Study of relationship among species ofVibrio,Photobacterium, and terrestrial enterobacteria by an immunological comparison of glutamine synthetase and superoxide dismutase

The amino acid sequence divergence of glutamine synthetase (GS) from species ofVibrio, Photobacterium, Aeromonas, Escherichia, Salmonella, Citrobacter, Enterobacter, Serratia, Proteus, Erwinia, Xenorhabdus, andPlesiomonas was determined by quantitative microcomplement fixation, using antisera to GS fromVibrio alginolyticus andEscherichia coli. A similar study was performed with superoxide dismutase (SOD), using antiserum to the enzyme fromV. alginolyticus. A comparison of the results for GS and SOD, relative to the enzymes fromV. alginolyticus, as well as a comparison of these data with the results of previous ribosomal RNA (rRNA)/DNA homology studies indicated a high degree of congruence (correlation coefficients≥0.9). The results with both enzymes suggested four major groupings among these genera: (i)Vibrio, (ii)Photobacterium, (iii)Aeromonas, (iv) a large and heterogeneous group which included the peritrichously flagellated terrestrial enterobacteria.     

Electrophoretic mobilities of superoxide dismutases from species ofPhotobacterium,beneckea, vibrio,and selected terrestrial enterobacteria

Cell-free extracts of 93 strains from 23 species and biotypes ofPhotobacterium, Beneckea, andVibrio were electrophoresed on polyacrylamide gels and then stained for superoxide dismutase (SOD) activity. All strains exhibited a single activity band with the exception ofPhotobacterium leiognathi, which had one major and two minor bands. Strains representative of all three genera were found to have SOD activities that were sensitive to treatment with H₂O₂, suggesting that they were iron-containing enzymes. Examination of the effect of gel concentration on relative mobility (Rm) suggested that all the iron-containing SODs had very similar molecular weight. Most species could be distinguished on the basis of differences in the Rm values of their SODs.Vibrio was readily separable fromBeneckea andPhotobacterium. A limited electrophoretic analysis of the SODs fromAeromonas, Serratia, Proteus, Erwinia, and other species of terrestrial enterobacteria indicated groupings that were in agreement with previous studies.     

Intracellular localization of glutamine synthetase in Chlorogloeopsis fritschii

After differential centrifugation of cell-free extracts of Chlorogloeopsis fritschii, 71% of the original glutamine synthetase (GS) activity was associated with the thylakoids, while little activity was detected in the cytoplasmic membranes. Monospecific antiserum to a purified GS inhibited 88% of the enzyme activity in solubilized thylakoid membranes. An antiserum raised against thylakoids gave 81% inhibition. However, using intact thylakoid membranes, only 7% inhibition was obtained with the GS antiserum, indicating that GS is located inside the thylakoid membranes.The author is with the Department of Biological Sciences, University of Science and Technology, Irbid, Jordan     

Regulation,purification and partial characterization of glutamine synthetase fromStreptomyces aureofaciens

The activity of glutamine synthetase (GS) fromStreptomyces aureofaciens was regulated by the availability of the nitrogen source. Rich nitrogen sources repressed GS synthesis and increased GS adenylylation. The enzyme was purified 270-fold to virtual homogeneity with 37% recovery. The molar mass of the native enzyme and its subunits was determined to be 620 and 55 kDa, respectively, indicating that GS is composed of 12 identical subunits. The enzyme has a hexagonal-bilayered structure as observed by electron microscopy. The isoelectric point of the purified GS was at pH 4.2. The enzyme was stable for 1 h at 50°C but lost activity rapidly when incubated at 65 and 70°C. Mg²⁺ supported relative synthetic activity of 100 and 72%, respectively, with the corresponding pH optima of 7.3 and 7.0. Mn²⁺ ions activated transferase activity at a pH optimum of 7.0. The temperature optimum for all GS activities was 50°C. Intermediates of the citric acid cycle exerted insignificant effects on the synthetic activities. There was no SH-group essential for the GS activity.     

