Partial purification and characterization of an ascorbate-reducible b-type cytochrome from the plasma membrane of Arabidopsis thaliana leaves.

The plant plasma membrane (PM) contains more than one b-type cytochrome. One of these proteins has a rather high redox potential (can be fully reduced by ascorbate) and is capable of transporting electrons through the PM. Four genes encoding proteins with considerable homology to the sequences of cytochrome b(561) proteins in the animal chromaffin granule membrane have recently been identified in the genome of Arabidopsis thaliana. In order to characterize the cytochrome b(561) located in the Arabidopsis PM, first PM vesicles were purified by aqueous polymer two-phase partitioning from the leaves of 9-week-old A. thaliana. PM proteins were solubilized by nonionic detergent, and the fully ascorbate-reducible b-type cytochrome was partially purified by anion-exchange chromatography steps. Potentiometric redox titration of the fraction, containing the fully ascorbate-reducible b-type cytochrome after the first anion-exchange chromatography step, revealed the presence of two hemes with redox potentials of 135 mV and 180 mV, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the fractions containing the fully ascorbate-reducible b-type cytochrome after the second anion-exchange chromatography step revealed the presence of a single polypeptide band at about 120 kDa. However, heat treatment (15 min, 90 degrees C) before electrophoresis was able to split the 120 kDa band into two bands with molecular masses of about 23 and 28 kDa. These values are lower than the apparent molecular mass for the fully ascorbate-reducible b-type cytochrome purified from Phaseolus vulgarishypocotyls (about 52 kDa) but are in good agreement with those characteristic for the cytochrome b(561) proteins purified from chromaffin granule membranes (about 28 kDa) and the four polypeptides predicted from the Arabidopsis genome (24-31 kDa).

b-type cytochromes in plasma membranes of Phaseolus vulgaris hypocotyls, Arabidopsis thaliana leaves, and Zea mays roots.

The plasma membrane of higher plants contains more than one kind of b-type cytochromes. One of these has a high redox potential and can be fully reduced by ascorbate. This component, the cytochrome b561 (cyt b561), has its characteristic alpha-band absorbance close to 561 nm wavelength at room temperature. Cyt b561 was first isolated from etiolated bean hook plasma membranes by two consecutive anion exchange chromatography steps. During the first step performed at pH 8, cyt b561 did not bind to the anion exchange column, but other b-type cytochromes did. In the second step performed at pH 9.9, cyt b561 was bound to the column and was eluted from the column at an ionic strength of about 100 mM KCl. However, when the same protocol was applied to the solubilized plasma membrane proteins from Arabidopsis thaliana leaves and maize roots, the ascorbate-reducible cyt b561 bound already to the first anion exchange column at pH 8 and was eluted also at an ionic strength of about 100 mM KCl. Other b-type cytochromes than the ascorbate-reducible cyt b561 from the plasma membranes of Arabidopsis leaves and maize roots showed similar chromatographic characteristics to that of bean hypocotyls. These results demonstrate particular differences in the chromatographic behavior of cyt b561 from different sources.

Higher-plant plasma membrane cytochrome b561: a protein in search of a function.

During the past twenty years evidence has accumulated on the presence of a specific high-potential, ascorbate-reducible b-type cytochrome in the plasma membrane (PM) of higher plants. This cytochrome is named cytochrome b561 (cyt b561) according to the wavelength maximum of its alpha-band in the reduced form. More recent evidence suggests that this protein is homologous to a b-type cytochrome present in chromaffin granules of animal cells. The plant and animal cytochromes share a number of strikingly similar features, including the high redox potential, the ascorbate reducibility, and most importantly the capacity to transport electrons across the membrane they are located in. The PM cyt b561 is found in all plant species and in a variety of tissues tested so far. It thus appears to be a ubiquitous electron transport component of the PM. The cytochromes b561 probably constitute a novel class of transmembrane electron transport proteins present in a large variety of eukaryotic cells. Of particular interest is the recent discovery of a number of plant genes that show striking homologies to the genes coding for the mammalian cytochromes b561. A number of highly relevant structural features, including hydrophobic domains, heme ligation sites, and possible ascorbate and monodehydroascorbate binding sites are almost perfectly conserved in all these proteins. At the same time the plant gene products show interesting differences related to their specific location at the PM, such as potentially N-linked glycosylation sites. It is also clear that at least in several plants cyt b561 is represented by a multigene family. The current paper presents the first overview focusing exclusively on the plant PM cyt b561, compares it to the animal cyt b561, and discusses the possible physiological function of these proteins in plants.

