Fabrication of a cell-adhesive protein imprinting surface with an artificial cell membrane structure for cell capturing

We proposed a new molecular imprinting procedure based on molecular integration for the purpose of cell capture. We selected the cell-adhesive protein fibronectin (FN) as the imprinting protein for preparing templates and evaluated selective cell adhesion on the FN imprinting substrate. Silica beads with a diameter of 15 μm were used as the stamp matrix and FN molecules were adsorbed as a monolayer. The FN recognition sites were constructed by integrating a surfactant as the ligand and immobilizing it with new biocompatible photoreactive phospholipid polymer composed of 2-methacryloyloxyethyl phosphorylcholine (MPC) units. As control substrates, imprinting procedures were carried out using albumin (BSA imprinting substrate) and without imprinting protein (non-imprinting substrate). The binding of FN from the cell culture medium with the fetal calf serum was achieved on the FN imprinting substrate, and induced the cell adhesion. On the other hand, on the non-imprinted and BSA imprinting substrates, the FN scarcely bound from the cell culture medium, and subsequent cell adhesion could not be observed on the substrate. These results indicate that the FN binding sites were well constructed by arranging the ligand surfactant to a suitable position and immobilized by the photoreactive MPC polymer. The MPC polymer prevented the nonspecific adsorption of proteins from the cell culture medium. We concluded that this procedure is convenient and can be potentially used for the preparation of surfaces for cell engineering devices.     

A quantitative study of pinocytosis and intracellular proteolysis in rat peritoneal macrophages.

A method for the culture of rat peritoneal macrophages in vitro is described, in which pinocytic uptake of colloidal [198 Au]gold, 125I--labelled poly(vinylpyrrolidone) and [14C]sucrose proceeds at contant and fairly reproducible rates for several hours. The rat of uptake of colloidal [198 Au]gold, which wxhibited some inter-batch variation, was approx. 100 times that of the other two substrates. Colloidal gold did not affect the rate of uptake of 125I-labelled poly(vinylpyrrolidone) and therefore its own high rate of uptake could not be attributed to a stimulation of the formation of pinocytic vesicles. It conclude that uptake of collodial gold is highly dependent on adsorption on binding sites on the plasma membrane. Uptake of formaldehyde-treated 125I-labelled bovine serum albumin was followed by the release of [125I]iodo-L-tyrosine into the culture medium and took place at a rate intermediate between those of collodial [198Au]gold and the other two non-digestible substrates, 125I-labelled poly(vinylpyrrolidone) and [14C]sucrose.     

Serum-activated T51B rat liver cells transiently accumulate cyclic AMP-dependent protein kinases on their surfaces during the G1 phase

Confluent T51B rat liver epithelial cells promptly began accumulating cyclic AMP-binding sites on their surfaces when they were stimulated from quiescence by serum growth factors in medium containing 1.8 mM Ca2+, but they began losing the accumulated binding sites shortly before initiating DNA replication. When the medium contained only 0.02 mM Ca2+, the cells still accumulated surface cyclic AMP-binding sites, but they did not initiate DNA replication and tended to continue accumulating the binding sites. The cyclic AMP-binding sites were eliminated completely by treating intact cells for 5 minutes with 0.005% trypsin (which did not damage the cells), and cyclic AMP caused them to be released from intact, undamaged cells into the medium. The binding sites also comigrated electrophoretically with purified regulatory subunits of type I cyclic AMP-dependent protein kinase, and to a lesser extent the regulatory subunit of type II cyclic AMP-dependent protein kinase. Therefore, it is likely that a transient accumulation of cyclic AMP-dependent protein kinases on the outer surface of the plasma membrane is part of the T51B rat liver cell's prereplicate program.     

