding for JIP-23. The expression pattern of the JIP-37 genes over time shows differential responses to jasmonate, abscisic acid (ABA), osmotic stress (such as sorbitol treatment) and desiccation stress. No expression was found under salt stress. From experiments using an inhibitor and intermediates of jasmonate synthesis such as α-linolenic acid and 12-oxophytodienoic acid, we hypothesize that there is a stress-induced lipid-based signalling pathway in which an endogenous rise of jasmonate switches on JIP-37 gene expression. Using immunocytochemical techniques, JIP-37 was found to be simultaneously located in the nucleus, the cytoplasm and the vacuoles.     

Evidence for an association between a 33 kDa extrinsic membrane protein,manganese and photosynthetic oxygen evolution. I. Correlation with the S2 multiline EPR signal

The removal of peripheral membrane proteins of a molecular mass of 17 and 23 kDa by washing of spinach Photosystem-II (PS II) membranes in 1 M salt between pH 4.5 and 6.5 produces a minimal loss of the S₁ → S₂ reaction, as seen by the multiline EPR signal for the S₂ state of the water-oxidizing complex, while reversibly inhibiting O₂ evolution. The multiline EPR signal simplifies from a ‘19-line’ spectrum to a ‘16-line’ spectrum, suggestive of partial uncoupling of a cluster of 3 or 4 to yield photo-oxidation of a binuclear Mn site. Alkaline salt washing progressively releases a 33 kDa peripheral protein between pH 6.5 and 9.5, in direct parallel with the loss of O₂ evolution and the S₂ multiline EPR signal. The 33 kDa protein can be partially removed (20%) at pH 8.0 prior to managanese release. Salt treatment releases four Mn ions between pH 8.0 and 9.5 with the first 2 or 3 Mn ions released cooperatively. A common binding site is thus suggested in agreement with earlier EPR spectroscopic data establishing a tetranuclear Mn site. At least two of these Mn ions bind directly at a site in the PS II complex for which photooxidation by the reaction center is controlled by the 33 kDa protein. The washing of PS II membranes with 1 M CaCl₂ to affect the release of the 33 kDa protein, while preserving Mn binding to the membrane (Ono, T.-A. and Inoue, Y. (1983) FEBS Lett. 164, 255–260), is found to leave some 33 kDa protein undissociated in proportion to the extent of O₂ evolution and S₂ multiline yield. These depleted membranes do not oxidize water or produce the normal S₂ state without the binding of the 33 kDa protein. A method for the accurate determination of relative concentrations of the peripheral membrane proteins using gel electrophoresis is presented.     

Calcium and photosynthetic oxygen evolution in cyanobacteria

Calcium activation of oxygen evolution from French-press preparations of Phormidium luridum is largely reversible upon removal of added Ca²⁺. Activation occurs via a first-order binding with a dissociation constant of 2.8 mM. An 8-fold increase in oxygen evolution rate observed upon Ca²⁺ addition is accounted for by a 4-fold increase in the number of active photosynthetic units, and a doubling of turnover rate. While both Ca²⁺ and Mg²⁺ stimulate turnover, unit activation is Ca²⁺ specific. Under optimal conditions, 30% of the units functioning in the intact cell can be recovered in the Ca²⁺-activated preparation.

The Ca²⁺ requirement of P. luridum preparations is not relieved by proton-carrying uncouplers, or by rate-saturating concentrations of the Hill acceptor, ferricyanide. Taken together with the reported stimulation by Ca²⁺ of oxygen evolution in the presence of DCMU (Piccioni, R.G. and Mauzerall, D.C. (1976) Biochim. Biophys. Acta 423, 605–609) these observations strongly suggest a site of Ca²⁺ action within Photosystem II.

The pronounced specificity of the Ca²⁺ requirement appears in preparations of other cyanobacteria (Anabaena flos-aquae and Anacystis nidulans) but not in the eucaryote Chlorella vulgaris. While milder cell-disruption methods bring about some Ca²⁺ dependence in P. luridum, French-press treatment is required for maximal expression of Ca²⁺-specific effects. French-press breakage causes a release of endogenous Ca²⁺ from cells, supporting the view that added Ca²⁺ restores oxygen evolution by satisfying a physiological requirement for the cation.     

Measurement of photosynthetic oxygen evolution with a new type of oxygen sensor

We measured the light response curve of photosynthetic oxygen evolution by illuminating a leaf disc in an air-tight windowed chamber. Oxygen production was measured by monitoring the quenching of luminescence of an organometallic ruthenium compound. A photodiode based chlorophyll a fluorometer was used to measure the luminescence intensity. Oxygen evolution measurements with a traditional oxygen electrode gave the same numerical values at different light intensities when the same leaf disk was tested. The quality of the measurement signal of the new method was found to be similar to that obtained with the oxygen electrode method. The new luminescence based system is more stable against electrical disturbances than an oxygen electrode, its response to oxygen pressure changes is very rapid, and the new method allows the same basic equipment to be used for chlorophyll fluorescence and oxygen measurements.     

