Cytochemical Method to Localize Acidic Nuclear Proteins

Fast green FCF was used to localize acidic nuclear proteins in sections of young flower buds of Limnophyton obtusifolium (L.) Miq. After extracting nucleic acids, the slides were stained at hydrogen ion concentrations ranging from pH 2.6 to 9.0. At pH 5.0 and 8.0 staining is confined to the nucleus with no cytoplasmic reaction. Staining intensity is greater at pH 5.0 than at pH 8.0. The proteins responding to fast green at pH 8.0 are basic proteins. The positive reaction at pH 5.0 is attributed to acidic nuclear proteins. These findings are confirmed by control preparations. Acetylated slides and slides treated with 0.25 N HCl were unstained at pH 8.0 but staining at pH 5.0 was undisturbed. Dilute alkali (0.003 N NaOH) reduced staining intensity at pH 5.0 but had no effect at pH 8.0. Methylated slides did not stain at pH 5.0, but at pH 8.0 staining was unaffected. Deamination blocked staining at both pH's. It is concluded that fast green at pH 5.0 specifically binds with acidic nuclear proteins.     

A new facultatively autotrophic hydrogen- and sulfur-oxidizing bacterium from an alkaline environment

An alkaliphilic bacterium, strain AHO 1, was isolated from an enrichment culture with hydrogen at pH 10 inoculated with a composite sample of sediments from five highly alkaline soda lakes (Kenya). This bacterium is a gram-negative, nonmotile, rod-shaped, obligately aerobic, and facultatively autotrophic hydrogen-oxidizing organism. It was able to oxidize reduced sulfur compounds to sulfate during heterotrophic growth. It utilized a wide range of organic compounds as carbon and energy sources and grew mixotrophically with hydrogen and acetate. With sulfur compounds, mixotrophic growth was observed only in acetate-limited continuous culture. The normal pH range for autotrophic growth with hydrogen was pH 8.0–10.25, with a pH optimum at 9–9.5. Growth at pH values lower than 8.0 was extremely slow. Heterotrophic growth with acetate was optimal at pH 10.0. The hydrogen-oxidizing activity of whole cells was maximal at pH 9.0 and still substantial up to pH 11. NAD-dependent hydrogenase activity was found in the soluble fraction of the cell-free extract, but no methylene blue-dependent activity in either the soluble or membrane fractions was observed. On the basis of its pH profile, the soluble hydrogenase of strain AHO 1 was a typical pH-neutral enzyme. Phylogenetic analysis revealed that strain AHO 1 belongs to the α-3 subgroup of the Proteobacteria with a closest relation to a recently described alkaliphilic aerobic bacteriochlorophyll a-containing bacterium "Roseinatronobacter thiooxidans." Received: February 29, 2000 / Accepted: April 3, 2000     

pH值对香蕉枯萎病菌4号生理小种生长的影响

【背景】香蕉枯萎病菌4号生理小种(镰刀菌)是香蕉产业的致命威胁。已有研究表明土壤pH值越高,香蕉枯萎病发病率越低,但是现有pH值对镰刀菌影响的研究大都是用强酸强碱调节pH值,pH值没有缓冲体系保护,而且尚未检测试验终点时介质的pH值。此外,关于pH值对香蕉枯萎病菌4号生理小种(Foc4)影响的研究尚不系统,难以用于指导生产实践。【目的】为系统地了解土壤酸碱度对Foc4生长的影响。【方法】在pH 3.0-11.0之间设定9个pH值梯度,模拟酸性到碱性土壤pH值条件,于室内培养条件下系统研究pH值对Foc4生长、产孢、孢子萌发的影响及其生长过程对环境pH值的影响。【结果】弱酸性至中性环境(pH 5.0-7.0)最适宜于香蕉枯萎病菌的生长、产孢和孢子萌发。弱碱性处理(pH8.0和pH9.0)孢子平均萌发率较弱酸性环境处理(pH5.0和pH6.0)下降了73.1%。与pH 6.0酸性处理相比,pH 8.0和pH 9.0处理的产孢量分别下降了52.3%和68.1%。【结论】香蕉枯萎病菌Foc4生长和萌发过程会产酸,但是在缓冲体系液体培养基中,除了pH 9.0和pH10.0处理终点培养液pH值分别下降了0.34和0.27个单位外,其它处理起始和终点的pH值无差异。说明在缓冲体系液体培养基中的研究结果可以反映环境pH值对Foc4生长和萌发的影响。在作物可以生长的pH值范围内(pH5.0-9.0),碱性和微碱性条件(pH8.0-9.0)能明显抑制Foc4生长、产孢和孢子萌发。     

