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.     

Isolation and characterization of photosystem II core complexes with high oxygen evolution activity from spinach using the non-ionic detergent 6-O-(N-heptylcarbamoyl)-methyl-<Emphasis Type="Italic">α</Emphasis>-D-glucopyranoside

Two different kinds of oxygen evolving photosystem II (PSII) core complexes were isolated in the present study by solubilization of PSII enriched thylakoid membranes from spinach with the non-ionic detergent 6-O-(N-heptylcarbamoyl)-methyl-_α-D-glucopyranoside (Hecameg) under different conditions. The PSII core complex isolated at higher ionic strength was similar to that isolated by using octyl-β-D-glucopyranoside (OGP) and lacked the 23 and 17 kDa extrinsic proteins of the oxygen evolving complex but retained the 22 kDa PsbS protein. Solubilization of the PSII membranes with Hecameg at lower ionic strength allowed the isolation of another PSII complex that retained all the three extrinsic proteins (33, 23 and 17 kDa) of the oxygen evolving complex but was depleted of the 22 kDa PsbS protein. This complex exhibited high rates of oxygen evolution and was found to be more sensitive to DCMU indicating a better structural and functional integrity and may be treated as the minimal functional unit required for PSII photochemistry. The detergent Hecameg is relatively inexpensive and the methodology remains simple since it does not require any chromatography or density gradient ultracentrifugation.     

EPR studies on the photosystem II donor side in salt-washed and reconstituted inside-out thylakoids

EPR measurements on inside-out thylakoids revealed that salt-washing, known to inhibit oxygen evolution and release a 23 and a 16 kDa protein, induced a Signal II_f and decreased the EPR signal from state S₂. Readdition of the released 23 kDa protein restored the oxygen evolution and decreased the Signal II_f, but did not relieve the decrease in the state S₂ signal. It is suggested that salt-washing inhibits the electron transfer from the oxygen-evolving site to Z, the physiological donor to P680. In inhibited photosystem II units lacking Signal II_f, Z⁺ is rapidly reduced, possibly by a modified S-cycle unable to evolve oxygen.     

Reversible alteration of nanosecond reduction of chlorophyll a+II in inside-out thylakoids correlated to inhibition and reconstitution of oxygen-evolving activity

The electron donation to Chl a⁺_II has been studied by measurement of absorbance changes at 824 nm under repetitive excitation conditions. For untreated inside-out thylakoids the electron donation was dominated by 35 and 220 ns kinetics. After salt-washing, both oxygen-evolution and nanosecond phases decreased drastically with corresponding increase in the microsecond time range. On addition of a purified 23 kDa protein, a restoration of the nanosecond phases up to 75% of the orginal level was obtained concomitant with a corresponding restoration of oxygen evolution. The results are consistent with a function of the 23 kDa protein at the oxidizing side of Photosystem II and that the nanosecond donation to Chl-a⁺_II is coupled to the natural path of electrons from water.     

Studies on oxygen evolution of inside-out thylakoid vesicles from mangroves: Chloride requirement, pH dependence and polypeptide composition

Inside-out thylakoid vesicles from the halophyte Avicennia marina were isolated by the aqueous polymer phase partition method. Oxygen-evolution activity measured with ferricyanide and phenyl-p-benzoquinone was absolutely dependent on added chloride, since the vesicles were almost completely depleted of the 23 and 16 kDa polypeptides of the O₂-evolving complex. Addition of the spinach 23 kDa protein to the mangrove inside-out vesicles lowered their chloride requirement for O₂ evolution at least 50-fold. In the absence of added chloride, the mangrove vesicles were very sensitive to inhibition by light, which could be prevented by high chloride or low chloride plus added purified spinach 23 kDa protein. The preparations were also inactivated by neutral or alkaline pH (greater than 7.2) in the absence of high chloride concentrations. This inactivation was not significantly influenced by addition of spinach 23 kDa protein. Chloride binding and alkaline inhibition may therefore be closely related, either directly via the manganese centers or, more likely, via pK_a changes in as yet unidentified proteins.     

