Effect of the Pore Size of a Support on Activity and Action Pattern of Immobilized Endopolygalacturonase

Endopolygalacturonase (E.C. 3.2.1.15) was covalently bound to silica supports of different porosity treated with 3-(2′,3′-epoxypropoxy)propyltrimethoxysilane. The activity and action pattern on sodium pectate and tetra(D-galactosiduronic acid) were investigated in batch and continuous flow-reactors. Pore size of the supports affected the loading of the enzyme as well as its action pattern and kinetics. A decrease in randomness of degradation of D-galacturonan, loss of specificity of (3 + 1) splitting of tetra(D-galactosiduronic acid) and decrease in K_m value were found with the supports containing predominantly micropores. The extent of the changes decreased with increasing pore size of the support. The catalytic behaviour of endopolygalacturonase bound on the supports with large pores was quite analogous to that of the free enzymes.     

Selective isolation of endo-D-galacturonanase of Aspergillus niger based on interaction with tri(D-galactosiduronic acid) covalently bound to poly(hydroxyalkyl methacrylate)

A selective affinity-adsorbent for the extracellular endo-D-galacturonanase (E.C. 3.2.1.15) of Aspergillus niger was prepared by covalent coupling of tri(D-galactosiduronic acid) to Separon, a poly(hydroxyalkyl methacrylate) gel. Complexing of the enzyme with the adsorbent is pH dependent; maximal interaction occurs at the optimum pH for enzyme activity. The enzyme was quantitatively displaced from the adsorbent either by changing the pH or by bioelution with soluble tri(D-galactosiduronic acid) or other substrate. Within the range of substitution of Separon examined [content of tri(D-galactosiduronic acid) 1.7-6.7%] the amount of endo-D-galacturonanase retained was proportional to the content of affinity ligand. Under the same conditions, unsubstituted carrier did not complex with endo-D-galacturonanase. The dissociation constant of the affinity complex, as determined by zonal analysis, kinetic measurements, and by means of the adsorption isotherm KL (0.54 mmol.L-1), is close to the value (KI 0.44 mmol.L-1) obtained by the two first methods with soluble tri(D-galactosiduronic acid). The results show that adsorption of endo-D-galacturonanase on tri(D-galactosiduronic acid)-Separon is due exclusively to active-site-directed interaction with bound affinity-ligand.     

Mode of action of endopolygalacturonase immobilized by adsorption and by covalent binding via amino groups

Action pattern of endopolygalacturonase (E.C.3.2.1.15) immobilized by adsorption on porous powdered poly(ethyleneterephthalate) and covalently bound via amino groups on poly(2, 6-dimethyl-p-phenyleneoxide) and poly(6-caprolactame), respectively, were investigated in suspension and packed columns using polymeric and oligomeric D-galactosiduronates as substrates. The covalent binding invariably led to a lowering of randomness of degradation of high-molecular substrates and loss of specificity of (3 + 1) splitting of tetra(galactosiduronic acid), typical of the free enzyme. In the adsorbed endopolygalacturonase the degree of randomness of degradation of D-galacturonan and K(m,app) value were dependent on the substrate transfer; the former parameter increased, the later decreased with increasing flow-rate of the substrate through the immobilized enzyme bed. The action pattern on low-molecular substrates was not altered. The changes in action pattern of the covalently immobilized endopolygalacturonase are ascribed to sterical limitations resulting from a binding of the enzyme molecule in the proximity of its active site. In endopolygalacturonase bound to the support by hydrophobic interactions external diffusion effects are regarded the factors governing the enzyme action.     

The Effects of the Porosity of a Support and of Attachment on the Mode of Action of Immobilized Endopolygalacturonase

Endopolygalacturonase of Aspergillus sp. was immobilized by three different methods; viz. (a) via amino groups, (b) via carboxyl groups and (c) by means of epoxy groups to a nonporous microparticular silicon dioxide (Cabosil), functionalized by 3-(amino)-propyl groups and 3-(2',3'-epoxypropoxy)-propyl groups, respectively. The conjugates were compared in their mode of action with corresponding immobilized preparations based on microporous ceramics. The binding via amino groups and via carboxyl groups lead, by itself, to changes in the mode of action of the enzyme, consisting of a decrease in randomness of glycosidic linkage splitting. The changes were greater in microporous support conjugates due to additional size-exclusion effects. The action pattern of endopolygalacturonase bound by means of epoxy groups was modulated exclusively by the porosity of the support, whereas the binding alone did not play any role.     