Purification of cytochrome b-561 from bean hypocotyls plasma membrane. Evidence for the presence of two heme centers.

The high potential, ascorbate-reducible b-type cytochrome of plant plasma membranes, named cytochrome b-561, has been purified to homogeneity from etiolated bean hypocotyls. The pure protein migrated in denaturing electrophoresis as a broad band of approximately 55 kDa, and was found to be glycosylated. Optical redox titrations of partially purified cytochrome b-561 indicated that it contains two hemes with similar spectral features, but distinct midpoint redox potentials (E(m7)+135 mV and +206 mV, respectively). The presence of two heme centers in cytochrome b-561 is consistent with its role in electron transfer across plant plasma membranes.

Rotational mobility of Ca2+-ATPase of sarcoplasmic reticulum in viscous media.

The rotational diffusion of Ca2(+)-ATPase [Ca2+,Mg2(+)-activated ATP phosphohydrolase E.C. 3.6.1.38] was studied in native sarcoplasmic reticulum membrane by saturation transfer ESR spectroscopy after covalent labelling of intramembranous sulfhydryl groups with nitroxyl derivative of maleimide (5-MSL) as a function of sucrose and glycerol in the suspending medium. The relative enzymatic activity of sarcoplasmic reticulum was followed by increasing the viscosity of the aqueous phase. The ATP hydrolysing activity of the enzyme decreased differently on adding sucrose and glycerol. In the case of sucrose the reciprocal of power dependence of viscosity was observed, whereas for glycerol an exponential decay law was obtained, indicating solvent-protein interaction. On increasing the viscosity of the aqueous phase by either sucrose or glycerol, no changes were observed in the intramembranous viscosity as measured using intercalated spin-labelled stearic acid (16-SASL). The effective rotational correlation time of the protein was measured, as a mobility parameter, using saturation transfer ESR spectroscopy and found to be increased linearly with the viscosity of the sucrose containing medium and for the extramembranous size a height of 6.8 nm was obtained, indicating that approx. 82% of the volume of Ca2(+)-ATPase protein is external to the sarcoplasmic reticulum. The addition of glycerol probably promoted protein-protein interaction, as indicated by the larger changes in rotational diffusion and non-linear viscosity dependence.

Purification and characterization of an NADH-hexacyanoferrate(III) reductase from spinach leaf plasma membrane.

Plasma membranes were purified from spinach (Spinacea oleracea L.) leaves by aqueous two-phase partitioning. The NADH-hexacyanoferrate(III) reductase was released from the membrane by Chaps solubilization and purified 360-fold by ion-exchange chromatography followed by affinity chromatography and size-exclusion chromatography on FPLC. A major band of 45 kDa and a minor contaminant of 66 kDa were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The band at 45 kDa cross-reacted with antibodies raised against an NADH-hexacyanoferrate(III) reductase from potato tuber microsomes. The native size of the enzyme was 160 kDa as determined by size-exclusion chromatography indicating that it is a tetramer. Two-dimensional gel electrophoresis, isoelectric focusing, followed by SDS-PAGE revealed three main bands of identical molecular weight with pI of 5.3-5.6. The enzyme contained about one flavin adenine dinucleotide (FAD) per 45-kDa subunit as determined by fluorescence spectroscopy, was specific for the beta-hydrogen of NADH, preferred NADH over NADPH as electron donor, and preferred hexacyanoferrate(III) as electron acceptor, e.g., it reduced Fe3+-EDTA, cytochrome c, oxygen, and duroquinone at < 10% of the rate with hexacyanoferrate(III). p-Chloromercurobenzoate, mersalyl, and dicumarol inhibited the activity by > 70% whereas FAD, flavin mononucleotide, duroquinone, and ubiquinone0 did not affect the activity.

H5D12 monoclonal antibody identifies a 24-kD immunosuppressor factor produced by human chorio carcinoma cells.