Reconstitution of oxygen evolution in high salt washed photosystem II particles

Photosystem II thylakoid particles possessing high rates of oxygen evolution, were shown to have a very simple polypeptide composition. Upon washing of these particles with 250 mM NaCl the oxygen evolution was inhibited up to 80% concomitant with a release of two polypeptides of 23 and 16 kDa. Readdition of the pure 23 kDa protein to the depleted thylakoids under low ionic strength reconstituted more than half of the lost activity. No stimulation was obtained with the 16 kDa protein alone or in combination with glycerol. The results give further strong evidence that the 23 kDa protein is an essential component in the oxygen evolving complex. The possible involvement of other proteins in this complex is discussed in light of the demonstrated simple polypeptide pattern of the photosystem II particles.     

Cross-reconstitution of various extrinsic proteins and photosystem II complexes from cyanobacteria, red algae and higher plants

Photosystem II (PSII) contains different extrinsic proteins required for oxygen evolution among different organisms. Cyanobacterial PSII contains the 33 kDa, 12 kDa proteins and cytochrome (cyt) c-550; red algal PSII contains a 20 kDa protein in addition to the three homologous cyanobacterial proteins; whereas higher plant PSII contains the 33 kDa, 23 kDa and 17 kDa proteins. In order to understand the binding and functional properties of these proteins, we performed cross-reconstitution experiments with combinations of PSII and extrinsic proteins from three different sources: higher plant (spinach), red alga (Cyanidium caldarium) and cyanobacterium (Synechococcus vulcanus). Among all of the extrinsic proteins, the 33 kDa protein is common to all of the organisms and is totally exchangeable in binding to PSII from any of the three organisms. Oxygen evolution of higher plant and red algal PSII was restored to a more or less similar level by binding of any one of the three 33 kDa proteins, whereas oxygen evolution of cyanobacterial PSII was restored to a larger extent with its own 33 kDa protein than with the 33 kDa protein from other sources. In addition to the 33 kDa protein, the red algal 20 kDa, 12 kDa proteins and cyt c-550 were able to bind to cyanobacterial and higher plant PSII, leading to a partial restoration of oxygen evolution in both organisms. The cyanobacterial 12 kDa protein and cyt c-550 partially bound to the red algal PSII, but this binding did not restore oxygen evolution. The higher plant 23 kDa and 17 kDa proteins bound to the cyanobacterial and red algal PSII only through non-specific interactions. Thus, only the red algal extrinsic proteins are partially functional in both the cyanobacterial and higher plant PSII, which implies a possible intermediate position of the red algal PSII during its evolution from cyanobacteria to higher plants.     

Cloning and sequencing of the 23 kDa mouse photoreceptor cell-specific protein.

The 23 kDa protein was localized by immunocytochemistry to photoreceptor cells of the mouse retina, and bovine and mouse cDNA clones were isolated and sequenced. The deduced amino acid sequences showed that the mouse 23 kDa protein is 91% identical to the bovine protein, and is the same as S-modulin, the CAR (cancer-associated retinopathy) protein and recoverin, the Ca(2+)-dependent activator of photoreceptor guanylate cyclase. The amino acid sequence reveals two Ca2+ binding sites, no internal repeats, 59% homology to the chicken visinin protein and 40% homology to calmodulin while Northern analysis demonstrated a single 1.0 kb mRNA species in bovine and mouse retina.     

A receptor for protein import into potato mitochondria

Five potential surface receptors for protein import into plant mitochondria were identified by gentle trypsin treatment of intact mitochondria from potato tubers and subsequent preparation of outer mitochondrial membranes. One of them, a 23 kDa protein, was purified to homogeneity and analysed by direct protein sequencing. Copy DNA clones encoding the corresponding polypeptide were isolated with labelled oligonucleotides derived from the amino acid data. The 23 kDa protein shares significant sequence similarity with protein import receptors from fungal mitochondria and contains one of their typical tetratricopeptide motifs. Its integration into the outer membrane is independent of protease accessible surface receptors and not accompanied by proteolytic processing. Monospecific antibodies against the 23 kDa protein significantly reduce import capacity of isolated mitochondria indicating that this component is indeed involved in the recognition or import of precursor proteins. As in fungi, immunological inhibition of protein import with IgGs against a single receptor is incomplete suggesting the existence of other receptors in the outer mitochondrial membrane of plant mitochondria.     