The Effects of the Porosity of a Support and of Attachment on the Mode of Action of Immobilized Endopolygalacturonase

Endopolygalacturonase of Aspergillus sp. was immobilized by three different methods; viz. (a) via amino groups, (b) via carboxyl groups and (c) by means of epoxy groups to a nonporous microparticular silicon dioxide (Cabosil), functionalized by 3-(amino)-propyl groups and 3-(2′,3′-epoxypropoxy)-propyl groups, respectively. The conjugates were compared in their mode of action with corresponding immobilized preparations based on microporous ceramics. The binding via amino groups and via carboxyl groups lead, by itself, to changes in the mode of action of the enzyme, consisting of a decrease in randomness of glycosidic linkage splitting. The changes were greater in microporous support conjugates due to additional size-exclusion effects. The action pattern of endopolygalacturonase bound by means of epoxy groups was modulated exclusively by the porosity of the support, whereas the binding alone did not play any role.     

Changes in the cell size of Escherichia coli M-17 during induced destruction

The autolysis of E. coli, induced by their deprivation of nutrition in combination with the action of oleic acid and a temperature of 45 degrees C, was studied. The study revealed that an increase in the number of cells during 4 hours after the induction of the process was accompanied by a decrease in their size and an increase in the surface/volume ratio. Changes in the size of bacteria in the course of induced destruction resulting from their deprivation of nutrition in combination the action of oleic acid and a temperature of 45 degrees C were found to occur in two phases: (1) a decrease in size with an increase in the surface/volume ratio; (2) an increase in size with a decrease in the surface/volume ratio.     

Mechanism of 3-mercaptopicolinic acid inhibition of hepatic phosphoenolpyruvate carboxykinase (GTP).

The hypoglycemic agent 3-mercaptopicolinic acid inhibits gluconeogenesis from lactate by isolated, perfused livers from fasted rats and guinea pigs. A 3-mercaptopicolinate concentration of 50 muM caused a sharp decrease in glucose synthesis, with virtually complete inhibition at 100 muM. This inhibitory effect was reversed completely when 3-mercaptopicolinate was removed and the rate of glucose synthesis returned to normal values within 2 min. Oxygen consumption was not altered, even at the highest concentration of inhibitor. Gluconeogenesis from glycerol by guinea pig liver was blocked completely by 100 muM 3-mercaptopicolinate but was inhibited only partially in rat liver. After removal of the inhibitor glucose synthesis returned to levels higher than noted before the addition of this compound. The formation of P-enolpyruvate bu isolated guinea pig liver mitochondria metabolizing alpha-ketoglutarate (State 3) was inhibited markedly by 3-mercaptopicolinate, but malate conversion to P-enolpyruvate was considerably less sensitive. Kinetic studies with purified P-enolpyruvate carboxykinase from rat liver cytosol indicate that 3-mercaptopicolinate is a noncompetitive inhibitor with respect to both oxalacetate and MnGTP2-, and that simulataeous saturation with both substrates does not diminish this inhibition. The inhibitory effects of 3-mercaptopicolinate occur primarily by decreasing the rate of product formation while having relatively minor effects on the apparent Michaelis constants for substrates. Inhibition constants for slope and intercept effects ranged from 3 to 9 muM 3-mercaptopicolinate, and the inhibition patterns were dependent on the concentration of free Mn2+ present. Comparison of the inhibition constants with the observed inhibition of gluconeogenesis in livers perfused with 3-mercaptopicolinate supports the contention that P-enolpyruvate carboxykinase is the site of action of this inhibitor. The possibility that 3-mercaptopicolinate inhibition occurs by binding either free or bound manganese was eliminated by determination of the dissociation constant of 0.51 mM for the manganese-3-mercaptopicolinate complex. In addition, no tightly bound, slowly exchanging metal was bound to purified enzyme protein. These results suggest that 3-mercaptopicolinate inhibits by the removal of a tightly bound, rapidly exchanging metal ion other than Mn2+.     

