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R Malkin

Publications and source records attributed to R Malkin.

At least 37 records · Page 2Linked to original sources
PubMed1990

Photosystem I reaction-centre proteins contain leucine zipper motifs. A proposed role in dimer formation.

The photosystem I (PS I) reaction-centre polypeptides, encoded by the psaA and psaB genes, are shown to contain several highly conserved leucine repeats, consisting of a leucine residue every seventh amino acid, similar to the leucine zipper motifs known to mediate DNA-binding polypeptide dimerisation. In each of the PSI reaction-centre subunits the leucine zipper motif precedes highly conserved cysteine residues which have been proposed to ligate the interpolypeptide [4Fe-4S] centre, Fx. We propose that PS I reaction-centre dimerisation and [4Fe-4S] centre formation are mediated through the leucine zipper.

Amino Acid Sequence↗
PubMed1989

Structural and functional properties of the cyanobacterial photosystem I complex.

Photosystem I (PSI) complexes have been isolated from two cyanobacterial strains, Synechococcus sp. PCC 7002 and 6301. These complexes contain six to seven low molecular mass subunits in addition to the two high molecular mass subunits previously shown to bind the primary reaction center components. Chemical cross-linking of ferredoxin to the complex identified a 17.5-kDa subunit as the ferredoxin-binding protein in the Synechococcus sp. PCC 6301-PSI complex. The amino acid sequence of this subunit, deduced from the DNA sequence of the gene, confirmed its identity as the psaD gene product. A 17-kDa subunit cross-links to the electron donor, cytochrome c-553, in a manner analogous to the cross-linking of plastocyanin to the higher plant PSI complex. Using antibodies raised against the spinach psaC gene product (a 9-kDa subunit which binds Fe-S centers A and B), we identified an analogous protein in the cyanobacterial PSI complex.

Amino Acid Sequence↗
PubMed1989

On the interaction between cytochrome f and plastocyanin.

The interaction between cytochrome f and its electron acceptor plastocyanin (PC) was studied. To address the question of which specific regions and which of the positively charged residues of cytochrome f are important for the interaction with the negatively charged residues of PC we have used two different experimental approaches. Cytochrome f was proteolytically cleaved and fragments that could bind to a PC-affinity column were isolated. The smallest of these fragments was analysed to give information on the minimum structural requirement for binding to PC. By this procedure, we identified a peptide of approx. 11 kDa, containing the heme binding site, and having an N-terminal sequence identical to that of the mature cytochrome f. This finding suggests that the first 90 amino acids of cytochrome f contain at least some of the residues interacting with PC. The second approach involved modification of Arg residues of cytochrome f with the specific chemical modifier, hydroxyphenylglyoxal (HPG). Cytochrome f modification was performed in the absence of PC to enable identification of residues that are protected from modification when PC is bound to cytochrome f. Two peptides containing Arg residues which are modified in the absence of PC, but are not modified when PC is present, were isolated. Sequence analysis of these two peptides revealed that Arg residues no. 88 and 154 of cytochrome f are the residues that are protected from modification when cytochrome f is bound to PC, suggesting a role for these residues in the binding of cytochrome f to PC.

Amino Acid Sequence↗
PubMed1989

Maize Photosystem I : Identification of the Subunit which Binds Plastocyanin.

Photosystem I (PSI) has been isolated from mesophyll chloroplasts of mature maize leaves. The isolated PSI (PSI-200) was used as starting material for preparing an antenna-depleted core (PSI-100). Both of these preparations appear to be quite analogous to PSI complexes isolated from other plant tissue sources, such as those from C3 plants, as judged from NADP photoreduction assays, immunoblotting, and the ability of the complexes to form a covalent crosslinked product with spinach plastocyanin. The study suggests that the PSI complex from a C4 plant is similar to that isolated from a C3 plant in that both contain the plastocyanin docking protein although the apparent molecular weight of these respective subunits differ slightly.

Journal Article↗
PubMed1988

Characterization of two operons encoding the cytochrome b6-f complex of the cyanobacterium Nostoc PCC 7906. Highly conserved sequences but different gene organization than in chloroplasts.

