Protein Mass Ratio Research Articles

1,3-Regiospecific ethanolysis of soybean oil catalyzed by crosslinked porcine pancreas lipase aggregates.

The preparation of crosslinked aggregates of pancreatic porcine lipase (PPL-CLEA) was systematically studied, evaluating the influence of three precipitants and two crosslinking agents, as well as the use of soy protein as an alternative feeder protein on the catalytic properties and stability of the immobilized PPL. Standard CLEAs showed a global yield (CLEA' observed activity/offered total activity) of less than 4%, whereas with the addition of soy protein (PPL:soy protein mass ratio of 1:3) the global yield was approximately fivefold higher. The CLEA of PPL prepared with soy protein as feeder (PPL:soy protein mass ratio of 1:3) and glutaraldehyde as crosslinking reagent (10 μmol of aldehyde groups/mg of total protein) was more active mainly because of the reduced enzyme leaching in the washing step. This CLEA, named PPL-SOY-CLEA, had an immobilization yield around 60% and an expressed activity around 40%. In the ethanolysis of soybean oil, the PPL-SOY-CLEA yielded maximum fatty acid ethyl ester (FAEE) concentration around 12-fold higher than that achieved using soluble PPL (34 h reaction at 30°C, 300 rpm stirring, soybean oil/ethanol molar ratio of 1:5) with an enzyme load around 2-fold lower (very likely due to free enzyme inactivation). The operational stability of the PPL-SOY-CLEA in the ethanolysis of soybean oil in a vortex flow type reactor showed that FAEE yield was higher than 50% during ten reaction cycles of 24 h. This reactor configuration may be an attractive alternative to the conventional stirred reactors for biotransformations in industrial plants using carrier-free biocatalysts. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:910-920, 2018.

A Systematic Approach to the Comparison of Cost Efficiency of Endopeptidases for the Hydrolysis of Atlantic Salmon (Salmo salar) By-Products.

The hydrolytic and cost efficiencies of five endopeptidases (Alcalase 2.4L, Corolase 7089, Neutrase 0.8L, Promod 671L and Protex 7L) to hydrolyze Atlantic salmon by-products were compared at standardized activity levels based on a casein assay. The substrate was characterized prior to the hydrolytic experiments (pH=6.5, t=50 °C) to obtain substrate--specific constants for nitrogen to protein mass (in g) ratio, i.e. conversion factor fN=5.23 and total amount of peptide bonds (htot)=9.3 mmol per g of protein. At low enzyme activity to substrate ratio, all enzymes were equally efficient in hydrolyzing the substrate. At highest enzyme activity to substrate ratio, Protex 7L, Alcalase 2.4L and Promod 671L gave higher degree of hydrolysis (DH=14.2-14.6%) than Corolase 7089 (13.2%) and Neutrase 0.8L (11.6%) after 120 min of hydrolysis. No differences were observed in protein recovery (yield of solubilized protein) relative to DH. Determination of DH was followed by the pH-STAT and o-phthaldialdehyde methods. Based on pH-STAT data, response surface regression models were established based on the combined effects of hydrolysis time and enzyme activity to substrate ratio on DH and protein recovery. The modelling approach was combined with enzyme cost to identify the most cost-efficient enzyme (Protex 7L).

β-Mannanase (Man26A) and α-galactosidase (Aga27A) synergism – A key factor for the hydrolysis of galactomannan substrates