Improving solubility of Shewanella oneidensis MR-1 and Clostridium thermocellum JW-20 proteins expressed into Esherichia coli

Low solubility of proteins overexpressed in E. coli is a frequent problem in high-throughput structural genomics. To improve solubility of proteins from mesophilic Shewanella oneidensis MR-1 and thermophilic Clostridium thermocellum JW20, an approach was attempted that included a fusion of the target protein to a maltose-binding protein (MBP) and a decrease of induction temperature. The MBP was selected as the most efficient solubilizing carrier when compared to a glutathione S-transferase and a Nus A protein. A tobacco etch virus (TEV) protease recognition site was introduced between fused proteins using a double polymerase-chain reaction and four primers. In this way, 79 S. oneidensis proteins have been expressed in one case with an N-terminal 30-residue tag and in another case as a fusion protein with MBP. A foreign tag might significantly affect the properties of the target polypeptide. At 37 degrees C and 18 degrees C induction temperatures, only 5 and 17 tagged proteins were soluble, respectively. In fusion with MBP 4, 34, and 38 proteins were soluble upon induction at 37 degrees, 28 degrees, and 18 degrees C, respectively. The MBP is assumed to increase stability and solubility of a target protein by changing both the mechanism and the cooperativity of folding/unfolding. The 66 C. thermocellum proteins were expressed as fusion proteins with MBP. Induction at 37 degrees, 28 degrees, and 18 degrees C produced 34, 57, and 60 soluble proteins, respectively. The higher solubility of C. thermocellum proteins in comparison with the S. oneidensis proteins under similar conditions of induction correlates with the thermophilicity of the host. The two-factor Wilkinson-Harrison statistical model was used to identify soluble and insoluble proteins. Theoretical and experimental data showed good agreement for S. oneidensis proteins; however, the model failed to identify soluble/insoluble Clostridium proteins. A suggestion has been made that the Wilkinson-Harrison model is not applicable to C. thermocellum proteins because it did not account for the peculiarities of protein sequences from thermophiles.     

Interaction of heme proteins with poly(propyleneimine) dendrimers in layer-by-layer assembly films under different pH conditions

With the isoelectric point at pH 7.4, hemoglobin (Hb) has net positive surface charges at pH 5.0 and overall negative charges at pH 9.0, and is essentially neutral at pH 7.0. The fifth-generation poly(propyleneimine) (PPI) dendrimer is usually positively charged in aqueous solution. The {PPI/Hb}n films under different pH conditions have been successfully fabricated on various solid surfaces by the layer-by-layer assembly technique, and the growth of films was monitored by ultraviolet-visible (UV-vis) spectroscopy, quartz crystal microbalance (QCM), and cyclic voltammetry (CV). Not only was the negatively charged Hb at pH 9.0 alternately adsorbed with positively charged PPI onto solid substrates by electrostatic attraction between them, but the positively charged Hb at pH 5.0 was also successfully assembled with like charged PPI into layer-by-layer {PPI/Hb(pH 5.0)}n films. For the latter, the localized electrostatic interaction or the charge reversal of proteins on PPI surface may be the main driving force. For {PPI/Hb(pH 7.0)}n films, however, the hydrophobic/hydrophilic interaction may play a more important role in the assembly, making the amount of adsorbed Hb even less than that of {PPI/Hb(pH 5.0)}n films. For comparison, negatively charged catalase (Cat) at pH 8.0 was used to assemble layer-by-layer films with positive PPI, but {PPI/Cat}n films showed quite different properties from {PPI/Hb}n films. UV-vis and infrared (IR) spectroscopy, QCM, ellipsometry, and voltammetry were utilized to characterize the {PPI/protein}n films. The results suggest that the proteins in the multilayer films retain their near-native structure and display good voltammetric response for heme Fe(III)/Fe(II) redox couples at underlying pyrolytic graphite (PG) electrodes. Electrocatalysis of oxygen and hydrogen peroxide based on direct electrochemistry of heme proteins at {PPI/protein}n film electrodes was also demonstrated.     