Identification and characterization of a heat-labile type I glutamine synthetase fromStreptomyces cinnamonensis

Streptomycetes have two distinct glutamine synthetases (GS): a heat-stable dodecameric GSI and a heat-labile octameric GSII. A heat-inactivated GS activity was detected in crude extracts ofStreptomyces cinnamonensis cells grown with nitrate or glutamate as the nitrogen source. The purified enzyme obtained from crude extracts of the nitrate-grown cells after affinity and anion-exchange chromatography was also heat-labile; it was inactivated by 80 % when incubated at 50 °C for 1 h. However, the enzyme has properties typical of GSI and similar with those of the heat-stable GSI purified fromS. aureofaciens: It is composed of twelve subunits, each ofM 55 kDa, and has a native molar mass of 625 kDa and an isoelectric point at pH 4.2. In addition, its activity is regulated by reversible adenylylation. Mg²⁺ and NaCl but not Mn²⁺ protected the purified enzyme from thermal inactivation, and both NaCl and Mn²⁺ or Mg²⁺ stabilized its activity at 4–8 °C. As compared with GSI fromS. aureofaciens, theS. cinnamonensis enzyme was cleaved more extensively during SDS-PAGE, was less sensitive to feedback inhibitors, and similarly affected by divalent cations. TheK _m values were 12.5 mmol/L forl-glutamate, 0.1 for NH ₄ ⁺ , 1.25 for ATP, 18.5 forl-glutamine, 3.3 for hydroxylamine and 0.087 for ADP. To our best knowledge, this is the first report of a heatlabile GSI from any source.     

Purification and characterisation of glutamine synthetase fromNocardia corallina

Glutamine synthetase (GS) (EC 6.3.1.2) has been purified 67-fold fromNocardia corallina. The apparentM _r of the GS subunit was approximately 56,000. Assuming the enzyme is a typical dodecamer this indicates a particle mass for the undissociated enzyme of 672,000. The GS is regulated by adenylylation and deadenylylation, and subject to feedback inhibition by alanine and glycine. The pH profiles assayed by the -glutamyl transferase method were similar for NH₄ ⁺-treated and untreated cell extracts and an isoactivity point was not obtained from these curves. GS activity was repressed by (NH₄)₂SO₄ and glutamate. Cells grown in the presence of glutamine, alanine, proline and histidine had enhanced levels of GS activity. The GS ofN. corallina cross-reacted with antisera prepared against GS from a Gram-negativeThiobacillus ferrooxidans strain but not with antisera raised against GS from a Gram-positiveClostridium acetobutylicum strain.     

Actinomycin D inactivating enzymes fromActinoplanes missouriensis and several other members of theActinoplanaceae family

Summary The enzymes for inactivating actinomycin D appear to be widely distributed amongst species belonging to the familyActinoplanaceae. Actinomycin D was completely or partially inactivated by cell-free extracts fromActinoplanes missouriensis, Streptosporangium viridogriseum, S. violaceocbromogenes, S. roseum, S. brasiliense, S. albidum, Spirillospora sp.,Sp. albida, Kitasatoa kauaiensis, Planobispora longispora, P. rosea, Dactylosporangium aurantiacum, andD. thailandense. No inactivation was obtained with extracts fromAmorphosphorangium auranticolor, Ampullariella lobata, Planomonospora parontospora, andP. venezuelensis. Actinomycin lactonase was partially purified by ultracentrifugation, ultrafiltration, and isoelectric focusing from noninduced cells ofActinoplanes missouriensis. The enzyme has a molecular weight of greater than 200,000 daltons and an isoelectric point of 4.3 to 4.4.     

