Structural and biochemical properties of glycosylated and deglycosylated glucose oxidase from Penicillium amagasakiense

Glucose oxidase from Penicillium amagasakiense was purified to homogeneity by ion-exchange chromatography and deglycosylated with endoglycosidase H. On the basis of gas chromatography and sodium dodecyl sulphate/polyacrylamide gel electrophoretic (SDS-PAGE) analyses, the protein-bound high-mannose-type carbohydrate moiety corresponded to 13% of the molecular mass of glycosylated glucose oxidase. A total of six N-glycosylation sites per dimer were determined from the N-acetylglucosamine content. The enzymatically deglycosylated enzyme contained less than 5% of the original carbohydrate moiety. A molecular mass of 130 kDa (gel filtration) and 133 kDa (native PAGE) was determined for the dimer and 67 kDa (SDS-PAGE) for the monomer of the deglycosylated enzyme. The N-terminal sequence, which has not been published for glucose oxidase from P. amagasakiense to date and which showed less than 50% homology to the N terminus of glucose oxidase from Aspergillus niger, and the amino acid composition were not altered by the deglycosylation. Deglycosylation also did not affect the kinetics of glucose oxidation or the pH and temperature optima. It also did not increase the susceptibility of the enzyme to proteolytic degradation. However, deglycosylated glucose oxidase exhibited decreased pH and thermal stability. The thermal stability of both enzymes was shown to be dependent on the buffer concentration and was enhanced by certain additives, particularly 1 M (NH₄)₂SO₄, which stabilised glucose oxidase 100- to 300-fold at 50 °C and pH 7–8, and 2 M KF, which stabilised the enzyme up to 36-fold at 60 °C and pH 6. In sodium acetate buffer, changes in pH (4–6) affected the affinity for glucose but had no effect on the V _max of the reaction. In contrast, in TRIS buffer, pH 8, a 10-fold decrease in V _max and a 2-fold decrease in K _m were observed. Received: 8 October 1996 / Received revision: 14 January 1997 / Accepted: 17 January 1997     

Alkaline unfolding and salt-induced folding of arginine kinase from shrimp Feneropenaeus chinensis under high pH conditions

The structural and functional properties of arginine kinase (AK) in alkaline conditions in the absence or presence of salt have been investigated. The conformational changes of AK during alkaline unfolding and salt-induced folding at alkaline pH were monitored using intrinsic fluorescence emission, binding of the fluorescence probe 1-anilino-8-naphthalenesulfonate and circular dichroism. The results for the alkaline unfolded enzyme showed that much lower pH (11.0) was required to cause the complete loss of AK activity than was required to cause an obvious conformational change of the enzyme. Compared with the completely unfolded state in 5 M urea, the high pH denatured enzyme had some residual secondary and tertiary structure even at pH 13.0. Increasing the ionic strength by adding salt at pH 12.75 resulted in the formation of a relatively compact tertiary structure and a little new secondary structure with hydrophobic surface enhancement. These results indicate that the partially folded state formed under alkaline conditions may have similarities to the molten globule state which is compact, but it has a poorly defined tertiary structure and a native-like secondary structure.     

Effect of pH,temperature and alcohols on the stability of glycosylated and deglycosylated stem bromelain

The biological significance of the carbohydrate moiety of a glycoprotein has been a matter of much speculation. In the present work, we have chosen stem bromelain fromAnanas comosus as a model to investigate the role of glycosylation of proteins. Stem bromelain is a thiol protease which contains a single hetero-oligosaccharide unit per molecule. Here, the deglycosylated form of the enzyme was obtained by periodate oxidation. The differences in the glycosylated and deglycosylated forms of the glycoprotein have been studied at various temperatures and pH values, using probes such as loss of enzyme activity and by the changes in fluorescence and circular dichroism spectra. Deglycosylated bromelain showed decreased enzyme activity and perturbed fluorescence and circular dichroism spectra. In addition to this, a comparative study of their activities in different organic solvents showed a marked decrease in case of deglycosylated form of the enzyme. It is thus concluded that glycosylation contributes towards the functional stability of glycoenzymes.     