Gum Arabic coated magnetic nanoparticles with affinity ligands specific for antibodies

A novel magnetic support based on gum Arabic (GA) coated iron oxide magnetic nanoparticles (MNP) has been endowed with affinity properties towards immunoglobulin G (IgG) molecules. The success of the in situ triazine ligand synthesis was confirmed by fluorescence assays. Two synthetic ligands previously developed for binding to IgG, named as ligand 22/8 (artificial Protein A) and ligand 8/7 (artificial Protein L) were immobilized on to MNPs coated with GA (MNP_GA). The dimension of the particles core was not affected by the surface functionalization with GA and triazine ligands. The hydrodynamic diameters of the magnetic supports indicate that the coupling of GA leads to the formation of larger agglomerates of particles with about 1 µm, but the introduction of the triazine ligands leads to a decrease on MNPs size. The non‐functionalized MNP_GA bound 28 mg IgG/g, two times less than bare MNP (60 mg IgG/g). MNP_GA modified with ligand 22/8 bound 133 mg IgG/g support, twice higher than the value obtained for ligand 8/7 magnetic adsorbents (65 mg/g). Supports modified with ligand 22/8 were selected to study the adsorption and the elution of IgG. The adsorption of human IgG on this support followed a Langmuir behavior with a Q_máx of 344 mg IgG/g support and K_a of 1.5 × 10⁵ M. The studies on different elution conditions indicated that although the 0.05 M citrate buffer (pH 3) presented good recovery yields (elution 64% of bound protein), there was occurrence of iron leaching at this acidic pH. Therefore, a potential alternative would be to elute bound protein with a 0.05 M glycine‐NaOH (pH 11) buffer. Copyright © 2010 John Wiley & Sons, Ltd.     

Separation of peptides by reverse-phase high-performance liquid chromatography using propyl- and cyanopropylsilyl supports

The separation of peptides and proteins by reverse-phase high-performance liquid chromatography with cyanopropylsilyl and large-pore propylsilyl supports, together with aqueous trifluoroacetic acid/acetonitrile gradients, was studied. Operating parameters (trifluoroacetic acid concentration, flow rate, and gradient slope) were evaluated using different enzymatic digests of horse cytochrome c and bovine serum albumin. Peptides ranging in size from five amino acids to 68 kDa could be separated on the propylsilyl column in a single chromatographic run. The cyanopropylsilyl column is suitable as a supplement to the use of the large-pore column for medium size (5-20 amino acids) peptides. The chromatographic supports and conditions presented here offer a simple, sensitive, and rapid separation system for a wide size range of peptides and proteins. They extend the versatility of separation methodology for these molecules.     

Photomodulation of conformational states. IV. Integrin-binding RGD-peptides with (4-aminomethyl)phenylazobenzoic acid as backbone constituent

In previous studies we have investigated octapeptides backbone-cyclized by (4-amino)phenyl azobenzoic acid (APB) or (4-aminomethyl)phenylazobenzoic acid (AMPB) and containing the active-site sequence Cys-Ala-Thr-Cys-Asp from the thioredoxin reductase. The conformational and redox properties of these peptides were strongly dependent on the isomeric state of the azobenzene chromophore. Using the same approach we were successful in constructing photoresponsive ligands for alphavbeta3 integrin containing the Arg-Gly-Asp (RGD) sequence as binding motif. For achieving maximal conformational restriction of the peptide a reduced ring size compared to our previous azobenzene peptides was employed in the cyclic peptide c[Asp-D-Phe-Val-AMPB-Lys-Ala-Arg-Gly-]. Conformational properties of the trans and cis isomers of this peptide in solution were investigated by CD and NMR and were found to differ markedly from the thioredoxin derived azobenzene peptides. In a second peptide, c[Asp-D-Phe-Val-Lys-AMPB-Ala-Arg-Gly-], shifting the position of the chromophore lead to a marked decrease in affinity. With the availability of the x-ray structure of a cyclic RGD-pentapeptide bound to alphavbeta3 integrin (PDB entry 1L5G) modeling of possible bound conformations for trans and cis isomers of both azobenzene peptides was possible. Notably, both peptides in either isomeric form share the same overall conformation in the bound state according to our molecular dynamics simulations.     

