pH induced increase in cyclic GMP reactivity with cyclic AMP-dependent protein kinases

Cyclic AMP-dependent protein kinases have been found which exhibit an enhanced capacity to bind cyclic GMP at acidic values of pH. The binding of cyclic GMP to a protein kinase from skeletal muscle, eluted as a single peak from DEAE cellulose columns, is inversely proportional to pH between the values of 7 to 4; the enzyme exhibits a 5 fold greater ability to bind cyclic [³H]-GMP (10^?8M) at pH 4.0 than 7.0. Protein kinases prepared from skeletal or uterine muscle, eluted as the first of two peaks from DEAE cellulose, exhibited similar pH dependent changes in specificity for cyclic GMP as determined by inhibition of cyclic [³H]-AMP binding. Acidic pH did not appreciably enhance the binding of cyclic [³H]-AMP to kinases prepared from aged skeletal muscle or kinase eluted as the second peak from DEAE cellulose.     

Human Rhinovirus Subviral A Particle Binds to Lipid Membranes over a Twofold Axis of Icosahedral Symmetry

Minor group human rhinoviruses bind low-density lipoprotein (LDL) receptors for endocytosis. Once they are inside endosomes, the acidic pH triggers their dissociation from the receptors and conversion into hydrophobic subviral A particles; these attach to the membrane and transfer their single-strand, positive-sense RNA genome into the cytosol. Here, we allowed human rhinovirus 2 (HRV2) A particles, produced in vitro by incubation at pH 5.4, to attach to liposomes; cryo-electron microscopy 3-dimensional single-particle image reconstruction revealed that they bind to the membrane around a 2-fold icosahedral symmetry axis.     

Isolation and partial characterization of a cystine-rich basic heparin-binding protein from bovine platelets

We report the isolation and partial characterization of a so far unidentified basic platelet protein. Delipidated bovine platelets were extracted at pH 2.1. The extract was subjected to differential precipitation at pH 5.4-5.5 and by ammonium sulfate, then it was further purified by ion exchange chromatography on DEAE and CM cellulose columns in an urea containing medium. The major protein peak eluted from the CM cellulose column by NaCl gradient contained a protein in electrophoretically homogeneous form. It consists of a single polypeptide chain with an Mr of 28,000 as estimated by SDS PAGE. It was shown to be extremely rich in lysine and cystine and possessed a highly basic character (pI 9.8-10.1). On this basis the term cystine-rich basic protein (CRBP) was proposed for the new protein. Unlike some other low Mr basic proteins it did not bind calmodulin and troponin C, however, it showed significant heparin neutralizing activity.     

Isolation of transfer RNA isoacceptors by chromatography on dihydroxyboryl-substituted cellulose,polyacrylamide, and glass

Polyacrylamide and porous-glass supports containing the dihydroxyborylphenyl group can be prepared by a method similar to that used in the synthesis of N-[N′-(m-dihydroxyborylphenyl)succinamyl]aminoethylcellulose. The reaction of aminoethylpolyacrylamide or amino-substituted glass with N-(m-dihydroxyborylphenyl)succinamic acid in the presence of N-cyclohexyl-N′-β-(4-methyl-morpholinium) ethylcarbodiimide yields products which, together with the cellulose derivative, are all capable of binding tRNA dissolved in buffers at pH 8.7. The demonstration that bound tRNA can be released with sorbitol solutions or with low pH buffers, together with studies on the binding of tRNA species that contain chemically modified 3′-terminals, indicate that the predominant binding mechanism consists of cyclic complex formation between the immobilized dihydroxyboryl groups and the 3′-terminal cis-diol groups of the tRNA molecules. Aminoacylated tRNA does not bind under the conditions necessary to bind tRNA and this permits the isolation of specific tRNA isoacceptors. The purification of tRNA^Phe and the partial purification of tRNA^Glu and tRNA^Trp are described.     