PROBLEM: The purpose of this study was to identify and characterize embryo-associated immunosuppressor factor (EASF) secreted by chorio carcinoma cells with the help of EASF-specific monoclonal antibody H5D12 (raised against EASF purified from embryo growth media of in vitro fertilized human ova). METHOD: Paraffin-embedded slides of human chorio carcinoma as well as control cell lines were prepared, and immunohistochemistry was done by the avidin-biotin-peroxidase technique. EASF was affinity purified using H5D12-Sepharose 4B from culture media of cell lines and analyzed for immunosuppressive activity (by Concanavalin-A-induced lymphocyte proliferation assay) and molecular weight identity (by metabolic-labelling studies with 35S-methionine followed by immunoprecipitation and SDS-PAGE). RESULTS: H5D12 showed intense immunostaining of BeWo chorio carcinoma cells. Biosynthetic labeling studies identifies this factor as a 24-kD molecule, and EASF bioassay indicates that this factor possesses immunosuppressive activity. No such immunosuppressive activity or similar molecules were identified when control cell lines were analyzed. CONCLUSIONS: Monoclonal antibody H5D12 recognizes a 24-kD factor with immunosuppressive activity that is secreted by chorio carcinoma cells, which suggests that this is a unique factor and may be one of the key regulators of reproductive functions.

Interaction of a doxorubicin-transferrin conjugate with isolated transferrin receptors.

It is shown that transferrin-doxorubicin conjugates bind to isolated transferrin receptors. The receptors were isolated from human full-term placenta by solubilization of trophoblast plasma membranes with the nonionic detergent C12E8 and then by affinity chromatography on a diferric transferrin-coupled Sepharose CL-4B column. The binding affinity of such conjugate was similar to that of transferrin. Dissociation of conjugate from the isolated receptor occurred with time-dependent kinetics similar to those of transferrin when the experimental conditions mimicking the physiological steps of transferrin recycling were consecutively applied. These results support the idea that a) binding of such conjugates is primarily governed by the interaction between the transferrin part of the conjugates and the transferrin receptor, and b) it is not the transferrin receptor itself which participates in the putative secondary interaction between the receptor-bound conjugates and plasma membrane.

Inhibition of lipid A- and lipopolysaccharide-induced cytokine secretion, B cell mitogenesis, and lethal shock by lipid A-specific murine monoclonal antibodies.

Three murine hybridomas secreting IgM monoclonal antibodies (MAbs) to lipid A (LA) of Salmonella minnesota R595 were generated. These MAbs serologically cross-reacted with LA and lipopolysaccharide (LPS) of unrelated gram-negative bacterial species. All three MAbs significantly suppressed the ability of LA and LPS from various gram-negative bacteria to induce tumor necrosis factor (TNF)-alpha (36%-67%) and interleukin-1 (30%-98%) in murine peritoneal macrophages and to stimulate B lymphocytes (37%-78%). Lipid A-induced TNF alpha production was also suppressed in mice (86%-88%). All three antibodies protected adrenalectomized mice against lethal shock induced by LA of S. minnesota R595. Optimal protection was achieved with one of the antibodies (MLA-1), if it was administered 2 h before injection of lipid A, and full protection persisted < or = 24 h. Moreover, MLA-1 was able to protect adrenalized or D(+)-galactosamine-sensitized mice against lethal shock induced by LPS derived from various gram-negative bacteria. This cross-protection could be predicted on the basis of serologic cross-reactivity and cross-neutralization by MLA-1 of the bioactivity of the heterologous LA or LPS in vitro.

Influence of conjugation of doxorubicin to transferrin on the iron uptake by K562 cells via receptor-mediated endocytosis.

