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Biomedical subjects

R Malkin

Publications and source records attributed to R Malkin.

At least 55 records · Page 3Linked to original sources
PubMed1986

Interaction of stigmatellin and DNP-INT with the Rieske iron-sulfur center of the chloroplast cytochrome b6-f complex.

Stigmatellin and DNP-INT are effective inhibitors of the catalytic activity of the plastoquinol-plastocyanin oxidoreductase complex (cytochrome b6-f complex). Both inhibitors alter the EPR spectrum of the Rieske iron-sulfur center but do not produce band-shifts of cytochrome b-563. The midpoint redox potential of the Rieske center is unaffected by either inhibitor, although both alter the DBMIB-induced g-value shifts of the Rieske center. The results are considered in terms of binding domains for inhibitors in the cytochrome b6-f complex.

Binding Sites↗
PubMed1986

The interactions between macrophages and developmental stages of Schistosoma mansoni: effect of macrophage function modulators on the viability of S. mansoni in vivo and in vitro.

ICR mice infected with Schistosoma mansoni developed sizable concomitant immunity to a challenge infection 10 weeks, but not 7 weeks, following the primary infection. At 7 weeks, postprimary-infection mice exhibited increased resistance to reinfection when treated with BCG or MDP. BCG even rendered noninfected mice resistant to infection. Macrophage function inhibitors such as silica and trypan blue did not abolish the concomitant immunity state, but they increased the worm burden due to a single infection, whether given before or after the infection. The onset of concomitant immunity in infected mice was paralleled by the appearance in their peritoneal exudate of schistosomulicidal-adherent macrophages. Such cells were evident at 9 but not 7 weeks of infection. The in vivo injection of MDP accelerated their appearance in infected mice, while silica, trypan blue, and carrageenan abolished it. The findings suggest that highly activated schistosomulicidal macrophages develop in infected mice, and might participate in the destruction of the invading parasite.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1985

Characterization of a photosynthetic mutant of Lemna lacking the cytochrome b6-f complex.

A photosynthetic mutant of Lemna perpusilla (no. 1073) has been examined by spectrophotometric and immunoblotting techniques in order to localize the site of defect. In contrast to previous conclusions (Shahak, Y., Posner, H.B. and Avron, M. (1976) Plant Physiol. 57, 577-679), neither cytochrome f nor cytochrome b6 could be detected spectrophotometrically in the mutant. Furthermore, immunoblotting using antibodies specific for each of the four constituent subunits of the cytochrome b6-f complex demonstrate that the entire complex is absent in the mutant. The light-harvesting chlorophyll-protein complex of Photosystem II is present in similar amounts in wild-type and mutant Lemna. However, the total amount of plastoquinone-9 is reduced by approx. 65% in the mutant strain, while the photoreducible plastoquinone-9 pool is comparable in wild-type and mutant Lemna.

Antibodies↗
PubMed1985

Topographical studies of the polypeptide subunits of the thylakoid cytochrome b6-f complex.

The orientation of specific polypeptides of the cytochrome b6-f complex with respect to the chloroplast stromal phase has been studied using trinitrobenzenesulfonate (TNBS) and pronase E as impermeant modifying reagents. Of the four polypeptides of the complex (33,23,20 and 17 kDa), only cytochrome f was labeled by 14C-TNBS in unfractionated membranes. However, to a varying degree, all of the constituent polypeptides were sensitive to pronase digestion and, in the case of cytochrome f, it was possible, by immunoblotting techniques to identify several degradation products. These results are discussed in relation to the organization of the cytochrome complex in thylakoid membranes and argue for an exposure to the stromal phase of all of the polypeptides, while functional considerations indicate that at least cytochrome f and the Rieske iron-sulfur protein have a possible transmembrane organization.

Animals↗
PubMed1985

Characterization of a cyanobacterial photosystem I complex.

A simple procedure is described for the preparation of photosystem I (PSI) particles from Triton X-100-solubilized thylakoid membranes of the unicellular cyanobacterium Synechococcus 6301. The purified PSI complex contained the full complement of antenna chlorophylls, 130 +/- 5/P700, displayed the electron paramagnetic resonance signals characteristic of iron-sulfur centers X, A, and B, and had a protein/chlorophyll ratio of 2.9. Determination of the polypeptide composition, utilizing a uniformly 14C-labeled complex, showed that it contained polypeptides of 70, 18, 17.7, 16, and 10 kDa, in a molar ratio of 4.0:0.7:1.0:0.5:1.6. The relative amount of the lower molecular weight polypeptides showed progressive decrease with increase in Triton X-100 concentration and time of exposure to detergent. Consequently, it is proposed that in vivo the composition of the complex is [70 kDa]4 [18 kDa]1 [17.7 kDa]1 [16 kDa]1 [10 kDa]2. Relative to 130 mol of chlorophyll a, the PSI complex contained 16 mol of carotenoids, 13.7 +/- 1.0 g atoms of Fe, and 12.2 +/- 1.1 g atoms of labile sulfide. The properties of complexes fully depleted of the low-molecular weight polypeptides by treatment with sodium dodecyl sulfate or with proteinase K are also described.