Binding of Colloidal MnO2 by Extracellular Polysaccharides of Pedomicrobium manganicum

The extracellular acidic polysaccharides of the manganese-oxidizing bacterium Pedomicrobium manganicum were able to bind preformed colloidal MnO₂. The capacity of the cells to bind MnO₂ was pH dependent. Enhanced binding capacity below pH 5 suggests that ionic bonding forces are involved in the binding mechanism and that there is a charge reversal on the acidic polysaccharides between pH 5 and 4 that is due to increased protonation of carboxyl groups.     

Purification and properties of neutral β-galactosidases from human liver

Two neutral β-galactosidase isozymes were purified from human liver. The initial step of purification was removal of the acidic β-galactosidases by adsorption on concanavalin A-Sepharose 4B conjugate. Subsequent purification steps included ammonium sulfate precipitation, diethylaminoethyl cellulose column chromatography, Sephadex G-100 gel filtration, and preparative polyacrylamide-gel isoelectric focusing. The final step of purification was affinity chromatography of the separated isoelectric forms on ?-aminocaproyl-β-d-galactosylamine-Sepharose 4B conjugate. The purified β-galactosidase isozymes had activity toward both β-d-galactoside and β-d-glucoside derivatives of 4-methylumbelliferone and p-nitrophenol with a pH optimum around 6.2. These enzyme forms were also found to possess lactosylceramidase II activity with a pH optimum in the range of 5.4 to 5.6, but not lactosylceramidase I activity and no activity toward galactosylceramide or GM₁-ganglioside. The molecular weight was found to be in the range of 37,500–39,500 for the two neutral isozymes and they had similar K_m and V values; the more acidic form (designated β-galactosidase N₁) was more heat stable than the other form (designated β-galactosidase N₂). Antibodies evoked against the N₁ and N₂ β-galactosidases gave identical precipitin lines retaining enzymatic activity. No cross-reactivity was observed between the neutral and the acidic isozymes when examined with the respective antisera.     

Solution structure and conformational heterogeneity of acylphosphatase from Bacillus subtilis

Acylphosphatase is a small enzyme that catalyzes the hydrolysis of acyl phosphates. Here, we present the solution structure of acylphosphatase from Bacillus subtilis (BsAcP), the first from a Gram-positive bacterium. We found that its active site is disordered, whereas it converted to an ordered state upon ligand binding. The structure of BsAcP is sensitive to pH and it has multiple conformations in equilibrium at acidic pH (pH < 5.8). Only one main conformation could bind ligand, and the relative population of these states is modulated by ligand concentration. This study provides direct evidence for the role of ligand in conformational selection.     

Continuous Cellulosic Bioethanol Fermentation by Cyclic Fed-Batch Cocultivation

Cocultivation of cellulolytic and saccharolytic microbial populations is a promising strategy to improve bioethanol production from the fermentation of recalcitrant cellulosic materials. Earlier studies have demonstrated the effectiveness of cocultivation in enhancing ethanolic fermentation of cellulose in batch fermentation. To further enhance process efficiency, a semicontinuous cyclic fed-batch fermentor configuration was evaluated for its potential in enhancing the efficiency of cellulose fermentation using cocultivation. Cocultures of cellulolytic Clostridium thermocellum LQRI and saccharolytic Thermoanaerobacter pseudethanolicus strain X514 were tested in the semicontinuous fermentor as a model system. Initial cellulose concentration and pH were identified as the key process parameters controlling cellulose fermentation performance in the fixed-volume cyclic fed-batch coculture system. At an initial cellulose concentration of 40 g liter⁻¹, the concentration of ethanol produced with pH control was 4.5-fold higher than that without pH control. It was also found that efficient cellulosic bioethanol production by cocultivation was sustained in the semicontinuous configuration, with bioethanol production reaching 474 mM in 96 h with an initial cellulose concentration of 80 g liter⁻¹ and pH controlled at 6.5 to 6.8. These results suggested the advantages of the cyclic fed-batch process for cellulosic bioethanol fermentation by the cocultures.     