Elevation has contrasting effects on avian and mammalian nest traits in the Andean temperate mountains

Nest building is a widespread breeding strategy across taxa. Nest composition and structure can play a critical role in the breeding success and/or adult survival of nest‐building vertebrates. Although nest traits are expected to vary adaptively across elevational gradients, few studies address this relationship. We studied the variation in nest traits (composition and structure) across elevation for two taxa with two different functions in the Andean temperate forests of southern Chile: a bird (Aphrastura spinicauda, Furnariidae, 170 breeding nests) and a marsupial mammal (Dromiciops gliroides, Microbiotheriidae, 91 winter torpor nests). For A. spinicauda, we further assessed how nest traits influenced clutch size and hatching success. Both species used fewer types of nest materials (items) with increasing elevation, and a greater proportion of leaves were used in highland compared to lowland forests. Aphrastura spinicauda used feathers and hair, and D. gliroides used bryophytes more frequently in lowland forests. The mass and volume of nests decreased with increasing elevation for A. spinicauda and increased for D. gliroides. Nest traits had subsequent fitness consequences for A. spinicauda, such that: (i) greater cup volume and depth were associated with larger clutch sizes, (ii) more items used during nest building were linked to improved clutch size at high elevation only, and (iii) nest wall thickness was negatively associated with hatching success. Thus, in temperate mountain ecosystems, elevation may be an important factor influencing nest‐building behaviour for cavity‐using vertebrates. However, the direction of the elevational effects may vary among taxa and nest functions in these ecosystems.     

氮素形态对专用小麦中后期根际土壤微生物和酶活性的影响

采用盆栽方法研究了酰胺态氮、铵态氮和硝态氮对强筋小麦(Triticum aestivum L.)"豫麦34"、中筋小麦"豫麦49"和弱筋小麦"豫麦50"生育中后期根际微生物和土壤酶活性的影响.结果表明,专用小麦根际真菌、细菌、放线菌数量和土壤脲酶、蛋白酶、硝酸还原酶活性以及根际pH值对氮素形态的反应不同."豫麦34"施用硝态氮,对根际土壤真菌、细菌(除成熟期外)和放线菌数量均具有明显的促进作用;"豫麦49"施用铵态氮,根际土壤细菌和放线菌数量最大,根际真菌数量在孕穗期和开花期以酰胺态氮处理最大,而成熟期以硝态氮处理最大;"豫麦50"施用硝态氮,对根际土壤真菌、细菌和放线菌数量均具有明显的促进作用.不同专用小麦品种均表现为在酰胺态氮处理下,根际土壤脲酶活性最高;在铵态氮处理下,根际土壤蛋白酶活性最高;在硝态氮处理下,根际土壤硝酸还原酶活性和pH值最高.     

Effects of carbohydrate removal on the structure and activity of bovine lutropin

Bovine lutropin was successfully deglycosylated by treatment with anhydrous HF at 0°C for 60 min. The overall loss of carbohydrate residues was about 70%. Fucose, mannose and galactosamine residues were removed completely. About six residues of N-acetylglucosamine were left in the deglycosylated hormone. This degree of deglycosylation did not reduce receptor binding ability but in vitro biological activity was completely lost. Consistent with these properties, the deglycosylated lutropin inhibited the in vitro biological action of the native lutropin. The results demonstrate that the full complement of carbohydrate residues including the sulfated hexosamines are not required for receptor binding but they are necessary to impart appropriate conformational features necessary for activation of the target cells.     

Effect of periodate oxidation on the structure and properties of glucose oxidase.

In order to elucidate the molecular structure of glucose oxidase (beta-D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4) and the roles of its carbohydrate moiety, chemical, physiochemical and immunological experiments were performed with enzyme samples before and after periodate oxidation. Hydrodynamic parameters indicated that the native enzyme was a globular protein with values of 1.21 for the frictional ratio and 43 A for the Stokes radius. The enzyme contained about 12% carbohydrate by weight, of which the main component was mannose. The periodate treatment decreased the carbohydrate content to about 40% of its original value. Slight modifications were detected in the absorbance spectrum and the content of arginyl residue. However, no significant alteration was brought about by this treatment in the catalytic parameters, immunological reactivities of the gross structure, not in the secondary and quaternary structures of the protein moity. Thermal denaturation temperature (about 72.5 degrees C) and the enthalpy of denaturation (about 450 kcal/mol) were common to the native and the periodate-oxodozed enzymes. The native was found to be quite resistant to sodium dodecyl sulfate and fairly stable to urea and heating. The periodate-oxidized enzyme was also stable to heat treatment, but it showed a diminished stability when denaturing agents were present. Kinetic analyses of the thermal inactivation processes showed that the entropy of activation was greatly decreased by the denaturing agents, especially in the case of the periodate-oxidized enzyme. It is concluded that the carbohydrate moiety of the enzyme plays a role in increasing the stability of the protein moiety, but does not directly participate in the catalytic activity, the immunological reactivity, or in maintaining the conformation of the enzyme protein.     