Two glucose transport systems in Bacillus licheniformis.

Bacillus licheniformis NCIB 6346 showed active accumulation of glucose which was inhibited by agents which affect the transmembrane proton gradient. Phosphotransferase (PTS) activity, identified as phosphoenolpyruvate-dependent phosphorylation of glucose, was found in cell extracts but could not be demonstrated in cells permeabilized with toluene when assays were conducted at pH 6.6. The same was true for mannitol and fructose phosphotransferase activities. Cells grown on fructose accumulated glucose at a slower rate than glucose-grown cells, and extracts prepared from them did not contain glucose PTS activity. Examination of the effects of analogs on glucose uptake and phosphorylation showed that 2-deoxyglucose was not a PTS substrate, but did markedly inhibit glucose uptake, with stronger inhibition in cells grown on fructose. Glucose accumulation by whole cells grown on glucose became less sensitive to the uncoupler tetrachlorosalicylanilide (TCS) as the pH was raised from 6.6 to 8.0, while in fructose-grown cells TCS was equally effective across this pH range. PTS activity was exhibited by toluene-treated cells at pH 7.5 and above, although the system itself in extracts was not affected by pH in the range of 5.0 to 8.0. The results are consistent with the presence of two glucose transport systems, one a PTS and the other operating by an alternative mechanisms, and suggest that the PTS in B. licheniformis may be regulated in a pH-dependent manner.     

Interactions between PEG and type I soluble tumor necrosis factor receptor: modulation by pH and by PEGylation at the N terminus

The effects of polyethylene glycol (PEG) on protein structure and the molecular details that regulate its association to polypeptides are largely unknown. These issues were addressed using type I soluble tumor necrosis factor receptor (sTNF-RI) as a model system. Changes in solution viscosity established that a truncated form of sTNF-RI bound free PEG in a pH-dependent manner. Above pH 5.3, the viscosity escalated as the pH increased, while no effect occurred below pH 5.0. Conjugation of 2 kD, 5 kD, or 20 kD PEG to the N terminus attenuated the viscosity at the higher pH values. Tryptophan phosphorescence spectroscopy correlated changes in the protein structure about Trp-107, at the C terminus, with the pH-dependent and PEGylation-dependent attenuation of the viscosity. The results indicate that specific interactions between PEG and the truncated form of sTNF-RI are elicited by an increased flexibility of the truncated protein combined perhaps with removal of steric or charge barriers. Covalently bound PEG at the N terminus reduced the protein affinity for the free polymer and induced a more rigid and polar configuration around Trp-107. Deprotonation of His-105, which is perpendicular to Trp-107, was integral to the binding mechanism producing a pH-dependent switching mechanism. These findings stress the importance of surface charge and structural plasticity in determining macromolecular binding affinities and demonstrate the ability of conjugated PEG to modify the localized surface structure in proteins away from the site of conjugation.     

