Characterization of in vitro proton pumping by microsomal vesicles isolated from corn coleoptiles

Corn (Zea mays L. cv Golden Cross Bantam) coleoptile microsomal vesicles have been isolated which are capable of ATP-driven H⁺-transport as measured by [¹⁴C]methylamine accumulation and quinacrine fluorescence quenching. Formation of the pH gradient in vitro shows a high specificity for ATP·Mg, is temperature-sensitive, exhibits a pH optimum at 7.5, and is inhibited by carbonyl cyanide-m-chlorophenylhydrazone. Of the divalent cations tested, Mn²⁺ is almost as effective as Mg²⁺, while Ca²⁺ is ineffective. Excess divalent cations, particularly Ca²⁺, reduces the pH gradient. H⁺ transport is strongly promoted by anions, especially chloride, while potassium does not affect pump activity. Studies with ³⁶Cl⁻ indicate that ATP-driven H⁺ transport into the vesicles is associated with chloride uptake. Both carbonyl cyanide-m-chlorophenylhydrazone and the anion transport inhibitor, 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene, inhibit methylamine accumulation and ³⁶Cl⁻ uptake. Proton pumping is also blocked by diethyl stilbestrol and N,N′-dicyclohexylcarbodiimide, but is insensitive to oligomycin and vanadate. These properties of the pump are inconsistent with either a mitochondrial or plasma membrane origin.     

细菌视紫红质中质子泵出的分子机理

细菌视紫红质(Bacteriorhodopsin,或bR)是盐生嗜盐菌(Halobacterium salinarium)等细菌的跨膜蛋白质,其色基视黄醛的光致异构化作用触发细菌视紫红质的一系列结构变化,把质子从细胞质泵到细胞外空间。对细菌视紫红质中质子泵出分子机理进行了描述。     

Apical endosomes isolated from kidney collecting duct principal cells lack subunits of the proton pumping ATPase

Endocytic vesicles that are involved in the vasopressin-stimulated recycling of water channels to and from the apical membrane of kidney collecting duct principal cells were isolated from rat renal papilla by differential and Percoll density gradient centrifugation. Fluorescence quenching measurements showed that the isolated vesicles maintained a high, HgCl2-sensitive water permeability, consistent with the presence of vasopressin-sensitive water channels. They did not, however, exhibit ATP-dependent luminal acidification, nor any N-ethylmaleimide-sensitive ATPase activity, properties that are characteristic of most acidic endosomal compartments. Western blotting with specific antibodies showed that the 31- and 70-kD cytoplasmically oriented subunits of the vacuolar proton pump were not detectable in these apical endosomes from the papilla, whereas they were present in endosomes prepared in parallel from the cortex. In contrast, the 56-kD subunit of the proton pump was abundant in papillary endosomes, and was localized at the apical pole of principal cells by immunocytochemistry. Finally, an antibody that recognizes the 16-kD transmembrane subunit of oat tonoplast ATPase cross-reacted with a distinct 16-kD band in cortical endosomes, but no 16-kD band was detectable in endosomes from the papilla. This antibody also recognized a 16-kD band in affinity-purified H+ ATPase preparations from bovine kidney medulla. Therefore, early endosomes derived from the apical plasma membrane of collecting duct principal cells fail to acidify because they lack functionally important subunits of a vacuolar-type proton pumping ATPase, including the 16-kD transmembrane domain that serves as the proton-conducting channel, and the 70-kD cytoplasmic subunit that contains the ATPase catalytic site. This specialized, non-acidic early endosomal compartment appears to be involved primarily in the hormonally induced recycling of water channels to and from the apical plasma membrane of vasopressin-sensitive cells in the kidney collecting duct.     

Effects of Triton X-100 on proton transfer and in the photocycle of archaerhodopsin 4

Archaerhodopsin 4 (AR4), a retinal-containing membrane protein, exhibits a reversed order of proton release and uptake at neutral pH, as compared to the well-known bacteriorhodopsin (BR). In a preceding report, we stated that Triton X-100 solubilized the claret membrane containing AR4 (CM) into monomeric proteins and altered the time order in AR4 at neutral pH. The present study examined the mechanism underlying this phenomenon. We employed a photoelectrochemical cell suitable for observation of the proton pumping behaviors of both the membrane patch and detergent-solubilized proteins over a wide pH range. The pK(a) values of the proton release complex (PRC) in the initial state and the M state were determined with this device. The pK(a) of PRC of monomeric AR4 decreased to a value lower than 7.0 in the photocycle, allowing early proton release at neutral pH. The pK(a) of PRC in the initial state was also strongly affected by solubilization.     

Characterization of the 7S RNA and its gene from halobacteria.