A highly thermostable endo-(1,4)-β-mannanase from the marine bacterium Rhodothermus marinus

Rhodothermus marinus ATCC 43812, a thermophilic bacterium isolated from marine hot springs, possesses hydrolytic activities for depolymerising substrates such as carob-galactomannan. Screening of expression libraries identified mannanase-positive clones. Subsequently, the corresponding DNA sequences were determined, eventually identifying a coding sequence specifying a 997 amino acid residue protein of 113 kDa. Analyses revealed an N-terminal domain of unknown function and a C-terminal mannanase domain of 550 amino acid residues with homology to known mannanases of glycosidase family 26. Action pattern analysis categorised the R. marinus mannanase as an endo-acting enzyme with a requirement for at least five sugar moieties for effective catalytic activity. When expressed in Escherichia coli, purified gene product with catalytic activity was mainly found as two protein fragments of 45 kDa and 50 kDa. The full-length protein of 113 kDa was only detected in crude extracts of R. marinus, while truncated protein-containing fractions of the original source resulted in a major active protein of 60 kDa. Biochemical analysis of the mannanase revealed a temperature and pH optimum of 85 °C and pH 5.4, respectively. Purified, E. coli-produced protein fragments showed high heat stability, retaining more than 70% and 25% of the initial activity after 1 h incubation at 70 °C and 90 °C, respectively. In contrast, R. marinus-derived protein retained 87% activity after 1 h at 90 °C. The enzyme hydrolysed carob-galactomannan (locust bean gum) effectively and to a smaller extent guar gum, but not yeast mannan. Received: 5 November 1999 / Received revision: 19 January 2000 / Accepted: 23 January 2000     

Study on an antimicrobial protein produced by <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis> JSa-9 isolated from soil

Paenibacillus polymyxa strain JSa-9, a soil isolate showed in vitro inhibitory effects on several pathogens. A protein of about 71.9 kDa, isolated from a culture of JSa-9, exhibited broad-spectrum antibacterial and antifungal activities. The purification procedure consisted of ion-exchange chromatography on DEAE-Sephacel and Sephadex G-100 column chromatography. The purified protein was characterized to be a glycoprotein, which contained 53% of amino acids and 28% of carbohydrate. Its N-terminal sequence was determined as KCATIPVVIKHL and the amino acid composition showed no significant homology with any known antagonistic proteins from P. polymyxa. Because of the excellent antimicrobial activity, this protein may be a good prospect for food preservation and plant disease control.     

Cleavage of the PO glycoprotein of the rat peripheral nerve myelin: Tentative identification of cleavage site and evidence for the precursor-product relationship

