Modification of galactose and N-acetylgalactosamine residues by oxidation of C-6 hydroxyls to the aldehydes followed by reductive amination: model systems and antifreeze glycoproteins

Amino acids and peptides have been attached to the C-6 hydroxyls of the galactose and the N-acetylgalactosamine by first oxidizing the C-6 hydroxyls to the aldehydes by galactose oxidase in the presence of small amounts of catalase, followed by reductive amination (-amino group) in the presence of cyanoborohydride. The activity of oxidized antifreeze glycoprotein was >70% of the original, and considerable activity has been retained with some substitutions on reductive amination using cyanoborohydride. The following were some activities retained (as compared with the oxidized antifreeze glycoprotein): Gly, 64; (Gly)₂, 88; (Gly)₃, 82; (Gly)₄, 70; Gly-Gly-NH₂, 44, Gly-Glu, 13; Gly-Leu, 40; Gly-Tyr, 57; Gly-Gly-Leu, 50; Gly-Gly-Phe, 30; and Gly-Gly-Val, 35. On amino acid analysis of acid hydrolysates, some release of the amino acid attached by amination occurred; e.g., Gly-Tyr gave 0.26 Gly and 0.49 Tyr per disaccharide.     

Modulation of lymphocyte nuclear matrix organization in vivo by 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole: an autoradiographic and immunofluorescence study

Assembly of active nuclei in lymphocytes stimulated by mitogen is paralleled by the elaboration of a structurally and biochemically complex nuclear matrix (NM). To examine the dynamics of individual NM polypeptide components during blastogenesis, we have applied immunofluorescence labelling with anti-NM antibodies to concanavalin A-stimulated mouse splenocytes. Whereas peripherin and PI2 antigens did not reorganize during stimulation, labelling of PI1 and small nuclear ribonucleoprotein (snRNP) antigens increased markedly in intensity and redistributed in concert with the previously reported NM restructuring. Double-labelling showed, furthermore, that snRNPs and the internal staining component of PI1 were largely co-localized. As an approach to studying the role of RNA and RNA synthesis in NM organization, we have further examined the effects of the inhibitor of RNA synthesis, 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB), on NM antigen distribution. The rapid inhibition of 3H-uridine incorporation by DRB was accompanied by coordinate aggregation of snRNPs and of the internal PI1 component into large, brightly stained patches. Both 3H-uridine incorporation levels and antigen localization were readily reversed upon removal of DRB. We conclude that NM antigens behave independently during nuclear and NM assembly and that NM organization, as reflected by NM antigen distribution, is modulated by con A- and DRB-induced alterations in RNA synthesis. We propose, furthermore, that the PI1 antigen plays a role in RNA metabolism, and is possibly involved in RNA transport to the nuclear periphery.     

Cloning and sequencing of the genes encoding the large and the small subunits of the H2 uptake hydrogenase (hup) of Rhodobacter capsulatus

Summary The structural genes (hup) of the H₂ uptake hydrogenase of Rhodobacter capsulatus were isolated from a cosmid gene library of R. capsulatus DNA by hybridization with the structural genes of the H₂ uptake hydrogenase of Bradyrhizobium japonicum. The R. capsulatus genes were localized on a 3.5 kb HindIII fragment. The fragment, cloned onto plasmid pAC76, restored hydrogenase activity and autotrophic growth of the R. capsulatus mutant JP91, deficient in hydrogenase activity (Hup^-). The nucleotide sequence, determined by the dideoxy chain termination method, revealed the presence of two open reading frames. The gene encoding the large subunit of hydrogenase (hupL) was identified from the size of its protein product (68108 dalton) and by alignment with the NH₂ amino acid protein sequence determined by Edman degradation. Upstream and separated from the large subunit by only three nucleotides was a gene encoding a 34 256 dalton polypeptide. Its amino acid sequence showed 80% identity with the small subunit of the hydrogenase of B. japonicum. The gene was identified as the structural gene of the small subunit of R. capsulatus hydrogenase (hupS). The R. capsulatus hydrogenase also showed homology, but to a lesser extent, with the hydrogenase of Desulfovibrio baculatus and D. gigas. In the R. capsulatus hydrogenase the Cys residues, (13 in the small subunit and 12 in the large subunit) were not arranged in the typical configuration found in [4Fe–4S] ferredoxins.     