Chlorophyll↗
PubMed1985

Identification of a 19-kDa polypeptide as an Fe-S center apoprotein in the photosystem I primary electron acceptor complex.

Treatment of Photosystem I (PSI) with sodium thiocyanate, a chaotropic agent, results in the selective depletion of certain low-molecular-weight polypeptides. A PSI complex obtained following treatment with 0.5 M sodium thiocyanate is significantly depleted of polypeptides of approximately 8, 10, 14, and 16 kDa, relative to an untreated control, but retains approximately 90% of the EPR signal amplitude associated with the iron-sulfur Centers A and B. The only peptides remaining that could not be depleted without a parallel decrease in the signal amplitude of the Fe-S Centers A and B are the 62-kDa reaction center-containing polypeptide and a 19-kDa polypeptide. These results are considered in relation to the identity of the apoprotein of the Fe-S Centers A and B.

Apoproteins↗
PubMed1985

Interactions between thylakoid electron transfer complexes. I. In vitro kinetic studies with isolated photosystem II and cytochrome b6-f complexes.

The cytochrome b6-f complex from spinach thylakoids has been reconstituted with an oxygen-evolving Photosystem II (PSII) preparation isolated from the same source to give oxygenic plastocyanin reductase activity. We observe that (i) mixing of the two complexes in concentrated form prior to dilution with the assay medium is necessary for optimal reconstitution of activity; (ii) incubation for longer times after dilution can also give substantial reconstitution if the two complexes are added separately to the assay mixture; (iii) either monovalent or divalent cations are required for optimum activity in the reconstituted system; (iv) titration of the cytochrome complex with varying amounts of the PSII complex gave a saturation of the plastocyanin reduction activity at a cytochrome complex/PSII ratio of 3-4; (v) kinetic analysis of plastocyanin photoreduction by Photosystem II shows nonlinearity, while first-order reduction kinetics are observed with duroquinol as electron donor; and (vi) as the concentration of plastocyanin is increased, the half-time of the reduction increases. These observations are considered in terms of a functional association between PSII and the cytochrome b6-f complex in this reconstituted system, and the relevance of these observations to the situation in vivo is discussed.

Chlorophyll↗
PubMed1985

Interactions between thylakoid electron transfer complexes. II. Modification studies with glutaraldehyde.

Photosystem I (PSI) and photosystem II (PSII) complexes have been isolated from stacked spinach thylakoid membranes that had been treated with varying amounts of glutaraldehyde. The concentrations of cytochrome f, Q, and P700 have been determined by spectrophotometric methods. It was found that at low concentrations of glutaraldehyde, the amount of cytochrome f associated with either PSII or PSI increased significantly while the amounts of Q and P700 stayed relatively constant. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting analyses indicated the presence of cytochrome f and other components of the cytochrome b6-f complex in the PSII and PSI preparations after glutaraldehyde treatment, but no intermolecular cross-linked polypeptides could be detected. Solubilization of the cytochrome b6-f complex was also inhibited after thylakoid membranes were treated with low concentrations of glutaraldehyde. These results are discussed in relation to current models for the organization of the membrane complexes, and relate to the location of the cytochrome b6-f complex in appressed and nonappressed membrane regions of thylakoids.

Aldehydes↗
PubMed1985

Topography of the protein complexes of the chloroplast thylakoid membrane : studies of photosystem I using a chemical probe and proteolytic digestion.

