Electron transport components of the MnO2 reductase system and the location of the terminal reductase in a marine Bacillus.

The response of MnO2 reduction by uninduced and induced whole cells and cell extracts of Bacillus 29 to several electron transport inhibitors was compared. MnO2 reduction with glucose by uninduced whole cells and cell extracts was strongly inhibited at 0.1 mM dicumarol, 100 mM azide, and 8 mM cyanide but not by atebrine or carbon monoxide, suggesting the involvement of a vitamin K--type quinone and a metalloenzyme in the electron transport chain. MnO2 reduction with ferrocyanide by uninduced cell extracts was inhibited by 5 mM cyanide and 100 mM azide but not by atebrine, dicumarol, or carbon monoxide, suggesting that the metalloenzyme was associated with the terminal oxidase activity. MnO2 reduction with glucose by induced whole cells and cell extracts, was inhibited by 1 mM atebrine, 0.1 mM dicumarol, and 10 mM cyanide but not by antimycin A, 2n-nonyl-4-hydroxyguinoline-N-oxide) (NOQNO), 4,4,4-trifluoro-1-(2-thienyl),1,3-butanedione, or carbon monoxide. Induced cell extract was also inhibited by 100 mM azide, but stimulated by 1 mM and 10 mM azide. Induced whole cells were stimulated by 10 mM and 100 mM azide. These results suggested that electron transport from glucose to MnO2 in induced cells involved such components as flavoprotein, a vitamin K-type quinone, and metalloenzyme. The stimulatory effect of azide on induced cells was explained on the basis of a branching in the terminal part of the electron transport chain, one branch involving a metalloenzyme for the reduction of MnO2 and the other involving a metalloenzyme for the reduction of oxygen. The latter was assumed to be the more azide sensitive. Spectral studies showed the presence of a-, b-, and c-type cytochromes in membrane but not in soluble fractions. Of these cytochromes, only the c type may be involved in electron transport of MnO2, owing to the lack of inhibition by antimycin A or 2n-nonyl-4-hydroxyquinoline-N-oxide. The terminal MnO2 reductase appears to be loosely attached to the cell membrane of Bacillus 29 because of cell fractionation it is found associated with both particulate and soluble fractions. Electron photomicrographs of bacilli attached to synthetic Fe-Mn oxide revealed an intimate contact of the cell walls with the oxide particles.     

Electron transfer from acetate to the surface of MnO2 particles by a marine bacterium

Summary Intact cells of marine pseudomonad strain BIII 88, grown in the presence of added MnSO₄ (induced cells), reduced MnO₂ aerobically and anaerobically with acetate. They did not reduce limonite (FeOOH) with acetate. Spectrophotometric evidence of respiratory pigments in the cell envelope and inhibition of MnO₂ reduction by antimycin A and NQNO indicated that a respiratory process was involved. Stimulation of MnO₂ reduction by the oxidative phosphorylation uncouplers CCCP and 2,4-DNP indicated energy conservation during the reduction. Intact cells of strain BIII 88 grown in the absence of added manganese (non-induced cells) showed marginal MnO₂-reducing activity. Cell envelope fractions from induced cells prepared with a French press exhibited higher specific MnO₂-reducing activity on average than those prepared by sonication. Cell envelope fractions from induced cells contained more manganese than cell envelope fractions from non-induced cells. Recombined cell fractions from induced cells were more active than recombined cell fractions from non-induced cells. MnO₂ reducing activity was correlated with manganese content in cell envelope fractions. Cell envelope fractions from two cultures that do not reduce. MnO₂ contained less manganese in their cell envelope fractions than similar fractions from non-induced strain BIII 88. Manganese in the cell envelope of strain BIII 88 appears to play a role in the transfer of reducing power from respiration on acetate across the cell envelope to the surface of MnO₂ particles.     

Characteristics of a succinate-di chlorophenolinophenol reductase reconstituted with bovine heart electron transport components.

The reconstituted succinate-dichlorophenolindophenol reductase complex described previously (1) has been further characterized. The complex is very sensitive to 4,4,4 trifluoro-1-(2-thienyl)-1,3 butanedione (TTB), being inhibited 50% by a concentration of 2.5 μM. Combinations of complex III (reduced ubiquinone-cytochrome c reductase) and the DCPIP reductase catalyze a TTB and antimycin A sensitive succinate-cytoohrome c reductase. These observations suggest that the reconstructed succinate-DCPIP reductase is very similar in properties to isolated complex II (succinate-ubiquinone reductase).     

Bacteriophage T4 baseplate components. II. Binding and location of bacteriophage-induced dihydrofolate reductase.

