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Biomedical subjects

G Drews

Publications and source records attributed to G Drews.

At least 91 records · Page 5Linked to original sources
PubMed1988

Properties of a Tn5 insertion mutant defective in the structural gene (fruA) of the fructose-specific phosphotransferase system of Rhodobacter capsulatus and cloning of the fru regulon.

In photosynthetic bacteria such as members of the genera Rhodospirillum, Rhodopseudomonas, and Rhodobacter a single sugar, fructose, is transported by the phosphotransferase system-catalyzed group translocation mechanism. Previous studies indicated that syntheses of the three fructose catabolic enzymes, the integral membrane enzyme II, the peripheral membrane enzyme I, and the soluble fructose-1-phosphate kinase, are coordinately induced. To characterize the genetic apparatus encoding these enzymes, a Tn5 insertion mutation specifically resulting in a fructose-negative, glucose-positive phenotype was isolated in Rhodobacter capsulatus. The mutant was totally lacking in fructose fermentation, fructose uptake in vivo, phosphoenolpyruvate-dependent fructose phosphorylation in vitro, and fructose 1-phosphate-dependent fructose transphosphorylation in vitro. Extraction of the membrane fraction of wild-type cells with butanol and urea resulted in the preparation of active enzyme II free of contaminating enzyme I activity. This preparation was used to show that the activity of enzyme I was entirely membrane associated in the parent but largely soluble in the mutant, suggesting the presence of an enzyme I-enzyme II complex in the membranes of wild-type cells. The uninduced mutant exhibited measurable activities of both enzyme I and fructose-1-phosphate kinase, which were increased threefold when it was grown in the presence of fructose. Both activities were about 100-fold inducible in the parental strain. Although the Tn5 insertion mutation was polar on enzyme I expression, fructose-1-phosphate kinase activity was enhanced, relative to the parental strain. ATP-dependent fructokinase activity was low, but twofold inducible and comparable in the two strains. A second fru::Tn5 mutant and a chemically induced mutant selected on the basis of xylitol resistance showed pleiotropic loss of enzyme I, enzyme II, and fructose-1-phosphate kinase. These mutants were used to clone the fru regulon by complementing the negative phenotype with a wild-type cosmid bank.

Biological Transport↗
PubMed1988

Localization of reaction center and B800-850 antenna pigment proteins in membranes of Rhodobacter sphaeroides.

The localization of the N- and C-terminal regions of pigment-binding polypeptides of the bacterial photosynthetic apparatus of Rhodobacter sphaeroides was investigated by proteinase K treatment of chromatophore and spheroplast-derived vesicles and amino acid sequence determination. Under conditions of proteinase K treatment of chromatophores, which left the in vivo absorption spectrum and the membrane intact, 15 and 46 amino acyl residues from the N-terminal regions of the L and M subunits, respectively, of the reaction center polypeptides were removed. The N termini are therefore exposed on the cytoplasmic surface of the membrane. The C-terminal domain of the light-harvesting B800-850 alpha and B870 alpha polypeptides was found to be exposed on the periplasmic surface of the membrane. A total of 9 and 13 amino acyl residues were cleaved from the B800-850 alpha and B870 alpha polypeptides, respectively, when spheroplasts were treated with proteinase K. The N-terminal regions of the alpha polypeptides were not digested in either membrane preparation and were apparently protected from proteolytic attack. Seven N-terminal amino acyl residues of the B800-850 beta polypeptide were removed after the digestion of chromatophores. C-terminal residues were not removed after the digestion of chromatophores or spheroplasts. The C termini seem to be protected from protease attack by interaction with the membrane. Therefore, the N-terminal regions of the beta polypeptides are exposed on the cytoplasmic membrane surface. The C termini of the beta polypeptides are believed to point to the periplasmic space.

Amino Acid Sequence↗
PubMed1987

Effects of chloramine-T on charge movement and fraction of open channels in frog nodes of Ranvier.