The incubation of sciatic nerve slices in Krebs Ringer bicarbonate (KRB) buffer (pH 7.4) at 37°C, or the incubation of freshly isolated myelin in ammonium bicarbonate buffer (pH 8), resulted in the generation of a 24kDa protein with a concomitant decrease of PO protein. The conversion of PO into 24kDa protein was blocked by heating isolated myelin at 100°C for 5 min suggesting that the reaction is enzyme mediated. Inclusion of the protease inhibitors and chelating agent to isolated myelin did not prevent the formation of 24kDa protein. Similarly, addition of CaCl₂ to isolated myelin did not accentuate the formation of 24kDa protein suggesting that the conversion of PO into 24kDa protein may not be due to Ca²⁺ activated protease. It is postulated that the formation of 24kDa protein may be due to neutral protease and/or metalloproteinase associated with the PNS myelin. 24kDa protein was purified and characterized. The N-terminal sequence of 1–17 amino acid residues of 24kDa protein was identical to PO. 24kDa protein was immunostained and immunoprecipitated with anti-PO antiserum indicating the immunological similarities between PO and 24kDa protein. Labeling of 24kDa protein with [³⁵S]methionine provided evidence that PO may be in all probability cleaved between Met-168 and Met-193. Further studies were carried out to demonstrate that 24kDa protein was phosphorylated, glycosylated and acylated like PO. Phosphorylation of 24kDa protein in the nerve slices was increased five-fold by phorbol esters and phosphoserine was the only phosphoamino acid identified after partial acid hydrolysis of 24kDa protein. These results suggested that serine residue phosphorylated by protein kinase C may be located in amino acid residues 1-168. 24kDa protein was stained with periodic Schiff reagent. In addition, 24kDa protein was fucosylated and the fucosylation of 24kDa protein was inhibited (70%) by tunicamycin, providing evidence that it is N-glycosylated. Recently, it was demonstrated that both PO and 24kDa protein were fatty acylated with [³H]palmitic acid in the nerve slices and fatty acids are covalently linked to these proteins (Agrawal, H.C. and Agrawal, D. 1989, Biochem. J. 263:173–177). The time course of inhibition of acylation by cycloheximide of 24kDa protein was identical to PO. Cycloheximide inhibited acylation of PO and 24kDa protein by 61% and 58% respectively, whereas, monensin had little affect on the fatty acylation of these proteins. Less [³H]palmitic acid and¹⁴C-amino acids were incorporated into 24kDa protein when compared to PO between 5–30 min after incubation of the nerve slices. However, more radioactivity was incorporated into 24kDa protein after 60 min when compared to PO under identical conditions. These results provided evidence of a precursor-product relationship between PO and 24kDa protein. Therefore, PO may be cleaved into 24kDa protein in the myelin membrane following its acylation and glycosylation in the Schwann cells.     

Structure and expression of the gene encoding ribosomal protein S1 from the cyanobacterium Synechococcus sp. strain PCC 6301: striking sequence similarity to the chloroplast ribosomal protein CS1

We isolated a 38 kDa ssDNA-binding protein from the unicellular cyanobacterium Synechococcus sp. strain PCC 6301 and determined its N-terminal amino acid sequence. A genomic clone encoding the 38 kDa protein was isolated by using a degenerate oligonucleotide probe based on the amino acid sequence. The nucleotide sequence and predicted amino acid sequence revealed that the 38 kDa protein is 306 amino acids long and homologous to the nuclear-encoded 370 amino acid chloroplast ribosomal protein CS1 of spinach (48% identity), therefore identifying it as ribosomal protein (r-protein) S1. Cyanobacterial and chloroplast S1 proteins differ in size from Escherichia coli r-protein S1 (557 amino acids). This provides an additional evidence that cyanobacteria are closely related to chloroplasts. The Synechococcus gene rps1 encoding S1 is located 1.1 kb downstream from psbB, which encodes the photosystem 11 P680 chlorophyll a apoprotein. An open reading frame encoding a potential protein of 168 amino acids is present between psbB and rps1 and its deduced amino acid sequence is similar to that of E. coli hypothetical 17.2 kDa protein. Northern blot analysis showed that rps1 is transcribed as a monocistronic mRNA.     