The influence of conjugation of doxorubicin to holotransferrin on the receptor-mediated endocytosis of and on the iron uptake from transferrin was studied using K562 cells. 125I-labelled transferrin and doxorubicin-transferrin conjugates were used in the binding, dissociation, and ligand-exchange experiments at 0 degree C, and 59Fe,125I-labelled (double-labelled) ligands were used in the endocytosis, iron uptake, and recycling experiments at 37 degrees C. The binding affinity of conjugates was about half of that of transferrin. Binding of 125I-labelled ligands was blocked by both unlabelled ligands to the same degree, however, it was not blocked at all by an 8000-fold excess of doxorubicin. After saturation bindings, slightly more 125I-labelled conjugates dissociated from the surface of cells than transferrin. Exchange of 125I-labelled ligands for unlabelled ligands resulted in different EC50 values (defined as the concentration of unlabelled ligand at which half as much radioligand is exchanged for unlabelled ligand as would be exchanged at infinitely high concentration of unlabelled ligand under similar assay conditions). While transferrin exchanged transferrin with an EC50 value close to the binding affinity, conjugates exchanged conjugates with much lower efficiency. The heterolog exchange experiments yielded EC50 values inbetween the two extrema. For studying iron uptake, K562 cells were loaded with the double-labelled ligands either at 37 degrees C (endosome-loading only) or at 0 degree C (surface-loading only). Results obtained for the endocytosis of, the iron uptake from, and the recycling of double-labelled ligands indicate that (a) the rate of iron uptake is smaller from conjugates than from transferrin, (b) there are at least two parallel recycling processes for both ligand.receptor complexes, and (c) each time constant characterizing the different steps of iron uptake via receptor-mediated endocytosis is smaller for conjugates than for transferrin (or, the half times characterizing the different steps are higher for conjugates than for transferrin). Endocytosis and iron uptake were unaffected by free doxorubicin (12.5 microM) or colchicine (1 mM). Benzyl alcohol (30 mM) slowed down the rate of both endocytosis and iron uptake, while dithiothreitol (5 mM) decreased the rate of iron uptake and increased the rate of endocytosis. N-Ethylmaleimide (1 mM) completely stopped both endocytosis and iron uptake. The results suggest that the binding of conjugates to the surface of cells is governed by the binding of the transferrin part of conjugates to the transferrin receptor. However, conjugation of doxorubicin to transferrin seems to influence all properties of transferrin.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding of doxorubicin-conjugated transferrin to U937 cells.

Binding of transferrin (Trf) and its doxorubicin-conjugated forms (Conj) to U937 cells at 0 degrees C were compared using 125I-labelled Trf or Conj. The apparent binding affinity (Ka) of Conj to the surface of U937 cells was (1.9 +/- 0.4).10(8) l/mol; it is about 40% of that of Trf [(5.0 +/- 1.2).10(8) l/mol]. Binding of 125I-labelled ligands was blocked by the unlabelled ligands to the same degree, however, it was not blocked by a great excess of doxorubicin (Dox). N-ethylmaleimide caused about 10% inhibition while dithiothreitol was without effect. Dissociation of 125I-labelled ligands in the presence of different concentrations of unlabelled ligands (Trf and Conj in the all 4 variations) resulted in different R50 values (the concentration of the unlabelled ligand where 50% of the radiolabelled ligand was released). While Trf displaced Trf with an R50 value close to the binding affinity, Conj displacement by Conj occurred with much lower efficiency. The heterolog displacement experiments yielded R50 values in between the two extrema. These results suggest that 1) binding of Conj to the surface of cells is governed by the binding of the Trf part of Conj to the transferrin receptor, 2) -SH groups are not involved in the binding, and 3) a second interaction between the Conj and some constituent(s) of the plasma membrane may modify the binding of Conj in comparison to that of Trf.

Adriamycin conjugates of human transferrin bind transferrin receptors and kill K562 and HL60 cells.

Adriamycin (ADR) was coupled to human transferrin (TRF) by using a glutaraldehyde crosslinking method. The TRF-ADR conjugates were separated by column chromatography and the molar ratio of ADR to TRF (i.e., conjugation number) for the studied conjugates was found to be 1.2. Analysis in sodium dodecyl sulfate-polyacrylamide gels demonstrated that TRF-ADR conjugates with this molar ratio had the same mobility as native TRF and contained few aggregates. The ADR remained conjugated to TRF under conditions of decreased pH known to occur in many intracellular compartments, and analysis by spectrofluorometry revealed that the conjugated ADR retained its ability to intercalate DNA. The TRF-ADR conjugates were shown by flow cytometry to preferentially bind tumor cells and cell-bound conjugates were found to be laterally mobile within plasma membranes. The binding of TRF-ADR conjugates was determined to be saturable, and competition experiments done with both radioiodinated and fluorescein-labeled TRF-ADR conjugates demonstrated dose-dependent inhibition of conjugate binding by unlabeled TRF, indicating that TRF-ADR conjugates were bound by TRF receptors. Cytotoxicity studies performed with tritiated thymidine incorporation and tetrazolium reduction assays revealed that TRF-ADR conjugates inhibited the proliferation of both K562 and HL60 cells in culture more effectively than free ADR. Such conjugates could provide a delivery system for ADR that would target the drug and possibly diminish its dose-associated complications.

