Nitrite activation of nitrate reductase in higher plants

Comparative studies of nitrate-activated nitrate reductase (NR-NO₂) and nitrate-induced nitrate reductase (NR-NO₃) (EC 1.6.6.2) indicate that the enzymes differ in structure, heat stability, and pH dependence, but have the same cofactor requirment. NR-NO₂ developes in barley (Hordeum vulgare L. var. Dvir) seedlings as NR-NO₃ disappears. A transition from the active to the inactive form of nitrate reductase takes place. Nitrite seems to activate the inactive form of the enzyme.     

Methanogenesis from acetate: a nonmethanogenic bacterium from an anaerobic acetate enrichment

A methanogenic acetate enrichment was initiated by inoculation of an acetate-mineral salts medium with domestic anaerobic digestor sludge and maintained by weekly transfer for 2 years. The enrichment culture contained a Methanosarcina and several obligately anaerobic nonmethanogenic bacteria. These latter organisms formed varying degrees of association with the Methanosarcina, ranging from the nutritionally fastidious gram-negative rod called the satellite bacterium to the nutritionally nonfastidious Eubacterium limosum. The satellite bacterium had growth requirements for amino acids, a peptide, a purine base, vitamin B12, and other B vitamins. Glucose, mannitol, starch, pyruvate, cysteine, lysine, leucine, isoleucine, arginine, and asparagine stimulated growth and hydrogen production. Acetate was neither incorporated nor metabolized by the satellite organism. Since acetate was the sole organic carbon source in the enrichment culture, organism(s) which metabolize acetate (such as the Methanosarcina) must produce substrates and growth factors for associated organisms which do not metabolize acetate.     

Inactivation of Glutamine Synthetase by Ammonia Shock in the Gram-Positive Bacterium Streptomyces cattleya

In cultures of the gram-positive bacterium Streptomyces cattleya, a rapid inactivation of glutamine synthetase was seen after ammonia shock. pH activity curves for ammonia-shocked and control cultures are shown. A peak of glutamine synthetase activity was seen during fermentation for production of the antibiotic thienamycin.     

Modeling the rod outer segment birefringence change correlated with metarhodopsin II formation.

A rapid birefringence loss associated with metarhodopsin II formation, delta (delta n) MII, is produced when frog rod outer segments are exposed to a bleaching light flash. To analyze the nature of the underlying structure change, measurements of delta (delta n) MII were made in rod outer segments perfused with glycerol solutions to increase the refractive index of the cytoplasmic and intradisk spaces. Comparisons of experimental results with computed changes in the form birefringence component using two- and three-dielectric outer segment models for several putative structure changes were made. It is concluded that delta (delta n) MII can be due to either a change in the intrinsic birefringence component caused by the reorientation of anisotropic molecules, or to a change in the form birefringence component caused by small changes in the cytoplasmic and/or intradisk volumes.     

Analysis of DNAs from two species of the virilis group of Drosophila and implications for satellite DNA evolution

The DNAs from two virilis group species of Drosophila, D. lummei and D. kanekoi, have been analyzed. D. lummei DNA has a major satellite which, on the basis of CsCl equilibrium centrifugation, thermal denaturation, renaturation and in situ hybridization is identical to D. virilis satellite I. D. kanekoi DNA has a major satellite at the same buoyant density in neutral CsCl gradients as satellite III of D. virilis. However, on the basis of alkaline CsCl gradients, the satellite contains a major and a minor component, neither one of which is identical to D. virilis satellite III. By in situ hybridization experiments, sequences complementary to the major component of the D. kanekoi satellite are detected in only some species and in a way not consistent with the phylogeny of the group. However, by filter hybridization experiments using nick-translated D. kanekoi satellite as well as D. lummei satellite I and D. virilis satellite III DNAs as probes, homologous sequences are detected in the DNAs of all virilis group species. Surprisingly, sequences homologous to these satellite DNAs are detected in DNAs from non-virilis group Drosophila species as well as from yeast, sea urchin, Xenopus and mouse.     