The location of T4D phage-induced dihydrofolate reductase (dfr) has been determined in intact and incomplete phage particles. It has been found that phage mutants inducing a temperature-sensitive dfr (dfrts) procude heat-labile phage particles. The structural dfr produced by these ts mutants was shown to assume different configurations depending on the temperature at which the phage is assembled. Morphogenesis of incomplete phage particles lacking the gene 11 protein on their baseplates was found to be inhibited by reagents binding to dfr, such as antibodies to dfr. Further, cofactor molecules for dfr, such as reduced nicotinamide adenine dinucleotide phosphate and reduced nicotinamide adenine dinucleotide, also inhibited the step in morphogenesis involving the addition of gene 11 product. On the other hand, inhibitors of dfr, such as adenosine dephosphoribose, stimulated the addition of the gene 11 protein. It has been concluded that the phage-induced dfr is a baseplate component which is partially covered by the gene 11 protein. The properties of phage particles produced after infection of the nonpermissive host with the one known T4D mutant containing a nonsense mutation in its dfr gene suggested that these progeny particles contained a partial polypeptide, which was large enough to serve as a structural element.     

Leucine transport system in a facultatively alkalophilic Bacillus

Some characterizations of the leucine transport system in a facultative alkalophile, which is able to grow over a wide pH range from 7.0 to 10.5, were attempted. Although the direction of a transmembrane pH gradient of the bacterium below pH 8.2 is opposite to that above pH 8.2 (N. Koyama and Y. Nosoh (1985) Biochim. Biophys. Acta 812, 206-212), leucine transport is likely to be driven only by sodium electrochemical potential irrespective of the external pH. It was suggested that histidine and sulfhydryl groups in the leucine transporter are involved in the translocation mechanism and the pK value of the histidine residue involved is approximately 7.0.     

Electron transport components involved in hydrogen oxidation in free-living Rhizobium japonicum.

Membranes from free-living Rhizobium japonicum were isolated to study electron transport components involved in H2 oxidation. The H2/O2 uptake rate ratio in membranes was approximately 2. The electron transport inhibitors antimycin A, cyanide, azide, hydroxylamine, and 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO) inhibited H2 uptake and H2-dependent O2 uptake significantly. H2-reduced minus O2-oxidized absorption difference spectra revealed peaks at 551.5, 560, and 603 nm, indicating the involvement of cytochromes c, b, and a-a3, respectively. H2-dependent cytochrome reduction was completely inhibited in the presence of 0.15 mM HQNO. This inhibition was relieved by the addition of 0.1 mM menadione. Evidence is presented for the involvement of two b-type cytochromes in H2 oxidation. One b-type cytochrome was not reduced by ascorbate and had an absorption peak at 560 nm. The reduction of this cytochrome by H2 was not inhibited by cyanide. A second b-type cytochrome, cytochrome b', was not reduced by H2 in the presence of cyanide. This cytochrome had an absorption peak at 558 nm. Carbon monoxide difference spectra with H2 as reductant provided evidence for the involvement of cytochrome o as well as cytochrome a3 in H2 oxidation. H2 uptake activity in cell-free extracts was inhibited by UV light irradiation. Most of the activity of the UV-treated extracts was restored with the addition of ubiquinone. The restored activity was inhibited by cyanide. A branched electron transport pathway from H2 to O2 is proposed.     

Coupled expression of Ca2+ transport ATPase and a dihydrofolate reductase selectable marker in a mammalian cell system.

Stable expression of a full-length cDNA encoding chicken fast muscle Ca2+ transport ATPase was obtained in a Chinese hamster lung cell line (DC-3F), using a dual-promoter expression vector (pH beta FCaA3) in which the ATPase was cloned downstream of a human beta-actin gene promoter, and a mutant dihydrofolate reductase cDNA (A3/DHFR) was cloned downstream of an SV40 promoter-enhancer. Owing to its essentially normal catalytic activity and modest (20-fold) resistance to the antifolate methotrexate (MTX), the A3/DHFR mutant enzyme served as an efficient dominant selection marker in transfected cell populations challenged with MTX and, within a broad range of drug concentrations, allowed subsequent amplification and overexpression of vector sequences. In stable transfectants, the expressed ATPase was targeted to intracellular membranes, and the microsomal fractions from those cells exhibited high rates of Ca2+ transport. In comparative experiments using transient expression in COS1 cells, the level of ATPase per transfected cell was greater, but less than 5% of the transfected population exhibited ATPase expression. Furthermore, as opposed to the stable lines, the transiently expressing cells could not be propagated. Overall, the yield of ATPase was 12-16 and 4-6 micrograms per milligram of microsomal protein in the stable and the transient expression systems, respectively. The advantages of the stably transfected cell lines therefore lie in the homogeneity of ATPase expression and its distribution in cells and microsomes, in the large yield of microsomes obtained by continuous cell propagation, and in the reproducible functional characteristics of the microsomes. Moreover, the microsomes derived from stably transfected cell lines provide a convenient system for studies of Ca2+ transport and ATPase partial reaction, eliminating the need to conduct repetitive transient transfections to obtain sufficient amounts of enzyme for functional studies.     

Third system for neutral amino acid transport in a marine pseudomonad.

