Distribution of platelet glycoproteins and phosphoproteins in hydrophobic and hydrophilic phases in triton Triton-114 phase partition

Platelets, either unlabelled, surface-labelled by the periodate NaB³H₄ method or metabolically labelled with ³²P were solubilized in Triton X-114 and partitioned into aqueous and detergent phases. The phases were analysed by two-dimensional polyacrylamide gel electrophoresis followed by silver-staining, fluorography or indirect autoradiography. Each of the phases contains a distinct set of proteins. The surface-labelled glycoproteins partition into the hydrophobic phase with the notable exceptions of glycoproteins Ib and GP₁₇^5.8–6.5 and minor amounts of a few others. The phosphoproteins which undergo increased phosphorylation on platelet activation in general separate in the hydrophobic phase, while higher molecular weight phosphoproteins were principally in the hydrophilic phase. This method might be used as a first step in purifying many platelet components.     

Glycoprotein Ib beta is the only phosphorylated major membrane glycoprotein in human platelets.

Platelets were metabolically labelled with 32P and the phosphoproteins examined by two-dimensional non-reduced/reduced gel electrophoresis and isoelectric-focusing/gel electrophoresis. Comparison with similar separations of surface-labelled platelets showed that the only major glycoprotein which is phosphorylated is the beta-subunit of glycoprotein Ib, indicating that this subunit contains a cytoplasmic segment. The identification was confirmed using immunoblotting with an antibody to the beta-subunit. Phosphoserine was the principal phosphorylation site, with some phosphothreonine, but phosphotyrosine was absent. No quantitative or qualitative differences could be detected in the phosphorylation of glycoprotein Ib beta from resting or activated platelets. These results exclude changes in phosphorylation of the major platelet membrane glycoproteins as a method of signal transmission by these receptors.     

Depolarisation-Dependent Protein Phosphorylation and Dephosphorylation in Rat Cortical Synaptosomes Is Modulated by Calcium

The effect of calcium on protein phosphorylation was investigated using intact synaptosomes isolated from rat cerebral cortex and prelabelled with 32Pi. For nondepolarised synaptosomes a group of calcium-sensitive phosphoproteins were maximally labelled in the presence of 0.1 mM calcium. The phosphorylation of these proteins was slightly decreased in the presence of strontium and absent in the presence of barium, consistent with the decreased ability of these cations to activate calcium-stimulated protein kinases. Addition of calcium alone to synaptosomes prelabelled in its absence increased phosphorylation of a number of proteins. On depolarisation in the presence of calcium certain of the calcium-sensitive phosphoproteins were further increased in labelling above nondepolarised levels. These increases were maximal and most sustained after prelabelling at 0.1 mM calcium. On prolonged depolarisation at this calcium concentration a slow decrease in labelling was observed for most phosphoproteins, whereas a greater rate and extent of decrease occurred at higher calcium concentrations. At 2.5 mM calcium a rapid and then a subsequent slow dephosphorylation was observed, indicating two distinct phases of dephosphorylation. Of all the phosphoproteins normally stimulated by depolarisation, only phosphoprotein 59 did not exhibit the rapid phase of dephosphorylation at high calcium concentrations. Replacing calcium with strontium markedly decreased the extent of change observed on depolarisation whereas barium decreased phosphorylation changes even further. Taken together these data suggest that an influx of calcium into synaptosomes initially activates protein phosphorylation, but as the levels of intrasynaptosomal calcium rise protein dephosphorylation predominates. Other phosphoproteins were dephosphorylated immediately on depolarisation in the presence of calcium. The fine control of protein phosphorylation levels exerted by calcium supports the idea that the synaptosomal phosphoproteins could play a role in modulating events such as neurotransmitter release in the nerve terminal.     

iTRAQ-based phosphoproteomic analysis reveals host cell's specific responses to Toxoplasma gondii at the phases of invasion and prior to egress