Interaction of LDS-751 with P-glycoprotein and mapping of the location of the R drug binding site

One cause of multidrug resistance is the overexpression of P-glycoprotein, a 170 kDa plasma membrane ABC transporter, which functions as an ATP-driven efflux pump with broad specificity for hydrophobic drugs, peptides, and natural products. The protein appears to interact with its substrates within the membrane environment. Previous reports suggested the existence of at least two binding sites, possibly overlapping and displaying positively cooperative interactions, termed the H and R sites for their preference for Hoechst 33342 and rhodamine 123, respectively. In this work, we have used several fluorescence approaches to characterize the molecular interaction of purified P-glycoprotein (Pgp) with the dye LDS-751, which is proposed to bind to the R site. A 50-fold enhancement of LDS-751 fluorescence indicated that the protein binding site was located in a hydrophobic environment, with a polarity lower than that of chloroform. LDS-751 bound with sub-micromolar affinity (K(d) = 0.75 microM) and quenched P-glycoprotein intrinsic Trp fluorescence by 40%, suggesting that Trp emitters are probably located close to the drub-binding regions of the transporter and may interact directly with the dye. Using a FRET approach, we mapped the possible locations of the LDS-751 binding site relative to the NB domain active sites. The R site appeared to be positioned close to the membrane boundary of the cytoplasmic leaflet. The location of both H and R drug binding sites is in agreement with the idea that Pgp may operate as a drug flippase, moving substrates from the inner leaflet to the outer leaflet of the plasma membrane.     

Separation of human plasma proteins HSA and HIgG using high-capacity macroporous gel-filled membranes

This paper discusses the separation of human serum albumin (HSA), the most abundant protein present in plasma from human immunoglobulin G (HIgG) by membrane chromatography using a novel macroporous gel-filled membrane (designated Q Type 2). The membrane was prepared by anchoring a quaternary ammonium salt macroporous gel within the pores of a non-woven, polypropylene fabric. Factors affecting HSA binding were examined and operating conditions suitable for separating it from human plasma were identified. At an optimized condition, the HSA binding capacity of this novel membrane under saturating conditions was in the range of 290–300 mg/ml. This was not only significantly higher than binding capacities reported for other chromatographic membranes, but also higher than binding capacities of conventional gel based chromatographic media. The protein binding capacity was also largely insensitive to the superficial velocity, indicating the dominance of convective protein transport to and from the binding sites. The suitability of using this membrane for plasma fractionation was demonstrated by the separation of a simulated feed solution consisting of HSA and HIgG.     

A receptor of dihydropyridine blockaders of Ca2+ influx in human embryonic fibroblasts

The plasma membrane of human embryonic fibroblasts was shown to contain receptor binding sites for 1,4-dihydropyridine (DHP) Ca2+ entry blockers. In a subconfluent culture grown in serum medium the content of the DHP receptor amounted to 1.2 +/- 0.3 pmol per 10(6) cells. With progression to confluency this value decreased up to 0.28 +/- 0.08 pmol per 10(6) cells. The DHP binding capacity was shown to be affected by the presence of growth factors in culture medium. In a subconfluent culture of serum-deprived cells the content of DHP binding sites increased 1.9 fold, the steady-state level being achieved within 3 days in culture. The serum gradually reversed this process, and the DHP receptor density approached the initial level within 3 days.     

Interaction of prostaglandins and fatty acids with native and acetaldehyde-alkylated proteins

Using intrinsic and probe fluorescence, microcalorimetry and isotopic methods, the interactions of prostaglandins (PG) E2 and F2 alpha and some fatty acids with native and alkylated proteins (human serum albumin (HSA) and rat liver plasma membrane PG receptors), were studied. The fatty acid and PG interactions with human serum albumin (HSA) resulted in effective quenching of fluorescence of the probe, 1.8-anilinonaphthalene sulfonate (ANS), bound to the protein. Fatty acids competed with ANS for the binding sites; the efficiency of this process increased with an increase in the number of double bonds in the fatty acid molecule. PG induced a weaker fluorescence quenching of HSA-bound ANS and stabilized the protein molecule in a lesser degree compared to fatty acids. The sites of PG E2 and F2 alpha binding did not overlap with the sites of fatty acid binding on the HSA molecule. Nonenzymatic alkylation of HSA by acetaldehyde resulted in the abnormalities of binding sites for fatty acids and PG. Modification of the plasma membrane proteins with acetaldehyde sharply diminished the density of PG E2 binding sites without changing the association constants. Alkylation did not interfere with the parameters of PG F2 alpha binding to liver membrane proteins.     