ADP-Ribosylation of Human Myelin Basic Protein

Abstract: When isolated myelin membranes were ADP-ribosylated by [³²P]NAD⁺ either in the absence of toxin (by the membrane ADP-ribosyltransferase) or in the presence of cholera toxin, the same proteins were ADP-ribosylated in both cases and myelin basic protein (MBP) was the major radioactive product. Therefore, cholera toxin was considered a good model for ADP-ribosylation of myelin proteins. Although purified human MBP migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 20 kDa, the microheterogeneity that is masked under these conditions can be clearly demonstrated on alkaline-urea gels at pH 10.6. At this pH, MBP is resolved into several components that differ one from the other by a single charge (charge isomers). These charge isomers can be resolved on CM52 columns at pH 10.6, and several can be ADP-ribosylated. Component 1 (C-1), the most cationic charge isomer, incorporated 1.79 mol of ADP-ribose/mol of protein. C-2 and C-3 (which differ from C-1 by the loss of one and two positive charges, respectively) incorporated slightly less at 1.67 and 1.63 mol of ADP-ribose/mol of protein, respectively, whereas C-8, the least cationic, incorporated less than 0.11 mol/mol of protein. In the presence of neutral hydroxylamine, the ADP-ribosyl bond was shown to have a half-life of about 80 min, suggesting an N-glycosidic linkage between ADP-ribose and an arginyl residue of the protein. As MBP contains several components that are ADP-ribosylated to different specific activities, the use of MBP, ADP-ribosylated in the natural membrane, to identify the sites involved would yield a mixture of peptides difficult to resolve. Therefore, to identify the sites ADP-ribosylated, an endoproteinase Lys-C digest of C-1 ADP-ribosylated by cholera toxin was prepared. Two radioactive peptides were isolated by reversed-phase HPLC. Amino acid and sequence analyses identified the radioactive peptides as residues 5–13 and 54–58 of the human sequence (sp. act., 0.89 and 0.62 nmol of ADP-ribose/nmol of peptide, respectively). The ADP-ribosylated residues were identified as Arg⁹ and Arg⁵⁴ by automated and manual Edman sequencing. Taken together with our previous observation that MBP binds GTP at a single site, these data suggest that MBP functions as part of a signal transduction system in myelin.     

Increase in stability of xylanase from an alkalophilic thermophilic Bacillus (NCIM 59)

Summary The cellulase-free xylanase from an alkalophilic thermophilic Bacillus was stable at pH 7.0 to 10.0 at 50 ° for 3 days.At 60 ° the enzyme showed a decrease in stability with a half- life of 3 h. Addition of various additives had no effect on the enzyme stability at 60 °. Glycine (0.5M) increased the enzyme half-life 6-fold at pH 7.0 to 9.0 and at 60 and 70 °. Xylan could offer protection against thermoinactivation of the xylanase at pH 7.0 and 8.0 at 60 ° and only a marginal increase at pH 9.0 at 70 ° was observed.     

Effect of pH on bile salt degradation by mixed fecal cultures

Stool specimens from 3 healthy volunteers were cultured under anaerobic conditions in brain heart infusion broth with and without the addition of cholate, deoxycholate or chenodeoxycholate. The initial pH of the medium was adjusted to 5.5, 6.3, 7.3 (unadjusted), 8.0, and 9.0. Cell-free extracts prepared from the resulting bacterial growth contained increased levels of NAD- and NADP-dependent 3α-, 7α-, and 12α-hydroxysteroid oxidoreductases when the initial pH was 8.0 or 9.0 and depressed levels of these activities when the initial pH was 5.5 or 6.3 (as compared to control values obtained at 7.3). At pH 5.5 all activities except NAD-dependent 7α-hydroxysteroid oxidoreductase were absent. A powerful selective effect was imposed on NAD-dependent 7α-hydroxysteroid oxidoreductase when deoxycholate or chenodeoxycholate were incorporated into the medium. Thin-layer chromatography of ether extracts of cholate-containing, acidified spent bacterial medium showed measurable amounts of deoxycholate only when the initial culture pH was alkaline or neutral (optimal at pH 8). The percent hydroxyl group estimations at the 3α-, 7α-, and 12α-positions revealed an increase in disappearance of OH groups at all three positions with increasing initial pH value. The order of extent of bioconversion was 7α-OH > 12α-OH > 3α-OH; at pH 8 and 9, approximately 90% 7α-OH bioconversion was observed. Spent bacterial media and a number of commercial secondary bile salts were all negative in the Ames' assay for mutagenicity.     

Characterization and amino acid sequence of IRT-4, a novel TEM-type enzyme with a decreased susceptibility to β-lactamase inhibitors

Abstract A novel procedure was used to purify a cytosolic chitinase from Candida albicans to electrophoretic homogeneity. The results represent the first demonstration of the purification of a fungal intracellular chitinase using the criterion of a single band detected following silver-staining of a polyacrylamide gel run under denaturing conditions. Purified chitinase had pH and temperature optima of 5.0 and 50°C, respectively. Inhibition of enzyme activity by allosamidin was pH-dependent occuring maximally at pH 8.0. Phospholipids had similar marked and highly specific effects on the activities of both the purified soluble enzyme and a solubilized microsomal chitinase from C. albicans . Evidence is provided for the existence of a complex chitinolytic system in this organism.     