The 7S RNA is an abundant nonribosomal RNA in H. halobium and other halobacteria. A specific 7S RNA gene probe shows high homology to genomic DNA of all halobacteria tested but not to those of several other archaebacteria, eubacteria and eukaryotes. All halobacterial genomes seem to carry a single copy of the 7S RNA gene. The coding region of the 7S RNA gene is highly G+C rich whereas the 5'- and 3'-noncoding regions possess a rather low G+C content. An extended double stranded structure for the 7S RNA is deduced from its nucleotide sequence. The 7S RNA of H. halobium (304 nucleotides) resembles in size and structure the 7S-L RNA from mammalian cells and shares with it a sequence homology of about 50% when arranged in a colinear fashion. The similarities in sequence are found particularly at the 3'- and 5'-termini. No similarity was detected between the 7S RNA from H. halobium and the nonribosomal 6S RNA from Escherichia coli.     

Heat treatment of cytochrome c oxidase perturbs the CuA site and affects proton pumping behavior

It has been previously reported that mild heat treatment (43 degrees C for ca. 60 min) abolishes the proton pumping activity of cytochrome c oxidase while leaving the oxidase activity and cytochromes a and a3 unperturbed [Sone, N., & Nicholls, P. (1984) Biochemistry 23, 6550-6554]. We herein describe the effects of this heat treatment on the electron paramagnetic resonance (EPR) and optical absorption signatures of the redox-active metal centers in the enzyme. We find that heat treatment of the oxidized enzyme causes a local structural perturbation at the CuA site. After heat treatment, the enzyme sample contains three subpopulations, each of which has a different structure at CuA. These include (i) native CuA, (ii) a type 2 copper species similar to the one produced by chemical modification by p-(hydroxymercuri)benzoate (pHMB) [Gelles, J., & Chan, S. I. (1985) Biochemistry 24, 3963-3972], and (iii) a novel type 1 copper species. In addition to changes at the CuA site, we find that heat treatment results in accelerated cyanide binding and the removal of subunit III. If the cytochrome c oxidase is heat treated while fully reduced, none of these changes are observed except for subunit III depletion. Furthermore, partial (CO mixed-valence derivative) reduction of the enzyme as well as ligand binding to cytochrome a3 also protects the enzyme against the heat-induced changes, indicating that the oxygen binding site plays a role in stabilizing the CuA site against structural perturbations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of a prokaryotic photoreceptor: sensory rhodopsin from halobacteria

The photoreceptor sensory rhodopsin was isolated from halobacterial cell membranes solubilized in laurylmaltoside. In the presence of retinal, detergent and salt the native protein was obtained in pure form by sucrose density gradient centrifugation, hydroxyapatite chromatography and gel filtration. The apparent mol. wt of the molecule was 24 kd if analyzed by SDS gel electrophoresis, and 49 kd by sedimentation and size-exclusion chromatographic analysis. The chromoprotein had an absorption maximum at 580 nm which was 8 nm blue-shifted compared to the membrane-bound state. The molecule was photochemically active and the action spectrum for formation of SR380, the long-lived intermediate, coincided with the absorption spectrum.     

Defective strain of Mycoplasma pneumoniae isolated from a patient

Abstract The isolation from a patient, for the first time, of a defective Mycoplasma pneumoniae strain (146-2) is herewith reported. The strain was isolated 28 days after onset of the disease. It is devoid of two characteristic properties: haemadsorption and adherence to glass. The absence of these two characteristics coincides with significant physiological modifications of the microbial cell. Cell multiplication of strain 146-2 in the logarithmic phase is more rapid and is followed by the culture's rapid decline and death. The sera obtained from the immunized rabbits showed that the strain is immunogenic.     

Stochastic rotational catalysis of proton pumping F-ATPase

F-ATPases synthesize ATP from ADP and phosphate coupled with an electrochemical proton gradient in bacterial or mitochondrial membranes and can hydrolyse ATP to form the gradient. F-ATPases consist of a catalytic F1 and proton channel F0 formed from the alpha3beta3gammadelta and ab2c10 subunit complexes, respectively. The rotation of gammaepsilonc10 couples catalyses and proton transport. Consistent with the threefold symmetry of the alpha3beta3 catalytic hexamer, 120 degrees stepped revolution has been observed, each step being divided into two substeps. The ATP-dependent revolution exhibited stochastic fluctuation and was driven by conformation transmission of the beta subunit (phosphate-binding P-loop/alpha-helix B/loop/beta-sheet4). Recent results regarding mechanically driven ATP synthesis finally proved the role of rotation in energy coupling.     