Iron-reducing activity of plasma membranes.

Human placental trophoblast plasma membranes were prepared by differential centrifugation and solubilized in nonionic detergent. Transferrin receptors were isolated from the solubilized membranes by affinity chromatography on diferric transferrin-coupled Sepharose 4B. The trophoblast plasma membrane vesicles demonstrated NADH-ferricyanide oxidoreductive activity. However, NADH-Fe(III) oxidoreductive activity was very weak when Fe(III)-ammonium citrate or diferric transferrin was used as electron acceptor in the presence of bathophenanthroline disulfonate as an indicator of the reaction. After solubilization, only NADH-ferricyanide oxidoreduction was recovered. Affinity chromatography-purified transferrin receptors did not exhibit any measurable oxidoreductase activity. Thus, when these receptors are present in plasma membranes they mediate redox reactions, but biochemically isolated receptors do not mediate such reactions. These observation suggest that transferrin receptors in plasma membranes bind diferric transferrin, and, in an undetermined way, facilitate Fe(III) release so that iron reduction can occur.

Cytotoxicity of a transferrin-adriamycin conjugate to anthracycline-resistant cells.

Conjugates of adriamycin coupled to transferrin by glutaraldehyde are cytotoxic to human promyelocytic (HL-60) and erythroleukemic (K562) cells. Growth inhibition of adriamycin-sensitive cells, as evaluated by thymidine incorporation and the MTT-assay, was higher for conjugates than for free adriamycin. The cytotoxicity toward adriamycin-resistant K562 and HL-60 cells was 3-fold and more than 10-fold higher, respectively, for the transferrin-adriamycin conjugate than for the free drug. The effect of the conjugate was dependent on its adriamycin content, i.e., on its conjugation number.

Diferric transferrin reduction by K562 cells. A critical study.

This paper critically examines the redox activity of K562 cells (chronic myelogenous leukemia cells) and normal peripheral blood lymphocytes (PBL). Ferricyanide reduction, diferric transferrin reduction, and ferric ion reduction were measured spectrophotometrically by following the time-dependent changes of absorbance difference characteristic for ferricyanide disappearance and for the formation of ferrous ion:chelator complexes. Bathophenanthroline disulfonate (BPS) and ferrozine (FZ) were used to detect the appearance of ferrous ions in the reaction mixtures when diferric transferrin or ferric reduction was studied. Special attention was devoted to the analysis of time-dependent absorbance changes in the presence and absence of cells under different assay conditions. It was observed and concluded that: (i) FZ was far less sensitive and more sluggish than BPS for detecting ferrous ions at concentrations commonly used for BPS; (ii) FZ, at concentrations of at least 10-times the commonly used BPS concentrations, seemed to verify the results obtained with BPS; (iii) ferricyanide reduction, diferric transferrin reduction and ferric ion reduction by both K562 cells and peripheral blood lymphocytes did not differ significantly; and (iv) earlier values published for the redox activities of different cells might be overestimated, partly because of the observation published in 1988 that diferric transferrin might have loosely bound extra iron which is easily reduced. It is suggested that the specific diferric transferrin reduction by cells might be considered as a consequence of (i) changing the steady-state equilibrium in the diferric transferrin-containing solution by addition of ferrous ion chelators which effectively raised the redox potential of the iron bound in holotransferrin, and (ii) changing the steady-state equilibrium by addition of cells which would introduce, via their large and mostly negatively charged plasma membrane surface, a new phase which would favor release and reduction of the iron in diferric transferrin by a ferric ion oxidoreductase. The reduction of ferricyanide is also much slower than activities reported for other cells which may indicate reduced plasma membrane redox activity in these cells.