The effects of chloramine-T, a substance which partially and irreversibly inhibits inactivation of the Na current (Wang 1984), on the on and off charge movement (Qon and Qoff) and on the relative position of the Qon-E and F-E curves were studied on voltage clamped frog nodes of Ranvier. The decrease of Qoff with increasing pulse duration which is seen in normal fibres was much less pronounced in chloramine-T-treated fibres, i.e. chloramine-T inhibited charge immobilization. Also, the decrease of the time constant tau off which normally accompanies the decrease of Qoff was absent in chloramine-T-treated fibres. A correlation (r2 = 0.80) between the relative tau off and the relative Qoff was observed in chloramine-T-treated fibres. The voltage dependence of the on charge movement, Qon-E, was compared with the voltage dependence of the fraction of open channels, F-E. In normal fibres, the F-E curve rose very steeply and crossed the normalized Qon-E curve so that F greater than Qon/Qon max for E greater than -40 mV. After chloramine-T treatment, the F-E curve rose less steeply and remained below the normalized Qon-E curve at all potentials. The effect of halazone was similar to that of chloramine-T but weaker. The observations about the Qon-E and F-E curve are compared with the findings on the squid giant axon.

Action Potentials↗
PubMed1987

Transverse membrane topography of the B875 light-harvesting polypeptides of wild-type Rhodobacter sphaeroides.

Purified B875 light-harvesting complex, chromatophores, and spheroplast-derived vesicles from wild-type Rhodobacter sphaeroides were treated with proteinase K or trypsin, and the alpha and beta polypeptides were analyzed by electrophoretic, immunochemical, and protein-sequencing methods. With the purified complex, proteinase K digested both polypeptides and completely eliminated the A875 peak. Trypsin digested the alpha polypeptide and reduced the A875 by 50%. Proteinase K cleaved the beta polypeptide of chromatophores and the alpha polypeptide of spheroplast-derived vesicles. Sequence analyses of polypeptides extracted from proteinase K-treated chromatophores revealed that the beta polypeptide was cleaved between amino acids 4 and 5 from the N terminus. The N terminus of the alpha polypeptide was intact. We concluded that the N terminus of the beta polypeptide is exposed on the cytoplasmic membrane surface, and the difference in the digestion patterns between the spheroplast-derived vesicles and chromatophores suggested that the C terminus of the alpha polypeptide is exposed on the periplasmic surface.

Amino Acid Sequence↗
PubMed1986

Localization of the exposed N-terminal region of the B800-850 alpha and beta light-harvesting polypeptides on the cytoplasmic surface of Rhodopseudomonas capsulata chromatophores.

Proteinase K and trypsin were used to determine the orientation of the light-harvesting B800-850 alpha and beta polypeptides within the chromatophores (inside-out membrane vesicles) of the mutant strain Y5 of Rhodopseudomonas capsulata. With proteinase K 7 amino acid residues of the B800-850 alpha polypeptide were cleaved off up to position Trp-7--Thr-8 of the N terminus, and 11 residues were cleaved off up to position Leu-11-Ser-12 of the beta chain N terminus. The C termini of the B800-850 alpha and beta polypeptides, including the hydrophobic transmembrane portions, remained intact. It is proposed that the N termini of the alpha and beta subunits, each containing one transmembrane alpha-helical span, are exposed on the cytoplasmic membrane surface and the C termini are exposed to or directed toward the periplasm.

Amino Acids↗
PubMed1986

Effect of uncoupler on assembly pathway for pigment-binding protein of bacterial photosynthetic membranes.

The uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) was used to investigate membrane protein assembly in the phototrophic bacterium Rhodobacter capsulatus. As found for Escherichia coli (T. Date, G. Zwizinsky, S. Ludmerer, and W. Wickner, Proc. Natl. Acad. Sci. 77:827-831, 1980) and mitochondrial proteins (N. Nelson and G. Schatz, Proc. Natl. Acad. Sci. USA 76:4365-4369, 1979), assembly across the bacterial photosynthetic membranes was sensitive to CCCP. At uncoupler concentrations which were sufficient to block the export of the periplasmic cytochrome c2 and an outer membrane protein, the integration of pigment-binding protein into the photosynthetic apparatus was abolished. The unassembled protein was detected on the inner surface of the intracytoplasmic membrane. After inactivation of CCCP, accumulated protein continued insertion into the membrane. The data suggest that after binding to the cytoplasmic face of the membrane, translocation of protein into a transmembrane orientation takes place, which is a prerequisite for the formation of a functional pigment-protein complex.

Bacterial Proteins↗
PubMed1985

Gene expression of pigment-binding proteins of the bacterial photosynthetic apparatus: Transcription and assembly in the membrane of Rhodopseudomonas capsulata.