We have isolated and determined the nucleotide and derived protein sequences for the four genes, petCA and BD, which encode the cytochrome b6-f, electron-transfer complex of the filamentous cyanobacterium, Nostoc PCC 7906. The primary structure and cotranscription of the petCA genes encoding the Rieske-FeS (nuclear encoded in plants) and apocytochrome f proteins has been described previously (Kallas, T., Spiller, S., and Malkin, R. (1988) Proc. Natl. Acad. Sci. U.S.A., in press). The petBD genes (645 and 480 protein-coding nucleotides, respectively) for the apocytochrome b6 (24.3 kDa) and subunit-IV (17.5 kDa) proteins comprise a second operon located at least 12 kilobases (kb) from petCA. The Nostoc petBD genes are not closely linked to the psbB gene (encoding the 51-kDa photosystem II polypeptide) and do not contain introns as do the closely related chloroplast genes. DNA probes specific for each of the Nostoc cytochrome-complex genes hybridized to single bands in genomic DNA blots at intensities expected for single copy genes. These data suggest that a single set of cytochrome b6-f proteins function in the different types of membranes found in Nostoc vegetative and heterocyst cells. RNA blot hybridizations identified an 1.8-kb mRNA common to cytochrome b6 and subunit IV, and an intensely hybridizing 0.8-kb mRNA specific to the subunit IV gene probe. The role of the latter RNA is not clear but it may represent a transcript from the opposite strand. The deduced Rieske, apocytochrome f, apocytochrome b6, and subunit IV proteins exhibit 59, 58-63, 84-85, and 79-83% sequence identity with the proteins from chloroplast cytochrome b6-f complexes. The Nostoc proteins show lower but still significant sequences identity with the corresponding proteins of the mitochondrial-type b-c1 complexes. The four probable heme-liganding His residues, and the approximate spacings between them, have been conserved in all of the available cytochrome b6 and b sequences from divergent sources. The Nostoc apocytochrome b6 and subunit IV proteins, as well as the Rieske, appear to be translated and thus inserted into the membrane as mature forms without cleavable presequences. Hydropathy analyses revealed five potential membrane spans in cytochrome b6 and three in the subunit IV protein, consistent with the profiles observed for the chloroplast proteins and the related cytochrome b proteins of cytochrome b-c1 complexes.

Amino Acid Sequence↗
PubMed1988

Green algal cytochrome b6-f complexes: isolation and characterization from Dunaliella saline, Chlamydomonas reinhardtii and Scenedesmus obliquus.

Cytochrome b6-f complexes have been isolated from Chlamydomonas reinhardtii, Dunaliella saline and Scenedesmus obliquus. Each complex is essentially free of chlorophyll and carotenoids and contains cytochrome b6 and cytochrome f hemes in a 2:1 molar ratio. C. reinhardtii and S. obliquus complexes contain the Rieske iron-sulfur protein (present in approx 1:1 molar ratio to cytochrome f) and each catalyzes a DBMIB- and DNP-INT-sensitive electron transfer from duroquinol to spinach plastocyanin. Immunological assays using antibodies to the peptides from the spinach cytochrome complex show varying cross-reactivity patterns except for the complete absence of binding to the Rieske proteins in any of the three complexes, suggesting little structural similarity between the Rieske proteins of algae with those from higher plants. One complex (D. salina) has been uniformly labeled by growth in NaH14CO3 to determine stoichiometries of constituent polypeptide subunits. Results from these studies indicate that all functionally active cytochrome b6-f complexes contain four subunits which occur in equimolar amounts.

Chlamydomonas↗
PubMed1988

Interaction of plastocyanin with photosystem I: a chemical cross-linking study of the polypeptide that binds plastocyanin.

Plastocyanin has been covalently cross-linked to photosystem I (PSI) by using a water-soluble cross-linker, N-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The cross-linking reaction is light stimulated and results in the disappearance of a single 19-kDa subunit of PSI with the formation of a new protein-staining component of 31 kDa. The new product at 31 kDa reacts with both plastocyanin and 19-kDa subunit antibodies. Carboxyl group modified plastocyanin does not form a cross-linked product with PSI, implying that the negatively charged surface-exposed groups on plastocyanin are necessary to stabilize binding. These results demonstrate a specific interaction of plastocyanin with PSI and further implicate a specific protein to which plastocyanin binds to facilitate electron transfer to the P700 reaction center.

Chlorophyll↗
PubMed1988

Evidence for the presence of a [2Fe-2S] ferredoxin in bean sprouts.

An iron-sulfur protein with properties similar to those of ferredoxins found in the leaves of higher plants has been isolated from bean sprouts--a non-photosynthetic plant tissue. The bean sprout protein has a molecular mass of 12.5 kDa and appears to contain a single [2Fe-2S] cluster. The absorbance and circular dichroism spectra of the bean sprout protein resemble those of spinach leaf ferredoxin and the bean sprout protein can replace spinach ferredoxin as an electron donor for NADP+ reduction, nitrite reduction and thioredoxin reduction by spinach leaf enzymes. Although the reduced bean sprout protein (Em = -440 mV) is a slightly stronger reductant than spinach ferredoxin and appears to be less acidic than spinach ferredoxin, the two proteins are similar enough so that the bean sprout protein is recognized by an antibody raised against spinach ferredoxin.

Circular Dichroism↗
PubMed1988

Subunit stoichiometry of the chloroplast photosystem I complex.

