The 2.3 angstrom X-ray structure of nitrite reductase from Achromobacter cycloclastes.

The three-dimensional crystal structure of the copper-containing nitrite reductase (NIR) from Achromobacter cycloclastes has been determined to 2.3 angstrom (A) resolution by isomorphous replacement. The monomer has two Greek key beta-barrel domains similar to that of plastocyanin and contains two copper sites. The enzyme is a trimer both in the crystal and in solution. The two copper atoms in the monomer comprise one type I copper site (Cu-I; two His, one Cys, and one Met ligands) and one putative type II copper site (Cu-II; three His and one solvent ligands). Although ligated by adjacent amino acids Cu-I and Cu-II are approximately 12.5 A apart. Cu-II is bound with nearly perfect tetrahedral geometry by residues not within a single monomer, but from each of two monomers of the trimer. The Cu-II site is at the bottom of a 12 A deep solvent channel and is the site to which the substrate (NO2-) binds, as evidenced by difference density maps of substrate-soaked and native crystals.

Mouse monoclonal antibody to embryonic antigen: development, cross-reactivity with rodent and human tumors, and preliminary polypeptide characterization.

Hybridomas producing IgM and IgG monoclonal antibodies (MoAb) to embryonic or fetal antigens (EA) were obtained in a completely syngeneic system. Lethally irradiated, 13-day-gestation, C57BL/6N mouse fetal cells or KCI extracts of these fetal cells obtained from primaparous donors were used as immunogens in several regimens to induce splenocytes in C57BL/6N mice that were utilized to form the hybridomas following fusion with a mouse myeloma line. Successful growth and cloning of the IgM-producing hybridomas required supplementation with factor(s) produced in the growth medium of the macrophage cell line RAW 264.7. An enzyme-linked immunosorbent assay (ELISA) was employed to screen the primary fusion hybridomas for antibody directed against fetal cell or adult cell determinants with the use of freshly explanted tissues. Glutaraldehyde-fixed fetal cells as well as crude fetal cell membranes were used as EA+ target cells (i.e., cell lines known to activate T-lymphocyte-mediated tumor resistance) in a solid-phase ELISA to perform quantitative ELISA adsorption tests of the MoAb. The anti-EA monoclonal IgM and the IgG detected common, embryo-specific antigen(s) on mouse, hamster, and human fetuses. Term fetal cells and adult normal tissues of the mouse, hamster, and human did not express cross-reactive determinants for the MoAb by absorption analysis and/or by direct binding in ELISA. EA expression as oncofetal antigens could also be detected with the monoclones on several rodent tumor cell lines tested as well as on a variety of human carcinomas but not on a spectrum of normal human tissues with the use of indirect ELISA absorption and affinity gel and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses. Fluorescence analysis with the monoclones demonstrated specific reactivity with the surface of EA+ tumor cells in the FACS IV flow cytometer. The responsible antigen was carried on a 44- and a 200-kilodalton polypeptide.

Differential extraction of tumor-specific transplantation antigen and embryonic antigen from simian virus 40- and adenovirus 7-induced sarcoma cells of hamsters with 1-butanol and 3 M potassium chloride.

Simian virus 40 (SV40)-induced sarcomas and adenovirus 7-induced sarcomas (Adv-7) exhibit both specific tumor-specific transplantation antigens (TSTA) and cross-protective embryonic antigens at the cell surface in the LAK:LVG(SYR) strain of Syrian golden hamsters. Specific SV40 TSTA could be released from the surfaces of living hamster sarcoma cells in a 2.5% crude 1-butanol extract (CBE) and served as immunogen to protect syngeneic recipients against subsequent homologous but not heterologous tumor cell challenge. The CBE-extracted SV40-induced TSTA (tumor-specific) was observed to be free of detectable, cross-protective embryonic antigens (EA) by tumor transplantation assays. The induction of cytotoxic lymphocyte-mediated immunity with the CBE-released TSTA was dependent on the administration of a single sensitizing injection of 12-20 micrograms antigen protein. Higher concentrations (50-1,000 micrograms) of the CBE tumor cell extract, given in a single injection, enhanced tumor growth as did two injections of 12.5 micrograms CBE-extracted SV40-induced TSTA at 1-week intervals. A cross-protective antigen(s), not detected in the CBE tumor extracts, was retained in the intact, 1-butanol-extracted SV40 and Adv-7-induced tumor cell lines after completion of the CBE extraction procedure and in similarly extracted 10-day hamster fetal cells. Some alterations in the normal immunogenicity of EA extracted with CBE followed by KCI from SV40-induced sarcoma cells could be detected in the transplantation assays and lymphocyte transformation assays, whereas EA extracted from CBE-KCI-treated Adv-7 cells or 10-day hamster fetal cells retained normal immunogenicity in vivo and in vitro. These procedures provide a means for successful separation of immunogenic SV40- and Adv-7-induced TSTA from detectable, biologically active, cross-protective EA from the surfaces of these sarcoma cells.