This study investigated the behavior of mannan-degrading enzymes, specifically focusing on differences with respect to their substrate specificities and their synergistic associations with enzymes from different glycoside hydrolase (GH) families. Galactosidases from Cyamopsis tetragonolobus seeds (Aga27A, GH27) and Aspergillus niger (AglC, GH36) were evaluated for their abilities to synergistically interact with mannanases from Clostridium cellulovorans (ManA, GH5) and A. niger (Man26A, GH26) in hydrolysis of guar gum and locust bean gum. Among the mannanases, Man26A was more efficient at hydrolyzing both galactomannan substrates, while among the galactosidases; Aga27A was the most effective at removing galactose substituents on both galactomannan substrates and galactose-containing oligosaccharides. An optimal protein mass ratio of glycoside hydrolases required to maximize the release of both reducing sugar and galactose residues was determined. Clear synergistic enhancement of locust bean gum hydrolysis with respect to reducing sugar release was observed when both mannanases at 75% enzyme dosage were supplemented with 25% enzyme protein dosage of Aga27A. At a protein ratio of 75% Man26A to 25% Aga27A, the presence of Man26A significantly enhanced galactose release by 25% Aga27A (2.36 fold) with locust bean gum, compared to when Aga27A was used alone at 100% enzyme protein dosage. A dosage of Aga27A at 75% and ManA at 25% protein content liberated the highest reducing sugar release on guar gum hydrolysis. A dosage of Man26A and Aga27A at 75–25% protein content, respectively, liberated reducing sugar release equivalent to that when Man26A was used alone at 100% protein content. From the findings obtained in this study, it was observed that the GH family classification of an enzyme affects its substrate specificity and synergistic interactions with other glycoside hydrolases from different families (more so than its EC classification). The GH26 Man26A and GH27 Aga27A enzymes appeared to be more promising for applications that involve the hydrolysis of galactomannan containing biomass. This method of screening for maximal compatibility between various GH families can ultimately lead to a more rational development of tailored enzyme cocktails for lignocellulose hydrolysis.

Efisiensi Penggunaan Protein pada Ayam Broiler dengan Pemberian Pakan Mengandung Tepung Daun Kayambang (Salvinia molesta)

(Protein efficiency of broiler chickens fed with diets containing kayambang leaves meal)ABSTRACT The aim of the experiment was to determine the utilization of kayambang (Salvinia molesta) leaves meal until 18% based on its effects on protein consumption, protein intake, meat protein mass and protein efficiency ratio. One hundred broiler CP 707 day-old unsexed chicks, with an average body weight 502,48± 6,99 g age 15 until 42 days. This study used a completely randomized design (CRD) with 4 treatments and 5 replications and each replication consisted of 5 chickens.. The treatments diets were T0 (basal diet), T1 (6% Salvinia molesta leaves meal), T2 (12% Salvinia molesta leaves meal), and T3 (18% Salvinia molesta leaves meal). The results showed that administration of Salvinia molesta leaf meal up to 18% level in the diet resulted in a decrease in the quality of the ration so that the protein utilization also decreased as indicated by the decline in the value of protein intake, protein intake, protein mass of meat and protein efficiency ratio.

Production of combi-CLEAs of glycosidases utilized for aroma enhancement in wine

Glycosidases are frequently used in winemaking to liberate glycosidically bound aroma compounds. Since most of the glycosidases used for diglycoside hydrolysis act sequentially, their co-immobilization is an attractive alternative from a technical and economical perspective. The enzymes α-l-arabinosidase (ARA) and β-d-glucosidase (βG) from the preparation Rapidase®AR2000 were co-immobilized in CLEAs (combi-CLEAs), evaluating the effect of bovine serum albumin (BSA) addition and the concentration of glutaraldehyde (Glu) on enzyme immobilization yield and expressed activity. Combi-CLEAs prepared with a Glu to Rapidase protein mass ratio of 0.053 and BSA to Rapidase protein mass ratios of 1, 0.33, and 0.2 were selected, evaluating their stability at simulated winemaking conditions: 25°C, pH 3.5, and 10% (v/v) of ethanol. All combi-CLEAs were more stable than the soluble enzymes, the best result being obtained at a BSA to Rapidase protein mass ratio of 0.33. Half-lives of βG and ARA in combi-CLEAs were 43.9 and 54.9 days, respectively, whereas in the case of the soluble enzymes were only 1.3 and 6.2 days, respectively. Immobilization yields were 79.1 and 47.1% in terms of βG and ARA activity, respectively. Combi-CLEAs of glycosidases are technologically relevant robust biocatalysts for their application as aroma enhancers in winemaking.