Rhizome dynamics and resource storage in Phragmites australis

Seasonal changes in rhizome concentrations of total nonstructural carbohydrates (TNC), water soluble carbohydrates (WSC), and mineral nutrients (N, P and K) were monitored in two Phragmites australis stands in southern Sweden. Rhizome biomass, rhizome length per unit ground area, and specific weight (weight/ length ratio) of the rhizomes were monitored in one of the stands.Rhizome biomass decreased during spring, increased during summer and decreased during winter. However, changes in spring and summer were small (< 500 g DW m-2) compared to the mean rhizome biomass (approximately 3000 g DW m^–2). Winter losses were larger, approximately 1000 g DW m-2, and to a substantial extent involved structural biomass, indicating rhizome mortality. Seasonal changes in rhizome length per unit ground area revealed a rhizome mortality of about 30% during the winter period, and also indicated that an intensive period of formation of new rhizomes occurred in June.Rhizome concentrations of TNC and WSC decreased during the spring, when carbohydrates were translocated to support shoot growth. However, rhizome standing stock of TNC remained large (> 1000 g m^–2). Concentrations and standing stocks of mineral nutrients decreased during spring/ early summer and increased during summer/ fall. Only N, however, showed a pattern consistent with a spring depletion caused by translocation to shoots. This pattern indicates sufficient root uptake of P and K to support spring growth, and supports other evidence that N is generally the limiting mineral nutrient for Phragmites.The biomass data, as well as increased rhizome specific weight and TNC concentrations, clearly suggests that reloading of rhizomes with energy reserves starts in June, not towards the end of the growing season as has been suggested previously. This resource allocation strategy of Phragmites has consequences for vegetation management.Our data indicate that carbohydrate reserves are much larger than needed to support spring growth. We propose that large stores are needed to ensure establishment of spring shoots when deep water or stochastic environmental events, such as high rhizome mortality in winter or loss of spring shoots due to late season frost, increase the demand for reserves.     

Disulfide bridges in tomato pectinesterase: variations from pectinesterases of other species; conservation of possible active site segments.

Analysis of tomato pectinesterase by carboxymethylation, with and without reduction, shows that the enzyme has two intrachain disulfide bridges. Analysis of fragments obtained from the native enzyme after digestion with pepsin identified bridges connecting Cys-98 with Cys-125, and Cys-166 with Cys-200. The locations of disulfide bridges in tomato pectinesterase are not identical to those in three distantly related pectinesterases (18-33% residue identities) from microorganisms. However, one half-Cys (i.e., Cys-166) position is conserved in all four enzymes. Sequence comparisons of the overall structures suggest a special importance for three short segments of the entire protein. One segment is at the N-terminal part of the tomato pectinesterase, another in the C-terminal portion near the distal end of the second disulfide loop, and the third segment is located in the central part between the two disulfide bridges. The latter segment, encompassing only 40 residues of the entire protein, appears to high-light a functional site in a midchain segment.     