Plasma and cellular zinc levels and membrane lipid composition in streptozotocin diabetic rats

1. Lipid and zinc analyses were conducted on liver mitochondrial and microsomal membranes as well as erythrocyte ghosts from streptozotocin (STZ) treated animals. 2. In STZ animals, analysis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) fatty acids revealed an increase in palmitic acid and a corresponding decrease in stearic acid. 3. Polyunsaturated fatty acids were also affected, with an increase in 18:2, decrease in 20:4 and an increase in 22:6 in STZ animals compared to controls. 4. The change in fatty acid composition was observed in all three membrane fractions. 5. Plasma zinc levels in STZ animals were elevated while no difference was observed in membrane bound zinc. 6. Thus, while there appears to be both altered trace metal as well as membrane lipid metabolism in STZ treated animals, membrane bound zinc does not seem to be affected.     

Activation of c-Raf kinase by ultraviolet light. Regulation by retinoids

The present study highlights retinoids as modulators of c-Raf kinase activation by UV light. Whereas a number of retinoids, including retinol, 14-hydroxyretroretinol, anhydroretinol (AR), and retinoic acid bound the c-Raf cysteine-rich domain (CRD) with equal affinity in vitro as well as in vivo, they displayed different, even opposing, effects on UV-mediated kinase activation; retinol and 14-hydroxyretroretinol augmented responses, whereas retinoic acid and AR were inhibitory. Oxidation of thiol groups of cysteines by reactive oxygen, generated during UV irradiation, was the primary event in c-Raf activation, causing the release of zinc ions and, by inference, a change in CRD structure. Retinoids modulated these oxidation events directly: retinol enhanced, whereas AR suppressed, zinc release, precisely mirroring the retinoid effects on c-Raf kinase activation. Oxidation of c-Raf was not sufficient for kinase activation, productive interaction with Ras being mandatory. Further, canonical tyrosine phosphorylation and the action of phosphatase were essential for optimal c-Raf kinase competence. Thus, retinoids bound c-Raf with high affinity, priming the molecule for UV/reactive oxygen species-mediated changes of the CRD that set off GTP-Ras interaction and, in context with an appropriate phosphorylation pattern, lead to full phosphotransferase capacity.     

Identification of two binding sites for wheat-germ agglutinin on polylactosamine-type oligosaccharides.

The carbohydrate-binding properties of wheat-germ agglutinin (WGA) have been studied by using glycopeptides isolated from the cell surfaces of a cultured murine myeloid cell line (416B). The glycopeptides were passed through affinity columns of lentil lectin (LCA), concanavalin A (Con A) and WGA arranged in series so that material reaching the WGA column had failed to bind to LCA or Con A. WGA-binding glycopeptides were step-eluted with 0.01 M, 0.1 M and 0.5 M-N-acetylglucosamine (GlcNAc), to yield weak (WGA-W), intermediate (WGA-I) and strong (WGA-S) affinity fractions. WGA-W and WGA-I contained 'N'- and 'O'-linked oligosaccharides bound to separate polypeptides. WGA-S consisted almost entirely of N-linked components. Our analytical work was concentrated mainly on the N-linked fractions. In these carbohydrates WGA affinity was directly proportional to molecular size but inversely related to N-acetylneuraminic acid content. The binding of the weak-affinity fraction was dependent on N-acetylneuraminic acid, but the intermediate- and strong-binding species interacted with the lectin by N-acetylneuraminic acid-independent mechanisms. N-linked glycopeptides in each WGA-binding class were almost totally degraded to monosaccharides by the concerted action of the exoglycosidases neuraminidase, beta-galactosidase and beta-N-acetylglucosaminidase. Treatment with endo-beta-galactosidase caused partial depolymerization, yielding some disaccharides but also a heterogeneous population of partially degraded components. These findings suggest that WGA binds with high affinity to internal GlcNAc residues in large oligosaccharides containing repeat sequences of Gal beta(1----4)GlcNAc beta(1----3) (i.e. polylactosamine-type glycans). N-Acetylneuraminic acid is involved only in low-affinity interactions with WGA. WGA therefore displays an intricate pattern of saccharide specificities that can be profitably utilized for structural analysis of complex carbohydrates.     

