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     

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.     

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.     

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.     

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.     

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.     

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.     

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 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.     

Differential expression of a gene for a methionine-rich storage protein in maize

Summary A methionine-rich 10 kDa zein storage protein from maize was isolated and the sequence of the N-terminal 30 amino acids was determined. Based on the amino acid sequence, two mixed oligonucleotides were synthesized and used to probe a maize endosperm cDNA library. A fulllength cDNA clone encoding the 10 kDa zein was isolated by this procedure. The nucleotide sequence of the cDNA clone predicts a polypeptide of 129 amino acids, preceded by a signal peptide of 21 amino acids. The predicted polypeptide is unique in its extremely high content of methionine (22.5%). The maize inbred line BSSS-53, which has increased seed methionine due to overproduction of this protein, was compared to W23, a standard inbred line. Northern blot analysis showed that the relative RNA levels for the 10 kDa zein were enhanced in developing seeds of BSSS-53, providing a molecular basis for the overproduction of the protein. Southern blot analysis indicated that there are one or two 10 kDa zein genes in the maize genome.     

Function of the 23 kDa extrinsic protein of Photosystem II as a manganese binding protein and its role in photoactivation

The function of the extrinsic 23 kDa protein of Photosystem II (PSII) was studied with respect to Mn binding and its ability to supply Mn to PSII during photoactivation, i.e. the light-dependent assembly of the tetramanganese cluster. The extrinsic proteins and the Mn cluster were removed by TRIS treatment from PSII-enriched membrane fragments and purified by anion exchange chromatography. Room temperature EPR spectra of the purified 23 kDa protein demonstrated the presence of Mn. Photoactivation was successful with low Mn concentrations when the 23 kDa protein was present, while in its absence a higher Mn concentration was needed to reach the same level of oxygen evolution activity. In addition, the rate of photoactivation was significantly accelerated in the presence of the 23 kDa protein. It is proposed that the 23 kDa protein plays an important role in providing Mn during the process of PSII assembly and that it acquires Mn during the light-induced turnover of D1 in the PSII damage-repair cycle and delivers Mn to repaired PSII.     

Function of the 23 kDa extrinsic protein of Photosystem II as a manganese binding protein and its role in photoactivation

The function of the extrinsic 23 kDa protein of Photosystem II (PSII) was studied with respect to Mn binding and its ability to supply Mn to PSII during photoactivation, i.e. the light-dependent assembly of the tetramanganese cluster. The extrinsic proteins and the Mn cluster were removed by TRIS treatment from PSII-enriched membrane fragments and purified by anion exchange chromatography. Room temperature EPR spectra of the purified 23 kDa protein demonstrated the presence of Mn. Photoactivation was successful with low Mn concentrations when the 23 kDa protein was present, while in its absence a higher Mn concentration was needed to reach the same level of oxygen evolution activity. In addition, the rate of photoactivation was significantly accelerated in the presence of the 23 kDa protein. It is proposed that the 23 kDa protein plays an important role in providing Mn during the process of PSII assembly and that it acquires Mn during the light-induced turnover of D1 in the PSII damage-repair cycle and delivers Mn to repaired PSII.     

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。     

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.     

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.     

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.     

Isolation of two isoforms of a 21,000-dalton Ca2+-binding protein of bovine brain

Using Ca2+-dependent hydrophobic interaction chromatography we have identified a novel bovine brain Ca2+-binding protein (CaBP) composed of 21 kDa and 23 kDa polypeptides. This calciprotein was further purified by heat-treatment in the presence of Ca2+ and ion-exchange chromatography. The isolated protein exhibits a number of properties in common with proteins belonging to the calmodulin family of CaBPs, including a Ca2+-dependent electrophoretic mobility shift on SDS-polyacrylamide gel electrophoresis, retention of the ability to bind 45Ca2+ after electrophoresis and Western blotting, and a high content of acidic amino acids. We have recently isolated and characterized a 21 kDa CaBP from bovine brain and conclude that the 21 kDa and 21/23 kDa CaBPs are isoforms since they have very similar U.V. absorption spectra and amino acid compositions, and polyclonal antibodies raised in rabbits against the 21 kDa CaBP cross-react to an identical degree with the 21/23 kDa CaBP as determined by the competitive enzyme-linked immunosorbent assay (ELISA). Both proteins contain carbohydrate, but they differ in the degree of glycosylation. Tissue distribution studies indicate the presence of both 21 kDa and 23 kDa Ca2+-binding polypeptides in bovine trachea, aorta, kidney, skeletal muscle and cardiac muscle, and chicken gizzard smooth muscle.     

Drosophila contains three genes that encode distinct isoforms of protein phosphatase 1

The sequences of two Drosophila and one rabbit protein phosphatase (PP) 1 catalytic subunits were determined from their cDNA. The sequence of Drosophila PP1 alpha 1 was deduced from a 2.2-kb cDNA purified from an embryonic cDNA library, while that for Drosophila PP1 beta was obtained from overlapping clones isolated from both a head cDNA library and an eye imaginal disc cDNA library. The gene for Drosophila PP1 alpha 1 is at 96A2-5 on chromosome 3 and encodes a protein of 327 amino acids with a calculated molecular mass of 37.3 kDa. The gene for Drosophila PP1 beta is localized at 9C1-2 on the X chromosome and encodes a protein of 330 amino acids with a predicted molecular mass of 37.8 kDa. PP1 alpha 1 shows 96% amino acid sequence identity to PP1 alpha 2 (302 amino acids), an isoform whose gene is located in the 87B6-12 region of chromosome 3 [Dombrádi, V., Axton, J. M., Glover, D.M. Cohen, P.T.W. (1989) Eur. J. Biochem. 183, 603-610]. PP1 beta shows 85% identity to PP1 alpha 1 and PP1 alpha 2 over the 302 homologous amino acids. These results demonstrate that at least three genes are present in Drosophila that encode different isoforms of PP1. Drosophila PP1 alpha 1 and PP1 beta show 89% amino acid sequence identity to rabbit PP1 alpha (330 amino acids) [Cohen, P.T.W. (1988) FEBS Lett. 232, 17-23] and PP1 beta (327 amino acids), respectively, demonstrating that the structures of both isoforms are among the most conserved proteins known throughout the evolution of the animal kingdom. The presence of characteristic structural differences between PP1 alpha and PP1 beta, which have been preserved from insects to mammals, implies that the alpha and beta isoforms may have distinct biological functions.     

The major soluble 19.6 kDa protein of the organic shell matrix of the freshwater snail Biomphalaria glabrata is an N-glycosylated dermatopontin

The major Biomphalaria glabrata shell matrix protein of 19.6 kDa was isolated by preparative electrophoresis and sequenced. The sequence of 148 amino acids showed 32% sequence identity to mammalian dermatopontin sequences and 34-37% identity to two invertebrate dermatopontins described previously. A unique feature of the shell matrix dermatopontin was the presence of a single N-glycosylation consensus sequence, the asparagine of which was completely modified with a pentasaccharide. Sequence analysis of this short N-glycan by mass spectrometry and carbohydrate composition analysis indicated that it was the ubiquitous N-glycan core oligosaccharide with the exception that the terminal mannoses were 3-O-methylated. Dermatopontin is widespread in mammalian extracellular matrices, including the matrix of biominerals such as bone and teeth. Its occurrence in an invertebrate biomineral indicates that such phylogenetically distant biomineral-forming systems as vertebrate bone and mollusk shell share components which have undergone surprisingly few changes during a long evolution.