The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass

Lignocellulosic biomass is a renewable and abundant resource with great potential for bioconversion to value-added bioproducts. However, the biorefining process remains economically unfeasible due to a lack of biocatalysts that can overcome costly hurdles such as cooling from high temperature, pumping of oxygen/stirring, and, neutralization from acidic or basic pH. The extreme environmental resistance of bacteria permits screening and isolation of novel cellulases to help overcome these challenges. Rapid, efficient cellulase screening techniques, using cellulase assays and metagenomic libraries, are a must. Rare cellulases with activities on soluble and crystalline cellulose have been isolated from strains of Paenibacillus and Bacillus and shown to have high thermostability and/or activity over a wide pH spectrum. While novel cellulases from strains like Cellulomonas flavigena and Terendinibacter turnerae, produce multifunctional cellulases with broader substrate utilization. These enzymes offer a framework for enhancement of cellulases including: specific activity, thermalstability, or end-product inhibition. In addition, anaerobic bacteria like the clostridia offer potential due to species capable of producing compound multienzyme complexes called cellulosomes. Cellulosomes provide synergy and close proximity of enzymes to substrate, increasing activity towards crystalline cellulose. This has lead to the construction of designer cellulosomes enhanced for specific substrate activity. Furthermore, cellulosome-producing Clostridium thermocellum and its ability to ferment sugars to ethanol; its amenability to co-culture and, recent advances in genetic engineering, offer a promising future in biofuels. The exploitation of bacteria in the search for improved enzymes or strategies provides a means to upgrade feasibility for lignocellulosic biomass conversion, ultimately providing means to a ''greener'' technology.     

Partial purification of microsomal signal peptidase from hen oviduct

Signal peptidase has been purified approximately 600-fold from hen oviduct microsomes. Treatment of microsomes with ice-cold sodium carbonate at pH 11.5 removes soluble and extrinsic membrane proteins prior to solubilization of signal peptidase with Nonidet P-40. After dialysis to pH 8.2, the solubilized enzyme is chromatographed on diethylaminoethyl cellulose at pH 8.2. More than 90% of contaminating proteins bind to the column while signal peptidase and endogenous phospholipid are eluted in the column void volume. Enzyme activity subsequently binds to carboxymethyl cellulose at pH 5.8 and is eluted by approximately 100 to 200 mM NaCl during a NaCl gradient. Polypeptides present in partially purified hen oviduct signal peptidase have relative molecular masses ranging from 54 kD to less than 11 kD with major bands at 29, 23, 22, 19, 18 and 13 kD. The purified peptidase requires phospholipid for activity and is maximally active in the presence of 2 mg/ml phosphatidylcholine.     

Cellulose digestion in a leaf eating insect,the mexican bean beetle,Epilachna varivestis

Adult Mexican bean beetles, Epilachna varivestis (Mulsant) (Coleoptera: Coccinellidae), are able to digest cellulose. Females digest nearly three times as much cellulose and retain food in their guts three times longer than do males. There is no sexual dimorphism in enzyme activity although pH exerts a profound effect. The approximate pH optima are 4.5 for C₁-cellulase, 5.5 for C_x-cellulase and 4.5–4.8 for β-glucosidase. The acidic gut of beetles (pH 4.9–5.8) should permit maximum cellulase activity.     

Aluminum as an endocrine disruptor in female Nile tilapia (Oreochromis niloticus)

The effects of aluminum on plasma ion, lipid, protein and steroid hormone concentration were evaluated in Oreochromis niloticus broodstock females. Lipid and protein concentrations from the gonads and liver were also measured. Experiments were performed at neutral and acidic water pH. Four groups of fish were tested for 96 h: 1) control conditions at neutral water pH; 2) control conditions at acidic water pH (CTR-Ac); 3) aluminum at neutral water pH (Al-N); and 4) aluminum at acidic water pH (Al-Ac). Aluminum and acidic water pH exposure caused no ionoregulatory disturbances. Total lipid concentration increased in the mature gonads and decreased in the liver, suggesting an acceleration of lipid mobilization to the ovaries in animals exposed to aluminum. However, a decreased protein concentration in ovaries was also observed. Exposure of control fish to acidic water pH caused an increased concentration of plasma 17α-hydroxyprogesterone. However, females exposed to aluminum at acidic water pH showed a decreased of plasma 17α-hydroxyprogesterone and cortisol. No differences in plasma 17β-estradiol were observed. The physiological mechanisms underlying the disturbances observed are discussed focusing on reproduction. We suggest that aluminum can be considered an endocrine disrupting compound in mature O. niloticus females.     