Evidence of sex hormone binding globulin binding sites in the medial preoptic area and hypothalamus.

We have demonstrated a high density of both radiolabeled progesterone and estradiol conjugated to bovine serum albumin binding sites in the medial preoptic area and hypothalamus. Infusions of sex hormone binding globulin into the medial preoptic area of rats increased their female sexual receptivity similarly to the effect of estradiol conjugated to bovine serum albumin, suggesting sex hormone binding globulin acts at binding sites for estradiol conjugated to bovine serum albumin. In this study sex hormone binding globulin was used to displace radiolabeled progesterone conjugated to bovine serum albumin from plasma membrane fractions from the medial preoptic area-anterior hypothalamus and medial basal hypothalamus of ovariectomized rats injected with either 5 microg estradiol benzoate or sesame oil vehicle. We found that sex hormone binding displaced radiolabeled progesterone conjugated to bovine serum albumin in both areas and that in vivo estradiol treatment greatly increased the relative displacement by sex hormone binding globulin in the medial preoptic area-anterior hypothalamus. We interpret these data as indicating the presence of sex hormone binding globulin receptors in brain plasma membranes and further suggest that endogenous steroid conditions may alter these receptors.     

Presence of membrane ecdysone receptor in the anterior silk gland of the silkworm Bombyx mori.

Nongenomic action of an insect steroid hormone, 20-hydroxyecdysone (20E), has been implicated in several 20E-dependent events including the programmed cell death of Bombyx anterior silk glands (ASGs), but no information is available for the mode of the action. We provide evidence for a putative membrane receptor located in the plasma membrane of the ASGs. Membrane fractions prepared from the ASGs exhibit high binding activity to [3H]ponasterone A (PonA). The membrane fractions did not contain conventional ecdysone receptor as revealed by Western blot analysis using antibody raised against Bombyx ecdysone receptor A (EcR-A). The binding activity was not solubilized with 1 m NaCl or 0.05% (w/v) MEGA-8, indicating that the binding sites were localized in the membrane. Differential solubilization and temperature-induced phase separation in Triton X-114 showed that the binding sites might be integrated membrane proteins. These results indicated that the binding sites are located in plasma membrane proteins, which we putatively referred to as membrane ecdysone receptor (mEcR). The mEcR exhibited saturable binding for [3H]PonA (Kd = 17.3 nm, Bmax = 0.82 pmol.mg(-1) protein). Association and dissociation kinetics revealed that [3H]PonA associated with and dissociated from mEcR within minutes. The combined results support the existence of a plasmalemmal ecdysteroid receptor, which may act in concert with the conventional EcR in various 20E-dependent developmental events.     

Characterization of gonadotropin binding sites in the intracellular organelles of bovine corpora lutea and comparison with plasma membrane sites

The specific binding of 125I-human choriogonadotropin (hCG) to plasma membranes, nuclear membranes, lysosomes, rough endoplasmic reticulum, heavy golgi, and medium and light golgi of bovine corpora lutea was dependent on the amount of protein, 125I-hCG concentration and incubation time. The bound hormone in all the organelles was able to rebind to fresh corresponding organelles. Scatchard analysis revealed a homogenous population of gonadotropin binding sites in plasma membrane, rough endoplasmic reticulum, heavy golgi, and medium and light golgi, whose binding affinities (Kd = 8.6-11.0 X 10(-11) M) were similar but whose number of available gonadotropin binding sites varied. Scatchard analyses of nuclear membranes and lysosome binding, on the other hand, were heterogenous (Nuclear membranes, 11 and 23 X 10(-11) M lysosomes, 3.4 and 130 X 10(-11) M). The rate constants for association (5.9 to 12.1 X 10(6) M-1 S-1) and dissociation (7.4 to 9.0 X 10(-4) S-1) were similar among different subcellular organelles except for nuclear membranes and lysosomes, where rate constants for association were significantly lower. The ligand binding specificity, lower effectiveness of human luteinizing hormone as compared to hCG in competition, the optimal pH, the lack of ionic requirements for binding, and the molecular size of 125I-hCG-gonadotropin binding site complexes solubilized from various intracellular organelles were similar to those observed for plasma membranes. Numerous differences were also observed between intracellular organelles and plasma membranes as well as among intracellular organelles themselves with respect to binding losses due to exposure to low and high pH values, di- and monovalent cations, increasing preincubation temperatures, and a variety of enzymes and protein reagents. The possible reasons for these similarities as well as differences observed are discussed. The differences are viewed as an additional indication that contamination cannot solely explain the presence of gonadotropin binding sites in various intracellular organelles.     