The mechanism of rotating proton pumping ATPases

Two proton pumps, the F-ATPase (ATP synthase, F_oF₁) and the V-ATPase (endomembrane proton pump), have different physiological functions, but are similar in subunit structure and mechanism. They are composed of a membrane extrinsic (F₁ or V₁) and a membrane intrinsic (F_o or V_o) sector, and couple catalysis of ATP synthesis or hydrolysis to proton transport by a rotational mechanism. The mechanism of rotation has been extensively studied by kinetic, thermodynamic and physiological approaches. Techniques for observing subunit rotation have been developed. Observations of micron-length actin filaments, or polystyrene or gold beads attached to rotor subunits have been highly informative of the rotational behavior of ATP hydrolysis-driven rotation. Single molecule FRET experiments between fluorescent probes attached to rotor and stator subunits have been used effectively in monitoring proton motive force-driven rotation in the ATP synthesis reaction. By using small gold beads with diameters of 40-60 nm, the E. coli F₁ sector was found to rotate at surprisingly high speeds (> 400 rps). This experimental system was used to assess the kinetics and thermodynamics of mutant enzymes. The results revealed that the enzymatic reaction steps and the timing of the domain interactions among the β subunits, or between the β and γ subunits, are coordinated in a manner that lowers the activation energy for all steps and avoids deep energy wells through the rotationally-coupled steady-state reaction. In this review, we focus on the mechanism of steady-state F₁-ATPase rotation, which maximizes the coupling efficiency between catalysis and rotation.     

Refolding and proton pumping activity of a polyethylene glycol-bacteriorhodopsin water-soluble conjugate.

Bacteriorhodopsin (BR), from the purple membrane (PM) of Halobacterium halobium, was chemically modified with methoxypolyethylene glycol (m-PEG; molecular weight = 5,000 Da) succinimidyl carbonate. The polyethylene glycol-bacteriorhodopsin (m-PEG-SC-BR33) conjugate, containing one polyethylene glycol chain, was water soluble. The secondary structure of the conjugate in water appeared partially denatured, but was shown to contain alpha-helical segments by circular dichroism spectroscopy. The isolated bacteriorhodopsin conjugate, with added retinal, was refolded in a mixed detergent-lipid micelle and had an absorption maximum at 555 nm. The refolded conjugate was transferred into vesicles that pumped protons, upon illumination, as efficiently as did native BR. Modification of the PM with m-PEG did not alter the native structure or inhibit proton pumping, and therefore it is suggested that the glycol polymer is present as a moiety covalently linked to residues unnecessary for proton pumping and proper folding. The site of attachment of m-PEG was determined to be at either Lys 129 or Lys 159, with position Lys 129 the most probable site of attachment. The m-PEG-SC-BR33 could be stepwise refolded to the native conformation by the addition of trifluoroethanol to lower the dielectric constant, simulating the insertion of the BR into the phospholipid bilayer.     

Genome Sequence of Campylobacter jejuni strain 327, a strain isolated from a turkey slaughterhouse

Campylobacter is one of the leading causes of food-borne gastroenteritis and has a high prevalence in poultry. Campylobacter jejuni subsp. jejuni 327 is a subspecies of the genus Campylobacter of the family Campylobacteraceae in the phylum Proteobacteria. The microaerophilic, spiral shaped, catalase positive bacterium obtains energy from the metabolism of amino acids and Krebs cycle intermediates. Strain 327 was isolated from a turkey slaughter production line and is considered environmentally sensitive to food processing (cold, heat, drying) and storage conditions. The 327 whole genome shotgun sequence of 1,618,613 bp long consists of 1,740 protein-coding genes, 46 tRNA genes and 3 rRNA operons. A protein based BLAST analysis places the turkey isolate 327 close to the human clinical strain 81116 (NCTC 11828).     

Characterization of a fungal strain isolated from a polyphenol polluted site

A group of fungal strains were isolated from a polyphenol polluted soil, taken from an olive oil processing plant in Attica, Greece. The fungi were tested for their ability to decolorize a polyaromatic dye Poly R-478, which was used as a model compound to test their ligninolytic activities. The strain K1.1 decolorized efficiently the dye on agar plates and was further studied. PCR amplification of the internal transcribed spacer (ITS) region of the ribosomal RNA genes from the genomic DNA isolated from mycelium grown in liquid culture resulted in amplified fragments. Via BLASTN search, the length of a 773 base pairs was identified as the basidiomycetes Coprinellus xanthothrix. The growth rates and the tolerance of the fungus were compared on solid media, containing four different concentrations of pentachlorophenol. Extracellular enzyme activities (lignin peroxidase, manganese peroxidase and laccase) were determined in defined liquid medium. The isolate expressed laccase and manganese peroxidase but not lignin peroxidase. The removal of the dye was also estimated in liquid medium. The fungus showed biosorption and biotransformation as removal mechanisms.     