Resonance Raman studies on protocatechuate 3,4-dioxygenase-inhibitor complexes

Resonance Raman spectra of a number of protocatechuate 3,4-dioxygenase-inhibitor complexes were studied by use of the available lines of an argon and a krypton laser. Three types of inhibitors were investigated-hydroxybenzoates, dicarboxylates, and 4-nitrocatechol. The hydroxybenzoate study shows that the hydroxy group in 3-hydroxybenzoate does not coordinate to the active site iron, in agreement with earlier suggestions, and confirms the coordination of the hydroxy group in the isomeric 4-hydroxybenzoate. The dicarboxylate study demonstrates that both glutarate and terephthalate perturb the active-site environment, shifting the charge-transfer interaction to lower energy. The pH dependence of terephthalate binding as well as the spectral similarities of the dicarboxylate complexes to the ESO2 intermediate provides further evidence for the suggestion that this intermediate is a tightly bound enzyme-product complex. The 4-nitrocatechol study indicates that, unlike the substrate catechols, 4-nitrocatechol does not bind to the iron; a binding configuration wherein the acidic phenolate group interacts with the carboxylate binding site has been suggested by others. Finally the spectra of the 4-hydroxybenzoate and terephthalate complexes demonstrate the presence of two tyrosines coordinated to the active-site iron as suggested by others; these tyrosines have different vCO's and excitation profiles.     

Cellulose Degradation by Micromonosporas Recovered from Freshwater Lakes and Classification of These Actinomycetes by DNA Gyrase B Gene Sequencing

A number of Micromonospora strains isolated from the water column, sediment, and cellulose baits placed in freshwater lakes were shown to be able to degrade cellulose in lake water without any addition of nutrients. A selective isolation method was also developed to demonstrate that CFU arose from both spores and hyphae that inhabit the lake environment. Gyrase B gene sequencing performed on the isolates identified a number of new centers of variation within Micromonospora, but the most actively cellulolytic strains were recovered in a single cluster that equated with the type species of the genus, M. chalcea.     

Purification of guanosine triphosphate cyclohydrolase I and dihydrofolate reductase on a dihydrofolate-Sepharose affinity column.

Folate was coupled to AH-Sepharose 4B, the gel poured into small columns, and the Sepharose-bound folate reduced in situ to dihydrofolate by dithionite/ascorbate at pH 6 to 7. The dihydrofolate-Sepharose column was used to purify guanosine triphosphate cyclohydrolase I (EC 3.5.4.16) and dihydrofolate reductase (EC 1.5.1.3). All steps were carried out in the cold and in the presence of 20 mm mercaptoethanol. GTP cyclohydrolase I bound strongly to the dihydrofolate-Sepharose column and was purified several-hundred-fold in a single step. It did not bind to folate-Sepharose. Binding to dihydrofolate-Sepharose is assumed to reflect a physiological role of dihydrofolate. GTP cyclohydrolase II did not bind to either folate- or dihydrofolate-Sepharose. Dihydrofolate reductase from Escherichia coli B and from rat liver did not bind to folate-Sepharose under the test conditions, but could be well purified on the dihydrofolate-Sepharose column. This column is judged to beuseful for the purification of other folate-converting enzymes.     