Raising the active site of factor VIIa above the membrane surface reduces its procoagulant activity but not factor VII autoactivation

Tissue factor, the physiologic trigger of blood clotting, is the membrane-anchored protein cofactor for the plasma serine protease, factor VIIa. Tissue factor is hypothesized to position and align the active site of factor VIIa relative to the membrane surface for optimum proteolytic attack on the scissile bonds of membrane-bound protein substrates such as factor X. We tested this hypothesis by raising the factor VIIa binding site above the membrane surface by creating chimeras containing the tissue factor ectodomain linked to varying portions of the membrane-anchored protein, P-selectin. The tissue factor/P-selectin chimeras bound factor VIIa with high affinity and supported full allosteric activation of factor VIIa toward tripeptidyl-amide substrates. That the active site of factor VIIa was raised above the membrane surface when bound to tissue factor/P-selectin chimeras was confirmed using resonance energy transfer techniques in which appropriate fluorescent dyes were placed in the active site of factor VIIa and at the membrane surface. The chimeras were deficient in supporting factor X activation by factor VIIa due to decreased k(cat). The chimeras were also markedly deficient in clotting plasma, although incubating factor VII or VIIa with the chimeras prior to the addition of plasma restored much of their procoagulant activity. Interestingly, all chimeras fully supported tissue factor-dependent factor VII autoactivation. These studies indicate that proper positioning of the factor VII/VIIa binding site on tissue factor above the membrane surface is important for efficient rates of activation of factor X by this membrane-bound enzyme/cofactor complex.     

Cultured hepatocytes bind and internalize bovine serum amine oxidase-gold complexes

Cultured hepatocytes express binding sites for bovine serum amine oxidase on their membrane surfaces as evaluated at the electron microscopic level by using enzyme-gold complexes. Hepatocytes show binding sites as small clusters of gold granules, not bound in a specialized region of the plasma membrane. The binding competition of enzyme-gold ligand to cells was achieved by preincubation with uncoupled bovine serum amine oxidase. In addition, N-acetyl-galactosamine, N-acetyl-glucosamine and Mannose, at the final concentration of 80 mM, partially inhibit the binding.     

Iron binding to, and release from, the basolateral membrane of mouse duodenal enterocytes

The basolateral membrane of mouse duodenal enterocytes can be selectively labelled in vitro with 59Fe by incubating intact enterocytes with 59Fe(III)-nitrilotriacetate at 0-4 degrees C. It has been proposed that this labelling represents binding to a site important in the transfer of intracellular Fe to the portal plasma (Snape, S., Simpson, R.J. and Peters, T.J. (1990) Cell Biochem. Funct. 8, 107-115). Studies presented here show binding to intact enterocytes in vitro was complete within 1 h and was proportional to enterocyte protein concentration. Binding to enterocytes isolated from both normal and chronically hypoxic mice showed a hyperbolic dependence on medium Fe(III) concentration, consistent with a single class of binding sites. Neither apparent binding constant nor maximal binding were increased by hypoxic exposure of mice, suggesting that the increased in vivo labelling of this site in hypoxia is not due to an increase in affinity or capacity of this site for iron. Release of iron from intact enterocytes, labelled at 0-4 degrees C, was measured at 37 degrees C and 0-4 degrees C. Release of 59Fe was extensive and more rapid at 37 degrees C with highest release to mouse serum. Iron released to serum was found to be bound to transferrin. Prior dialysis of serum against buffer led to complete failure of enterocytes to release iron. Reconstituting serum by adding back the dialysate restores release to levels seen in fresh serum, suggesting that low molecular weight serum components, notably bicarbonate, mediate iron transfer from the basolateral membrane to serum transferrin. The properties of the basolateral membrane iron binding site described here are consistent with a role in the iron transfer process.     