Protein phosphorylation plays a key role in host cell-T. gondii interaction. However, the phosphoproteome data of host cell at various phases of T. gondii infection has not been thoroughly described. In this study, we assessed the host phosphoproteome data with isobaric tags for relative and absolute quantification (iTRAQ) method during the phases of T. gondii invasion (30 min post infection, PI) and prior to egress (28 h PI). Our iTRAQ analysis revealed a total of 665 phosphoproteins, among which the significantly regulated phosphoproteins in different between-group comparisons were further analyzed. Functional analysis of these significantly regulated phosphoproteins suggested that T. gondii modulated host cell processes through phosphorylation including cell cycle regulation, inducing apoptosis, blocking the synthesis of some inflammatory factors, mediating metabolism to support its proliferation at the infection phase prior to egress, and utilizing membrane and energy from host cell, reorganizing cytoskeleton to favor its invasion and PV formation at the phase of invasion. The phosphorylation level of Smad2, CTNNA1, and HSPB1 identified with western blot revealed a consistent trend of change with iTRAQ result. These newly identified and significantly regulated phosphoproteins from our phosphoproteome data may provide new clues to unravel the host cell's complex reaction against T. gondii infection and the interaction between the host cell and T. gondii.     

Phase separation of the receptor for immunoglobulin E and its subunits in Triton X-114

Above its critical micelle concentration, Triton X-114 in solution forms two phases at room temperature: a lower phase containing supramicellar aggregates and an upper phase largely depleted of detergent. This property of the detergent is potentially useful for separating under mild conditions proteins that bind detergent from those that do not (Bordier, C. (1981) J. Biol. Chem. 256, 1604-1607). We studied the distribution of the receptor for immunoglobulin E (IgE) and its subunits in the two phases. IgE and IgE complexed either with intact receptors or with the alpha chains of the receptor alone are principally partitioned into the upper phase, whereas the unliganded receptor as well as the isolated alpha, and especially the beta and gamma chains of the receptor, preferentially partition into the lower detergent phase. Chromatography of IgE and of the subunits of the receptor on a hydrophobic support showed that the beta and gamma chains have a considerably greater hydrophobic surface than the alpha chains or IgE. These results indicate that the distribution of a protein in the two phases of phase-separated Triton X-114 is not an all-or-none effect based upon whether it binds detergent or not. Rather, it reflects the overall balance between the hydrophobic and hydrophilic properties of the protein's surface.     

Mixed-mode hydrophobic ion exchange for the separation of oligonucleotides and DNA fragments using HPLC

Hydrophobic anion exchangers were formed by cobonding both ionic and hydrophobic ligands to silica gel. These phases were used to separate single-stranded oligonucleotides and double-stranded DNA restriction fragments. By varying the ratio of n-octyldimethylsilane and either 3-chloropropyldimethylsilane or 4-chlorobutyldimethylsilane added during silanization a series of mixed-ligand or mixed-mode stationary phases was created. Concentration and ratio of bonded ligands were determined using a new gas chromatography fluorination method. Total ligand coverage was found to approach 2.1 ligands nm-2 for n-octyldimethylsilane. Bonding reproducibility for mixed-mode phases was good. Nucleic acid separations were achieved under gentle mobile phase conditions by using the stationary phase as an easily modifiable variable.     

A method to measure receptor binding of ligands with low affinity : Application to plasma proteins binding assay with hemopoietic cells

A gradient has been developed for separating free ligand from ligand bound to cells growing in suspension. This method can be used with all kinds of ligand but it is particularly useful for those ligands having the tiresome tendency to adhere to the cells non-specifically or to polymerize by themselves. This is the case of fibronectin, fibrinogen, immunoglobulins and many other plasma proteins. The gradient consists of two layers: an upper aqueous phase and a lower hydrophobic organic phase. The aqueous phase, a sucrose buffer, allows the cells to become well dispersed before they enter the hydrophobic phase which excludes the free ligand efficiently. This reduces non-specific binding and allows the accurate measurement of specific binding which could not be obtained with a gradient made of a single phase. Depending upon the size and the density of the cells, and the nature of the ligand, the assay method can be modified by changing the density or the nature of the hydrophilic and hydrophobic phases.     