Immunological Discrimination of Phytophthora cinnamomi from other Phytophthorae Pathogenic on Chestnut

A polyclonal antiserum (A379) against water soluble proteins from Phytophthora cinnamomi mycelium was produced in rabbit. In ELISA, the 1 : 10 000 diluted antiserum revealed only Phytophthora isolates, not allowing a clear‐cut discrimination among congenerous species, in spite of a generally higher reactivity on P. cinnamomi proteins. The antiserum gave positive reactions in Western blot analyses against mycelial proteins from nine species of Phytophthora and Pythium sp. (grown on rich media), but not with Rhizoctonia solani, binucleate Rhizoctonia, Verticillium dahliae, Fusarium oxysporum and Cryphonectria parasitica. All Phytophthora species showed common epitopes on proteins of molecular masses 77, 66, 51 and 48 kDa. However, a species‐specific protein of 55 kDa was immunodecorated only in P. cinnamomi samples, thus allowing univocal identification of this species. When tested against total proteins from the same fungi grown on water, the antibody revealed diagnostic bands of 55 and 51 kDa in P. cinnamomi only. The antiserum is therefore suitable for the specific identification of P. cinnamomi emerging in distilled water from infected tissues of chestnut, blueberry and azalea.     

Acquired resistance to ixodid ticks induced by tick cement antigen

Antisera from guinea pigs made resistant to infestation with an ixodid tick of east and central Africa,Rhipicephalus appendiculatus, were used to identify the tick antigens they recognized by immunoblotting. Most of the antigens were found in tick salivary glands and in tick attachment cement. Antisera fromR. appendiculatus-resistant guinea pigs also recognized some salivarygland antigens in ticks of other species (R. pulchellus, R. evertsi, Amblyomma variegatum andA. gemma). Antibodies against the most strongly recognizedR. appendiculatus antigen, a 20-kDa molecule, were only poorly reactive with similar-sized molecules in the other ticks. A 94-kDa antigen, which appeared to have broader cross-reactivity, was purified fromR. appendiculatus attachment cement, and a monospecific rabbit serum was raised against it. This antiserum clearly recognized a molecule of similar molecular weight inR. pulchellus andR. evertsi. Intravenous inoculation of rabbits with the purified molecule elicited delayed-type hypersensitivity to the antigen. The hypersensitive rabbits demonstrated resistance to feeding ofR. appendiculatus ticks but slight enhanced feeding ofR. pulchellus ticks. These results are discussed with respect to their relevance for artificial induction of tick-feeding resistance.     

Removal of Mn2+ induces dissociation of glutamine synthetase fromStreptomyces aureofaciens

Removal of Mn²⁺ by EDTA treatment converted dodecameric glutamine synthetase (GS) fromStreptomyces aureofaciens into inactive subunits but did not affect significantly their conformation. However, when fractionated by gel filtration FPLC, the Mn²⁺-free subunits showed a 7-fold increase ofA ₂₈₀, probably due to a significant alteration in their tertiary structure. Mn²⁺ reduced theA ₂₈₀ of the subunits and promoted their reaggregation to form active GS. Mg²⁺ or Ca²⁺ but not Co²⁺ or Zn²⁺ might have similar effects. The results suggest that specific divalent cations might play a crucial role in stabilizing subunit interactions as well as the conformation of the individual subunits inStreptomyces GS. The role of specific divalent cations in the regulation of GS turnover is discussed.     

CHARACTERIZATION OF POLLEN ANTIGENS FROM AMBROSIA L. (COMPOSITAE) AND RELATED TAXA BY IMMUNOELECTROPHORESIS AND RADIAL IMMUNODIFFUSION