Gene pho-2 codes for the multiple active forms of Pi-repressible alkaline phosphatase in the mould Neurospora crassa

The mycelial Pi-repressible alkaline phosphatase of the wild-type strain 74A of Neurospora crassa was separated into at least ten isoforms by isoelectric focusing. The components visualized by activity with sodium -naphthyl phosphate as the substrate were predominantly acidic proteins with isoelectric points ranging from pH 4.5 to 7.6. The number of these isoforms was a function of growth pH. Strain pho-2A did not produce active Pi-repressible alkaline phosphatase (the pho-2 gene codes for its amino acid sequence), which gives an indication that these isoforms are encoded by the same structural gene.     

The effects of shear flow on protein structure and function

Protein molecules are subjected to potentially denaturing fluid shear forces during processing and in circulation in the body. These complex molecules, involved in numerous biological functions and reactions, can be significantly impaired by molecular damage. There have been many studies on the effects of hydrodynamic shear forces on protein structure and function. These studies are reviewed and the implications to bioprocessing and pathophysiology of certain diseases are discussed.     

Steady state and time resolved effects of guanidine hydrochloride on the structure of Humicola lanuginosa lipase revealed by fluorescence spectroscopy

Effects of guanidine hydrochloride (GdnHCl) on the structure and dynamics of wild-type Humicola lanuginosa lipase (HLL) and its two mutants were studied. The latter were S146A (with the active site Ser replaced by Ala) and the single Trp mutant W89m, with substitutions W117F, W221H, and W260H. Steady-state, stopped-flow, and time-resolved laser-induced fluorescence spectroscopy were carried out as a function of [GdnHCl]. The maximum emission wavelength and fluorescence lifetimes revealed the microenvironment of the tryptophan(s) in these lipases to become more polar upon increasing [GdnHCl]. However, significant extent of tertiary structure in GdnHCl is suggested by the observation that both wild-type HLL and W89m remain catalytically active at rather high GdnHCl concentrations of >6 and 4.0 M, respectively. Changes in steady-state emission anisotropy, as well as variation in rotational correlation times and residual anisotropy values, demonstrate that upon increasing [GdnHCl] the structure of the lipases became more loose, with increasing amplitude of structural fluctuations. Finally, intermediate states in the course of exposure of the proteins to GdnHCl were revealed by stopped-flow fluorescence measurements.     

Biochemical and immunologic studies on the native and deglycosylated forms of gamma-glutamyltranspeptidase of rat kidney

We have deglycosylated the enzyme gamma-glutamyl transpeptidase by treatment of the protein with anhydrous hydrofluoric acid at 0 degree C. After deglycosylation, the heavy and light subunits showed a molecular weight of 43 and 23 Kd respectively. Whereas the antiserum against the native enzyme recognized both proteins, the antiserum against the deglycosylated enzyme failed to recognize the native enzyme, indicating that some of the determinants of the native enzyme are masked by the carbohydrate moiety.     

Spatial heterogeneity of biomass turnover has contrasting effects on synchrony and stability in trophic metacommunities

Spatial heterogeneity is a fundamental feature of ecosystems, and ecologists have identified it as a factor promoting the stability of population dynamics. In particular, differences in interaction strengths and resource supply between patches generate an asymmetry of biomass turnover with a fast and a slow patch coupled by a mobile predator. Here, we demonstrate that asymmetry leads to opposite stability patterns in metacommunities receiving localized perturbations depending on the characteristics of the perturbed patch. Perturbing prey in the fast patch synchronizes the dynamics of prey biomass between the two patches and destabilizes predator dynamics by increasing the predator's temporal variability. Conversely, perturbing prey in the slow patch decreases the synchrony of the prey's dynamics and stabilizes predator dynamics. Our results have implications for conservation ecology and suggest reinforcing protection policies in fast patches to dampen the effects of perturbations and promote the stability of population dynamics at the regional scale.     