Inhibition of Fc-receptor dependent platelet aggregation by monoclonal antibodies against the glycoprotein IIb-IIIa complex]

A murine monoclonal antibody (MoAb) VM16a specifically binding to human platelets has been produced. Approximately 56,000 molecules of VM16a bound per platelet at saturation (Kd = 7.9 nM) but no binding to platelets from Glanzmann's thrombasthenia patients was detected. VM16a precipitated two proteins with molecular masses corresponding to those of glycoproteins (GP) IIb and IIIa from solubilized surface-labelled platelets. However, after dissociation of the GPIIb--IIIa complex with EDTA VM16a did not bind to platelets and precipitated nothing from their lysate, thus evidencing that its determinant is complex-dependent. VM16a had no effect on ADP-, thrombin- and ristocetin-induced platelet aggregation but inhibited the aggregation induced by collagen. This inhibitory effect was more pronounced in the presence of plasma. VM16a completely blocked the Fc-receptor-mediated aggregation induced by aggregated human IgG, aggregated murine IgG1 and the previously described MoAb VM58. F(ab')2 fragments of VM16a were also able to inhibit this aggregation by decreasing the rate of aggregation induced by aggregated IgG and by extending the lag phase of VM58-induced aggregation. These results suggest that the platelet Fc-receptor may be topographically associated with the GPIIb-IIIa complex.     

Protein organization in mouse liver peroxisomes.

Peroxisomes from mouse liver were fractionated with Triton X-114, a procedure which yields a detergent phase consisting of proteins containing hydrophobic binding sites, and a nondetergent, or aqueous, phase containing hydrophilic proteins. When this method was applied to peroxisomes from control mice, catalase and fatty acyl-CoA oxidase distributed to the aqueous phase, whereas the integral membrane protein, PMP68, and the bifunctional protein were recovered exclusively in the detergent phase. Urate oxidase distributed intermediate between these two phases. With peroxisomes from mice treated with the peroxisome proliferator clofibrate, the bifunctional protein was recovered in both the detergent and the aqueous phases, and urate oxidase was shifted toward the aqueous phase. Other analyses of the subperoxisomal distribution of the bifunctional protein were consistent with a proportion of this protein being tightly associated with the peroxisomal membrane, or with some other uncharacterized, poorly soluble, component. Sucrose gradient centrifugation of the aqueous phase resulting from Triton X-114 fractionation of peroxisomes revealed that a major proportion of catalase, fatty acyl-CoA oxidase, the bifunctional protein, and other unidentified proteins behaved as if associated under these conditions. In this respect, use of a higher concentration of Triton X-114 for peroxisome fractionation led to the partitioning of some catalase and fatty acyl-CoA oxidase to the detergent phase, indicating the presence of some detergent-accessible hydrophobic binding sites even on these proteins. These data have been interpreted as indicating matrix protein associations in vivo, associations which may be responsive to proliferator treatment.     

Proton magnetic resonance and viscosity studies of the interaction of local anesthetics and micelles.

This paper reports the interesting physical phenomenon of the formation of a very high viscosity phase when sodium dodecyl sulfate (SDS) micelles interact with the local anesthetics tetracaine and lidocaine. Charge neutralization by mobile counterions or interaction of the neutral form of the local anesthetic molecule with SDS micelles does not lead to the formation of high viscosity phases. The hydrocarbon chains of the surfactant appear to become extremely immobilized, as detected by proton magnetic resonance; likewise, the chemical shift of the aromatic protons of the local anesthetics as well as the broadening of the CH₂ protons of the SDS suggest that charge neutralization and the hydrophobic contribution of the anesthetic are the factors responsible for estabilizing the micellar aggregates. The induction of high viscosity phases can be utilized as a simple and economical method to estimate hydrophobic contributions.     

A novel approach for estimating growth phases and parameters of bacterial population in batch culture

Using mathematical analysis, a new method has been developed for studying the growth kinetics of bacterial populations in batch culture. First, sampling data were smoothed with the spline interpolation method. Second, the instantaneous rates were derived by numerical differential techniques and finally, the derived data were fitted with the Gaussian function to obtain growth parameters. We named this the Spline-Numerical-Gaussian or SNG method. This method yielded more accurate estimates of the growth rates of bacterial populations and new parameters. It was possible to divide the growth curve into four different but continuous phases based on changes in the instantaneous rates. The four phases are the accelerating growth phase, the constant growth phase, the decelerating growth phase and the declining phase. Total DNA content was measured by flow cytometry and varied depending on the growth phase. The SNG system provides a very powerful tool for describing the kinetics of bacterial population growth. The SNG method avoids the unrealistic assumptions generally used in the traditional growth equations.     