Fusion of carbohydrate binding modules from <Emphasis Type="Italic">Thermotoga neapolitana</Emphasis> with a family 10 xylanase from <Emphasis Type="Italic">Bacillus halodurans</Emphasis> S7

Xylanase A of Thermotoga neapolitana contains binding domains both at the N- and C-terminal ends of the catalytic domain. In the N-terminal position it contains two carbohydrate-binding modules (CBM) which belong to family 22. These CBMs bind xylan but not to cellulose. The gene encoding the mature peptide of these CBMs was fused with an alkaline active GH10 xylanase from Bacillus halodurans S7 and expressed in Escherichia coli. The (His)₆ tagged hybrid protein was purified by immobilized metal affinity chromatography and characterized. Xylan binding by the chimeric protein was influenced by NaCl concentration and pH of the binding medium. Binding increased with increasing salt concentration up to 200 mM. Higher extent of binding was observed under acidic conditions. The fusion of the CBM structures enhanced the hydrolytic efficiency of the xylanase against insoluble xylan, but decreased the stability of the enzyme. The optimum temperature and pH for the activity of the xylanase did not change.     

Clostridium difficile Cell Attachment Is Modified by Environmental Factors

Adherence of Clostridium difficile to Vero cells under anaerobic conditions was increased by a high sodium concentration, calcium-rich medium, an acidic pH, and iron starvation. The level of adhesion of nontoxigenic strains was comparable to that of toxigenic strains. Depending on the bacterial culture conditions, Vero cells could bind to one, two, or three bacterial surface proteins with molecular masses of 70, 50, and 40 kDa.     

Recombinant Protein Purification using Composite Polyacrylamide-Nanocrystalline Cryogel Monolith Column and Carbohydrate-Binding Module Family 64 as Affinity Tag

Background:In the field of recombinant protein production, downstream processing, especially protein purification, is critical and often the most expensive step. Carbohydrate binding module 64 (CBM64) was shown in 2011 to bind efficiently to a broad range of cellulose materials.Methods:In this study, we developed a protein purification method using nanocrystalline cellulose embedded in a polyacrylamide monolith cryogel and CBM64 affinity tag linked by intein to PD1 as a model protein. The CBM64-Intein-PD1 gene cassette was expressed in E. coli. Following cell lysis, CBM64-Intein-PD1 protein bound to the monolith PA-NCC cryogel. After washing and reducing the pH from 8.0 to 6.5, the intein underwent self-cleavage, resulting in the release and elution of pure PD1 protein.Results:The synthesized monolith column had a porous structure with an average pore size of 30 µm and a maximum binding capacity of 497 µg per gram of dried column. The yield of this purification method was 84%, while the yield of the His tag-acquired CBM64-Intein-PD1 method was 89%.DiscussionWe used cellulose as support for affinity chromatography, which can be used as a cost-effective method for protein purification.Key Words: Affinity tag, CBM64, PD1, Protein purification     

Crayfish carboxypeptidase. Affinity chromatography, characterization and amino-terminal sequence.

In an effort to trace the evolutionary history of the pancreatic metalloexopeptidases, carboxypeptidase has been isolated from the cardia of the crayfish Astacus fluviatilis. The isolation procedure included affinity chromatography on a column of potato carboxypeptidase inhibitor covalently linked to Sepharose. Approximately 25 mg of pure enzyme can be obtained by the present procedure from 50 ml of cardia fluid. The pure enzyme resembles bovine carboxypeptidase B in specificity and is inhibited both by 3-phenyllactate and by 6-aminohexanoate. The pH optimum of activity is about pH 6.5, and the isoelectric point,pH 4.0. Inhibition by typical metal chelating agents (i.e. ethylenediamine tetraacetate and 1,10-phenanthroline) and neutron activation analysis indicate that, like the mammalian enzyme, crayfish carboxypepetidase is a zinc metalloenzyme. The purified enzyme migrates as a single band in cellulose acetate, disc gel and sodium dodecylsulfate gel electrophoresis. The amino acid composition is similar to that of pancreatic carboxypeptidases except for a higher content of acidic amino acid residues. The amino acid sequence of the first 19 amino-terminal residues reveals significant homology to that of pancreatic carboxypeptidases A and B.