Lowering of oxygen partial pressure in chemotrophic cultures or reduction of light intensity in phototrophic cultures of Rhodopseudomonas capsulata induced formation of the photosynthetic apparatus. A maximum of mRNA coding for the reaction center (RC) and the light-harvesting 1 B870 antenna complex polypeptides occurred 30 min after induction. Maximal expression of mRNA for B800-B850 antenna proteins appeared with a lag time of about 25 min after RC/B870 mRNA. Pigment-binding polypeptides were inserted into the membrane immediately after mRNA synthesis. It is concluded that the delayed formation of the B800-B850 complex compared to the RC and the B870 complex is caused by sequential expression of the corresponding genes. Biological activity of pigment-protein complexes increased after the incorporation of their polypeptides parallel to the maximum of bacteriochlorophyll synthesis. Studies on mutant strains defective in the formation of pigment-protein complexes suggested that pigment synthesis is of importance for assembly of stable complexes.

Journal Article↗
PubMed1985

Fusion of liposomes and chromatophores of Rhodopseudomonas capsulata: effect on photosynthetic energy transfer between B875 and reaction center complexes.

The photosynthetic chromatophore membranes of Rhodopseudomonas capsulata were fused with liposomes to investigate the effects of lipid dilution on energy transfer between the bacteriochlorophyll-protein complexes of this membrane. Phosphatidylcholine-containing liposomes were mixed with chromatophores at pH 6.0 to 6.2, and the mixture was fractionated on discontinuous sucrose gradients into four membrane fractions with lipid-to-protein ratios that varied 11-fold. Freeze-fracture electron microscopy revealed that the fractions contained closed vesicles formed by the fusion of liposomes to chromatophores. Particles with 9-nm diameters on the P fracture faces did not appear to change in size with increasing lipid content, but the number of particles per membrane area decreased proportionally with increases in the lipid-to-protein ratio. The bacteriochlorophyll-to-protein ratios, electrophoretic polypeptide profiles on sodium dodecyl sulfate-polyacrylamide gels, and light-induced absorbance changes at 595 nm caused by photosynthetic reaction centers were not altered by fusion. The relative fluorescence emission intensities due to the B875 light-harvesting complex increased significantly with increasing lipid content, but no increases in fluorescence due to the B800-B850 light-harvesting complex were observed. Electron transport rates, measured as succinate-cytochrome c reductase activities, decreased with increased lipid content. The results indicate an uncoupling of energy transfer between the B875 light-harvesting and reaction center complexes with lipid dilution of the chromatophore membrane.

Bacterial Chromatophores↗
PubMed1985

Kinetic studies on formation of cytochrome oxidase of Rhodopseudomonas capsulata after a shift from phototrophic to chemotrophic growth.

Rhodopseudomonas capsulata cells were shifted from phototrophic (anaerobic, light) to chemotrophic (semiaerobic, dark, 10% air saturation) growth conditions. During the adaptation period of 4 h, the bacteriochlorophyll content of cells and membranes decreased, and a newly synthesized 65-kilodalton polypeptide of the cytochrome oxidase was incorporated into the membrane fraction. The enzymatic activity of the cytochrome oxidase increased strongly after a lag time of 2 h. The amount of cytochrome oxidase protein does not follow the same kinetics. The relative amount of a membrane-bound cytochrome c of low molecular weight, which has been proposed to be a donor for the cytochrome oxidase, increased during adaptation.

Bacterial Proteins↗
PubMed1984

Isolation and complete amino-acid sequence of the small polypeptide from light-harvesting pigment-protein complex I (B870) of Rhodopseudomonas capsulata.

The small bacteriochlorophyll-binding polypeptide of the light-harvesting complex B870 was extracted from the intracytoplasmic membrane of the strain A1a+ of Rhodopseudomonas capsulata with chloroform/methanol/ammonium acetate and separated by chromatography on Sephadex LH60 using the same solvent. The polypeptide obtained from the peak fraction III was found to be homogeneous and identical with the small polypeptide isolated from the B870 complex as shown by dodecyl sulfate/polyacrylamide gel electrophoresis, amino acid composition and N-terminal sequence. The complete amino acid sequence is given. The relative molecular mass based on the amino acid sequence is 5341. The polarity of amino acids is 35.42%. The C-terminal part of the peptide chain from residue 29 to 48 is hydrophobic and includes one His residue.

Amino Acid Sequence↗
PubMed1984

Construction of a gene bank of Rhodopseudomonas capsulata using a broad host range DNA cloning system.

