Alajos Bérczi

The negative surface charge density of plasmalemma vesicles from wheat and oat roots

Plasmalemma vesicles were isolated in a sucrose‐containing medium from wheat and oat roots and the net negative surface charge density was determined with 9‐aminoacridine fluorescence [Chow, W.S. and Barber, J. (1980) J. Biochem. Biophys. Methods 3, 173‐185]. The outer surface of the vesicles (measured in the presence of sucrose) had densities of − 16 to − 20 mC·m−2 and −29 mC·m−2 for wheat and oat roots, respectively. The inner surface ‐ presumed to be the cytoplasmic side and calculated from the values measured in the presence and absence of sucrose ‐ was more negative, and its size depended on the salt status of the roots.

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

SummaryDuring the past twenty years evidence has accumulated on the presence of a specific high-potential, ascorbate-reducibleb-type cytochrome in the plasma membrane (PM) of higher plants. This cytochrome is named cytochromeb561 (cytb561) according to the wavelength maximum of its α-band in the reduced form. More recent evidence suggests that this protein is homologous to ab-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 cytb561 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 cytochromesb561 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 cytochromesb561. 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 cytb561 is represented by a multigene family. The current paper presents the first overview focusing exclusively on the plant PM cytb561, compares it to the animal cytb561, and discusses the possible physiological function of these proteins in plants.

Oxygen consumption by purified plasmalemma vesicles from wheat roots: Stimulation by NADH and salicylhydroxamic acid (SHAM)

Plasmalemma vesicles from wheat (Triticum aestivum L.) roots consumed O2 and the addition of 1 mM NADH increased the rate ~ 3‐fold (to 15‐30 nmol O2·mg−1·min−1). The NADH‐dependent O2 uptake was abolished by catalase. In the presence of salicylhydroxamic acid (SHAM), an inhibitor of the alternative oxidase pathway in plant mitochondria, NADH‐dependent O2 consumption was stimulated 10–20‐fold (to 200–400 nmol·mg1̄·min−1). Catalase also abolished this stimulation, which was KCN‐sensitive but antimycin A‐insensitive, and the production of H2O2 during SHAM‐stimulated NADH‐dependent O2 uptake was demonstrated. Irrespective of the mechanism, SHAM‐stimulated respiration by root plasmalemma makes it difficult to interpret results on root respiration obtained using KCN and SHAM.

Structure prediction for the di-heme cytochrome b561 protein family.

Summary. Atomic models possessing the common structural features identified for the cytochrome b561 (cyt b561) protein family are presented. A detailed and extensive sequence analysis was performed in order to identify and characterize protein sequences in this family of transmembrane electron transport proteins. According to transmembrane helix predictions, all sequences contain 6 transmembrane helices of which 2–6 are located closely in the same regions of the 26 sequences in the alignment. A mammalian (Homo sapiens) and a plant (Arabidopsis thaliana) sequence were selected to build 3-dimensional structures at atomic detail using molecular modeling tools. The main structural constraints included the 2 pairs of heme-ligating His residues that are fully conserved in the family and the lipid-facing sides of the helices, which were also very well conserved. The current paper proposes 3-dimensional structures which to our knowledge are the first ones for any protein in the cyt b561 family. The highly conserved His residues anchoring the two hemes on the cytoplasmic side and noncytoplasmic side of the membrane are in all proteins located in the transmembrane helices 2, 4 and 3, 5, respectively. Several highly conserved amino acids with aromatic side chain are identified between the two heme ligation sites. These residues may constitute a putative transmembrane electron transport pathway. The present study demonstrates that the structural features in the cyt b561 family are well conserved at both the sequence and the protein level. The central 4-helix core represents a transmembrane electron transfer architecture that is highly conserved in eukaryotic species.

Localization of an Ascorbate-Reducible Cytochrome b561 in the Plant Tonoplast

As a free radical scavenger, and cofactor, ascorbate (ASC) is a key player in the regulation of cellular redox processes. It is involved in responses to biotic and abiotic stresses and in the control of enzyme activities and metabolic reactions. Cytochromes (Cyts) b561 catalyze ASC-driven trans-membrane electron transport and contribute to ASC-mediated redox reactions in subcellular compartments. Putative Cyts b561 have been identified in Arabidopsis (ecotype Columbia) on the basis of sequence similarity to their mammalian counterparts. However, little is known about the function or subcellular localization of this unique class of membrane proteins. We have expressed one of the putative Arabidopsis Cyt b561 genes (CYBASC1) in yeast and we demonstrate that this protein encodes an ASC-reducible b-type Cyt with absorbance characteristics similar to that of other members of this family. Several lines of independent evidence demonstrate that CYBASC1 is localized at the plant tonoplast (TO). Isoform-specific antibodies against CYBASC1 indicate that this protein cosediments with the TO marker on sucrose gradients. Moreover, CYBASC1 is strongly enriched in TO-enriched membrane fractions, and TO fractions contain an ASC-reducible b-type Cyt with α-band absorbance maximum near 561 nm. The TO ASC-reducible Cyt has a high specific activity, suggesting that it is a major constituent of this membrane. These results provide evidence for the presence of trans-membrane redox components in this membrane type, and they suggest the coupling of cytoplasmic and vacuolar metabolic reactions through ASC-mediated redox activity.

Benzylaminopurine-induced coupling between calmodulin and Ca-ATPase in wheat root microsomal membranes

The properties of the Ca‐ATPase prepared from roots of wheat seedlings treated with benzylaminopurine were studied. The affinity of the ATPase towards Ca2+, plant or erythrocyte calmodulin increased after the hormonal treatment. It seems that in the membrane calmodulin‐bonding sites were induced by benzylaminopurine, contributing to an increased affinity of the ATPase. The lower Ca‐content of the hormone‐treated plants suggests that in vivo the Ca‐ATPase is involved in a Ca‐extrusion process.

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.

An Arabidopsis cytochrome b561 with trans‐membrane ferrireductase capability

Ascorbate‐reducible cytochromes b561 (Cyts‐b561) are a class of intrinsic trans‐membrane proteins. Tonoplast Cyt‐b561 (TCytb), one of the four Cyt‐b561 isoforms in Arabidopsis was localized to the tonoplast. We demonstrate here that the optical spectra, EPR spectra and redox potentials of recombinant TCytb are similar to those of the well characterized bovine chromaffin granule Cyt‐b561. We provide evidence for the reduction of ferric‐chelates by the reduced TCytb. It is also shown that TCytb is capable of trans‐membrane electron transport from intracellular ascorbate to extracellular ferric‐chelates in yeast cells.

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

SummaryThe plasma membrane of higher plants contains more than one kind ofb-type cytochromes. One of these has a high redox potential and can be fully reduced by ascorbate. This component, the cytochromeb561 (cytb561), has its characteristic α-band absorbance close to 561 nm wavelength at room temperature. Cytb561 was first isolated from etiolated bean hook plasma membranes by two consecutive anion exchange chromatography steps. During the first step performed at pH 8, cytb561 did not bind to the anion exchange column, but otherb-type cytochromes did. In the second step performed at pH 9.9, cytb561 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 fromArabidopsis thaliana leaves and maize roots, the ascorbate-reducible cytb561 bound already to the first anion exchange column at pH 8 and was eluted also at an ionic strength of about 100 mM KCl. Otherb-type cytochromes than the ascorbate-reducible cytb561 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 cytb561 from different sources.

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.