The pollen antigens of various Ambrosia and related species were studied to learn whether substances closely related to antigen E (the major allergen of Ambrosia artemisiifolia) were present. After conventional immunoelectrophoresis, pollen extracts from six Ambrosia species each produced at least one pronounced precipitin line with antiserum for purified antigen E. Electrophoretic mobility was the same for several species (A. artemisiifolia, A. bidentata, A. psilostachya, and A. trifida) but was relatively lower for A. acanthicarpa and A. ambrosioides. Precipitin rings were also produced when pollen extracts of the various Ambrosia species were subjected to radial immunodiffusion in agarose which contained antiserum for purified antigen E. There was great variation among the Ambrosia species with respect to precipitin ring diameters. The variation may be due to differences among species in content of the antigen E-like substances or to altered interaction with the immobilized antibody. Crossed (2-dimensional) immunoelectrophoresis was shown to be useful for characterizing Ambrosia pollen antigens. Pollen extracts from A. artemisiifolia produced eight pronounced precipitin bands and at least eight faint, relatively fast-moving bands after crossed immunoelectrophoresis with antiserum against a whole pollen extract from the same species. One of the pronounced bands contained antigen E.     

Electrophoretic heterogeneity and age-related change of serine proteinase fromAspergillus sojae

Five forms of serine proteinase (EC 3.4.21.14) have been purified fromAspergillus sojae. This paper reports heterogeneity of electrophoretic mobilities, isoelectric points, and kinetic parameters of multiple forms of serine proteinase fromAspergillus sojae, including agerelated changes in thek _cat /K _m of electrophoretic species.     

Relationship between epitope density and immunoprecipitation of multivalent antigens by bivalent antibody: immunoprecipitation of adenylylated glutamine synthetase by anti-AMP antibodies

Escherichia coli glutamine synthetase (GS) preparations composed of 12 adenylylated subunits (GS₁₂^?) are almost completely precipitated by sheep Anti-AMP immunoglobulin G (IgG), whereas glutamine synthetase preparations containing 6 adenylylated subunits (GS₆^?) are only partially precipitated by the antibodies (R.J. Hohman, S.G. Rhee, and E.R. Stadtman, 1980, Proc. Nat. Acad. Sci. USA77, 7410–7414). By means of ¹²⁵I-labeled anti-AMP antibodies and double immunoprecipitation techniques, in which rabbit antiserum to sheep IgG or anti-GS antibodies were used to precipitate soluble immune complexes, it was demonstrated that under optimal conditions, both the soluble and insoluble immune complexes obtained with either GS₆^? or GS₁₂^? contain 0.5 mol antibody/mol adenylylated subunit. In agreement with the lattice theory of immuno-precipitation, soluble immune complexes are formed in antibody excess. Scatchard plots of binding data indicate that under conditions of antibody excess, one antibody molecule is bound to each AMP moiety of GS₁₂^?, whereas GS₆^? binds a maximum of only 0.68 antibody molecule/adenylylated subunit. We propose that with some species of GS₆^?, the distribution of adenylylated subunits favors monogamous interactions of the bivalent antibody with two subunits within the same GS molecule and thereby leads to the formation of small, soluble, immune complexes. Other explanations are considered. Only 30% of the antibody population that recognizes unconjugated 5′-AMP binds to the AMP moiety of adenylylated GS. Anti-AMP antiserum can be fractionated on a GS₁₂^?-Sepharose matrix into two subpopulations of antibody with strikingly different immunoprecipitation characteristics. Conversely, species of GS with various states of adenylylation ranging from 0 to 8 were separated from a GS₆^? preparation by means of affinity chromatography on an anti-AMP antibody-Sepharose matrix. Under optimal conditions, antibodies purified by affinity chromatography precipitated a smaller fraction of a GS₆^? preparation than did unfractionated antiserum. Competence of the purified antibody was nearly restored to that of the unfractionated serum by the addition of an enhancement factor present in the IgG fraction of nonimmune serum. The enhancement factor was not required for complete precipitation of GS^?₁₂ by purified antibodies. Contrary to most antibody-antigen reactions, immunoprecipitation of GS₆^? with anti-AMP antibodies is greater at 30 °C than at 4 °C.     