A novel approach for estimating growth phases and parameters of bacterial population in batch culture

Traditionally, microbiologists divided bacterial growth in batch cultures into lag, exponential, station-ary and death phases[1], following the Logistic equa-tion that has been applied to the growth of human populations. The growth curves can always be ch…     

Characteristics of Ca2+/calmodulin- and Ca2+/phosphatidylserine-stimulated phosphoproteins in rat striatum

The characteristics of endogenous Ca²⁺/calmodulin (CaM)- and Ca²⁺/phosphatidylserine (PS)-stimulated phosphorylated proteins in the striatum of rat were partially determined and compared in this study. The Ca²⁺/CaM-dependent phosphoproteins were associated with serine and threonine residues. The sensitivity of these proteins for phosphorylation by Ca²⁺/CaM was not affected by pretreatment of tissue with Ca²⁺ chelating agent, EGTA or with non-ionic detergent, Triton X-114. Triton X-114 phase separation experiments revealed that these Ca²⁺/CaM-dependent phosphoproteins were partitioned in the detergent rich phase suggesting that they are integral proteins of the striatal membrane. On the other hand, the Ca²⁺/PS-dependent phosphorylated proteins were primarily associated with the serine residue. Phosphorylation of these proteins by Ca²⁺/PS were abolished after the treatment with EGTA or Triton X-114. These results suggest that Ca²⁺/PS-dependent striatal phosphoproteins are biochemically unstable in maintaining their state of phosphorylation.     

Two detergent-insoluble proteins of the human lymphocyte membrane are enriched in an isolated membrane fraction

Human lymphocytes isolated from peripheral blood on Ficoll/Paque density gradients were surface-labelled by 125I/lactoperoxidase or 3H/reductive alkylation and lysed in buffer solutions containing non-ionic or amphoteric detergents (octylphenylpolyoxyethylenes, octylglucoside, cholylamidopropyldimethylammoniopropane sulfonate) under a variety of conditions. The cell lysate was fractionated by sedimentation or by density gradient centrifugation. The large majority of the labelled proteins is solubilized by the detergents. Two proteins of 45 000 and 30 000 molecular weight are the main detergent-insoluble, surface-labelled components. They can be fractionated from detergent lysates of cells in relatively pure form from the other membrane proteins and from nuclear material on density gradients. The same two proteins are specifically enriched in a membrane fraction isolated from a detergent-free cell homogenate by density gradient centrifugation. Cytoskeletal and other intracellular proteins remain associated with these two proteins when fractionated by either of these two independent methods.     

The influence of protein-lipid interactions on the order-disorder conformational transitions of the hydrocarbon chain.

The phases of simple systems involving one type of protein (lysozyme or cytochrome c) and one type of lipid (phosphatidic acid) have been characterized by X-ray crystallography, chemical analysis and spin-labeling technique as a function of temperature. They are of the lamellar type with alternative protein monolayers and lipid bilayers. According to the pH, two types of lamellar phases are obtained, one where the lipid-protein interactions are mainly hydrophobic, the other where they are electrostatic. In both cases, a phase transition occurs as temperature is lowered, between a high temperature phase, where all the lipids are in the liquid-like state, and another phase where some lipid chains are rigid. In the case of the phases with electrostatic interaction, it is shown that the onset of the order-disorder transition is shifted towards low temperature as compared with the homologous lipid-water phase and that the protein content of the phase decreases as the ratio of the liquid to rigid hydrocarbon chains decreases. This leads us to suggest that in the systems studied in this work the proteins interact only with lipid in the liquid-like state. In the case of the phases with hydrophobic interaction, it is shown that the extent of hydrophobic interaction between protein and lipid increases as the unsaturation of the hydrocarbon chains increases. The onset of the order-disorder transition shows a greater shift towards low temperature than the one observed in the case of the phase with electrostatic interaction.     

Cytosolic ionized calcium in human platelets: the influence of collagen and a novel antiplatelet agent

Two phases of calcium mobilization were observed when aequorin-loaded human platelets, suspended in a nominally calcium-free medium containing 0.1 mM EGTA, were stimulated with collagen. The first phase coincided with platelet shape change, and the second phase corresponded to aggregation. On the other hand, only one [Ca2+]i peak was found in systems containing 1.0 mM Ca, or 1.0 or 2.0 mM EGTA. A novel antiplatelet compound alpha,alpha'-bis [3-(N,N-diethylcarbamoyl)piperidino]-p-xylene dihydrobromide, inhibited both [Ca2+]i peaks. It is suggested that inhibition of the mobilization of intraplatelet calcium stores as well as the blocking of transmembrane calcium flux may be responsible for the platelet aggregation-inhibitory action of this compound.     