Kinetic properties of sorbitol dehydrogenase from calf liver cell cytoplasm

The kinetic properties of sorbitol dehydrogenase from calf liver cell cytoplasm during sorbitol oxidation were studied at pH 7.0, 7.5, 8.0, 9.0 and 10.0. It was found that the shape of kinetic curves for NADH accumulation depends on pH and substrate concentration. At pH 7.0, 7.5 and 8.0 the enzymatic reaction obeys the Michaelis-Menten kinetics with Km of 3.3 x 10(-3) M. 2.3 x 10(-3) M and 2.08 x 10(-3) M, respectively. At pH 9.0 and 10.0 the vovs [So] curves have an "intermediate plateau". The Hill plots for this reaction reveal two slopes that are dependent on substrate concentration. The nH values for sorbitol (up to 2 mM) are 1.0 and 1.16 at pH 9.0 and 10.0, respectively. With a further rise in the substrate concentration, the nH value increases up to 2.4 and 2.18 at pH 9.0 and 10.0, respectively. This is suggestive of the existence of a slowly dissociating enzymatic system of the Np in equilibrium P type (where P is the oligomeric and p the monomeric forms of the enzyme); N approximately greater than 2. The vovs NAD plots are S-shaped at all pH values studied. The data obtained are discussed in terms of regulatory effects of sorbitol and acidity on association-dissociation of sorbitol dehydrogenase from liver cell cytoplasm.     

The preparation of antibodies reactive against citrulline-containing charge isomers of myelin basic protein but not against the arginine-containing charge isomers.

Human myelin basic protein (MBP) is composed of several charge isomers, the result of various post-translational modifications. One of the charge isomers C-8, has been shown in our laboratory to contain six citrullinyl residues which replace arginyl residues at selected sites in the MBP. In order to determine the disposition of the citrulline-containing charge isomers in the myelin stack, we prepared specific antisera against the citrullinyl group. Since 9-fluorenylmethoxycarbonyl (Fmoc)-citrulline, required for the preparation of the synthetic peptides to be used for antibody production, was not commercially available, synthesis of the Fmoc-citrulline was a necessary prerequisite. The synthesis and purification of the N-9-fluorenylmethyloxycarbonyl derivative of citrulline is described. It was characterized by thin layer chromatography, 1H and 13C NMR spectroscopy, fast-atom bombardment mass spectroscopy, and thermal analyses. It was used in the automated peptide synthesis of a peptide Ala-Cit-His-Gly-Phe-Leu-Pro-Cit-His-Arg corresponding to residues 24-33 and Gly-Cit-Asp-Ser-Arg-Ser-Gly-Ser-Pro-Met-Ala-Cit-Arg, corresponding to residues 158-170 of the C-8 sequence, a naturally occurring charge isomer of human myelin basic protein, and a tetracitrulline peptide, Cit-Cit-Cit-Cit-Gly. The tetracitrulline peptide was used for the production of an antibody shown to react only with synthetic peptides and proteins containing citrulline. This antibody was used to distinguish between a citrulline-containing protein, C-8, a naturally occurring charge isomer of MBP, and a non-citrulline-containing charge isomer of MBP, C-1.     

Identification and partial characterisation of a chitinase from Nile tilapia, Oreochromis niloticus

Measurement of chitinase activity in extracts from stomach, intestine, and serum of Nile tilapia with the artificial substrates 4-methylumbelliferil beta-D-N,N'-diacetylchitobioside and 4-methylumbelliferil beta-D-N,N'N"-triacetylchitotrioside (4MU[GlcNAc](2,3)) showed that an endochitinase was involved in the liberation of the fluorophore 4-methylumbelliferone (MU). Enzymes were isolated from tilapia serum by a combination of gel filtration, ion exchange, and reverse-phase chromatography. The molecular mass of the enzyme was estimated to be 75 kDa by SDS-PAGE, suggesting that the enzyme occurs as a monomer. The partially purified enzyme showed maximal activity at pH 7.0 when assayed with 4MU[GlcNAc](2) and lost its activity below pH 5.0 and above pH 8.0. The optimal pH of the purified enzyme toward the substrate 4MU[GlcNAc](3) was pH 9.0 and activity was lost below pH 8.0 and above pH 9.0. Our study has revealed the presence of a chitinolytic enzyme in the gastrointestinal tract and serum that may play a role in digestion and/or defense.     