Cloning and expression of a toxin gene from Pseudomonas fluorescens GcM5-1A

Pseudomonas fluorescens GcM5-1A was isolated from the pine wood nematode (PWN), Bursaphelenchus xylophilus, obtained from wilted Japanese black pine, Pinus thumbergii, in China. In this paper, a genomic library of the GcM5-1A strain was constructed and a toxin–producing clone was isolated by bioassay. Nucleotide sequence analysis revealed an open reading frame of 1,290 bp encoding a protein of 429 amino acids with N-terminal putative signal peptide of 36 amino acids, which shared a similarity of 83, 82 and 80% identity with hypothetical protein PFLU2919 from P. fluorescens SBW25, Dyp-type peroxidase family protein from P. fluorescens Pf-5 and Tat-translocated enzyme from P. fluorescens Pf0-1, respectively. The gene encoding a full-length protein or without the putative signal peptide was cloned and expressed as a soluble protein in E. coli. The recombinant protein was purified to electrophoretic homogeneity by affinity chromatography using a Ni²⁺ matrix column. Its relative molecular weight was estimated to be 48.5 kDa by SDS-PAGE for full-length protein, and 45.0 kDa for the recombinant protein without putative signal peptide. Bioassay results showed that the recombinant protein with or without the putative signal peptide was toxic to both suspension cells and P. thunbergii seedlings. HPLC analysis demonstrated that components in branch extracts of P. thunbergii were significantly changed after addition of the recombinant full-length protein and hydrogen peroxide, which indicated that it is probably a peroxidase. This study offers information that can be used to determine the mechanism of pine wilt disease caused by the PWN.     

Structure and expression of the gene encoding ribosomal protein S1 from the cyanobacterium Synechococcus sp. strain PCC 6301: striking sequence similarity to the chloroplast ribosomal protein CS1

We isolated a 38 kDa ssDNA-binding protein from the unicellular cyanobacterium Synechococcus sp. strain PCC 6301 and determined its N-terminal amino acid sequence. A genomic clone encoding the 38 kDa protein was isolated by using a degenerate oligonucleotide probe based on the amino acid sequence. The nucleotide sequence and predicted amino acid sequence revealed that the 38 kDa protein is 306 amino acids long and homologous to the nuclear-encoded 370 amino acid chloroplast ribosomal protein CS1 of spinach (48% identity), therefore identifying it as ribosomal protein (r-protein) S1. Cyanobacterial and chloroplast S1 proteins differ in size from Escherichia coli r-protein S1 (557 amino acids). This provides an additional evidence that cyanobacteria are closely related to chloroplasts. The Synechococcus gene rps1 encoding S1 is located 1.1 kb downstream from psbB, which encodes the photosystem 11 P680 chlorophyll a apoprotein. An open reading frame encoding a potential protein of 168 amino acids is present between psbB and rps1 and its deduced amino acid sequence is similar to that of E. coli hypothetical 17.2 kDa protein. Northern blot analysis showed that rps1 is transcribed as a monocistronic mRNA.     

Heat inactivation of oxygen evolution in Photosystem II particles and its acceleration by chloride depletion and exogenous manganese

Heat inactivation of oxygen evolution by isolated Photosystem II particles was accelerated by Cl⁻ depletion and exogenous Mn²⁺. Weak red light also accelerated heat inactivation. Heat treatment released the 33, 24 and 18 kDa proteins and Mn from the Photosystem II particles. The protein release was stimulated by Cl⁻ depletion and exogenous Mn²⁺, and the Mn release was also stimulated by Cl⁻ depletion. A 50% loss of Mn corresponded to full inactivation of oxygen evolution, whereas no direct correlation seemed to exist between the loss of any one protein and inactivation of oxygen evolution. Removal of the 24 and 18 kDa proteins from photosystem II particles only slightly decreased the heat stability of oxygen evolution.     

Cloning and expression of the bioluminescent photoprotein pholasin from the bivalve mollusc Pholas dactylus

Pholasin is the photoprotein responsible for luminescence in the bivalve Pholas dactylus and consists of a luciferin tightly bound to a glycosylated protein. It is a sensitive indicator of reactive oxygen species. A full-length clone encoding apopholasin was isolated from a P. dactylus light organ cDNA library. The unprocessed apoprotein contained 225 amino acids, starting with a signal peptide of 20 amino acids, 3 predicted N-linked glycosylation sites, 1 O-linked site, no histidines, and 7 cysteines. The recombinant apoprotein was expressed in cell extracts and insect cells. The size of the apoprotein expressed in cell extracts and the cytosol of insect cells was 26 kDa but that of the fully processed protein was 34 kDa, as was native pholasin. Both the processed and unprocessed recombinant apoproteins were recognized by a polyclonal antibody raised against native pholasin. Acid methanol extracts from Pholas added to recombinant apoprotein resulted in chemiluminescence triggered by sodium hypochlorite but not photoprotein formation. These results have important implications in understanding the molecular evolution of bioluminescence and will allow the development of recombinant pholasin as an intracellular indicator of reactive oxygen species.     