Preparation of bovine serum albumin imprinting sensitive hydrogels using ionic liquid as co-monomer and stabilizer

Through consulting the Hofmeister series, a novel biocompatible and polymerizable ionic liquid (IL) was designed and used as stabilizer and co-monomer to prepare bovine serum albumin (BSA) imprinted hydrogels. N-isopropylacrylamide (NIPA) was chosen as the assistant monomer for imparting environmental sensitivity to the hydrogels. The stabilizing effect of the IL was verified by circular dichroism. Several parameters, such as the mass ratio of the template protein, IL and crosslinker, the drying method of hydrogels and the elution method of MIHs that could affect the performance of molecular imprinted hydrogels (MIHs) were investigated. The optimum mass ratio of BSA, IL and crosslinker was found to be 200:30:6. The best drying and preferred elution method for the MIHs was achieved by slowly evaporating and washing with 0.5M NaCl solution at 15 °C, respectively. The MIHs prepared under optimized conditions were subsequently used in the adsorption isotherm, adsorption dynamics, adsorption selectivity, and competition test. The adsorption isotherm revealed that the MIHs showed the best imprinted effect at a BSA concentration of 0.2 mg mL(-1) and their imprinting factor at 2.66. The adsorption dynamic studies revealed that the adsorptive rate of the MIHs was much faster than the non-imprinted hydrogels (NIHs), and both of them could be equilibrated in 1h. The adsorption selectivity and competition tests were conducted to estimate the specific recognition property of the MIHs for BSA. The MIHs showed excellent selectivity and recognition ability to BSA. The strategy of applying biocompatible and polymerizable ILs to imprinting technology may provide a new approach for effective biomacromolecular imprinting.

Antigenicity and structural properties of soybean protein-glucose conjugates

Soybean is a high-quality protein source and one of eight major food allergens. Glycation is an effective way to remove protein allergens. In this paper, soy protein isolate(SPI) and glucose were used as raw materials for Maillard reactions, which were performed with various protein to carbohydrate ratios, temperatures, and experimental time, to prepare glycated conjugates. The inhibition of antigen β-conglycinin in SPI-glucose conjugates was used as an indicator, and indirect competitive enzyme-linked immunosorbent assay(ELISA) was employed to measure the antigenicity of glycated conjugates. When the mass ratio of protein to sugar was 3:1 at a temperature of 55, the antigenicity of the prepared glycated conjugates was relatively low. When the reaction time was 72 h, the inhibition rate of antigen β-conglycinin decreased from 93.54% to 22.58%. The Maillard reaction was confirmed by the trinitrobenzene sulfonic acid(TNBS) method and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). Changes in protein structure were studied by ultraviolet scans and Fourier transform infrared spectroscopy(FT-IR). These basic studies might provide research methods and a theoretical basis for determining the mechanism mediating the effect of glycation on antigenicity and developing soy protein products with low allergen contents.

Closteroviridae: the beginning

Closteroviridae: the beginning

Understanding complex coacervation in serum albumin and pectin mixtures using a combination of the Boltzmann equation and Monte Carlo simulation

A combination of turbidimetric titration, a sigmoidal Boltzmann equation approach and Monte Carlo simulation has been used to study the complex coacervation in serum albumin and pectin mixtures. The effects of the mass ratio of protein to polysaccharide on the critical pH values, the probability of complex coacervation and the electrostatic interaction from charge patches in serum albumin were investigated. Turbidimetric titration results showed an optimum pH for complex coacervation (pHm), which corresponded to the maximum turbidity in the protein/polysaccharide mixture. The pHm monotonically decreased as the ratio decreased, and could be fitted using the sigmoidal Boltzmann equation. It suggests that pHm could be a good ordering parameter to characterize the phase behavior associated with protein/polysaccharide complex coacervation. Qualitative understanding of pHm by taking into account the minimization of electrostatic interaction, as well as quantitative matching of pHm according to the concept of charge neutralization were both achieved. Our results suggest that the serum albumin/pectin complexes were ultimately neutralized by the partial charges originated from the titratable residues in protein and polysaccharide chains at pHm. The Monte Carlo simulation provided consistent phase boundaries for complex coacervation in the same system, and the intermolecular association strength was determined to be several kBT below the given ionic strength. The strongest binding site in the protein is convergent to the largest positive charge patch if pure electrostatic interaction was considered. Further inclusion of contribution from excluded volume resulted in the binding site distribution over five different positive charge patches at different protein/polysaccharide ratios and pH values.