pH-Dependent Solubility and Dissolution Behavior of Carvedilol—Case Example of a Weakly Basic BCS Class II Drug

The objective of this study was to investigate the pH-dependent solubility and dissolution of weakly basic Biopharmaceutical Classification Systems (BCS) class II drugs, characterized by low solubility and high permeability, using carvedilol, a weak base with a pK _a value of 7.8, as a model drug. A series of solubility and in vitro dissolution studies was carried out using media that simulate the gastric and intestinal fluids and cover the physiological pH range of the GI from 1.2 to 7.8. The effect of ionic strength, buffer capacity, and buffer species of the dissolution media on the solubility and dissolution behavior of carvedilol was also investigated. The study revealed that carvedilol exhibited a typical weak base pH-dependent solubility profile with a high solubility at low pH (545.1–2591.4 μg/mL within the pH range 1.2–5.0) and low solubility at high pH (5.8–51.9 μg/mL within the pH range 6.5–7.8). The dissolution behavior of carvedilol was consistent with the solubility results, where carvedilol release was complete (95.8–98.2% released within 60 min) in media simulating the gastric fluid (pH 1.2–5.0) and relatively low (15.9–86.2% released within 240 min) in media simulating the intestinal fluid (pH 6.5–7.8). It was found that the buffer species of the dissolution media may influence the solubility and consequently the percentage of carvedilol released by forming carvedilol salts of varying solubilities. Carvedilol solubility and dissolution decreased with increasing ionic strength, while lowering the buffer capacity resulted in a decrease in carvedilol solubility and dissolution rate.     

Basic pH-induced modification of excitation-energy dynamics in fucoxanthin chlorophyll a/c-binding proteins isolated from a pinguiophyte,Glossomastix chrysoplasta

Photosynthetic organisms have diversified light-harvesting complexes (LHCs) to collect solar energy efficiently, leading to an acquisition of their ecological niches. Herein we report on biochemical and spectroscopic characterizations of fucoxanthin chlorophyll a/c-binding protein (FCP) complexes isolated from a marine pinguiophyte Glossomastix chrysoplasta. The pinguiophyte FCP showed one subunit band in SDS-PAGE and one protein-complex band with a molecular weight at around 66 kDa in clear-native PAGE. By HPLC analysis, the FCP possesses chlorophylls a and c, fucoxanthin, and violaxanthin. To clarify excitation-energy-relaxation processes in the FCP, we measured time-resolved fluorescence spectra at 77 K of the FCP adapted to pH 5.0, 6.5, and 8.0. Fluorescence curves measured at pH 5.0 and 8.0 showed shorter lifetime components compared with those at pH 6.5. The rapid decay components at pH 5.0 and 8.0 are unveiled by fluorescence decay-associated (FDA) spectra; fluorescence decays occur in the 270 and 160-ps FDA spectra only at pH 5.0 and 8.0, respectively. In addition, energy-transfer pathways with time constants of tens of picoseconds are altered under the basic pH condition but not the acidic pH condition. These findings provide novel insights into pH-dependent energy-transfer and energy-quenching machinery in not only FCP family but also photosynthetic LHCs.     

Effect of initial pH independent of operational pH on hydrogen fermentation of food waste

The effect of initial pH from 5.0 to 9.0 on H₂ fermentation of food waste was investigated. In this batch experiment, however, unlike previous studies for initial pH, operational pH was maintained at 5.0 by the addition of alkaline solution. Although the period for pH drop from the initial values to 5.0 was less than one-tenth of the entire fermentation, this short period significantly affected the H₂ production performance. At initial pH 6.0-9.0, successful H₂ yield of 1.3-1.9 mol H₂/mol hexose_added was achieved with a peak value at pH 8.0. The H₂ yield achieved at initial pH 8.0 was corresponded to the 8.13% of total energy content in the substrate. At initial pH 5.0, the smallest butyrate production, but the highest ethanol production was detected, indicating unfavorable conditions for H₂ production. There was no significant relationship between total required amount of alkaline solution and initial pH values.     