Erythrocyte Mr 28,000 transmembrane protein exists as a multisubunit oligomer similar to channel proteins

A novel Mr 28,000 erythrocyte transmembrane protein was recently purified and found to exist in two forms, "28kDa" and "gly28kDa," the latter containing N-linked carbohydrate (Denker, B. M., Smith, B. L., Kuhajda, F. P., and Agre, P. (1988) J. Biol. Chem. 263, 15634-15642). Although 28kDa protein resembles the Rh polypeptides biochemically, structural homologies were not identified by immunoblot or two-dimensional iodopeptide maps. The NH2-terminal amino acid sequence for the first 35 residues of purified 28kDa protein is 37% identical to the 26-kDa major intrinsic protein of lens (Gorin, M. B., Yancey, S. B., Cline, J., Revel, J.-P., and Horwitz, J. Cell 39, 49-59). Antisera to a synthetic peptide corresponding to the NH2-terminus of 28kDa protein gave a single reaction of molecular mass 28kDa on immunoblots of erythrocyte membranes. Selective digestions of intact erythrocytes and inside-out membrane vesicles with carboxypeptidase Y indicated the existence of a 5-kDa COOH-terminal cytoplasmic domain. Multiple studies indicated that 28kDa and gly28kDa proteins exist together as a multisubunit oligomer: 1) similar partial solubilizations in Triton X-100; 2) co-purification during ion exchange and lectin affinity chromatography; 3) cross-linking in low concentrations of glutaraldehyde; and 4) physical analyses of purified proteins and solubilized membranes in 1% (v/v) Triton X-100 showed 28kDa and gly28kDa proteins behave as a large single unit with Stokes radius of 61 A and sedimentation coefficient of 5.7 S. These studies indicate that the 28kDa and gly28kDa proteins are distinct from the Rh polypeptides and exist as a multisubunit oligomer. The 28kDa protein has NH2-terminal amino acid sequence homology and membrane organization similar to major intrinsic protein and other members of a newly recognized family of transmembrane channel proteins.     

枸杞子糖蛋白的分离纯化、物化性质及糖肽键特征

从宁夏枸杞子提取得到的粗多糖,经ＤＥＡＥ－Ｃｅｌｌｕｌｏｓｅ和ＳｅｐｈａｄｅｘＧ－１００柱层析,得到均一的枸杞子糖蛋白ＬｂＧＰ。分子量由ＳＤＳ－ＰＡＧＥ测定为８８ｋｄ,糖含量为７０％,糖组成为Ａｒａ：Ｇａｌ：Ｇｌｃ＝２．５：１．０：１．０（摩尔比）,并含有其他１８种天然氨基酸。初步分析表明ＬｂＧＰ是Ｏ－连接的糖蛋白。     

Effect of Irradiance on the Thermal Stability of Thylakoid Membrane Isolated from Acclimated Wheat Leaves

Thermal stability of thylakoid membranes isolated from acclimated and non-acclimated wheat (Triticum aestivum L. cv. HD 2329) leaves under irradiation was studied. Damage to the photosynthetic electron transport activity was more pronounced in thylakoid membranes isolated from non-acclimated leaves as compared to thylakoid membrane isolated from acclimated wheat leaves at 35 °C. The loss of D1 protein was faster in non-acclimated thylakoid membrane as compared to acclimated thylakoid membranes at 35 °C. However, the effect of elevated temperature on the 33 kDa protein associated with oxygen evolving complex in these two types of thylakoid membranes was minimal. Trypsin digestion of the 33 kDa protein in the thylakoid membranes isolated from control and acclimated seedlings suggested that re-organisation of 33 kDa protein occurs before its release during high temperature treatment.