Different localization of inositol 1,4,5-trisphosphate and ryanodine binding sites in rat liver.

The distribution of inositol 1,4,5-trisphosphate and ryanodine binding sites between plasma membrane, microsomal, and mitochondrial fractions of rat liver were compared. IP3 bound mostly to the plasma membrane fraction (Kd = 6 nM; Bmax = 802 fmol/mg protein). Some IP3 binding sites were also present in the microsomal and mitochondrial fractions (Kd = 2.5 and 2.9 nM; Bmax = 35 and 23 fmol/mg protein respectively). The possibility that these binding sites are due to contamination of the fractions with plasma membrane cannot be excluded. Binding of IP3 to the plasma membrane was inhibited by heparin but not by either caffeine or tetracaine. High-affinity ryanodine binding sites were present mostly in the microsomal fraction (Kd = 13 nM; Bmax = 301 fmol/mg protein). Lower affinity binding sites were also found to be present in the mitochondrial and plasma membrane fractions. Binding of ryanodine to the microsomal fraction was inhibited by both caffeine and tetracaine but not by heparin. These data demonstrate that IP3 and ryanodine binding sites are present in different cellular compartments in the liver. These differences in the localization of the binding sites might be indicative of their functional differences.     

Binding of norgestrel to human plasma proteins.

Binding of [14, 15-3H](+/-)-norgestrel to human plasma proteins has been investigated. Norgestrel showed greater affinity to plasma than to human serum albumin indicating specific norgestrel binding protein(s) in the plasma. alpha1-acid glycoprotein showed high affinity for norgestrel when compared with human serum albumin. The binding protein was eluted at pH 5.8 by step by step elution on a DEAE-cellulose column. Norgestrel binding to plasma proteins was not affected at 60 degrees C. The optimal binding occurred between pH 7 and 8. Ligand specificity of the binding protein revealed that progesterone was able to compete for the norgestrel binding sites, whereas corticosterone, testosterone, oestradiol, and norethindrone acetate did not show much competition. The molecular weight of the binding protein was found to be approximately 43 000. Sucrose density gradient analysis indicated that norgestrel bound to a macromolecular component of sedimentation coefficient 2.9 S. The association constant (Kass) and dissociation constant (Kdiss) of norgestrel-binding plasma protein was found to be 1.4-10(6) M-1 and 0.7-10(-6) M respectively. The number of binding sites was 0.5-10(-9) mol/mg protein. Norgestrel-binding protein in the plasma appeared to be a protein different from human serum albumin, corticosteroid-binding globulin and sex-steroid-binding protein. This binding protein showed some similarities to alpha1-acid glycoprotein.     

Electron spin-echo studies of spin-labelled lipid membranes and free fatty acids interacting with human serum albumin

Human serum albumin (HSA) is an abundant plasma protein that transports fatty acids and also binds a wide variety of hydrophobic pharmacores. Echo-detected (ED) EPR spectra and D(2)O-electron spin echo envelope modulation (ESEEM) Fourier-transform spectra of spin-labelled free fatty acids and phospholipids were used jointly to investigate the binding of stearic acid to HSA and the adsorption of the protein on dipalmitoyl phosphatidylcholine (DPPC) membranes. In membranes, torsional librations are detected in the ED-spectra, the intensity of which depends on chain position at low temperature. Water penetration into the membrane is seen in the D(2)O-ESEEM spectra, the intensity of which decreases greatly at the middle of the membrane. Both the chain librational motion and the water penetration are only little affected by adsorption of serum albumin at the DPPC membrane surface. In contrast, both the librational motion and the accessibility of the chains to water are very different in the hydrophobic fatty acid binding sites of HSA from those in membranes. Indeed, the librational motion of bound fatty acids is suppressed at low temperature, and is similar for the different chain positions, at all temperatures. Correspondingly, all segments of the bound chains are accessible to water, to rather similar extents.     

Identification and characterization of parathyroid hormone receptors on dog kidney, human kidney, chick bone and human dermal fibroblast. A comparative study of functional and structural properties.