Application of high-performance liquid chromatography to competitive labeling studies: The chemical properties of functional groups of glucaton

A competitive-labeling study of glucagon was carried out using [³H]- and [¹⁴C]-1-fluoro-2,4-dinitrobenzene to determine simultaneously the chemical properties of the α-amino and imidazole groups of the N-terminal histidine residue, and the lysine and tyrosine residues, under conditions where glucagon is in its physiologically active monomer form. The dinitrophenyl derivatives of these groups were purified by high-performance liquid chromatography which greatly simplified the separation steps of the procedure. The results showed the α-amino and tyrosine groups to have relatively normal behavior, with pK values of 7.98 and 10.22, respectively, while the lysine had a low pK of 8.46. The imidazole function had an apparent pK of 7.84, substantially higher than previous estimates. This difference may be accounted for by the effect of the charged form of the adjacent α-amino group on the nucleophilicity of the imidazole group.     

Lymphocyte activation and phospholipid pathways. 31P magnetic resonance studies

31P NMR spectra of perfused lymphocytes, embedded in alginate capsules and activated by interleukin-2, were remarkably different from those of control lymphocytes. The main differences were the appearance and gradual increase in phosphodiester signals, glycerophosphocholine and glycerophosphoethanolamine. These metabolic changes also occurred following perfusion with phorbol ester and after incubation with phytohemagglutinin (PHA) and were not dependent on a special growth medium. Nifedipine, a calcium channel blocking drug, inhibited the effects of phytohemagglutinin, but not of interleukin-2. There were no NMR spectral differences between peripheral lymphocytes, stimulated for 3 weeks, and tumor-infiltrating lymphocytes. Thus, sustained accelerated turnover of phosphatidylcholine and phosphatidylethanolamine is an inherent feature of the activation process. 31P NMR spectra of lymphocytes are characterized by a low signal of phosphocholine. Perfusion studies with high concentrations of choline and the use of dapsone, an inhibitor of cytidylyltransferase, indicated that choline kinase plays a key role in regulating phosphaditylcholine synthesis in human lymphocytes.     

Substrate properties of C1 inhibitor Ma (alanine 434----glutamic acid). Genetic and structural evidence suggesting that the P12-region contains critical determinants of serine protease inhibitor/substrate status

The serine protease inhibitor (serpin) C1 inhibitor inactivates enzymes involved in the regulation of vascular permeability. A patient from the Ma family with the genetic disorder hereditary angioedema inherited a dysfunctional C1 inhibitor allele. Relative to normal plasma, the patients's plasma contained an additional C1 inhibitor immunoreactive band, which comigrated with normal C1 inhibitor cleaved by plasma kallikrein, C1s, or factor XIIa. C1 inhibitor Ma did not react with a monoclonal antibody to a neoepitope that is present in complexed and cleaved normal C1 inhibitor, suggesting conformational differences between cleaved normal C1- inhibitor and cleaved C1 inhibitor Ma. Molecular cloning and sequencing of exon 8 of the C1 inhibitor Ma allele revealed a single C to A mutation, changing alanine 434 to glutamic acid. Ala 434 of C1 inhibitor aligns with the P12 residue of the prototypical serpin alpha 1-antitrypsin. The P12 amino acid of all inhibitory serpins is alanine, and it is present in a highly conserved region on the amino-terminal side of the serpin-reactive center loop. Whereas normal C1 inhibitor expressed by transfected COS-1 cells formed complexes with and was cleaved by kallikrein, fXIIa, and C1s, COS-1-expressed Ala434---Glu C1 inhibitor was cleaved by these enzymes but did not form complexes with them. These results, together with evidence from other studies, suggest that serpin protease inhibitor activity is the result of protein conformational change that occurs when the P12 region of a serpin moves from a surface location, on the reactive site loop of the native molecule, to an internal location within sheet A of the complexed inhibitor.     

Continuous culture system for production of biopolymer levan using erwinia herbicola

The optimal production of the fructan biopolymer levan by the bacterium Erwinia herbicola was investigated, including variations in nitrogen, carbon and phosphorous sources, pH, incubation time, culture yields up to 19% by weight produced based on conversion of sucrose as the carbon source when grown in a continuous culture system and processed by tangential flow filtration. Product identity was confirmed with gas chromatography (GC) and (13)C nuclear magnetic resonance (NMR). Gel permeation chromatography (GPC) and low-angle laser light scattering (LALLS) determination of the molecular weight of the product showed a significant difference in molecular weight values dependent on the method of analysis. Analysis by GPC resulted in molecular weight one order of magnitude lower than LALLS independent of sample, underscoring the unusual nature of this biopolymer.