Surface-coating montmorillonite nanoclay by water-soluble proteins extracted from hominy feed

Montmorillonite (MMT) nanoclay was surface-coated by water-soluble proteins precipitated from hominy feed extract at pH 6.0, 5.5, and 4.3 to modify its native structure. Surface-coating was performed at 60°C, pH 2.0–10.0, and MMT:protein mass ratios from 49:1 to 2:1, followed by lyophilizing the triple-washed precipitate for characterization. Proteins precipitated at pH 5.5 were the most effective in creating intercalated and/or exfoliated MMT structures that were also impacted by the protein level and pH during coating. At pH 2.0, X-ray diffraction peaks of pristine MMT were no longer observed at an MMT:protein mass ratio of 4:1 or lower, while different degrees of intercalation were achieved at other pH conditions. The adsorption of proteins on MMT at all studied pH conditions was confirmed by FTIR, analysis of adsorbed proteins, and zeta-potential. These results suggest that interactions involved in coating MMT include coulombic and non-coulombic forces.

Seed Oil Bodies from Gevuina avellana and Madia sativa

In this study, oil bodies (OBs) from Gevuina avellana (OBs-G) and Madia sativa (OBs-M) were isolated and characterized. Microscopic inspection revealed that the monolayer on OB-G was thinner compared to that on OB-M. Cytometric profiles regarding size, complexity, and staining for the two OB sources were similar. Fatty acid to protein mass ratio in both OBs was near 29, indicating high lipid enrichment. OBs-G and OBs-M showed a strong electrostatic repulsion over wide ranges of pH (5.5-9.5) and NaCl concentration (0-150 mM). Proteins displaying highly conserved sequences (steroleosins and aquaporins) in the plant kingdom were identified. The presence of oleosins was immunologically revealed using antibodies raised against Arabidopsis thaliana oleosins. OBs-G and OBs-M exhibited no significant cytotoxicity against the cells. This is the first report about the isolation and characterization of OBs-G and OBs-M, and this knowledge could be used for novel applications of these raw materials.

Experimental design to optimize an Haemophilus influenzae type b conjugate vaccine made with hydrazide-derivatized tetanus toxoid

The introduction of type b Haemophilus influenzae conjugate vaccines into routine vaccination schedules has significantly reduced the burden of this disease; however, widespread use in developing countries is constrained by vaccine costs, and there is a need for a simple and high-yielding manufacturing process. The vaccine is composed of purified capsular polysaccharide conjugated to an immunogenic carrier protein. To improve the yield and rate of the reductive amination conjugation reaction used to make this vaccine, some of the carboxyl groups of the carrier protein, tetanus toxoid, were modified to hydrazides, which are more reactive than the ε -amine of lysine. Other reaction parameters, including the ratio of the reactants, the size of the polysaccharide, the temperature and the salt concentration, were also investigated. Experimental design was used to minimize the number of experiments required to optimize all these parameters to obtain conjugate in high yield with target characteristics. It was found that increasing the reactant ratio and decreasing the size of the polysaccharide increased the polysaccharide:protein mass ratio in the product. Temperature and salt concentration did not improve this ratio. These results are consistent with a diffusion controlled rate limiting step in the conjugation reaction. Excessive modification of tetanus toxoid with hydrazide was correlated with reduced yield and lower free polysaccharide. This was attributed to a greater tendency for precipitation, possibly due to changes in the isoelectric point. Experimental design and multiple regression helped identify key parameters to control and thereby optimize this conjugation reaction.

Temperature-dependent complexation between sodium caseinate and gum arabic

Complex formation between sodium caseinate and gum arabic as a function of temperature was investigated using dynamic light scattering, fluorescence and NMR. At neutral pH, the turbidity and the particle size increased when sodium caseinate and gum arabic mixtures were heated in situ at temperatures above a critical temperature. The increases in turbidity and particle size were reversible. This effect was considered to be due to hydrophobic interactions, leading to the formation of a complex between sodium caseinate and gum arabic. 1H NMR spectroscopy showed that ANS, which bound to caseinate at low temperatures in caseinate solution or a caseinate–gum arabic mixture, was released at high temperatures upon formation of a caseinate or caseinate–gum arabic complex. This supported changes observed in the fluorescence of 8-anilino-1-naphthalene sulfonate upon binding to caseinate, which decreased at high temperatures for caseinate alone or when sodium caseinate was mixed with gum arabic. Light-scattering (turbidity) and dynamic light-scattering studies show that the temperature-dependent complexation between sodium caseinate and gum arabic was sensitive to the mass ratio of protein to gum arabic (greater complexation at a 1:5 ratio than a 1:1 ratio) and the pH (maximum complexation at pH 6.5).