Physicochemical aspects of carbohydrate binding to some plant lectins with binding preference forN-acetylgalactosamine and galactose

This contribution illustrates the advantages of some chromophoric and fluorophoric carbohydrate derivatives such asp-nitrophenyl (pNO₂Phe) or 4-methylumbelliferyl (MeUmb) glycosides andN-dansylgalactosamine in studies of the binding equilibrium and kinetics with some plant lectins. The methods used involve continuous titrations of changes in ligand or protein absorption and ligand fluorescence, including substitution titrations as well as stopped-flow, temperature-jump or pressure-jump relaxation kinetics. When monitored by temperature-jump relaxation, binding of MeUmbαGal to the bloodgroup A specific lectin GSAI-A₄ fromGriffonia simplicifolia is a simple bimolecular association with parametersk ₊ = 9.4 × 10⁴ M^-1 s^-1 andk _-1 = 5.3 s^-1 at 23°C, but binding to the GSAI-B₄ lectin is biphasic. The complementarity of the peanut agglutinin binding site with Galβ1 → 3GalNAc that occurs in manyO-glycoproteins follows from enthalpic considerations and also from the value of the dissociation-rate parameterk _-1 = 0.24 s^-1 of the MeUmbβGalβl → 3GalNAc.lectin complex. This value, obtained by stopped-flow kinetics is 100 times smaller than for other mono-and disaccharides investigated. The binding mechanism is simple and the derivatisation of Galβ1 → 3GalNAc does not affect the affinity to a considerable degree. The binding preference of tetravalentsoybean agglutinin for MeαGalNAc over MeαGal by a factor of 25 is mainly of enthalpic origin with an additional 7 kJ mol^-1; the NAc group causes perturbation of a tryptophanyl residue, evidenced by protein difference absorption spectrometry. In the glycosides, a large aglycon likeβpNO₂ Phe orβMeUmb hardly affects the affinity of SBA but a largeN-dansyl group increases the affinity by a factor 20 as compared to GalNAc. The 10-fold increase in carbohydrate-specificN-dansylgalactosamine fluorescence, together with a very favourable entropic contribution point at the presence of a hydrophobic region in the vicinity of the carbohydrate-binding site. The dissociation-rate parameter of the MeUmbβGalNAc SBA complex is slower than for any reported monosaccharide-lectin complex: 0.4 s^-1. The divalent lectin fromErythrina cristagalli preferentially binds the Galβ1 → 4GlcNAc structure that occurs in manyN-glycoproteins. The combining site was mapped thermodynamically with carbohydrates ranging from mono-to pentasaccharides as derived fromN-glycoproteins. Here, N-dansylgalactosamine was used as a fluorescent indicator ligand in substitution titrations. When Galβ1 → 4GlcNAc was linkedα1 → 2 orα1 → 6 to Man, the binding enthalpy and entropy remained practically constant. Application of stopped flow kinetics and pressure-jump relaxation withN-dansylgalactosamine gave mono-exponential signal changes with a concentration dependence corresponding tok ₊ = 4.8 x 10⁴ M^-1 s^-1 k _- = 0.4 to 0.66 s^-1 and a change in reaction volume of+7ml/mol.     

Cloning and sequencing the genes encoding uptake-hydrogenase subunits of Rhodocyclus gelatinosus

Summary Rhodocyclus gelatinosus grew photosynthetically in the light and consumed H₂ at a rate of about 665 nmol/min per mg protein. The uptake-hydrogenase (H₂ase) was found to be membrane bound and insensitive to inhibition by CO. The structural genes of R. gelatinosus uptake-H₂ase were isolated from a 40 kb cosmid gene library of R. gelatinosus DNA by hybridization with the structural genes of uptake-H₂ase of Bradyrhizobium japonicum and Rhodobacter capsulatus. The R. gelatinosus genes were localized on two overlapping DNA restriction fragments subcloned into pUC18. Two open reading frames (ORF1 and ORF2) were observed. ORF1 contained 1080 nucleotides and encoded a 39.4 kDa protein. ORF2 had 1854 nucleotides and encoded a 68.5 kDa protein. Amino acid sequence analysis suggested that ORF1 and ORF2 corresponded to the small (HupS) and large (HupL) subunits, respectively, of R. gelatinosus uptake-H₂ase. ORF1 was approximately 80% homologous with the small, and ORF2 was maximally 68% homologous with the large subunit of typical membrane-bound uptake-H₂ases.     