A protein kinase system from platelet rich plasma

Treatment of platelet rich plasma (PRP) at pH 5 results in the precipitation of a protein kinase system. The protein kinase is associated with the platelet fraction and is capable of phosphorylation of several plasma proteins. Analysis of the ³²P-labeled phosphoproteins by two dimensional gel electrophoresis showed the existence of three major phosphoproteins: 72K and 80K proteins with identical isoelectric points (pI) of 6.0 and another 72K protein with a pI of 6.8–7.0. This latter 72K phosphoprotein has recently been identified as the α-chain of fibrinogen. The identity of the other 2 proteins remains to be shown. The activity of the protein kinase is markedly enhanced by Mn²⁺, it phosphorylates calf thymus histone as an exogenous substrate and is independent of cAMP or cGMP. This protein kinase activity is inhibited competitively by ADP.     

Mapping of phosphorylated proteins on two-dimensional polyacrylamide gels using protein phosphatase

This study developed an enzymatic method for high-throughput mapping of phosphoproteins on two-dimensional (2-D) polyacrylamide gels. Proteins of cultured rat skin fibroblasts were divided into two aliquots, one of which was dephosphorylated using recombinant lambda protein phosphatase and the other was not treated with the enzyme. The two aliquots were then subjected to 2-D electrophoresis. Phosphoproteins could be mapped on the 2-D gel of the nontreated aliquot by comparing the gels of the two aliquots, because the phosphoproteins in the treated aliquot shifted to more basic positions on the gel. This technique revealed that approximately 5% of the detectable proteins were phosphorylated. Fourteen phosphoproteins were identified by mass spectrometry, including proteasome component C8 and small glutamine-rich tetratricopeptide repeat-containing protein. Furthermore, the extent of phosphorylation of two actin modulating proteins, destrin and cofilin, was found to be significantly reduced when the cells were chemically or enzymatically detached from the culture dishes. The method developed by this study can generally be applied to all biological materials and is useful for high-throughput mapping of phosphoproteins in proteome research.     

The influence of protein-lipid interactions on the order-disorder conformational transitions of the hydrocarbon chain

The phases of simple systems involving one type of protein (lysozyme or cytochrome $\text{c}$) and one type of lipid (phosphatidic acid) have been characterized by X-ray crystallography, chemical analysis and spin-labeling technique as a function of temperature. They are of the lamellar type with alternative protein monolayers and lipid bilayers. According to the pH, two types of lamellar phases are obtained, one where the lipid-protein interactions are mainly hydrophobic, the other where they are electrostatic. In both cases, a phase transition occurs as temperature is lowered, between a high temperature phase, where all the lipids are in the liquid-like state, and another phase where some lipid chains are rigid. In the case of the phases with electrostatic interaction, it is shown that the onset of the order-disorder transition is shifted towards low temperature as compared with the homologous lipid-water phase and that the protein content of the phase decreases as the ratio of the liquid to rigid hydrocarbon chains decreases. This leads us to suggest that in the systems studied in this work the proteins interact only with lipid in the liquid-like state. In the case of the phases with hydrophobic interaction, it is shown that the extent of hydrophobic interaction between protein and lipid increases as the unsaturation of the hydrocarbon chains increases. The onset of the order-disorder transition shows a greater shift towards low temperture than the one observed in the case of the phase with electrostatic interaction.     

Refolding molecular dynamics simulations of small- and middle-sized proteins in an explicit solvent

In order to elucidate the protein folding problem, we performed molecular dynamics simulations for small- and middle-sized two unfolding and six refolding proteins in an explicit solvent. Histidine-containing phosphocarrier protein and small designed protein were chosen for the simulations. We found that the protein folding process of these proteins was divided into three phases: an α -helix formation phase, a packing phase and a β -sheet formation phase. In the α -helix formation phase, an α -helix was developed from a β -turn structure through a 3₁₀-helix state. In the packing phase, proteins became compact, and tertiary structures (α / α or pre- β / β packing) were formed. Formation of a hydrophobic nucleus occurred concomitant with the α -helix formation and packing phase. Finally, in the β -sheet formation phase, a β -sheet was developed owing to the sequential formation of hydrogen bonds between two neighbouring strands, just like a "closing zipper".