Nutrition-induced differences in body composition, compensatory growth and endocrine status in growing pigs ,

In this experiment, we assessed the effect of amino acid (AA) intake restriction in entire male Yorkshire pigs between 15 and 38 kg BW (restriction phase) on BW gain, body composition and plasma levels of blood urea nitrogen (BUN), cortisol, insulin-like growth factor I (IGF-I), growth hormone (GH) and leptin during the subsequent re-alimentation phase. During the restriction phase, 36 pigs were allotted to one of two dietary treatments: adequate AA intake (control) or AA-limiting diets (AA-30%). Thereafter, pigs were fed common non-limiting diets up to 110 kg BW. Throughout the experiment, pigs were scale-fed at 90% of the estimated voluntary daily digestible energy intake. At the end of the restriction phase, pigs on AA-30% had lesser BW gain (650 v. 784 g/day; P < 0.001), loin area (LA; 12.2 v. 14.2 cm2; P < 0.001), BUN (4.6 v. 6.3 mg/dl; P < 0.02), lesser plasma levels of IGF-I (440 v. 640 ng/m; P < 0.001) and cortisol (8.2 v. 19.2 μg/dl; P < 0.001), greater backfat thickness (BF; 7.56 v. 6.56 mm; P < 0.02), and greater plasma levels of leptin (2.7 v. 1.8 ng/ml; P = 0.027) and GH (3.3 v. 2.0 ng/ml; P = 0.05) than pigs on control. During the re-alimentation phase, previously restricted pigs showed full compensatory growth (CG) in terms of BW gain (1170 v. 1077 g/day; P < 0.002), whole-body protein deposition (Pd) (179 v. 163 g/day; P < 0.001) as well as physical and chemical body composition (whole-body lipid to body protein mass ratio, LB/PB; 1.14 v. 1.15; P > 0.10). Besides GH at 45 kg BW (4.2 v. 2.4 ng/ml; P = 0.066), there were no effects of previous AA intake restriction on leptin, IGF-I and BUN during the re-alimentation phase (P > 0.10). Plasma cortisol and IGF-I levels may act as an indicator of AA-induced restriction in Pd in growing pigs. Plasma BUN level does not appear as a sensitive indicator for compensatory Pd. Plasma leptin and GH levels allow for the involvement of the brain in controlling chemical body composition. Full CG was observed during the energy-dependent phase of Pd in growing pigs and might be driven by a target LB/PB, possibly mediated via plasma leptin, IGF-I and GH levels.

Glass-immobilized glycated-trypsin: A novel modified trypsin that is remarkably thermostable

Porcine trypsin was glycated with glucose and covalently immobilized through its carboxyl groups onto aminated glass beads to produce porcine immobilized glycated-trypsin (IGT). On incubation at 60 °C and pH 8, IGT retained its full activity for 8 h and 50% of its activity after 24 h. In comparison, under the same conditions porcine native trypsin lost 80% of its activity in 2 h and was completely inactivated in less than 4 h. The rate of autolysis of porcine glycated-trypsin at 37 °C was 40% that of native trypsin and with IGT there was no significant autolysis, even at elevated temperatures as high as 60 °C. Glycation significantly increased the stability of trypsin and immobilization also significantly increased the stability of trypsin. The remarkable thermostability of IGT is attributed to a synergistic effect when these two modifications are combined. Tryptic fragmentation of denatured proteins with IGT can be performed at 60 °C for shorter digestion times and with smaller amounts of enzyme than normally employed to achieve complete digestion with soluble forms of trypsin. Prior denaturation of proteins for tryptic digestion is not required with IGT as in situ denaturation and digestion can be achieved simultaneously at 60 °C with an enzyme:protein mass ratio as low as 1:1000.

An insight into the mechanism of protein separation by colloidal gas aphrons (CGA) generated from ionic surfactants