Effect of vitamin B3-active compounds on the content of free and combined gamma-aminobutyric acid and glutamic acid in the brain of mice

The bound and free GABA and glutamic acid content in the brain of F1 (CBA X C57B1/6) hybrid mice was investigated by the Eliott method. A tendency to a decrease of GABA and glutamate content in the brain with their practically constant bound/free ratio was observed 24 h after calcium-D-pantothenate injections (150 mumole/kg, 9 injections for 3 days). Calcium-D-homopantothenate injected in the same way caused a significant decrease in the GABA content, and a sharp drop of the bound/free GABA ratio. The effect is not associated with the influence of calcium ions in the composition of the injected compounds.     

The source of the iron binding with nitric oxide in activated macrophages]

No decrease in iron-sulphur centers was found in cultured macrophage cells (J774) after the treatment with nitric oxide (10(-7) M NO/10(7) cells) during 5 min. The center content was controlled by the electron spin resonance (ESR) method. The macrophages pretreated with dithionite + methyl viologen showed the formation of dinitrosyl iron complexes (DNIC) with a characteristic ESR signal at g approximately 2.03. The data suggest that loosely bound nonheme iron (free iron) mostly contributes to the formation of these complexes. Iron from iron-containing proteins does not release from these centers under the direct action of nitric oxide. The iron-sulphur centers can be destroyed by the products of nitric oxide oxidation (NO2, N2O3, etc.) as oxidizing and acid agents.     

Repeated-batch fermentation in biofilm reactors with plastic-composite supports for lactic acid production

Novel plastic supports consisting of polypropylene blended with oat hulls/soybean flour or oat hulls/zein were evaluated as supports for mixed- and pure-culture, repeated-batch, lactic acid fermentations in biofilm reactors. Streptomyces viridosporus T7A (ATCC 39115) was used to form a biofilm for mixed-culture fermentations, and Lactobacillus casei subsp. rhamnosus (ATCC 11443) was used for L-lactic acid production. The pure- and mixed-culture biofilm reactors were operated as repeated-batch fermentors with pH controlled at 5 for more than 2 months in which each reactor's medium was changed every 3 days for 24 batches. The plastic-composite supports performed better than polypropylene-alone supports. Significantly (P<0.05) higher concentrations of lactic acid were produced by the mixed- and pure-culture biofilm bioreactors with corresponding plastic-composite supports (55 g/l and 60 g/l respectively) than with polypropylene-alone supports (48 g/l for both mixed and pure culture). However, the percentage yields, maximum productivity, glucose consumption rates, and growth rates (based on the mass of suspended cells only) were not significantly different between reactors. Maximum lactic acid concentration was consistently greater for the plastic-composite support biofilm reactors. In the suspension culture at pH 5 without plastic supports, maximum lactic acid concentration at days 3 and 5 was 48 g/l and 60 g/l, respectively. These results confirm that the use of plastic-composite supports is recommended for pure-culture lactic acid production in long-term repeated-batch fermentation, and that cell immobilization was occurring.Journal Paper No. J-15813 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, Projects No. 3253 and 0178     

Fatty Acid Composition of Human Myelin Proteolipid Protein in Peroxisomal Disorders