Recent advances in cancer research: drug targeting without the use of monoclonal antibodies.

Cancer research in drug targeting has focused on the use of monoclonal antibody conjugates of drugs. This paper discusses the use of ligand conjugates of drugs to deliver to receptors on cancer cells. We have used transferrin coupled to adriamycin, and report these conjugates specifically bind and kill cancer cells in culture. Our studies of the mechanism show targeted plasma membranes are compromised for NADH ferricyanide reduction, and targeted cells lose diferric transferrin reductase activity. These results indicate that the binding of transferrin-adriamycin conjugates to transferrin receptors on either isolated plasma membranes or viable tumor cells causes an inhibition of redox reactions that are essential for growth. Since transferrin receptors are endocytosable, ligand-drug conjugates also are delivered to the interior of targeted cells where other mechanisms of killing can be employed. This novel method of drug delivery circumvents the need for monoclonal antibodies, and more investigation of the system may allow a controlled clinical study of its effectiveness.

Plasma membrane purification from roots of sunflower by phase partitioning.

Plasma membrane vesicles were purified from the roots of sunflower (Helianthus annuus L. cv. Topflor) by aqueous polymer two-phase partitioning. The optimal conditions for separation were determined by systematic variation of the polymer concentration and salt composition. The phase system containing 6% (w/w) dextran T-500, 6% (w/w) polyethylene glycol 3350, 250 mM sucrose, 5 mM potassium phosphate, pH 7.8, without added salts proved to be the best. The ATPase activity had a pH optimum at 6.5 and it was stimulated by Mg2+, but not by Ca2+. The plasma membrane MgATPase activity was inhibited by vanadate but not by nitrate, an inhibitor of tonoplast ATPase. Only 10% of the microsomal protein was responsible for 36% of the total MgATPase activity. Moreover IDPase activity, a Golgi marker, appeared to be very low indicating the high purity of the preparation.

Influence of EDTA on the ATPase activities of plasma membranes from winter wheat roots.

Plasma membrane vesicles were purified from roots of winter wheat seedlings (Triticum aestivum L. cv. Marton-vásári-8) by aqueous polymer two-phase partitioning. The ATPase activity of vesicles in the presence of Ca was independent of but in the presence of Mg depended on the EDTA concentration in the ATPase assay. The potassium stimulation and vanadate inhibition of the MgATPase also depended on the EDTA concentration. Consequences of these results are briefly discussed.

Interactions of haemoglobin with erythrocyte membrane phospholipids in monomolecular lipid layers.

The role of red cell membrane lipids in the membrane-haemoglobin interaction was studied by measuring the surface potential and surface pressure of monomolecular lipid layers interacting with haemoglobin. Lipids of the outer and inner half of the red cell membrane were compared in respect to their haemoglobin-binding capacity. It was shown, that haemoglobin molecules interacted readily with the inner layer lipid film in acidic pH regions. This interaction is reduced as pH is increasing but still exists in the physiological pH range. It is in contrast with the increasing but still exists in the physiological pH range. It is in contrast with the findings for the outer layer lipid film, where only a partial interaction could be shown at pH 4, which was reduced to zero reaching the physiological pH range. It can be concluded from titration experiments that the process of haemoglobin binding as reflected in the measured parameters is irreversible. The result of this model experiments support the hypothesis on phosphatidylserine binding sites for haemoglobin in the inner side of red cell membrane.

Incorporation of brain membrane proteins into planar bilayer lipid membranes.

Am method is presented which enables the incorporation of solubilized brain membrane proteins into bilayer lipid membranes (BLM) from brain lipids. The digitonin solubilized proteins are first incorporated into liposomes which undergo fusion with the BLM in a second step. The incorporation results in a large increase in membrane conductivity which maintains at a stable level for hours. Identical electrical phenomena were observed after incorporation of the membrane fragments which were used as starting material of protein extraction. The method can be used to reconstitute membrane proteins to the their natural lipid environment in a planar BLM. Our results argue against the essential role of Ca2+ and osmotic gradients during fusion of bilayer structures.