Time-resolved infrared detection of the proton and protein dynamics during photosynthetic oxygen evolution

Photosynthetic oxygen evolution by plants and cyanobacteria is performed by water oxidation at the Mn(4)CaO(5) cluster in photosystem II. The reaction is known to proceed via a light-driven cycle of five intermediates called S(i) states (i = 0-4). However, the detailed reaction processes during the intermediate transitions remain unresolved. In this study, we have directly detected the proton and protein dynamics during the oxygen-evolving reactions using time-resolved infrared spectroscopy. The time courses of the absorption changes at 1400 and 2500 cm(-1), which represent the reactions and/or interaction changes of carboxylate groups and the changes in proton polarizability of strong hydrogen bonds, respectively, were monitored upon flash illumination. The results provided experimental evidence that during the S(3) → S(0) transition, drastic proton rearrangement, most likely reflecting the release of a proton from the catalytic site, takes place to form a transient state before the oxidation of the Mn(4)CaO(5) cluster that leads to O(2) formation. Early proton movement was also detected during the S(2) → S(3) transition. These observations reveal the common mechanism in which proton release facilitates the transfer of an electron from the Mn(4)CaO(5) cluster in the S(2) and S(3) states that already accumulate oxidizing equivalents. In addition, relatively slow rearrangement of carboxylate groups was detected in the S(0) → S(1) transition, which could contribute to the stabilization of the S(1) state. This study demonstrates that time-resolved infrared detection is a powerful method for elucidating the detailed molecular mechanism of photosynthetic oxygen evolution by pursuing the reactions of substrate and amino acid residues during the S-state transitions.     

Isolation and partial characterization of a 39 kDa major outer membrane protein of Actinobacillus actinomycetemcomitans Y4

The outer membrane fractions of Actinobacillus actinomycetemcomitans, which were extracted from whole cells with cetyl trimethyl ammonium bromide and CaCl2, contained four major outer membrane proteins (MOMP) of 39, 37, 36 and 30 kDa. The 39 kDa MOMP of A. actinomycetemcomitans was sequentially purified by extraction with Zwittergent 3-14 detergent, anion-exchange chromatography and gel filtration chromatography. Analysis of amino acid composition and N-terminal amino acid sequence of 20 residues of purified 39 kDa MOMP was performed. Although some of the periodontitis patient sera reacted strongly with 39 kDa and 30 kDa MOMP in crude outer membrane fractions, purified 39 kDa MOMP showed decreased immunoreactivity with the human sera.     

Isolation and characterization of viridin, a new 65 kDa antifungal protein from the mould Trichoderma viride

A new extracellular antifungal protein with a yield of 10 mg per liter was isolated from the culture medium of the mould Trichoderma viride. The protein, which we named viridin, was purified by carboxymethyl-cellulose cation-exchange chromatography and Superose 12 HR 10/30 high-performance liquid chromatography. Viridin, a basic protein of approximately 65 kDa as determined by SDS-PAGE, inhibits the growth of the cotton pathogen Verticillum dahliae, the IC50 being 6 microM.     

Calcium and photosynthetic oxygen evolution in cyanobacteria.

Calcium activation of oxygen evolution from French-press preparations of Phormidium luridum is largely reversible upon removal of added Ca(2+). Activation occurs via a first-order binding with a dissociation constant of 2.8 mM. An 8-fold increase in oxygen evolution rate observed upon Ca(2+) addition is accounted for by a 4-fold increase in the number of active photosynthetic units, and a doubling of turnover rate. While both Ca(2+) and Mg(2+) stimulate turnover, unit activation is Ca(2+) specific. Under optimal conditions, 30% of the units functioning in the intact cell can be recovered in the Ca(2+) -activated preparation. The Ca(2+) requirement of P. luridum preparations is not relieved by proton-carrying uncouplers, or by rate-saturating concentrations of the Hill acceptor, ferricyanide. Taken together with the reported stimulation by Ca(2+) of oxygen evolution in the presence of DCMU (Piccioni, R.G. and Mauzerall, D.C. (1976) Biochim. Biophys. Acta 423, 605--609) these observations strongly suggest a site of Ca(2+) action within Photosystem II. The pronounced specificity of the Ca(2+) requirement appears in preparations of other cyanobacteria (Anabaena flos-aquae and Anacystis nidulans) but not in the eucaryote Chlorella vulgaris. While milder cell-disruption methods bring about some Ca(2+) dependence in P. luridum, French-press treatment is required for maximal expression of Ca(2+) -specific effects. French-press breakage causes a release of endogenous Ca(2+) from cells, supporting the view that added Ca(2+) restores oxygen evolution by satisfying a physiological requirement for the cation.     