A gene bank of the phototrophic bacterium Rhodopseudomonas capsulata was constructed using the binary plasmid system pRK290/pRK2013. Fragments of about 20 kb of chromosomal DNA of R. capsulata strain 37b4 were inserted into the cloning vector pRK290. The hybrid plasmids of the gene bank, maintained in Escherichia coli HB101 were transferred by conjugation to R. capsulata strains defective in the photosynthetic apparatus with frequencies of 5 X 10(-4) to 5 X 10(-2). Phototrophically growing transconjugants occurred with frequencies of 5 X 10(-7) to 5 X 10(-6). Recombination between the hybrid plasmids and the R. capsulata chromosome was shown. The hybrid plasmid pRCF1002, carrying a 25 kb insert of R. capsulata wild type DNA, was isolated from one E. coli clone of the gene bank. It reconstituted some bacteriochlorophyll- and photosynthetic negative mutants to phototrophic growth.

Cloning, Molecular↗
PubMed1984

Transverse topography of the photochemical reaction center polypeptides in the Rhodopseudomonas capsulata membrane.

The exposure of the three polypeptide subunits H, M, and L of the photochemical reaction center (RC) on both surfaces of the membrane of Rhodopseudomonas capsulata was studied by partial proteolysis with proteinase K and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of of degradation products. The possible association of RC subunits with bacteriochlorophyll a and bacteriopheophytin was investigated by spectroscopical measurements. Chromatophores (inside-out oriented) and spheroplasts (right-side-out oriented), as well as purified, detergent-solubilized RCs and RCs reconstituted into phosphatidyl choline liposomes, were used. Subunit H of the RC was degraded to fragments with apparent MrS of 15,000 and 12,500, which were possibly derived from cleavage of a loop exposed on the cytoplasmic surface. Polypeptide M was digested at a comparable rate. The apparent Mr of M decreased by roughly 4,000 upon proteolytic cleavage. Subunit L was relatively insensitive to protease attack, except that a small peptide was clipped off. The primary donor P870 was also found to be only slightly affected proteinase K. All three RC subunits appear to be exposed on the chromatophore surface.

Antigen-Antibody Complex↗
PubMed1984

Protein on the cell surface of the moderately halophilic phototrophic bacterium Rhodospirillum salexigens.

A cell surface protein (Mr 68,000) of the moderately but obligately halophilic phototrophic bacterium Rhodospirillum salexigens was identified by two independent methods: first, by labeling the cell surface with radioactive iodine and lactoperoxidase, and second, by washing cells in 30% sucrose to remove proteins attached to the cell surface by ionic bonds. The identified protein very likely represents the outermost layer of the cell envelope of R. salexigens as observed by electron microscopy. The protein was isolated. Its isoelectric point was determined to be 4.4; the excess of acidic over basic amino acids was found to be 18.3 mol%; and its average hydrophobicity was 2.26 kJ per residue.

Cell Membrane↗
PubMed1983

Characterization of a new membrane-bound cytochrome c of Rhodopseudomonas capsulata.

A cytochrome c (cyt. c) was solubilized with Triton-X-100 and co-purified with cytochrome c oxidase from membranes of chemotrophically grown cells of Rhodopseudomonas capsulata. Cyt. c and cytochrome oxidase were separated on Sephadex G-50 columns. Antibodies against cytochrome c2 from the same bacterium did not cross react with the membrane-bound cyt. c. The IEP of the membrane-bound cyt. c was found to be pH 8.2, the midpoint potential was 234 +/- 11 mV at pH 7.0. This cyt. c binds CO. The native cyt. c is a dimer with an apparent Mr of 25000 containing 2 mol heme per mol dimer, which is believed to function as an electron donor for the high-potential cytochrome c oxidase.

Cell Membrane↗
PubMed1983

The complete amino-acid sequence of the large bacteriochlorophyll-binding polypeptide from light-harvesting complex II (B800-850) of Rhodopseudomonas capsulata.

The large bacteriochlorophyll-a-binding polypeptide of the light-harvesting complex II (B800-850), having an apparent Mr with sodium dodecyl sulfate/polyacrylamide electrophoresis of 10000, has been isolated and purified from intracytoplasmic membranes of the phototrophically negative mutant strain Y5 of Rhodopseudomonas capsulata. The primary structure of this polypeptide has been determined. The polypeptide consists of 60 amino acid residues yielding an Mr of 7322. The hydrophobic stretch in positions 16-35 with a histidine in position 31 might be of importance for interaction with bacteriochlorophyll. The C-terminal part is also hydrophobic while the N-terminal part consists of hydrophilic amino acids. The polarity of the total amino acids was determined to be 28.3%.

Amino Acid Sequence↗