Uptake of leucine by the marine pseudomonad B-16 is an energy-dependent, concentrative process. Respiratory inhibitors, uncouplers, and sulfhydryl reagents block transport. The uptake of leucine is Na+ dependent, although the relationship between the rate of leucine uptake and Na+ concentration depends, to some extent, on the ionic strength of the suspending assay medium and the manner in which cells are washed prior to assay. Leucine transport can be separated into at least two systems: a low-affinity system with an apparent Km of 1.3 X 10(-5) M, and a high-affinity system with an apparent Km of 1.9 X 10(-7) M. The high-affinity system shows a specificity unusual for bacterial systems in that both aromatic and aliphatic amino acids inhibit leucine transport, provided that they have hydrophobic side chains of a length greater than that of two carbon atoms. The system exhibits strict stereospecificity for the L form. Phenylalanine inhibition was investigated in more detail. The Ki for inhibition of leucine transport by phenylalanine is about 1.4 X 10(-7) M. Phenylalanine itself is transported by an energy-dependent process whose specificity is the same as the high-affinity leucine transport system, as is expected if both amino acids share the same transport system. Studies with protoplasts indicate that a periplasmic binding protein is not an essential part of this transport system. Fein and MacLeod (J. Bacteriol. 124:1177-1190, 1975) reported two neutral amino acid transport systems in strain B-16: the DAG system, serving glycine, D-alanine, D-serine, and alpha-aminoisobutyric acid; and the LIV system, serving L-leucine, L-isoleucine, L-valine, and L-alanine. The high-affinity system reported here is a third neutral amino acid transport system in this marine pseudomonad. We propose the name "LIV-II" system.     

Characterization of components of the anaerobic ribonucleotide reductase system from Escherichia coli.

Anaerobic growth of Escherichia coli induces an oxygen-sensitive ribonucleoside triphosphate reductase system, different from the aerobic ribonucleoside diphosphate reductase (EC 1.17.4.1) of aerobic E. coli and higher organisms (Fontecave, M., Eliasson, R., and Reichard, P. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2147-2151). We have now purified and characterized two proteins from the anaerobic system, provisionally named dA1 and dA3. dA3 is the actual ribonucleoside triphosphate reductase; dA1 has an auxiliary function. From gel filtration, dA1 and dA3 have apparent molecular masses of 27 and 145 kDa, respectively. In denaturing gel electrophoresis, dA3 gives two bands of closely related polypeptides with apparent molecular masses of 77 (beta 1) and 74 (beta 2) kDa. Immunological and structural evidence suggests that beta 2 is a degradation product of beta 1 and that the active enzyme is a dimer of beta 1. dA1 activity coincides on denaturing gels with a band of 29 kDa and thus appears to be a monomer. The reaction requires, in addition, an extract from E. coli heated for 30 min at 100 degrees C. Potassium is one required component, but one or several others remain unidentified and are provisionally designated fraction RT. With dA3, dA1, RT, and potassium ions, CTP reduction shows absolute requirements for S-adenosylmethionine, NADPH (with NADH as a less active substitute), dithiothreitol, and magnesium ions, and is strongly stimulated by ATP, probably acting as an allosteric effector. Micromolar concentrations of several chelators inhibit CTP reduction completely, suggesting the involvement of (a) transition metal(s).     

Electron transport system of an aerobic carbon monoxide-oxidizing bacterium.

Experiments with crude extracts of Pseudomonas carboxydohydrogena revealed that a quinone is necessary for CO oxidation, and that cytochromes of the a, b, and c types and functional terminal oxidase(s) are found in cells grown on CO as the sole source of carbon and energy. CO dehydrogenase delivers electrons to the electron transport system at the level of quinone, and nicotinamide adenine dinucleotide (phosphate) is not involved in CO oxidation.     

Pathways of terminal respiration in marine invertebrates. II. The cytochrome system of Aplysia

The classic spectrophotometric method for identification and characterization of respiratory enzymes has been used for the study of the cytochrome system of Aplysia. Particles have been prepared from the buccal mass and the gizzard muscles. Difference spectra taken on isolated particle suspensions show the presence of a complete cytochrome system composed of five components: cytochrome a, b, c, c(1), and a(3). As indicated by the peaks of the sharp absorption bands of their reduced forms, they are very similar to the cytochromes of mammals and yeast. Cytochrome a(3) has been identified as the terminal oxidase of Aplysia muscle by means of the spectrophotometric study of its carbon monoxide compound. Further evidence for the presence of a cytochrome system in Aplysia was obtained by assays of the catalytic activities of the isolated particles: succinic dehydrogenase, cytochrome oxidase, DPNH cytochrome c reductase. The cytochrome oxidase activity was strongly inhibited by carbon monoxide in the dark; the inhibition was totally relieved by light. Cytochrome c has been extracted and purified from muscle tissue. Its spectrum is almost identical with that of the mammalian pigment both in the oxidized and reduced forms. From the hepatopancreas a new respiratory enzyme has been extracted which has many physical and chemical properties in common with cytochrome h from terrestrial snails.