Colloidal gas aphrons (CGA), which are surfactant stabilised microbubbles, have been previously applied for the recovery of proteins from model mixtures and a few studies have demonstrated the potential of these dispersions for the selective recovery of proteins from complex mixtures. However there is a lack of understanding of the mechanism of separation and forces governing the selectivity of the separation. In this paper a mechanistic study is carried out to determine the main factors and forces influencing the selectivity of separation of whey proteins with CGA generated from ionic surfactants. Two different separation strategies were followed: (i) separation of lactoferrin and lactoperoxidase by anionic CGA generated from a solution of sodium bis-(2-ethyl hexyl) sulfosuccinate (AOT); (ii) separation of beta-lactoglobulin by cationic CGA generated from a solution of cetyltrimethylammonium bromide (CTAB). Separation results indicate that electrostatic interactions are the main forces determining the selectivity however these could not completely explain the selectivities obtained following both strategies. Protein-surfactant interactions were studied by measuring the zeta potential changes on individual proteins upon addition of surfactant and at varying pH. Interestingly strongest electrostatic interactions were measured at those pH and surfactant to protein mass ratios which were optimum for protein separation. Effect of surfactant on protein conformation was determined by measuring the change in fluorescence intensity upon addition of surfactant at varying pH. Differences in the fluorescence patterns were detected among proteins which were correlated to differences in their conformational features which could in turn explain their different separation behaviour. The effect of conformation on selectivity was further proven by experiments in which conformational changes were induced by pre-treatment of whey (heating) and by storage at 4 degrees C. Overall it can be concluded that separation of proteins by ionic CGA is driven mainly by electrostatic interactions however conformational features will finally determine the selectivity of the separation with competitive adsorption having also an effect.

Mass-dependent signaling between G protein coupled receptors

The present study provides evidence that G protein coupled receptor (GPCR) signaling pathways participate in an interactive signaling network governed by the principles of mass action. Using an inducible thromboxane A 2 receptor (TPR)/platelet activating factor receptor (PAFR) co-expressing cell model, TPR or PAFR expression was independently up-regulated. Immunostaining and radioligand binding experiments demonstrated that this receptor up-regulation resulted in increased GPCR : G protein mass ratios. This increase in mass ratio impacted both TPR and PAFR ligand affinity. Specifically, up-regulating TPR expression not only decreased TPR ligand affinity, but also decreased the ligand affinity of PAFRs. A similar effect on ligand affinities was observed when PAFRs were up-regulated. In addition, increasing the GPCR : G protein mass ratio for TPRs led to desensitization of the calcium mobilization response to PAFR activation, and increasing PAFR mass desensitized the TPR-mediated calcium response. Finally, it was observed that an increased TPR : G protein mass ratio was associated with a shift in the TPR signaling response, and revealed an additional TPR signaling pathway through G S. Collectively, these results describe a novel mechanism, i.e., mass-dependent GPCR signaling, by which cells can modulate their GPCR signaling pathways and signaling priorities.

Dynamic and Differential in Vivo Modifications of the Isoform HMGA1a and HMGA1b Chromatin Proteins

Most naturally occurring mammalian cancers and immortalized tissue culture cell lines share a common characteristic, the overexpression of full-length HMGA1 (high mobility group A1) proteins. The HMGA1 protooncogene codes for two closely related isoform proteins, HMGA1a and HMGA1b, and causes cancerous cellular transformation when overexpressed in either transgenic mice or "normal" cultured cell lines. Previous work has suggested that the in vivo types and patterns of the HMGA1 post-translational modifications (PTMs) differ between normal and malignant cells. The present study focuses on the important question of whether HMGA1a and HMGA1b proteins isolated from the same cell type have identical or different PTM patterns and also whether these isoform patterns differ between non-malignant and malignant cells. Two independent mass spectrometry methods were used to identify the types of PTMs found on specific amino acid residues on the endogenous HMGA1a and HMGA1b proteins isolated from a non-metastatic human mammary epithelial cell line, MCF-7, and a malignant metastatic cell line derived from MCF-7 cells that overexpressed the transgenic HMGA1a protein. Although some of the PTMs were the same on both the HMGA1a and HMGA1b proteins isolated from a given cell type, many other modifications were present on one but not the other isoform. Furthermore, we demonstrate that both HMGA1 isoforms are di-methylated on arginine and lysine residues. Most importantly, however, the PTM patterns on the endogenous HMGA1a and HMGA1b proteins isolated from non-metastatic and metastatic cells were consistently different, suggesting that the isoforms likely exhibit differences in their biological functions/activities in these cell types.

Mass-Dependent Cross-Signaling between TXA2 and PAF Receptors.