Myelin proteolipid protein (PLP) is an acylated protein which contains approximately 2 mol of ester-bound fatty acids. In this study, the amount and composition of fatty acids covalently bound to human myelin PLP were determined during development and in peroxisomal disorders. Palmitic, oleic, and stearic acids accounted for most of the PLP acyl chains. However, in contrast to PLP in other species, human PLP contains relatively more very long chain fatty acids (VLCFA). The fatty acid composition remained essentially unchanged between 1 day and 74 years of age. The total amount of fatty acid bound to PLP was not altered in any of the pathological cases examined. However, in the peroxisomal disorder adrenoleukodystrophy, the proportions of saturated and, to a lesser extent, monounsaturated VLCFA bound to PLP were increased at the expense of oleic acid. Smaller, but significant, changes were observed in adrenomyeloneuropathy. The reduction in the levels of oleic acid was also observed in two other peroxisomal disorders, the cerebrohepatorenal (Zellweger) syndrome and neonatal adrenoleukodystrophy, as well as in the lysosomal disorder Krabbe globoid cell leukodystrophy. However, in these disorders, the decrease in oleic acid occurred at the expense of stearic acid, and not VLCFA. The results indicate that, although a characteristic PLP fatty acid pattern is normally maintained, changes in the acyl chain pool can ultimately be reflected in the fatty acid composition of the protein. The altered PLP-acyl chain pattern in peroxisomal disorders may contribute to the pathophysiology of these devastating disorders.     

Incorporation of fucose and glucosamine into cell bound and medium released macromolecules.

(1) Determinations were carried out on the incorporation of fucose-6-(3H) and glucosamine-6-(3H) into trichloracetic acid insoluble macromolecules which remained bound to the cells or were released into the medium of chick embryo muscle cell cultures. The radioactivity determined in the medium was corrected for unspecific binding of label to components of the medium. (2) During an incorporation period of six hours the incorporation per microgram DNA with fucose as label into cell bound macromolecules is about twice as high as the incorporation into macromolecules released into medium. With glucosamine about twice as much is incorporated into medium released into the cell bound macromolecules. (3) The incorporation per microgram DNA increased during a culture period of three days but the increase ceases at different times during this culture period when determined with fucose or glucosamine or for cell bound and medium released material. (4) An increase in cell density increases the incorporation per DNA of fucose and to a much slighter extent that of glucosamine. Reduction of cell density by addition of cytosine arabinoside to the medium does not increase the incorporation per microgram DNA. (5) The effect of changes of fibroblast/myoblast ratios on the incorporation of fucose and glucosamine were examined. No significant effect was observed for a ratio of 10-30% fibroblasts when control cultures or cultures after cell sedimentation were maintained in complete medium. Marked changes were observed after culture in medium without protein components. Under these conditions an increase in the fibroblast/myoblast ratios were observed as well as an increase in the incorporation of label into medium released and a decrease into cell bound macromolecules.     

Mossbauer spectroscopy of cells of cyanobacteria Synechocystis sp. PCC 6803 devoid of photosystem I and containing inactive phycobilisomes]

Mossbauer spectra of the psaAB mutant of Synechocystis sp. PPC 6803 devoid of photosystem I grown in a 57Fe-containing medium were measured. The spectrum is a broadened doublet whose size (about 20%) and parameters (isomeric shift delta = 0.3 mm/s and quadrupole splitting delta = 0.8 mm/s) suggest the presence of abundant nanoclusters of Fe3+ oxides in a superparamagnetic state tightly bound to the membrane. Treatment of cells with EDTA was accompanied by a substantial (tenfold) decrease in the amount of iron nonspecifically bound to the membrane and the appearance of Fe2+ localized, probably, inside cells and/or cell membranes. In addition, the spectrum of washed cells exhibited superfine magnetic splitting due to iron oxide clusters greater in size than nanoclusters present in the membrane prior to EDTA treatment.     

Clearance of senescent erythrocytes: wheat germ agglutinin distribution on young and old human erythrocytes

To add an additional aspect to the process of recognition and removal of senescent human erythrocytes from the circulation, the binding of wheat germ agglutinin (WGA) to separated young, old and sialidase-treated human erythrocytes is evaluated with the immune-electron microscopical method. WGA/gold conjugate binding to old erythrocytes was lower (27%) than to young erythrocytes and even lower following treatment with sialidase (82%), exhibiting a clustered, non-continuous labeling pattern in all three erythrocyte populations, thus showing a possible redistribution of WGA binding sites. The decrease in bound WGA/gold particles correlates well with the previously reported decrease in surface sialic acid on old erythrocytes. The binding of WGA/gold are indicative of the changes occurring on erythrocyte membrane surfaces when interacting with different agglutinins.