To evaluate the functional and structural characteristics of the parathyroid hormone (PTH) receptors on different tissues and the possible heterogeneity in structure and function, PTH receptors on dog kidney membrane, human kidney membrane, chick bone cell membrane and human dermal fibroblast membrane were evaluated. The results showed that human kidney plasma membrane, canine kidney plasma membrane and chick bone cell membrane possess one single class of PTH receptor with a Kd (dissociation constant) of 1-5 nM and an IC50 also of 1-5 nM. The number of binding sites was 800 fmol per mg of protein for chick bone cell particulate membrane, 1-5 pmol per mg of protein for human kidney plasma membrane and 2.2 pmol per mg of protein for dog kidney plasma membrane. Photoaffinity labelling identified a major binding component with a molecular mass of 70 kDa in all three types of membrane. The plasma membrane fraction from human dermal fibroblast contained two different binding sites for PTH with high (Kd = 2 nM) and low (Kd = 580 nM) affinities respectively. The IC50 for the adenylate cyclase is about 2 nM, which is similar to the Kd of the high-affinity site. Photoaffinity labelling also demonstrated a major binding component with a molecular weight of 70 kDa. We conclude that structural and functional similarity exists among the PTH receptors present on chick bone cell membrane, dog kidney membrane and human kidney membrane. The human dermal fibroblast possesses two different binding sites, one of which is coupled to adenylate cyclase.     

Involvement of sperm proteases in the binding of sperm to the vitelline envelope in Xenopus laevis

Sperm binding to the vitelline envelope in dejellied Xenopus laevis eggs was effectively inhibited by inhibitors for trypsin (soybean trypsin inhibitor and p-toluenesulfonyl-L-lysine chloroethyl ketone) and aminopeptidase B (o-phenanthroline, bestatin, and arphamenine B). Likewise, synthetic 4-methylcoumaryl-7-amide (MCA) substrates (t-butoxycarbonyl-GlyArgArg-MCA, benzyloxycarbonyl-ArgArg-MCA, and Arg-MCA) inhibited binding. Consistently, when jellied eggs were inseminated in the presence of these substrates or inhibitors for proteases, fertilization was effectively blocked. The medium in which live sperm or the sperm membrane fraction were suspended exhibited hydrolyzing activities against the synthetic substrates mentioned above, and these activities were effectively inhibited by the protease inhibitors. Ultracentrifugal fractionation of the sperm suspension following induction of the acrosome reaction by a calcium ionophore, A23187, indicated that a considerable amount of the total tryptic and aminopeptidase B activity was released into the medium. On this occasion, part of the tryptic and aminopeptidase B activity was definitely estimated to be discharged in association with a vesiculated membrane, supporting the notion that the proteases involved in binding to the vitelline envelope are present on the sperm plasma membrane.     

Mouse SLLP1, a sperm lysozyme-like protein involved in sperm-egg binding and fertilization

This study demonstrates the retention of mouse sperm lysozyme-like protein (mSLLP1) in the equatorial segment of spermatozoa following the acrosome reaction and a role for mSLLP1 in sperm-egg binding and fertilization. Treatment of cumulus intact oocytes with either recmSLLP1 or its antiserum resulted in a significant (P < or = 0.05) inhibition of fertilization. Co-incubation of zona-free mouse oocytes with capacitated mouse spermatozoa in the presence of varying concentrations of anti-recmSLLP1 serum or recmSLLP1 also inhibited sperm-oolemma binding. A complete inhibition of binding and fusion of spermatozoa to the oocyte occurred at 12.5 muM concentration of recmSLLP1, while conventional chicken and human lysozymes did not block sperm-egg binding. mSLLP1 showed receptor sites in the perivitelline space as well as on the microvillar region of the egg plasma membrane. The retention of mSLLP1 in the equatorial segment of acrosome-reacted sperm, the inhibitory effects of both recmSLLP1 and antibodies to SLLP1 on in vitro fertilization with both cumulus intact and zona-free eggs, and the definition of complementary SLLP1-binding sites on the egg plasma membrane together support the hypothesis that a c lysozyme-like protein is involved in the binding of spermatozoa to the egg plasma membrane during fertilization.