A native photosystem I (PS I) complex and a PS I core complex depleted of antenna subunits has been isolated from the uniformly 14C-labeled aquatic higher plant, Lemna. These complexes have been analyzed for their subunit stoichiometry by quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis methods. The results for both preparations indicate that one copy of each high molecular mass subunit is present per PS I complex and that a single copy of most low molecular mass subunits is also present. These results suggest that iron-sulfur center X, an early PS I electron acceptor proposed to bind to the high molecular mass subunits, contains a single [4Fe-4S] cluster which is bound to a dimeric structure of high molecular mass subunits, each providing 2 cysteine residues to coordinate this cluster.

Carbon Radioisotopes↗
PubMed1988

Quinone interactions with the chloroplast cytochrome b6-f complex.

The requirements for reconstitution of electron transfer activity with a plastoquinone (PQ)-depleted cytochrome b6-f complex from spinach have been considered. Full restoration of activity measured as plastocyanin reduction with either duroquinol in the dark or Photosystem II (PSII) in the light requires both PQ-9 and phospholipid. However, a substantial dark activity can be observed with duroquinol and phospholipid in the absence of any added PQ-9. PSII, with its associated PQ molecules, can also donate electrons in the light to the cytochrome complex which has been depleted of plastoquinone. Electron donation by duroquinol in the dark to the PQ-depleted cytochrome complex is stimulated by PSII, and this stimulation is dependent on the presence of the two PQ molecules in the PSII preparation. Measurements of proton translocation with the PQ-depleted complex indicate this quinone is not required for the observed H+/e- ratio of 2. Studies of cytochrome b6 kinetics with the free and liposome-incorporated PQ-depleted complex show this cytochrome undergoes redox reactions similar to those of a control complex which contains PQ. These results indicate the PQ that copurifies with the cytochrome complex is not essential for any of the measured activities. These findings are considered in relation to a quinone binding site(s) in the cytochrome complex which is not specific to PQ but can bind other quinones, such as duroquinol, in a lipid-dependent process.

Binding Sites↗
PubMed1988

Primary structure of cotranscribed genes encoding the Rieske Fe-S and cytochrome f proteins of the cyanobacterium Nostoc PCC 7906.

The thylakoid membrane cytochrome b6-f complex (plastoquinol:oxidized-plastocyanin oxidoreductase, EC 1.10.99.1) catalyzes electron-transfer and proton-translocation reactions essential for oxygenic photosynthesis. We have isolated and determined the nucleotide sequences of the petC and petA genes encoding the Rieske Fe-S and cytochrome f polypeptides from the filamentous cyanobacterium Nostoc PCC 7906. These genes occur as single genomic copies, are tightly linked, and, as indicated by hybridization of gene-specific probes to Nostoc RNA, are cotranscribed as a 2.0-kilobase message. The Rieske Fe-S/cytochrome f gene pair thus represents an example of clustering and cotranscription in cyanobacteria of functionally related genes that, in photosynthetic eukaryotes, reside on separate nuclear and plastid genomes. These data are consistent with the progressive degeneration of the modern chloroplast genome from the ancestral, cyanobacterial-like genome of an endosymbiont. The Rieske Fe-S and the mature cytochrome f apoproteins are encoded by 537 and 867 nucleotides and have molecular masses of 19.2 and 31.2 kDa, respectively. They show 59% and 60% protein sequence identity, respectively, relative to spinach. Forty-four amino acids (4.7 kDa) resembling a prokaryotic signal sequence precede apocytochrome f. In contrast, the Rieske Fe-S protein appears to be translated without a presequence. The 183 bases separating the Rieske Fe-S and preapocytochrome f genes contain two families of 7- to 9-base tandem repeats, and some part of this sequence is highly reiterated in the genome. The C terminus of the Rieske Fe-S protein contains cysteine and histidine residues (probable ligands for the Fe2S2 center) in two peptides, Cys-Thr-His-Leu-Gly-Cys-Val and Cys-Pro-Cys-His-Gly-Ser, which have been conserved in spinach and in the five available Rieske Fe-S sequences from the mitochondrial-type cytochrome b-c1 complexes. Cytochrome f shows the heme binding residues Cys-Xaa-Xaa-Cys-His near its N terminus. Single, long hydrophobic stretches occur near the N and C termini, respectively, of the Rieske Fe-S and cytochrome f proteins and may form membrane-spanning helices.

Amino Acid Sequence↗
PubMed1988

Ferredoxin Cross-Links to a 22 kD Subunit of Photosystem I.