Buffered diffusion around a spherical proton pumping cell: a theoretical analysis

H⁺ ions are a substrate of many active and passive membrane transporters in all cells. Absolute proton fluxes are often quantified using intracellular pH sensitive microelectrodes or pH sensitive dyes. These measurements, however, rely on a priori estimates of the intracellular buffer capacity and on the assumption of diffusive equilibrium inside the cell. Here, assuming local equilibrium of protons with a single mobile buffer, we model the diffusion of H⁺ in the extracellular medium around an H⁺ pumping cell to estimate the expected pH changes as a function of time, distance from the cell, extracellular buffer capacity, and the absolute proton flux across the membrane. In particular, using accurate numerical simulation, we gauge the range of validity of an explicit, analytical solution of the linearized, nonstationary diffusion equation. Our results provide a framework to quantify the absolute membrane proton flux, if spatiotemporal information about the extracellular pH change is available, e.g., using imaging of pH dependent fluorescent dyes.     

Production and characterization of xylanases of a Bacillus strain isolated from soil

Xylanase production was performed by growing a Bacillus isolate on agricultural by-products, wheat straw, wheat bran, corn cobs and cotton bagasse. A maximum xylanase activity of 180 U/ml was obtained together with a cellulase activity of 0.03 U/ml on 4 (w/v) corn cobs. Electrophoretic analysis showed the presence of three endo--1, 4-xylanases having molecular weights of about 22, 23 and 40 kDa. Xylanolytic activity was stable up to 50 °C in the pH range of 4.5–10 and the highest activity was observed at 70 °C and pH 6.5.     

Comparative ultrastructure of a mucoid strain of Pseudomonas aeruginosa isolated from cystic fibrosis patient and its spontaneous non-mucoid mutant

The ultrastructure of a mucoid strain of Pseudomonas aeruginosa of cystic fibrosis origin and its spontaneous non-mucoid variant was compared by transmission electron microscopy. Negatively-stained preparations of the mucoid strain obtained from plate cultures demonstrated dense, fibrous material projecting from the cell. No such material was observed in thin-sections or in negatively-strained preparations from liquid cultures. Thin-sections of ethanol-precipitated extracellular material from liquid cultures of the mucoid-strain revealed a cottony mesh of thin electron dense fibres. The non-mucoid strain did not produce such material. When prefixed with glutaraldehyde/malachite green mixture, cells of both strains demonstrated electron dense intracellular and extracellular malachite green-stainable structures. The internal complexes were frequently associated with the nucleoid or cell membrane and were replaced by electron transparent areas in cells prefixed with glutaraldehyde alone. Aeruginocins of the R-type were observed in mitomycin C induced cultures of both strains. Bacteriophages with 'claw-shaped' tail-tips were observed in the mucoid strain. Crystalline material was produced by the mucoid strain but only when plated on certain media.     

Allosteric interactions and proton conducting pathways in proton pumping aa3 oxidases: Heme a as a key coupling element

In this paper allosteric interactions in protonmotive heme aa₃ terminal oxidases of the respiratory chain are dealt with. The different lines of evidence supporting the key role of H⁺/e^? coupling (redox Bohr effect) at the low spin heme a in the proton pump of the bovine oxidase are summarized. Results are presented showing that the I-R54M mutation in P. denitrificans aa₃ oxidase, which decreases by more than 200 mV the E_m of heme a, inhibits proton pumping. Mutational aminoacid replacement in proton channels, at the negative (N) side of membrane-inserted prokaryotic aa₃ oxidases, as well as Zn^2 + binding at this site in the bovine oxidase, uncouples proton pumping. This effect appears to result from alteration of the structural/functional device, closer to the positive, opposite (P) surface, which separates pumped protons from those consumed in the reduction of O₂ to 2 H₂O. This article is part of a Special Issue entitled: Respiratory Oxidases.     

Characterization of cellobiohydrolase from a newly isolated strain of Agaricus arvencis

A highly efficient cellobiohydrolase (CBH)-secreting basidiomycetous fungus, Agaricus arvensis KMJ623, was isolated and identified based on its morphological features and sequence analysis of internal transcribed spacer rDNA. An extracellular CBH was purified to homogeneity from A. arvencis culture supernatant using sequential chromatography. The relative molecular mass of A. arvencis CBH was determined to be 65 kDa by SDSPAGE and 130 kDa by size-exclusion chromatography, indicating that the enzyme is a dimer. A. arvencis CBH showed a catalytic efficiency (kcat/Km) of 31.8 mM?1 s?1 for p-nitrophenyl-beta-D-cellobioside, the highest level seen for CBH-producing microorganisms. Its internal amino acid sequences showed significant homology with CBHs from glycoside hydrolase family 7. Although CBHs have been purified and characterized from other sources, A. arvencis CBH is distinguished from other CBHs by its high catalytic efficiency.