A comparative immunocytochemical investigation of the crustacean hyperglycemic hormone (CHH) in the eyestalks of some decapod crustacea

The eyestalk of the astacideans Orconects limosus, Nephrops norvegicus, and Homarus gammarus, and the palinuran Palinurus vulgaris, was examined with an antiserum raised against purified crustacean hyperglycemic hormone (CHH) of the astacidean species Astacus leptodactylus. A distinct immunopositive reaction occurs in a group of neurosecretory cells in the medulla terminalis ganglionic X-organ (MTGX), in the MTGX-sinus gland tractus, and in a considerable part of the sinus gland. The immunoreactive sites in the eyestalk of the investigated species correspond to the site of production, storage, and release of the CHH. Preliminary investigations with this antiserum also indicate that a positive immunoreaction can be obtained in the eyestalk of other decapod crustaceans, for example, of the brachyuran Macropipus puber and the caridean Palaemon serratus.     

Identification of the yeast genusSchizosaccharomyces by a murine monoclonal antibody

A precise analysis for the identification of the yeast genusSchizosaccharomyces by immunoenzymatic methods is presented. By use of the murine monoclonal antibody JHF13–17, two proteins were recognized in cell extracts ofS. pombe, S. malidevorans, andS. japonicus in Western blot analyses and were termed SSP-A and SSP-B (Schizosaccharomyces-specific proteins). The migration pattern of the proteins was identical in 18 strains of these species that were tested. An analogous protein doublet was also visualized in cell extracts of ten strains ofS. octosporus, and, owing to the migration pattern, these proteins were termed SSP-B and SSP-C. A polyclonal antiserum raised against the SSP-A and SSP-B fromS. pombe cross-reacted with SSP-A and SSP-B fromS. malidevorans andS. japonicus and SSP-B and SSP-C fromS. octosporus.     

Evolutionary relationships of superoxide dismutases and glutamine synthetases from marine species of Alteromonas,Oceanospirillum, Pseudomonas and Deleya

Evolutionary relationships among marine species assigned to the genera Alteromonas, Oceanospirillum, Pseudomonas, and Alcaligenes were determined by an immunological study of their Fe-containing superoxide dismutases (FeSOD) and glutamine synthetases (GS), two enzymes with differentially conserved amino acid sequences which are useful for determining intermediate and distant relationships, respectively. Five reference antisera were prepared against the FeSODs from Alteromonas macleodii, A. haloplanktis, Oceanospirillum commune, Pseudomonas stanieri, and Deleya pacifica. For GS, a previously prepared antiserum to the enzyme from Escherichia coli was employed. Amino acid sequence similarities for both enzymes were determined by the quantitative microcomplement fixation technique and the Ouchterlony double diffusion procedure. Six evolutionary groups were detected by FeSOD sequence similarities: three subgroups within the genus Alteromonas, the genera Oceanospirillum and Pseudomonas, and a new genus, Deleya (to accommodate marine Alcaligenes). Only four groupings were delineated by the GS data: the latter three genera and one group composed of all the species of Alteromonas. Evidence that all of these subgroups are derived from the evolutionary lineage defined by the purple sulfur photosynthetic bacteria is presented.Abbreviations Alt Alteromonas - anti-Amac, anti-Ahal, anti-Ocom, anti-Psta, anti-Dpac antisera to the Fe-containing superoxide dismutases from Alteromonas macleodii 107, Alteromonas haloplanktis 121, Oceanospirillum commune 8, Pseudomonas stanieri 146, Deleya pacifica 62 - FeSOD Fe-containing superoxide dismutase - G+C guanine plus cytosine - GS glutamine synthetase - ImD immunological distance - MnSOD Mn-containing superoxide dismutase - Oce Oceanospirillum - Pse Pseudomonas - Rm relative mobility - rRNA ribosomal RNA - SOD superoxide dismutase Dedicated to the memory of Professor Roger Y. Stanier     

Recruitment of lysozyme as a major enzyme in the mouse gut: Duplication,divergence, and regulatory evolution