We have used a cross-linking approach to study the interaction of ferredoxin (Fd) with photosystem I (PSI). The cross-linking reagent N-ethyl-3-(3-dimethylaminopropyl) carbodiimide was found to cross-link spinach Fd to a 22 kilodalton subunit of PSI in both isolated spinach (Spinacia oleracea) PSI complexes and spinach thylakoid membranes. The product had an apparent molecular weight of 38 kilodaltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was identified as a cross-linked product using specific antibodies to Fd and the 22 kilodalton subunit. In both a native PSI complex (200 Chl/P700) and a PSI core complex (100 Chl/P700), a second cross-linked product at 36 kilodaltons was seen. The latter cross-reacted with an antibody to Fd but did not cross-react with antibodies directed against the 24.3, 22, 19, 17.3 or 8.5 kilodalton, or psaC subunits of PSI. Its composition remains to be determined. In thylakoids only the 38 kilodalton product was observed along with a cross-linked complex of Fd and Fd:NADP(+) reductase.

Journal Article↗
PubMed1988

Structural Aspects of Photosystem I from Dunaliella salina.

A native PSI complex and a PSI core complex have been isolated from the halophilic green alga, Dunaliella salina. The composition and properties of these complexes are similar to previously described PSI complexes from spinach membranes. By growth on (14)C-NaHCO(3), it has been possible to isolate uniformly labeled (14)C-PSI complexes in order to determine PSI subunit stoichiometry. This analysis has shown a ratio of one copy of three low molecular weight subunits (22,000; 15,000; 8,000) per two copies of high molecular weight subunits (84,000). Using a (14)C-labeled cytochrome b(6)-f complex as an internal protein standard, it has been possible to estimate the molecular weight of a PSI core complex as about 330,000. This complex contains one P700, two 84,000 subunits, and one subunit of 22,000, 15,000, and 8,000.

Journal Article↗
PubMed1987

Reconstitution of isolated Rieske Fe-S protein into a Rieske-depleted cytochrome b6-f complex.

The Rieske Fe-S protein can be isolated from the cytochrome b6-f complex by means of chromatography on a hydroxyapatite column in the presence of detergent. Depletion of the cytochrome complex from the Rieske protein results in the loss of oxidoreductase activity, as well as the ability to reduce cytochrome b6. The Rieske Fe-S protein can be reconstituted into the Rieske-depleted complex by removal of the Triton X-100 molecules associated with the protein fractions, and their substitution by lipids. Upon reconstitution the complex is reactivated, and the role of the Rieske Fe-S protein in the reduction of both plastocyanin and cytochrome b6 can be demonstrated.

Chromatography↗
PubMed1987

Oxidation-reduction reactions of cytochrome b6 in a liposome-incorporated cytochrome b6-f complex.

The chloroplast cytochrome b6-f complex, incorporated into phospholipid vesicles, shows proton translocation with an observed H+/e- ratio of approximately 2. The oxidation-reduction behavior of cytochrome b6 during electron transport from duroquinol to plastocyanin is affected by incorporation. The most obvious effect of incorporation is an increase in the duration of a steady-state level of cytochrome b6 that persists during electron transport. Reagents that decrease activity increase the duration of the steady state while reagents that stimulate activity decrease this time. Uncoupling conditions yield cytochrome kinetics similar to those in the unincorporated complex. 2,5-Dibromo-3-methyl-6-isopropyl-p-benzoquinone and 5-n-undecyl-4,7-dioxobenzothiazole inhibited reduction of cytochrome b6 in the incorporated complex, but this apparent inhibition was due to a rapid oxidation of the cytochrome by these compounds.

Carbonyl Cyanide m-Chlorophenyl Hydrazone↗
PubMed1987

On the interactions between macrophages and developmental stages of Schistosoma mansoni: the cytotoxic mechanisms involved in macrophage-mediated killing of schistosomula in vitro.

In an in vitro cytotoxicity assay, mouse adherent peritoneal exudate macrophages (APEM), harvested 8-10 weeks post Schistosoma mansoni infection caused sizable (greater than 90%) specific killing of schistosomula. This cidal effect was not diminished by the addition of scavengers of oxidative burst products to the cytotoxicity assay, albeit macrophages from schistosome-infected mice produced more H2O2 than did macrophages from non-infected mice. Of inhibitors of lysosomal enzyme function and release added to the cytotoxicity assay, trypan blue (1 mg/ml) fully abolished the schistosomulicidal effect; hydrocortisone (100 micrograms/ml) was partly effective, and gold salts (1 mg/ml) were ineffective. A cidal effect was not apparent in the absence of L-arginine nor in the presence of excess (greater than 400 micrograms/ml) L-arginine, L-lysine or L-ornithine. Arginase (5 U/ml) totally abrogated the schistosomulicidal effect. The findings suggest that a macrophage protein of a lysosomal origin, dependent on arginine for its reaction and/or production, may be involved in the in vitro killing of schistosomula by macrophages from S. mansoni-infected mice.

Animals↗