Response of seeds ofLupinus termis andVicia faba to the interactive effect of salinity and ascorbic acid or pyridoxine

The interactive effect of salinity and presoaking in ascorbic acid or phyridoxine on germination, seedling growth, and some relevant metabolic changes ofLupinus termis andVicia faba seeds were studied. Germination studies indicated that broad bean tolerated NaCl salinity up to 240mM NaCl and lupin to 200mM NaCl. The lengths of roots and shoots and their water content, as well as dry matter yield, remained more or less unchanged up to the level of 80mM NaCl. Salinity induced marked progressive increases of carbohydrates and proline in broad bean and soluble protein in lupin seedlings, irrespective of the salinity level used. The other organic solutes (soluble protein in broad bean and carbohydrates in lupin seedlings) remained more or less unchanged at low and moderate levels of NaCl. However, under the higher salinity levels, in lupin the losses in carbohydrates were accompanied by increases in soluble protein, whereas in broad bean an opposite effect was obtained. The level of 40mM NaCl had a pronounced stimulatory effect on the all the variables studied. Presoaking seeds in either ascorbic acid or pyridoxine counteracted the adverse effects of salinity on germination and seedling growth as well as on some metabolic mechanisms of lupin and broad bean plants. The importance of these processes to the salinity tolerance of broad bean and lupin have been discussed.     

Morphological and structural adaptation of nodules of cowpea to functioning under sub- and supra-ambient oxygen pressure

Nodules of cowpea (Vigna unguiculata (L.) Walp. cv. Vita 3:Bradyrhizobium CB 756) from 28-d-old plants cultured for 23 d with their root systems maintained in O₂ levels from 1 to 80% (v/v, in N₂) in the external gas phase showed a range of structural changes which have been interpreted in relation to an over- or under-supply of O₂. A response to the partial pressure of O₂ in the gas phase (pO₂) was noted with respect to nodule size, lenticel development, the relative distributions of cortical and infected central tissue, the differentiation of cortex, especially the inner cortex, the frequency and size of infected and uninfected interstitial cells, the volume of extracellular spaces both in cortex and infected tissue, and in the frequency of bacteroids. As a consequence of these changes the surface area of inner cortex relative to the nitrogenase-containing units of fixing tissue (infected cells or bacteroids) was increased by as much as 20-fold. Effectiveness of bacteroid functioning increased from 0.10 ± 0.02 · 10^-9 μmol acetylene reduced per bacteroid in air-grown nodules to 0.9 ± 0.16 · 10^-9 (same units) per bacteroid in those cultured in 1% O₂. This work was supported by a grant from the Australian Research Council (to C.A.A.) and an Australian International Development Assistance Bureau postgraduate fellowship (to F.D.D.). The authors wish to thank Dr. W.F.C. Blumer for his considerable help with morphometric analysis, Dr. J. Kuo for guidance in the use of histological techniques, and to Dr. J.S. Pate for the suggestion that lenticel development might be quantified by surface staining of nodules.     

The influence of polyalcohols and carbohydrates on the thermostability of alpha-amylase

The influence of polyhydric alcohols and carbohydrates on the thermostability, i.e., the heat inactivation kinetics, of Bacillus licheniformis alpha-amylase was studied in the temperature range 96 degrees to 130 degrees C. High concentrations (from 9 to 60 weight percent) of glycerol, sorbitol, mannitol, sucrose, or starch can markedly decrease the inactivation rate constant, k, and in the studied cases, this stabilizing effect grows stronger with increasing additive concentration. Statements about stabilization should, however, be specified carefully with respect to temperature, because E(A) is mostly altered likewise. For dissolved enzyme E(A) was almost always decreased in the presence of polyol or carbohydrate, whereas for immobilized enzyme it was augmented in each studied instance. The inactivation of dissolved enzyme can, in all the studied cases, be adequately described as a first-order process. Immobilized enzyme, however, shows biphasic then first-order inactivation kinetics, depending on the additive concentration and temperature.