The transverse heterogeneity of the polypeptides associated with the Photosystem I (PSI) complex in spinach thylakoid membranes and in a highly resolved PSI preparation has been studied using the impermeant chemical modifier, 2,4,6-trinitrobenzenesulfonate (TNBS) and the proteolytic enzyme, Pronase E. The present study has shown that the PSI reaction center polypeptide of approximately 62 kilodaltons and the 22 and 20 kilodalton polypeptides of the PSI light-harvesting chlorophyll protein (LHCPI) complex are not labeled by [(14)C]TNBS in unfractionated thylakoids. On the other hand, the 23 kilodalton polypeptide of the PSI LHCP and the 19 and 14 kilodalton polypeptides associated with the PSI primary electron acceptor complex are readily labeled by [(14)C]TNBS and are exposed to the stromal side of the thylakoid. Differences and similarities in the labeling of polypeptides associated with the PSI complex in thylakoids and in the isolated PSI complex are also noted. Treatment of thylakoids with pronase had no effect on the organization of the polypeptides in the LHCPI or the reaction center core complex, as manifested by the separation of these two subcomplexes from pronase-treated membranes. The 62, 19, and 14 kilodalton polypeptides associated with the reaction center core complex and the 23 and 22 kilodalton polypeptides associated with LHCPI are sensitive to pronase treatment while the 20 kilodalton polypeptide of LHCPI was inaccessible to the protease. The proteolysis of the 62 kilodalton polypeptide generated first a single immunodetectable fragment at about 48 kilodaltons, and further proteolytic digestion generated two other fragments at 30 and 17 kilodaltons respectively. These results are discussed in relation to the organization of the PSI complex in spinach thylakoids. A model for the transmembrane topography of the polypeptide constituents of PSI has been developed.

Journal Article↗
PubMed1985

Topography of the Protein Complexes of the Chloroplast Thylakoid Membrane : Studies of Photosystem II using Pronase Digestion and Chemical Labeling.

The accessibility of various Photosystem II (PSII)-associated polypeptides to the protease pronase and the chemical modifier trinitrobenzene-sulfonic acid (TNBS) has been investigated. Three polypeptides with apparent molecular weight of 32, 21, and 16 kilodaltons, known to be associated with O(2) evolution, are all resistant to pronase digestion and TNBS labeling in intact thylakoids. All the polypeptides in the isolated PSII preparation were labeled with TNBS while a different pattern of labeling was observed when the PSII complex was isolated from TNBS-modified thylakoids. Attempts to prepare PSII particles from pronase-treated thylakoids using the Triton X-100 solubilization method were unsuccessful. Pronase-treated thylakoids were probed with antisera against the chlorophyll proteins of PSII using immunoblotting techniques. This allowed for a positive identification of proteolytic fragments from the respective proteins. The results are discussed in relation to the transmembrane organization of PSII in spinach thylakoids.

Journal Article↗
PubMed1984

Light inhibition of respiration is due to a dual function of the cytochrome b6-f complex and the plastocyanin/cytochrome c-553 pool in Aphanocapsa.

Studies of the respiratory electron transport pathway in the blue-green alga, Aphanocapsa, demonstrated the presence of cytochrome oxidase and a cytochrome complex. The use of antimycin A showed only the occurrence of a plastidal type of cytochrome complex (the cytochrome b6-f complex), which is insensitive to this inhibitor. Determination of the extent of photooxidation of cytochromes c-553 and f-556 under conditions of high and low cytochrome oxidase activities indicated an electron flow through both cytochromes to cytochrome oxidase. Direct evidence for a common segment of photosynthetic and respiratory electron transport from plastoquinone via the cytochrome b6-f complex to the soluble plastocyanin/cytochrome c-553 pool, as well as a competition between cytochrome oxidase and Photosystem I for reductants in this pool in the light, was obtained by measurements of electron transport with suitable electron donors in this alga.

Cyanobacteria↗
PubMed1984

Isolation and Characterization of a Light-Harvesting Chlorophyll a/b Protein Complex Associated with Photosystem I.

A chlorophyll a/b protein complex has been isolated from a resolved native photosystem I complex by mildly dissociating sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The chlorophyll a/b protein contains a single polypeptide of molecular weight 20 kilodaltons, and has a chlorophyll a/b ratio of 3.5 to 4.0. The visible absorbance spectrum of the chlorophyll a/b protein complex showed a maximum at 667 nanometers in the red region and a 77 K fluorescence emission maximum at 681 nanometers. Alternatively, by treatment of the native photosystem I complex with lithium dodecyl sulfate and Triton, the chlorophyll a/b protein complex could be isolated by chromatography on Sephadex G-75. Immunological assays using antibodies to the P(700)-chlorophyll a-protein and the photosystem II light-harvesting chlorophyll a/b protein show no cross-reaction between the photosystem I chlorophyll a/b protein and the other two chlorophyll-containing protein complexes.

Journal Article↗
PubMed1983

Characterization of electron transfer from water to plastocyanin catalyzed by resolved electron transfer complexes from chloroplasts.