Detergent inhibition of nitric-oxide reductase activity.

Gas chromatography revealed that exposure of extracts of the denitrifiers 'Achromobacter cycloclastes', Paracoccus denitrificans, Pseudomonas aeruginosa and Pseudomonas perfectomarina to Triton X-100 inhibited reduction of NO to N2O, and thus concomitantly inhibited reduction of NO2- to N2O. After exposure of extracts to Triton X-100, the ratio of H+ consumed to NO2- added decreased from approx. 2.0 (for untreated extracts) to approx. 1.5, which indicated that NO2- was reduced to NO by the treated extracts. Addition of a CHAPS-soluble extract (devoid of nitrite reductase activity but rich in nitric-oxide reductase activity) to the Triton X-100-treated extract of P. denitrificans restored capacity for reduction of NO2- on to N2O. Exposure to either the NO that accumulated from reduction of NO2- or to enthetic NO transiently inhibited rates of NO2- reduction in Triton X-100-treated extracts. Use of an Oxides of Nitrogen analyzer indicated that only 5-33% of NO2- reduced by untreated extracts appeared in the stripping gas as NO, whereas 80-95% of NO2- reduced by Triton X-100-treated extracts was recovered as NO.

Crystallization of nitrite reductase from Achromobacter cycloclastes.

Crystals of a green copper-containing nitrite reductase from Achromobacter cycloclastes, which diffract to high resolution, belong to the cubic space group P213, with a = b = c = 98.4 A. Crystals of a nitrite reductase from Alcaligenes faecalis S-6 have been made, and belong to space group P212121, with a = 77.3 A, b = 94.6 A and c = 141 A. Crystals of the blue copper protein from Ac. cycloclastes have also been obtained: these belong to space group P21212, with cell dimensions a = 34.9 A, b = 91.1 A and c = 36.7 A (1 A = 0.1 nm).

Comparative EPR studies on the nitrite reductases from Escherichia coli and Wolinella succinogenes.

Hexaheme nitrite reductases purified to homogeneity from Escherichia coli K-12 and Wolinella succinogenes were studied by low-temperature EPR spectroscopy. In their isolated states, the two enzymes revealed nearly identical EPR spectra when measured at 12 K. Both high-spin and low-spin ferric heme EPR resonances with g values of 9.7, 3.7, 2.9, 2.3 and 1.5 were observed. These signals disappeared upon reduction by dithionite. Reaction of reduced enzyme with nitrite resulted in the formation of ferrous heme-NO complexes with distinct EPR spectral characteristics. The heme-NO complexes formed with the two enzymes differed, however, in g values and line-shapes. When reacted with hydroxylamine, reduced enzymes also showed the formation of ferrous heme-NO complexes. These results suggested the involvement of an enzyme-bound NO intermediate during the six-electron reduction of nitrite to ammonia catalyzed by these two hexaheme nitrite reductases. Heme proteins that can either expose bound NO to reduction or release it are significant components of both assimilatory and dissimilatory metabolisms of nitrate. The different ferrous heme-NO complexes detected for the two enzymes indicated, nevertheless, their subtle variation in heme reactivity during the reduction reaction.

Regulation of the hexaheme nitrite/nitric oxide reductase of Desulfovibrio desulfuricans, Wolinella succinogenes and Escherichia coli. A mass spectrometric study.

Dissimilatory nitrite reduction, carried out by hexaheme proteins, gives ammonia as the final product. Representatives of this enzyme group from 3 bacterial species can also reduce NO to either ammonia or N2O. The redox regulation of the nitrite/nitric oxide activities is discussed in the context of the denitrifying pathway.

Localization and specificity of cytochromes and other electron transfer proteins from sulfate-reducing bacteria.