Deglycosylation of glucoamylase from Aspergillus niger: Effects on structure,activity and stability

A comparative structure–function study was performed to establish possible roles of carbohydrates in stabilization of glycoproteins, using glucoamylase (GA) as a model system. In addition to kinetic properties, stability toward elevated temperatures, extremes of pH, high salt concentrations together with circular dichroism, intrinsic/extrinsic fluorescence studies, proteolysis and affinity for interaction with hydrophobic ligands were investigated. Related to all the main properties examined, with one exception, glycosylation provided improvement in functional characteristics of the enzyme, especially in relation to its thermostability. Results are explained in terms of provision of stabilizing intermolecular interactions by the sugar molecules. The improvement in protein rigidity together with reduction of surface hydrophobicity appear to be especially important in relation to prevention of aggregation, an important mechanism of irreversible thermoinactivation, occurring at elevated temperatures.     

Alkaline pre-treatment of oilseed rape straw for bioethanol production: evaluation of glucose yield and pre-treatment energy consumption

The objective of the research was to investigate the effect of biomass loading, alkali (NaOH) concentration and pre-treatment time on the yield of glucose obtained following alkaline pre-treatment and enzymatic hydrolysis of oilseed rape (OSR) straw. A maximum glucose yield of (440.6 ± 14.9) g glucose kg⁻¹ biomass was obtained when OSR straw was pre-treated at a biomass loading of 50 g kg⁻¹ and an alkali concentration of 0.63 mol dm⁻³ NaOH for 30 min. The energy efficiency of glucose extraction (0.39 kg glucose MJ⁻¹ consumed) was highest when OSR straw was pre-treated at a biomass loading of 50 g kg⁻¹ and an alkali concentration of 0.63 or 0.75 mol dm⁻³ for 30 min. The study demonstrated alkaline pre-treatment of OSR straw is superior to acid pre-treatment in terms of glucose yield and energy efficiency.     

Plant diversity has contrasting effects on herbivore and parasitoid abundance in Centaurea jacea flower heads

High biodiversity is known to increase many ecosystem functions, but studies investigating biodiversity effects have more rarely looked at multi‐trophic interactions. We studied a tri‐trophic system composed of Centaurea jacea (brown knapweed), its flower head‐infesting tephritid fruit flies and their hymenopteran parasitoids, in a grassland biodiversity experiment. We aimed to disentangle the importance of direct effects of plant diversity (through changes in apparency and resource availability) from indirect effects (mediated by host plant quality and performance). To do this, we compared insect communities in C. jacea transplants, whose growth was influenced by the surrounding plant communities (and where direct and indirect effects can occur), with potted C. jacea plants, which do not compete with the surrounding plant community (and where only direct effects are possible). Tephritid infestation rate and insect load, mainly of the dominant species Chaetorellia jaceae, decreased with increasing plant species and functional group richness. These effects were not seen in the potted plants and are therefore likely to be mediated by changes in host plant performance and quality. Parasitism rates, mainly of the abundant chalcid wasps Eurytoma compressa and Pteromalus albipennis, increased with plant species or functional group richness in both transplants and potted plants, suggesting that direct effects of plant diversity are most important. The differential effects in transplants and potted plants emphasize the importance of plant‐mediated direct and indirect effects for trophic interactions at the community level. The findings also show how plant–plant interactions critically affect results obtained using transplants. More generally, our results indicate that plant biodiversity affects the abundance of higher trophic levels through a variety of different mechanisms.     