An Antibody Specific for Component 8 of Myelin Basic Protein from Normal Brain Reacts Strongly with Component 8 from Multiple Sclerosis Brain

Myelin basic protein (MBP) consists of several components or charge isomers (C-1 through C-8) generated by one or a combination of posttranslational modifications. One of these, C-8, has been shown to contain citrulline (Cit) at defined sites formed by deimination of six arginyl residues. This unusual modification has allowed us to raise antibodies specific for this charge isomer only. To do this, a synthetic peptide, Gly-Cit-Cit-Cit-Cit, was coupled to keyhole limpet hemocyanin and injected into rabbits. The antibodies so generated reacted only with C-8 and not with any of the other charge isomers. A second antibody fraction was raised against the synthetic peptide ACitHGFLPCitHR naturally occurring between residues 24 and 33 of C-8 (all other charge isomers contain R instead of Cit at positions 25 and 31). These antibodies preferred C-8 but reacted with the other charge isomers, to the extent of approximately 25-30% of the reactivity shown with C-8. In studies with C-8 from multiple sclerosis (MS) MBP, much greater reactivity was obtained with these antibodies when compared with their reactivity with C-8 from normal MBP. Because the total number of Cit residues in C-8 from MS and normal MBP is the same, the difference in reactivity may be related to structural factors. The antibodies raised with the tetra-Cit peptide were reacted with three pairs of synthetic peptides: 24ARHGFLPRHR33 and ACitHGFLPCitHR; 120GQRPGFGYGGRAS132 and GQCitPGFGYGGCitAS; and 157GGRDSRSGSPMARR170 and GGCitDSRSGSPMACitR. They reacted only with the Cit-containing peptides in the order 157-170 greater than 120-130 greater than 24-33.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Ability to Enhance the Solubility of Its Fusion Partners Is an Intrinsic Property of Maltose-Binding Protein but Their Folding Is Either Spontaneous or Chaperone-Mediated

Escherichia coli maltose binding protein (MBP) is commonly used to promote the solubility of its fusion partners. To investigate the mechanism of solubility enhancement by MBP, we compared the properties of MBP fusion proteins refolded in vitro with those of the corresponding fusion proteins purified under native conditions. We fused five aggregation-prone passenger proteins to 3 different N-terminal tags: His₆-MBP, His₆-GST and His₆. After purifying the 15 fusion proteins under denaturing conditions and refolding them by rapid dilution, we recovered far more of the soluble MBP fusion proteins than their GST- or His-tagged counterparts. Hence, we can reproduce the solubilizing activity of MBP in a simple in vitro system, indicating that no additional factors are required to mediate this effect. We assayed both the soluble fusion proteins and their TEV protease digestion products (i.e., with the N-terminal tag removed) for biological activity. Little or no activity was detected for some fusion proteins whereas others were quite active. When the MBP fusions proteins were purified from E. coli under native conditions they were all substantially active. These results indicate that the ability of MBP to promote the solubility of its fusion partners in vitro sometimes, but not always, results in their proper folding. We show that the folding of some passenger proteins is mediated by endogenous chaperones in vivo. Hence, MBP serves as a passive participant in the folding process; passenger proteins either fold spontaneously or with the assistance of chaperones.     

pH-Dependent Interactions between Pea Cell Wall Polymers Possibly Involved in Wall Deposition and Growth

In an effort to detect a pH-dependent release of polymers such as xyloglucans, thought to be involved in auxin-induced cell wall expansion during growth, radioactively labeled cell walls from pea stem tissue were incubated at different pH values, and changes in water-soluble, ethanol- or trichloroacetic acid-insoluble components were determined. This revealed the occurrence, at neutral pH, of a time- and pH-dependent binding of soluble pectin, in the walls, to a heat-labile, presumably protein, wall component, yielding a trichloroacetic acid-insoluble pectin-protein complex. This reaction, which can also be observed between polymers in water extracts of cell walls, is inhibited at low pH and by Ca²⁺, and appears to be of a physical, possibly lectin-like, nature. Progressive binding of pectin or of the pectin-protein complex to the insoluble wall structure is also observed. These reactions may be involved in wall assembly during its deposition, and may participate in, or be analogous to pH-dependent physical interactions that participate in, wall extension during cell growth.