Recently data have accumulated concerning the electron transfer chains of sulfate-reducing bacteria in general and of the genus Desulfovibrio in particular. Because of the ever growing number of newly discovered individual redox proteins, it has become essential to try to assign them to physiologically relevant chains. This work presents some new data concerning the localization of these proteins within the bacterial cell and the specificity of electron transfer between the three types of hydrogenases which have been found so far in Desulfovibrio, namely the iron-only, the iron-nickel and the iron-nickel-selenium enzymes. The iron-only hydrogenase reduces cytochromes which have bis-histidinyl heme ligation or histidinyl-methionyl heme ligation. In contrast, the iron-nickel and iron-nickel-selenium hydrogenases cannot reduce cytochromes having a His-Met heme ligation, but are very active toward the cytochromes having a bis-histidinyl ligand. This observation has been used to demonstrate that the tetraheme cytochrome c3 can exchange electrons with the monoheme cytochrome c553. No clear specificity has been established for the reaction of hydrogenases toward the hexadecaheme cytochromes from either D vulgaris or D gigas.

Microbial and plant metabolism of NO.

During microbial denitrification, NO is produced by reduction of nitrite by either the reduced high spin d1 hemes in a unique reductase (NIR) or at the expense of a blue copper protein that transfers electrons that move first to a type I copper and then to a type II copper in a unique trimeric NIR. This latter type of NIR is also produced by several denitrifying filamentous fungi. Reduction of NO is then carried out by either a specific cytochrome be complex NOR in denitrifying bacteria or a unique cytochrome P-450 in denitrifying filamentous fungi. NO is also produced by an anomalous reaction of a molybdoprotein, nitrate reductase (NAR), acting on an odd substrate, NO2-. NO is also reduced by a multiheme NIR that serves physiologically for reduction of NO2- to NH3. This type NIR reduces NO to either N2O, if only partially reduced, or NH3, if fully reduced, when it encounters NO. This multiheme NIR is very sensitive to cyanide. Transcription of the genes for NIR and NOR production in a denitrifier is activated by NO, a process that also requires the presence of the gene product, a transcriptional activator, NnrR.

Role of the Salt Marsh Grass Spartina alterniflora in the Response of Soil-Denitrifying Bacteria to Glucose Enrichment.

Long-term incubations of salt marsh soil systems in the presence of glucose resulted in a decrease in the soils' denitrification potential. Addition of nitrate or the presence of living Spartina alterniflora reversed this effect, indicating that Spartina, through the establishment of an oxidized rhizosphere where nitrification can occur, enables the denitrifying bacteria to adequately compete with the less energetically efficient components of the anaerobic soil microbial community.

Effect of the Spartina alterniflora Root-Rhizome System on Salt Marsh Soil Denitrifying Bacteria.

Nitrous oxide (N(2)O) reductase activity was used as an index of the denitrification potential in salt marsh soils. In a short Spartina alterniflora marsh, the seasonal distribution of N(2)O reductase activity indicated a causal relationship between S. alterniflora root-rhizome production and the denitrification potential of the soil system. The relationship was not discerned in samples from a tall S. alterniflora marsh. To further examine the in situ plant-denitrifier interaction in the short S. alterniflora marsh, plots with and without living S. alterniflora were established and analyzed for N(2)O reductase activity 5 and 18 months later. In the plots without living Spartina there was a significant reduction in the soil denitrification potential after 18 months, indicating that in the SS marsh the denitrifiers are tightly coupled to the seasonal production of below-ground Spartina macroorganic matter. In plots with intact Spartina, the soil denitrification potential was not altered by NH(4)NO(3) or glucose enrichment. However, in plots without living Spartina, there were significant changes in soil N(2)O reductase activity, thus indicating that the plants can serve as a "buffer" against this form of pulse perturbation.

Respiration-linked proton flux in Wolinella succinogenes during reduction of N-oxides.

Formate uncoupled proton translocation in formate-grown Wolinella succinogenes cells supplied with N-oxides as terminal electron acceptors. In suspensions containing KSCN (but not valinomycin), H2 supported proton translocation when NO3-, NO2-, and NO were provided as oxidants. H+/N-oxide ratios were 4.77 for NO3-, 2.49 for NO2-, and 1.75 for NO. KSCN inhibits N2O reduction thus precluding use of N2O as oxidant. Repeated exposure of cells to NO inhibited their ability to translocated protons with NO as oxidant but only slightly diminished and did not eliminate their capacity for NO3(-)- or NO2(-)-dependent proton flux. Substituting reduced benzyl viologen for H2 and measuring proton uptake provided results consistent with an extramembranal location for the N- oxide reductases. The uncoupler, carbonyl cyanide m-chlorophenylhydrazone, collapsed proton gradients, permitted uptake of 2 mol H+/mol NO3- or NO2-, but unaccountably inhibited NO3- reduction by 50% while leaving H+ uptake stoichiometry of the cells unaffected.