南方鲇碱性磷酸酶的分离纯化及部分性质的研究

用正丁醇抽提,硫酸铵分级沉淀,ＤＥＡＥ－纤维素和ＳｅｐｈａｃｒｙｌＳ－２００柱层析,从南方鲇（Ｓｉｌｕｒｕｓ　ｍｅｒｉｄｉｏｎａｌｉｓ　Ｃｈｅｎ）肠粘膜中提取出碱性磷酸酶（ＡＫＰ）。提纯倍数为３９．５０倍,比活为６８．３５μ／ｍｇ蛋白,提取酶液经ＰＡＧＥ和ＳＤＳ－ＰＡＧＥ只呈现一条区带。该酶的分子量为１３２１４０,Ｎ末端氨基酸为门冬氨酸,最适ｐＨ为１０．１０,７．５＞ｐＨ＞１１．５时不稳定,最适温度为４０℃左右,对热不很稳定,以磷酸苯二钠为底物其Ｋ_ｍ值为１．７２×１０~（－３）ｍｏｌ／Ｌ。Ｍｇ~（２＋）、Ｍｎ~（２＋）为该酶的激活剂,ＫＨ_２ＰＯ_４、Ｌ－ＣｙＳ、ＭＥ、ＤＦＰ、ＥＤＴＡ－Ｎａ_２为抑制剂。选用ＫＨ_２ＰＯ_４和ＤＦＰ作抑制类型的判断,结果表明,ＫＨ_２ＰＯ_４属竞争性掏剂,其抑制常数为２．３ｍｍｏｌ／Ｌ;ＤＦＰ为非竞争性抑制剂,抑制常数为１．０５ｍｍｏｌ／Ｌ。     

Flight metabolic rate has contrasting effects on dispersal in the two sexes of the Glanville fritillary butterfly

Evolution of dispersal is affected by context-specific costs and benefits. One example is sex-biased dispersal in mammals and birds. While many such patterns have been described, the underlying mechanisms are poorly understood. Here, we study genetic and phenotypic traits that affect butterfly flight capacity and examine how these traits are related to dispersal in male and female Glanville fritillary butterflies (Melitaea cinxia). We performed two mark–recapture experiments to examine the associations of individuals’ peak flight metabolic rate (MR_peak) and Pgi genotype with their dispersal in the field. In a third experiment, we studied tethered flight in the laboratory. MR_peak was negatively correlated with dispersal distance in males but the trend was positive in females, and the interaction between MR_peak and sex was significant for long-distance dispersal. A similar but nonsignificant trend was found in relation to molecular variation at Pgi, which encodes a glycolytic enzyme: the genotype associated with high MR_peak tended to be less dispersive in males but more dispersive in females. The same pattern was repeated in the tethered flight experiment: the relationship between MR_peak and flight duration was positive in females but negative in males. These results suggest that females with high flight capacity are superior in among-population dispersal, which facilitates the spatial spreading of their reproductive effort. In contrast, males with high flight capacity may express territorial behaviour, and thereby increase the number of matings, whereas inferior males may be forced to disperse. Thus, flight capacity has opposite associations with dispersal rate in the two sexes.     

The relationship of structure of glucoamylase and glucose oxidase to antigenicity

Glucoamylase and glucose oxidase fromAspergillus niger have been purified to homogeneity by chromatography on DEAE-cellulose and the purified enzymes have been used to investigate structural and antigenicity relationships. In structure, glucoamylase and glucose oxidase are glycoproteins containing 14% and 16% carbohydrate. Earlier methylation and reductive -elimination results have shown that glucoamylase has an unusual arrangement of carbohydrate residues, with 20 single mannose units and 25 di-, tri-, or tetrasaccharide chains of mannose, glucose, and galactose, all attached O-glycosidically to serine and threonine residues of the protein moiety. The antigenicity of the glucoamylase has now been found to reside predominantly in the types and arrangement of the carbohydrate chains. Glucose oxidase contains mannose, galactose, and glucosamine in the N-acetyl form in the native enzyme, but the complete structure of the carbohydrate chains has not yet been determined. The antigenicity of this enzyme does not reside in the carbohydrate units, but rather in the polypeptide chains of the two subunits of the enzyme. Glucose oxidase can be dissociated into subunits by mercaptoethanol and sodium dodecyl sulfate treatment, while glucoamylase cannot be dissociated, but undergoes only an unfolding of the polypeptide chain under these conditions. The subunits of glucose oxidase do not react with the anti-glucose oxidase antibodies, but the unfolded molecule and peptide fragments produced from glucoamylase by cyanogen bromide cleavage do react with antiglucoamylase antibodies.