Isolation and characterization of the 32.5 kDa protein from the venom of an endoparasitic wasp

The major venom proteins from the endoparasitic wasp were analyzed for distribution in the venom gland. A 32.5 kDa protein was purified from the venom gland of the Chelonus near curvimaculatus wasp. The protein accounts for about 25% of the total protein content of the venom and each gland contains 3-6 pmol of this component. The protein is acidic in nature and anion-exchange chromatography facilitated the purification of the protein to apparent homogeneity. On testing the purified protein by in vivo bioassay, it was found to elicit an effect comparable with the complete venom. The protein does not appear to have any disulfide bonds of major structural importance exposed under SDS-denaturing conditions. Products of chemical partial digest of the purified protein at the methionyl residues by cyanogen bromide were analyzed by SDS-PAGE. The 27.6 kDa fragment retained an epitope to an antibody raised against total Chelonus venom proteins, whereas no epitopes were detected for 4.9 and 0.6 kDa fragments.     

Structural predictions on the 33 kDa extrinsic protein associated to the oxygen evolving complex of photosynthetic organisms

Modern computational methods for protein structure prediction have been used to study the structure of the 33 kDa extrinsic membrane protein, associated to the oxygen evolving complex of photosynthetic organisms. A multiple alignment of 14 sequences of this protein from cyanobacteria, algae and plants is presented. The alignment allows the identification of fully conserved residues and the recognition of one deletion and one insertion present in the plant sequences but not in cyanobacteria. A tree of similarity, deduced from pair-wise comparison and cluster analysis of the sequences, is also presented. The alignment and the consensus sequence derived are used for prediction the secondary structure of the protein. This prediction indicates that it is a mainly-beta protein (25–38% of -strands) with no more than 4% of -helix. Fold recognition by threading is applied to obtain a topological 2D model of the protein. In this model the secondary structure elements are located, including several highly conserved loops. Some of these conserved loops are suggested to be important for the binding of the 33 kDa protein to Photosystem II and for the stability of the manganese cluster. These structural predictions are in good agreement with experimental data reported by several authors.     

Bicarbonate ion as a critical factor in photosynthetic oxygen evolution

Bicarbonate ion, not dissolved CO₂ gas, is shown to increase 4- to 5-fold the rate of dichlorophenol indophenol reduction by isolated maize (Zea mays) chloroplasts. Glutaraldehyde fixed chloroplasts continue to exhibit bicarbonate-dependent 2,6-dichlorophenol indophenol reduction. Bicarbonate is shown to act close to the oxygen-evolving site, i.e. prior to the electron donation site of diphenyl carbazide to photosystem II. Dark incubation and light pretreatment of chloroplasts in various concentrations of bicarbonate, just prior to assay, indicate that bicarbonate binds to chloroplasts in the dark and is released again as the Hill reaction proceeds in the light. It is suggested that bicarbonate ions may play a critical role in the oxygen-evolving process in photosynthesis.     

Isolation and partial characterization of a 30 kDa adhesin from Paracoccidioides brasiliensis

The virulence of Paracoccidioides brasiliensis can be attenuated or lost after long periods of repeated subculturing and reestablished after animal inoculation. Only one adhesin (gp43) has been described until now, among the various identified components of P. brasiliensis, and gp43 shows adhesion to laminin. Thus, the present study was designed to isolate and characterize factors putatively related to the capacity of this fungus to adhere to the host by comparing P. brasiliensis samples, taken before and after animal inoculation. The two samples differed in their pattern of adhesion and invasion. The sample recently isolated from animals (Pb18b) demonstrated a greater capacity to adhere and to invade the Vero cells than the one subcultured in vitro (Pb18a). Extract from Pb18b also showed higher levels of protein expression than that from Pb18a, when two-dimensional electrophoresis gels were compared. A protein species of 30 kDa, pI 4.9, was more evident in the Pb18b extract and had properties of adhesin. Laminin, but none of the other extracellular matrix (ECM) components, such as fibronectin, collagen I and IV, bound specifically to the P. brasiliensis 30 kDa protein. The roles of 30 kDa and gp43 in cellular interactions were investigated and the adhesion of P. brasiliensis yeast cells was intensively inhibited by pre-treatment of epithelial cells with 30 kDa protein and gp43. Thus, this study presents evidence that adhesion capacity could be related to virulence, and that a 30 kDa adhesin accumulated differentially in samples with different levels of pathogenicity. This protein and its adhesion characteristics are being published for the first time and may be related to the virulence of P. brasiliensis.