Influence of Na+ on synthesis of macromolecules by a marine bacterium.

Resting cells of Vibrio natriegens acquired the ability to take up (14)C-labeled mannitol in media containing Na(+) and K(+). But, the cells took up a significant quantity of the label as well in the presence of 0.3 m K(+) and no Na(+). The label was distributed throughout the cells in both systems. Cells incubated in mannitol minimal culture medium proliferated and synthesized approximately nine times as much protein in the presence of Na(+) and K(+) as those incubated in the presence of mannitol and 0.3 m K(+). The bacteria did not proliferate in the absence of Na(+). Cells incubated in medium containing mannitol and Na(+) and K(+) synthesized approximately twice the quantity of deoxyribonucleic acid and ribonucleic acid as those incubated in medium containing mannitol and 0.3 m K(+) but no Na(+). A significant amount of mannitolbinding protein was synthesized in the membranes of V. natriegens incubated in the presence of mannitol and Na(+) and K(+), but only a small quantity was produced in medium containing mannitol and 0.3 m K(+) but no Na(+). A binding fraction comprising at least two proteins (both with molecular weight near 34,000) was isolated by gel electrophoresis from other components of a K(2)CO(3)-extract of membrane protein from mannitol-grown cells. This binding fraction mediated phosphorylation of mannitol at the expense of either adenosine triphosphate or phosphoenolpyruvate. It was then found that mannitol-grown, but not broth-grown, cells contained nicotinamide adenine dinucleotide-linked mannitol-1-phosphate dehydrogenase. Neither contained mannitol dehydrogenase.

Flavin-linked dehydrogenation of ether glycols by cell-free extracts of a soil bacterium.

Payne, W. J. (University of Georgia, Athens), and R. L. Todd. Flavin-linked dehydrogenation of ether glycols by cell-free extracts of a soil bacterium. J. Bacteriol. 91:1533-1536. 1966.-Cell-free extracts of bacterium TEG-5 grown on tetraethylene glycol dehydrogenated a variety of ether glycols and nonylphenoxy and secondary alcohol ethoxy derivatives. Nicotinamide nucleotides did not serve as electron acceptors, but ferricyanide was effective. Dialysis of crude extract depressed activity with tetraethylene glycol, which was restored then by flavine adenine dinucleotide (FAD) or boiled extract supernatant fluid (BES) but not by other flavins. Precipitatation of extract protein at pH 4.0 at 80% ammonium sulfate saturation dissociated FAD and yielded an inactive fraction. Activity was restorable by FAD and BES but not by other flavins. Ethylene glycol was not dehydrogenated by the acid ammonium sulfate fraction with FAD. Atabrine inhibited tetraethylene glycol oxidation, and the inhibition was relieved by FAD but not by other flavins. Tergitols which have sulfated ethoxy side chains on secondary alcohols were not dehydrogenated, but those with free ethoxy side chains on identical alcohols were.

Influence of cations on spheroplasts of marine bacteria functioning as osmometers.

Penetration of substrates into marine bacteria as influenced by cations has been demonstrated by the effects of increased osmotic pressure in spheroplasts of these cells. Spheroplasts of Pseudomonas natriegens, stabilized with lactose, underwent a metabolic swelling in the presence of a substrate to which they had been induced. Maximal and persistent swelling was achieved only by addition of catabolizable substrate and both Na(+) and K(+). Addition, along with substrate, of Na(+) alone or K(+) alone did not stimulate swelling; no metabolic swelling occurred in the presence of a sugar to which the cells had not been induced. Confirmation of rapid uptake by induced cells of the inducer sugar, l-arabinose, but not the d-isomer, was obtained with (14)C-labeled substrate. Addition of NaN(3) completely inhibited swelling, and 2, 4-dinitrophenol and ouabain each suppressed it by 50%, indicating requirement for energy metabolism and involvement of an adenosine triphosphatase in the penetration phenomena of these cells.