We have previously demonstrated in human platelets that G-protein coupled receptors (GPCRs) which share a common G” subunit have the capacity to cross-signal when exposed to their respective ligands. This ligand-dependent cross-signaling was shown to involve a redistribution of GPCR-coupled G” subunits, a consequent shift in GPCR ligand affinity and a synergistic effector response (Djellas, et al. 1998; PNAS , 95:10944 and Djellas, et al. 2000; Biochem.Pharmacol. 59:1521). Based on these findings, we proposed that the phenomenon of ligand-dependent cross-signaling represents one manifestation of a more general process by which cells can dynamically regulate their different GPCR signaling priorities in response to various stimuli. In this model, the relative signaling capacity through different GPCRs is determined by the distribution of G proteins amongst the different GPCRs at a given point in time. The model further predicts that this G protein distribution is not only modulated by ligand-induced shifts in GPCR:G protein coupling affinities (ligand-dependent cross-signaling), but also by shifts in GPCR:G protein mass ratios (mass-dependent cross-signaling), such as those that may occur by up- or down-regulation of GPCRs or their respective G protein partners. The present study examined this mass-dependent GPCR cross-signaling process.Specifically, we established a series of cell lines in which the expression levels of thromboxane A2 receptors (TPR) and platelet activating factor receptors (PAFR) can be progressively altered. These expression systems allowed an alteration of the GPCR:G protein mass ratio for specific GPCRs, i.e., TPR and PAFR. Using radioligand binding analysis and Western blot, we provide evidence that up-regulating TPR expression levels increased the TPR:G-protein mass ratio. This TPR up-regulation resulted in a reduced TPR ligand affinity for [3H] SQ29548. Thus increasing the mass of TPR relative to its G protein partners (Gq or G13) shifted TPRs to a lower ligand affinity state. A similar reduction in PAFR ligand affinity was observed when the PAFR:G-protein mass ratio was increased by elevating PAFR expression levels. Moreover, using an inducible TPR/PAFR co-expressing cell line, increasing the expression level of one of the receptors resulted in reduced ligand affinity for both receptors. Taken in combination with our previous studies, these findings suggest that increasing TPR: G protein mass ratio results in a shift in the TPR-G protein coupling status, such that a larger fraction of total TPR is in the uncoupled state. This reduction in TPR-G protein coupling in turn leads to lower TPR ligand affinity. The finding that expression of TPR also causes reduced ligand affinity for a separate GPCR, i.e. PAFR, suggests that there is a dynamic equilibrium between the distribution of G proteins amongst GPCRs, and that this equilibrium can be altered by changes in the GPCR: G protein mass ratio. In summary, both ligand-dependent and mass-dependent GPCR cross-signaling appear to result from a general mechanism by which cells modulate their GPCR-G protein coupling status, and hence their GPCR ligand affinities and signaling priorities.

Water-soluble covalent conjugates of bovine serum albumin with anionic poly( N-isopropyl-acrylamide) and their immunogenicity

We have conjugated bovine serum albumin (BSA) to poly( N-isopropylacrylamide-co-acrylic acid) (poly(NIPAAm-AA)) by using water-soluble carbodiimide, and the effects of the bulk mass ratio of protein to polymer ( r) on the formation of polymer–protein conjugates have been studied. HPLC, electrophoresis, viscosimetry and fluorescence spectroscopy suggest that the mode of covalent binding of BSA to poly(NIPAAm-AA) depends upon the weight concentration ratio ( r) of BSA to poly(NIPAAm-AA). At r⩽1, free poly(NIPAAm-AA) molecules coexist with conjugate, and when r reaches 1 the amount of free polymer is too small to be observed. It is shown that depending on the ratio r, two types of conjugate particles were formed: at r<1, the protein molecules in the structure of conjugate particles are densely covered as a shell by polymer chain and practically “fenced off” from water environment; at r>1 the conjugate-forming particles possess more friable structures in which protein molecules are practically exposed to the solvent. The complex formation involving electrostatic interactions between BSA and carbodiimide activated polymer are proposed as the driving force for the covalent binding of BSA to polymer macromolecules. The coil-globule transition of macromolecules in low and thermally induced precipitation in more concentrated solutions of bioconjugates was observed. The immunogenic properties of covalent conjugates of CP–BSA were investigated and the temperature-modulated solubility–immunogenicity alterations was analyzed. A single immunization of mice with conjugates at the thermally precipitating concentration without an adjuvant evoked increased specific immune response to BSA, which practically did not depend on the initial conjugation ratio of components. Such a modulated system is attractive for application as a novel immunogenic system in vaccine technology.