Summary Two major types of lysozymec (M and P) occur in the mouse genus,Mus, and have been purified from an inbred laboratory strain (C58/J) ofM. domesticus. They differ in physical, catalytic, and antigenic properties as well as by amino acid replacements at 6 of 49 positions in the amino-terminal sequence. Comparisons with four other mammalian lysozymesc of known sequence suggest that M and P are related by a gene duplication that took place before the divergence of the rat and mouse lineages. M lysozyme is present in most tissues; achieves its highest concentration in the kidney, lung, and spleen; and corresponds to the lysozyme partially sequenced before from another strain ofM. domesticus. InM. domesticus and several related species, P lysozyme was detected chiefly in the small intestine, where it is probably produced mainly by Paneth cells. A survey of M and P levels in 22 species of muroid rodents (fromMus and six other genera) of known phylogenetic relationships suggests that a mutation that derepressed the P enzyme arose about 4 million years ago in the ancestor of the housemouse group of species. Additional regulatory shifts affecting M and P levels have taken place along lineages leading to other muroid species. Our survey of 187 individuals of wild house mice and their closest allies reveals a correlation between latitude of origin and level of intestinal lysozyme.     

Characterization of Plasmodium berghei glutathione synthetase

Plasmodium berghei contained 0.454 ± 0.031 U/mg of glutathione synthetyase (GS). GS was purified using solid ammonium sulfate and Sephadex G-200 from P. berghei infected mouse erythrocytes. SDS-PAGE showed purified GS as a single band protein of 70 kDa and its Km for γ-glutamylcysteine, glycine and ATP being 0.33 mM, 8.3 mM and 0.43 mM respectively with noncompetitive inhibition by GSH. The malaria parasite enzyme was optimally active at 37 °C and pH 8.0–8.5. Heavy metals significantly inhibited parasite GS activity.     

Properties of membrane-bound ATPase of some acidophilic heterotrophic bacteria

Membrane-bound ATPase (EC 3.6.1.4) of acidophilic heterotrophic bacteria from mine environment was isolated and characterized. The enzyme preparations fromAcidiphilium symbioticum KM2 and the strains GS18h and GS19h have a pH optimum of 7.7, 8.2 and 7.7, respectively, in the presence of Mg²⁺ which is required for activity. In an assay system containing Mn²⁺ or Ca²⁺ only, some activity was also evident. These enzymes hydrolyzed inorganic diphosphate (PPi), guanosine triphosphate (GTP) and inosine triphosphate (ITP) as the better substrate than ATP and theK _m values of the enzymes with respect to ATP were determined to be 238, 157 and 228 μmol/L forA. symbioticum KM2 and the strains GS18h and GS19h, respectively. The activity was stimulated by sulfite while Zn²⁺, Hg²⁺, 4-chloromercuribenzoic acid (p-CMB) and the specific inhibitors of F₀F₁ type ATPase,viz. N,N′-dicyclohexylcarbodiimide (DCCD), oligomycin and azide reduced the activity of the enzyme preparations.     

Chemical examinations of some non-vascular Paleozoic plants

The organic chemical constituents of compression fossils ofNematothallus,Spongiophyton, Orestovia andEohostimella are identified and compared with those isolated from living and fossil forms ofBotryococcus (a green alga) andTaeniocrada (a vascular plant fossil). The range and maxima in the carbon numbers observed in the normal, saturated acids isolated fromNematothallus, Orestovia, andSpongiophyton are similar to those of fossilBotryococcus, while those acids contained within compression fossils ofEohostimella are similar to the hydrocarbon composition ofTaeniocrada. Isoprenoid, branched hydrocarbons and steroids identified fromNematothallus, Orestovia, andSpongiophyton suggest these genera have algal affinities, while the presence of thick cuticles and in some cases cutin-like compounds appear to show adaptation to a terrestrial environment. Phenolic compounds retained within rock matrices associated withEohostimella are similar to those isolated fromTaeniocrada suggesting chemical, as well as morphological parallels with the land plant habit. These data are interpreted as indicating an early polyphyletic exploitation of the terrestrial habitat during the Paleozoic.