The photoreduction of soluble plastocyanin in a homologous reconstituted system requires a resolved Photosystem II preparation and the cytochrome b6-f complex from spinach chloroplasts. Oxygen evolution is linked quantitatively to plastocyanin reduction in the reconstituted system, indicating that water is the electron donor for the photoreduction. The photoreduction is sensitive to inhibitors which specifically interact with the Photosystem II preparation [DCMU: 3-(3,4-dichlorophenyl)-1,1-dimethylurea] as well as those which interact with the cytochrome complex (DNP-INT: 2-iodo-6-isopropyl-3-methyl-2',4,4'-trinitrodiphenyl ether; and DBMIB: 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone). The plastocyanin requirement in the reconstituted system indicated a Km of 4.6 microM and a Vmax of 4.2 mumols plastocyanin reduced per nmol cytochrome f per h. Inhibition curves for DNP-INT and DBMIB show that both are effective inhibitors (50% inhibition at a ratio of inhibitor to cytochrome f of 1.0). The rate of reduction of plastocyanin was found to be stimulated by cations. This dependency was localized in the transfer of electrons from the Photosystem II preparation to the cytochrome complex. These results are considered in terms of organizational aspects of integral protein complexes in thylakoid membranes.

Catalysis↗
PubMed1983

Iron-sulfur centers and activities of the photosynthetic electron transport chain in iron-deficient cultures of the blue-green alga aphanocapsa.

Cultures of the blue-green alga, Aphanocapsa, were grown under iron-limiting conditions and changes in concentration of redox components of the photosynthetic electron transport chain, particularly iron-sulfur centers, were monitored by spectroscopic methods. A moderate iron depletion (1/10 of the normal concentration) had little effect on photosynthetic electron transport reactions and growth. Nevertheless, the amount of membrane-bound non-heme iron decreased sharply, and ferredoxin was nearly totally replaced by a flavin-containing protein, flavodoxin. Severe iron-deficiency (1/100 of the normal concentration) was accompanied by growth inhibition and decreased rates of photosynthetic electron flow. The Photosystem I reaction center was most affected by iron depletion as evidenced by a decrease in the amounts of iron-sulfur centers A, B, and X. However, formation of other redox proteins, even those that do not contain iron, was also inhibited by severe iron deficiency.

Journal Article↗
PubMed1982

Interaction of photosynthetic electron transport inhibitors and the Rieske Iron-Sulfur center in chloroplasts and the cytochrome b6-f complex.

The interaction of electron transport inhibitors with the Rieske iron-sulfur center in chloroplast membranes and in a purified chloroplast cytochrome complex (the b6-f complex) has been studied by using electron paramagnetic resonance (EPR) spectroscopy. Several quinone inhibitors, all of which contain a halogen substituent and a bulky alkyl side chain, cause a shift in the EPR signal of the reduced Rieske iron-sulfur center from g = 1.90 to g = 1.94. This g-value shift occurs in untreated membranes as well as in the cytochrome complex, which contains the Rieske center and no other iron-sulfur centers. Other compounds known to inhibit electron transport in the region of the iron-sulfur center cause a smaller alteration in the EPR signal of the Rieske center and are able to interact with the quinone-binding sites as evidenced by their displacement of quinones from the Rieske center. One substrate, plastoquinone 9, was also able to displace quinones from the Rieske center while other, such as plastoquinone 1 and duroquinone, did not show this effect. These results are considered in relation to the mode of interaction of quinones with the Rieske center in the photosynthetic membrane.

Binding Sites↗
PubMed1982

Reconstruction of the chloroplast noncyclic electron transport pathway from water to NADP with three integral protein complexes.

RECONSTRUCTION OF PHOTOSYNTHETIC NONCYCLIC ELECTRON TRANSPORT FROM WATER TO NADP HAS BEEN ACCOMPLISHED BY USING THREE INTEGRAL PROTEIN COMPLEXES ISOLATED FROM CHLOROPLAST THYLAKOID MEMBRANES: photosystems I and II and the cytochrome b(6)-f complex. This system shows an absolute dependence on the presence of all three protein complexes for NADP reduction, in addition to plastocyanin, ferredoxin, and ferredoxin-NADP reductase. The reconstructed system was found to be sensitive to low concentrations of known inhibitors of noncyclic electron transport. Depletion of the Rieske iron-sulfur center and bound plastoquinone from the cytochrome b(6)-f complex resulted in an inhibition of the photoreduction of NADP.

Journal Article↗